Project Grant R21CA274064

INVESTIGATING PROTAC-MEDIATED ANTIGEN DEGRADATION AS A NOVEL STRATEGY TO ENHANCE CYTOTOXIC T-CELL RESPONSES AGAINST CANCER IN VIVO. - ABSTRACT CYTOTOXIC T LYMPHOCYTES (CTLS) ARE KEY IMMUNE EFFECTORS AND ABLE TO KILL CANCER CELLS UPON RECOGNITION OF ANTIGENS PRESENTED ON MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLASS I MOLECULES. PROTEASOMAL DEGRADATION OF ENDOGENOUS PROTEINS INTO PEPTIDES IS ESSENTIAL FOR THIS PROCESS AND A RATE-LIMITING STEP IN MHC CLASS I PRESENTATION. CANCER CELLS OFTEN DOWNREGULATE MHC CLASS I PRESENTATION TO AVOID RECOGNITION AND DESTRUCTION BY CTLS. DESPITE OF SIGNIFICANT PROGRESS IN CANCER IMMUNOTHERAPY, THERE IS CURRENTLY NO THERAPEUTIC STRATEGY AVAILABLE TO SPECIFICALLY ENHANCE THE MHC CLASS I PRESENTATION OF CANCER-ASSOCIATED ANTIGENS. THIS, HOWEVER, COULD BE GAME-CHANGING BY COMPLEMENTING CURRENT THERAPIES INCLUDING CHECKPOINT BLOCKADE AND THERAPEUTIC CANCER VACCINES THAT REMAIN INEFFECTIVE IN A MAJORITY OF CANCER PATIENT. THIS PROJECT PROPOSES A NOVEL TYPE OF IMMUNOTHERAPY UTILIZING PROTEOLYSIS TARGETING CHIMERAS (PROTACS) TO INCREASE THE IMMUNOLOGICAL VISIBILITY OF CANCER TOWARDS CTL-MEDIATED KILLING BY SPECIFICALLY BOOSTING MHC CLASS I PRESENTATION. PROTACS ARE HETERO-BISPECIFIC MOLECULES THAT LINK A SPECIFIC E3 LIGASE COMPLEX WITH A TARGET PROTEIN THAT IS CONSEQUENTLY DEGRADED BY THE UBIQUITIN/PROTEASOME SYSTEM. SO FAR, PROTACS HAVE EXCLUSIVELY BEEN ENVISIONED AS CHEMOTHERAPIES TO DEGRADE ESSENTIAL TUMOR PROTEINS (DIRECT ANTI-CANCER EFFECT). INTERESTINGLY, FIRST CLINICAL TRIALS SHOW THE EFFICACY OF CUSTOM- DESIGNED PROTACS TARGETING ENDOGENOUS PROTEINS, THEREBY OPENING THE DOOR TO A MYRIAD OF NOVEL CLINICAL APPLICATIONS. PREVIOUS STUDIES DEMONSTRATE THAT PROTAC-MEDIATED DEGRADATION OF MODEL ANTIGENS SIGNIFICANTLY ENHANCES THEIR MHC CLASS I PRESENTATION. THIS PROJECT EXPLORES PROTACS AS NOVEL TOOLS TO IMPROVE CTL-MEDIATED CANCER CELL DEPLETION IN VIVO, USING PRECLINICAL MOUSE MODELS FOR MELANOMA. AS PROTACS TARGETING CLINICALLY-RELEVANT CANCER ANTIGENS ARE YET TO BE DEVELOPED, IT UTILIZES THE RECENTLY INTRODUCED DEGRADATION TAG (DTAG) SYSTEM TO DEGRADE PROTEINS EXPRESSED IN LINEAR FUSION WITH THE DEGRADATION TAG FKBP12 (F12). TWO NOVEL CLONES OF MURINE B16F10 MELANOMA CELLS HAVE ALREADY BEEN GENERATED FOR THESE EXPERIMENTS: B16F10 CELLS EXPRESSING A) AN F12-TAGGED MODEL ANTIGEN, AND B) A CLINICALLY-RELEVANT MELANOMA ANTIGEN THAT HAS BEEN ENDOGENOUSLY TAGGED WITH THE DEGRADATION TAG F12 USING CRISPR/CAS9-MEDIATED GENOME EDITING. T-CELL RECEPTOR TRANSGENIC CTLS SPECIFIC FOR THESE ANTIGENS WILL BE USED TO STUDY PROTAC-MEDIATED EFFECTS ON CANCER GROWTH AND REGRESSION IN MICE. SPECIFIC AIMS ARE (1) TO DEMONSTRATE THAT PROTACS CAN IMPROVE CANCER-SPECIFIC CTL RESPONSES AND THEREBY PROMOTE TUMOR REGRESSION IN VIVO, (2) TO SHOW THAT THE PROTAC-MEDIATED INCREASE IN MHC CLASS I PRESENTATION CAN COMPLEMENT EXISTING CANCER IMMUNOTHERAPIES, SUCH AS IMMUNE CHECKPOINT BLOCKADE, AND (3) TO TRANSLATE THESE FINDINGS INTO THE HUMAN SYSTEM BY PROVIDING EVIDENCE THAT PROTACS CAN ENHANCE THE KILLING OF HUMAN MELANOMA CELLS BY PATIENT-DERIVED CTLS IN VITRO. THIS PROJECT WILL IMPACT THE FIELD OF CANCER IMMUNOTHERAPY BY PROVIDING FIRST IN VIVO EVIDENCE THAT PROTACS CAN BE USED AS NOVEL TOOLS TO ENHANCE MHC CLASS I-MEDIATED CTL RESPONSES IN CANCER. IT THEREFORE EXTENDS PREVIOUS DISCOVERIES IN THE PROTAC FIELD TOWARDS NEW DIRECTIONS AND APPLICATIONS AND PROVIDES THE RATIONALE TO DESIGN PROTACS DIRECTLY TARGETING ENDOGENOUS CANCER ANTIGENS, PERMITTING THE CLINICAL TRANSLATION OF THIS NOVEL APPROACH.