trispecific SbCAR

Stages

1. 1.
   Phage display identification of sherpabodies(broad_screen)

   This stage generates antigen-binding sherpabodies that can serve as targeting modules for downstream CAR engineering.

   Selection: Identification of sherpabodies against a panel of popular tumor-associated antigens.

2. 2.
   Incorporation into second-generation CAR constructs(library_build)

   This stage converts selected binders into functional T-cell receptor constructs for downstream testing.

   Selection: Selected sherpabodies were incorporated into second-generation CAR constructs termed SbCAR.

3. 3.
   In vitro functional characterization(functional_characterization)

   This stage tests whether engineered SbCARs function specifically and kill target cells before in vivo evaluation.

   Selection: Potent in vitro specificity and cytotoxicity against solid cancer TAAs without cross-reactivity to closely related proteins.

4. 4.
   Logic and multispecific architecture characterization(secondary_characterization)

   This stage extends baseline CAR function into more advanced circuit behaviors enabled by sherpabody modularity and small size.

   Selection: Assessment of trispecific OR-logic activation and IF-THEN logic circuits in combination with synthetic Notch.

5. 5.
   Xenograft mouse validation(in_vivo_validation)

   This stage tests whether SbCAR T cells retain antitumor function in vivo after in vitro characterization.

   Selection: Dose-dependent antitumor response in xenograft mouse models.

Steps

1. 1.
   Identify sherpabodies against tumor-associated antigens by phage displayengineered binder being selected

   Recover sherpabody binders against a panel of tumor-associated antigens.

   Binder identification is required before the binders can be incorporated into CAR constructs.

2. 2.
   Incorporate selected sherpabodies into second-generation CAR constructs to create SbCARsbinder converted into CAR targeting module

   Build sherpabody-guided CAR constructs for T-cell testing.

   The selected binders must be embedded in a CAR architecture before functional T-cell assays can be performed.

3. 3.
   Test SbCARs for in vitro specificity and cytotoxicity and assess cross-reactivityCAR construct being functionally screened

   Determine whether SbCARs specifically kill target cells while avoiding recognition of closely related proteins.

   In vitro testing provides functional evidence before more complex logic designs or in vivo validation.

4. 4.
   Build and test multispecific and logic-gated SbCAR variantsadvanced SbCAR variants being characterized

   Evaluate whether sherpabody modularity supports trispecific OR logic, synthetic Notch IF-THEN logic, and inducible control formats.

   Advanced circuit formats are enabled after establishing that the base sherpabody-guided CAR architecture functions.

5. 5.
   Evaluate SbCAR T cells in xenograft mouse models for antitumor responsetherapeutic CAR T-cell product under in vivo validation

   Test whether the engineered SbCAR platform produces antitumor activity in vivo.

   Mouse validation follows in vitro functional evidence to assess therapeutic potential in a higher-fidelity setting.