Most solid tumors are comprised of multiple clones that express orthogonal antigens, suggesting that novel strategies must be developed in order to adapt chimeric antigen receptor (CAR) T-cell therapies to treat heterogeneous solid tumors. Here, we utilized a cocktail of low-molecular-weight bispecific adapters, each comprised of fluorescein linked to a different tumor-specific ligand, to bridge between an antifluorescein CAR on the engineered T cell and a unique antigen on the cancer cell. This formation of an immunologic synapse between the CAR T cell and cancer cell enabled use of a single antifluorescein CAR T cell to eradicate a diversity of antigenically different solid tumors implanted concurrently in NSG mice. Based on these data, we suggest that a carefully designed cocktail of bispecific adapters in combination with antifluorescein CAR T cells can overcome tumor antigen escape mechanisms that lead to disease recurrence following many CAR T-cell therapies. Significance: A cocktail of tumor-targeted bispecific adapters greatly augments CAR T-cell therapies against heterogeneous tumors, highlighting its potential for broader applicability against cancers where standard CAR T-cell therapy has failed.

Purpose: The ability to locate and remove all malignant lesions during radical prostatectomy leads not only to prevent biochemical recurrence (BCR) and possible side effects but also to improve the life expectancy of prostate cancer (PCa) patient. Fluorescence-guided surgery (FGS) has emerged as a technique that uses fluorescence to highlight cancerous cells and guide surgeons to resect tumors in real-time. Thus, development of tumor-specific near-infrared (NIR) agents that target biomarkers solely expressed on PCa cells will enable to assess negative tumor margins and affected lymph nodes. Experimental Design: Since PSMA is overexpressed in PCa cells in > 90% of PCa patient population, a PSMA-targeted NIR agent (OTL78) was designed and synthesized. Optical properties, in vitro and in vivo specificity, tumor-to-background ratio (TBR), accomplishment of negative surgical tumor margins using FGS, pharmacokinetics (PK) properties, and preclinical toxicology of OTL78 were then evaluated in requisite models. Results: OTL78 binds to PSMA-expressing cells with high affinity, concentrates selectively to PSMA-positive cancer tissues, and clears rapidly from healthy tissues with a half-time of 17 min. It also exhibits an excellent TBR (5:1) as well as safety profile in animals. Conclusions: OTL78 is an excellent tumor-specific NIR agent for use in fluorescence-guided radical prostatectomy and FGS of other cancers.

Multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors. Such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein. However, the lack of a monovalent folic acid–polymer material has prevented a full binding analysis of these conjugates, as multivalent binding mechanisms and polymer-mass mechanisms are convoluted in samples with broad distributions of folic acid-to-dendrimer ratios. In this work, the synthesis of a monovalent folic acid–dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid–polymer conjugate and a folate binding protein surface. The increased avidity is due to a folate-keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow-onset, tight-binding mechanisms of ligand/protein interactions.