Abstract Background: Antifolates targeting glycinamide ribonucleotide formyltransferase (GARFTase) disrupt cell division (mitosis) by inhibiting the de novo purine biosynthesis pathway. Recently, novel GARFTase inhibitors, exhibiting high folate receptor (FR) binding affinity and low affinity for the reduced folate carrier (RFC), have been explored as chemotherapeutic agents. Theoretically, these GARFTase inhibitors could also be used as FR-targeting ligands for delivering additional payloads to tumor types over-expressing the FR. In this poster, we will present our practical approach to explore the dual role of these new GARFTase inhibitors as cytotoxic agents and as targeting ligands for delivering tubulysins. Tubulysins are natural products isolated from myxobacterial species and are highly potent mitotic poisons that inhibit tubulin polymerization. Methods: We developed a general method for preparing dual drug/ligand conjugates possessing two disulfide-based self-immolative linker systems. In our synthetic protocol we relied on consecutive introduction of two heterosubstituted disulfide bonds. A GARFTase inhibitor was first connected to a hydrophilic spacer unit through the first disulfide-based linker system. The obtained conjugate contains also an S-9-fluorenylmethyl (Fm)-protected thiol in its molecular architecture. Treatment with a strong organic base resulted in spontaneous removal of the Fm moiety. The deprotected thiol reacted in situ with a propreatary thiophilic tubulysin derivative, present in the reaction mixture, and a second self-immolative disulfide-based linker system was formed. Results: Novel conjugates of GARFTase inhibitor with tubulysins were prepared using newly designed synthetic strategies. In a high-yielding chemoselective reaction we introduced a second disulfide bond into a molecular framework without destroying the pre-existing disulfide linker system. The versatility of this synthetic protocol was demonstrated by synthesizing several related drug conjugates. Conclusion: In the poster to be presented, we will disclose, for the first time, the structure and synthesis of novel conjugates of GARFTase inhibitors with tubulysins. The target compounds contain two self-immolative disulfide linker systems. The observed excellent in vivo activity in tumor xenografts demonstrates the dual role of GARFTase inhibitors as therapeutic agents and as FR-targeting ligands for simultaneously delivering two highly potent payloads. Citation Format: Iontcho R. Vlahov, Fei You, Hanna F. Klein, Paul J. Kleindl, Melissa Nelson, Marilynn Vetzel, Joseph A. Reddy, Christopher P. Leamon, Larry H. Matherly, Aleem Gangjee. Design and synthesis of novel conjugates for targeting the folate receptor: Exploiting cytotoxic GARFTase inhibitors for delivering additional chemotherapeutic payloads to cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2015-4393