The disclosure relates generally to alpha polyglutamated pralatrexate, formulations containing liposomes filled with alpha polyglutamated pralatrexate, methods of making the alpha polyglutamated pralatrexate and liposome containing formulations, and methods of using polyglutamated alpha polyglutamated pralatrexate and liposome containing formulations to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., an autoimmune disease such as rheumatoid arthritis).