Compositions and methods for adoptive cell therapy for cancer
Cellular, cancer technology, applied in the field of compositions and methods for adoptive cell therapy of cancer
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Embodiment 1
[0465] Example 1. Synthesis of prodrugs of cytotoxic and targeting small molecules for use with SEAKER cells.
[0466] This example describes adenosine-sulfamoyl (AMS) (structure 1c)-based Figure 2C Synthesis of an exemplary prodrug of ) to be cleaved by the bacterial hydrolase CPG2. AMS is a close analogue of ribozin and a potent cytotoxic molecule (HepG2 IC 50 = 9 nM). A detailed structure-activity relationship (SAR) study was performed, which indicated that, through the 6-amino (Me 2 , >500 μM) C2 (Ph, ≈500 μM), C8 (n-Pr, >250 μM) or substitution at the sulfamate position (salicyl, >500 μM), the cytotoxicity of AMS was significantly reduced or eliminated. Based on these SAR data, 6-N-acylation, desilylation (TBAF), sulfamylation (H 2 NSO 2 Cl, DMA) and acetonide hydrolysis (TFA, H 2 O, 15% after 3 steps) from 2′,3′-O-isopropylidene-5′-O-TBS-adenosine (structure 3, Figure 2D ) synthesis of AMS prodrug 1a ("P-AMS"; Figure 2C ). Two alternative N-sulfamate-linked p...
Embodiment 2
[0529] Example 2. Method of Testing Prodrug Candidates
[0530] Candidate prodrugs for use with selected prodrug converting enzymes can be tested by in vitro assays and filtered at each step to achieve target product profiles (Table 2).
[0531] Table 2
[0532]
[0533]These results can be used to design improved prodrugs. The kinetics (Km, kcat) of CPG2-mediated prodrug cleavage were determined using recombinant, purified CPG2 (Jeyaharan et al. Protein Expr Purif. 127:44-52 (2016)). As previously for ZD2767P (Springer et al., J Med Chem.37 (15): 2361-70 (1994) and Springer et al., J Med Chem. 38 (26): 5051-65 (1995)) and methotrexate ( The reaction was monitored based on decreased UV absorbance as described by Sherwood et al., Eur J Biochem. 148(3):447-53 (1985)). The stability of the prodrug was confirmed under physiological conditions. Prodrugs with kcat / Km≥1s–1 μM–1 (ideally kcat / Km≥10) were developed.
[0534] The cytotoxicity of each prodrug / drug pair can be tes...
Embodiment 3
[0538] Example 3. Construction of Cells Expressing Chimeric Antigen Receptors
[0539] This example describes constructs for cells comprising a CAR (eg, with an antigen-binding domain specific for CD19, WT1, or PRAME). In this exemplary embodiment, CPG2 is engineered to have a transmembrane domain and localize to the extracellular side of the cell membrane, where it can hydrolyze and activate a prodrug in contact with the cell to the active drug (e.g., the prodrug of Example 1 , which are hydrolyzed to active cytotoxic drugs). Activation of the drug locally enhances cytolysis within the tumor at the site of targeting of CAR T cells via the antigen-binding domain.
[0540] CD19 CAR T cells and WT1 CAR T cells have been made and initial publications describing methods for generating them and characterizing their activity (Brentjens et al., Sci.Trans.Med.5(177):177ra38(2013), Pegram et al Leukemia 29(2):415-22(2015)). PRAME-reactive CAR T cells will be generated and characteri...
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