There remains a significant need for immunotherapeutics to drive clinically effective anti-tumor T cell responses. Adoptive T cell therapies (ACT) have demonstrated impressive clinical results for several cancers using patient-derived T cells activated ex vivo with potent T cell receptor (TCR), costimulatory and cytokine stimulation. However, cellular therapies reach few patients due to complex manufacturing and reinfusion requirements, patient conditioning regimens and safety considerations. In contrast, systemic costimulation represents a scalable pharmacologic approach to cancer immunotherapy which has the intent of activating and expanding tumor-specific T cells directly within the patient. However, attempts to induce anti-tumor T cell responses using systemic agonism with agents such as anti-CD137 antibodies and high dose interleukin-2 (IL-2) are associated with significant risk of toxicity. IL-2 is of particular interest as a potent cytokine capable of inducing the proliferation and differentiation of CD8 effector T cells (Teff), as well as other T, B, and NK lineages with anti-tumor potential. In the contexts of metastatic renal cell carcinoma and malignant melanoma, high-dose IL-2 can induce curative remissions in a minority of patients, associated with elevated levels of AgS Teff, but is dose limited by severe and potentially life-threatening toxicities such as vascular leak syndrome and cytokine release syndrome. The anti-tumor effects of IL-2 are also indirectly limited by regulatory T cells (Treg), which expand efficiently to IL-2 in vivo due to high level expression of the high affinity IL-2 receptor. Elevated Treg counts, which can limit Teff responses, are associated with poor prognosis in cancer patients. In addition, cancer immunotherapeutic combinations targeting distinct costimulatory pathways hold the potential of greatly amplifying T cell responses. However, as illustrated by the combined inhibition of CTLA-4 and PD-1 pathways in the treatment of metastatic melanoma, toxicities may also compound, limiting their utility. Broader therapeutic use of IL-2 and other costimulatory axes in cancer will likely require focusing their effects on those T cells which stand to deliver the greatest therapeutic effect, in particular cancer AgS T cells.
Cancer vaccines represent another scalable pharmacologic approach to cancer immunotherapy. However, vaccine efficacy depends on antigen-presenting cell (APC) function, including trafficking, antigen-processing, costimulation versus co-inhibition, and susceptibility to tumor immunosuppression. For example, dendritic cells are APC which play critical roles in the priming and maintenance of anti-tumor CD8 T cell responses via delivery of peptide-HLA and potent costimulatory ligands such as CD80, CD86, and CD137L. However, dendritic cells are also replete with inhibitory ligands such as TIM-3, PD-L1, PD-L2, HVEM, B7-H3, B7-H4, IL-T3, and IL-4, which blunt T cell responses and are subject to local and distal tumor influence. Therefore, safe, scalable and APC-independent immunotherapeutics are needed that will enable clinically effective levels of anti-tumor T cell activation and associated tumor cell killing. We have identified a potential solution to this challenge based on the natural signals governing T cell function: peptide-HLA and costimulatory ligands, embodied in the Immuno-STAT (Selective Targeting and Alteration of T cells) and Neo-STAT immunotherapeutics platforms. Immuno-STAT and Neo-STAT utilize compact, Fc-based architectures amenable to clinical manufacturing, and are designed to focus optimized signals for potent costimulatory axes such as IL-2 directly to AgS T cells in vivo, thereby enhancing anti-tumor T cell responses while avoiding indiscriminate immune activation.
The Immuno-STAT framework comprises a covalent fusion of peptide epitope, MHC class I allele, co-modulator, and Fc, which imparts avidity and symmetrical multivalency, sufficient for cognate T cell activation. Potential co-modulators, costimulatory and co-inhibitory ligands, may be fused to the N- or C-terminus of the MHC-Fc heavy chain or the C-terminus of β2m, allowing organizational, compositional and stoichiometric flexibility. Our initial exploration of the Immuno-STAT platform utilized IL-2 as the co-modulator to activate and expand AgS cytotoxic effector T cells (Teff).
Immuno-STAT design, optimization and activity. (a) Immuno-STAT frameworks comprising peptide (pep) epitope, β2m, MHC heavy chain, Fc, and co-modulatory domain (MOD) in different relative positions, covalently linked by engineered (ss) and native ( =) disulfide bonds. (b) In vitro pSTAT5 activity of P14 transgenic versus C57BL/6 CD8 splenocytes challenged with construct LCMV-IST-IL2.FH 4. Inset: Fc fusion bearing four IL2.FH but without pMHC. %pSTAT5+ responses within each independent dose response titration were normalized as: normalized response = (sample − minimum)/(maximum − minimum). Data represent mean ± SD of duplicate samples from three independent experiments. (c) Humanized Immuno-STAT framework comprising two copies of pHLA, four copies of IL2.FH and human IgG1 Fc. (d) Representative dual-tetramer plots of human PBMC stimulated in vitro for ten days with media, CMV-IST-IL2.FH 4, or MART-IST-IL2.FH 4. (e) Peak fold expansion of AgS T cells from human PBMC incubated with CMV-IST-IL2.FH 4 and IST-MART-IL2.FH 4, from 1 to 4 independent expansions per donor. (f) Representative IFN-γ, TNF-α, CD107a, and granzyme B staining following cognate peptide challenge of Immuno-STAT-expanded CMV- or MART-specific T cells. (g) Paired TCRαβ sequence frequencies from individual CMV- or MART-specific CD8 T cells expanded with cognate Immuno-STAT or peptide. Lines connect Immuno-STAT- and peptide-expanded clones having TCRαβ sequence identity. Three donors per specificity. Total clones surveyed in parentheses.
To identify an optimized IL-2-based Immuno-STAT framework, we evaluated a panel of constructs for relative potency, AgS selectivity, and manufacturability. Constructs comprised LCMV gp 33-41/H-2D b, recognized by the murine TCR P14, fused at its N-terminus to variants of human IL-2 and C-terminally to an effector-attenuated murine IgG2a Fc. IL-2-attenuating mutations were included to limit IL-2Rα-dependent toxicity and Treg engagement as well as to reduce IL-2 affinity in favor of pMHC selectivity. IL-2 stoichiometries were limited to two or four based on manufacturability which showed a significant drop in protein titer beyond four copies of IL-2. Human IL-2 exhibits potent activity on both human and mouse cells including phosphorylation of STAT5, an IL-2 receptor (IL-2R) proximal signaling molecule and phosphorylated STAT5 (pSTAT5) serves as an index of IL-2R engagement which correlates well with downstream consequences of IL-2R agonism such as proliferation and phenotypic marker expression. We compared pSTAT5 induction for purified CD8 splenocytes from AgS P14 TCR transgenic mice to non-AgS C57BL/6 mice and ranked constructs based on logEC50 P14 (potency index), P14 minus C57BL/6 signal at EC50 P14 (selectivity index), and protein expression titer. Construct LCMV-IST-IL2.FH 4 ranked highest followed by LCMV-IST-IL2.F 4, bearing four copies of IL-2 F42 A, H16A or IL-2 F42A, respectively. Dose responses for top candidates LCMV-IST-IL2.FH 4 and LCMV-IST-IL2.F 4 from this initial screen were reevaluated over a broader concentration range and with greater resolution and compared with reference constructs bearing two or four copies of wildtype IL-2, LCMV-IST-IL2 2 or LCMV-IST-IL2 4, respectively. AgS potency, as indicated by the logarithm of the pSTAT5 EC50 for responding P14 splenocytes (logEC50 P14), was not significantly different between LCMV-IST-IL2.FH 4, LCMV-IST-IL2.F 4, LCMV-IST-IL2 2 and LCMV-IST-IL2 4. Likewise, non-AgS potency (logEC50 B6) was also similar for these constructs. AgS selectivity was measured first by the difference in normalized pSTAT5 signal at EC50 P14 for AgS (P14) splenocytes (i.e. 50%) relative to non-AgS (C57BL/6) splenocytes as well as by the difference in the logEC50 for AgS versus non-AgS splenocytes. Both measures of AgS selectivity were not significantly different across constructs LCMV-IST-IL2.FH 4, LCMV-IST-IL2.F 4, LCMV-IST-IL2 2 and LCMV-IST-IL2 4. Thus, significant F42A- and H16A-mediated decreases in IL-2R signaling potency or selectivity were not observed relative to reference constructs bearing two or four copies of wildtype IL-2 (LCMV-IST-IL2 2 or LCMV-IST-IL2 4), presumably masked in part by the increased IL-2 stoichiometry.
We next generated and tested humanized Immuno-STATs bearing IL2.FH 4, effector-attenuated human IgG1, HLA-A*0201 and model epitopes CMV pp65 495-503 or MART1 26-35. For multiple donors, we determined the frequency of dual-tetramer-positive AgS CD8 T cells following a 10 day incubation of human PBMC with specific IST-IL2.FH 4. CMV-IST-IL2.FH 4, but not CMV-IST (no IL-2 fusion) was able to expand CMV-specific CD8 T cells from PBMC, indicating that proliferation was dependent on the presence of IL2.FH 4. Similarly, CMV-IST in the presence, but not absence, of recombinant human IL-2 (wildtype)-expanded CMV-specific CD8 T cells in vitro, further supporting the requirement for IL-2R agonism for Immuno-STAT activity. Peak expansions (> 30 fold) were similar for PBMC CD8 T cells responsive to CMV-IST-IL2.FH 4 or MART-IST-IL2.FH 4. In addition, peak expansions for PBMC CD8 T cells responsive to CMV-IST-IL2.FH 4 or MART-IST-IL2.FH 4 were achieved at similar Immuno-STAT concentrations. However, higher CMV- versus MART-specific frequencies were prese
