(M1330-04-20) Immunomodulation Improves Cancer Cell Clearance During Multi-Antigen T Cell Hybridizer Therapy

Purpose: Multi-antigen T cell hybridizer (MATCH) therapy is a novel treatment paradigm which uses hybridization of complementary morpholino oligonucleotides to recruit patient’s autologous T cells to target and eliminate B cell malignancies [1]. Heterodimerization of a cancer-targeting Fab’_x-MORF1 with a T cell directed Fab’_CD3-MORF2 [2] creates a construct that resembles a bispecific antibody. MATCH is a split antibody technology; its advantage is the possibility to create a targeting motif library based on the expression of receptors on patient cancer cells. Thus, a combination of Fab’_CD38-MORF1, Fab’_CD20-MORF1, Fab’_BCMA-MORF1, Fab’_CD319-MORF1 or Fab’_CD19-MORF1 with Fab’_CD3-MORF2 can be generated and T cells activated against leukemia, lymphoma, and myeloma cells. The system is active both in vitro and in vivo [1]; following hybridization, effector cells attack the cancer cells. However, upon repeated activation T cells experience a state of cellular exhaustion [3]. This exhaustion state is characterized by a reduction of effector output (such as interferon gamma (IFN-γ) and granzyme B release), limiting the extent of the therapy. The purpose of this study is to quantify and assess exhaustion patterns during MATCH therapy and to evaluate the success of potential combinatorial treatments to reduce T cell exhaustion and improve the efficacy of cancer therapy.

Methods: Two complementary 25 base pair phosphorodiamidate MORFs were customized from Gene Tools. Fab’-MORF conjugates were synthesized as previously described (Figure 1) [2]. Raji (B cell lymphoma) cells were purchased from American Type Culture Collection. Healthy human T cells were isolated from peripheral blood mononuclear cells (PBMC)s purchased from StemCell. To quantify T cell exhaustion trends in response to MATCH therapy, T cells were initially co-cultured with Raji cells and MATCH conjugates. At 24 h, and again at 48 h, additional Raji cells and MATCH conjugates were added to the co-culture to simulate repeated antigenic exposure and activation. At each time point, an aliquot of cells was removed and flow cytometry was used to assess the expression of exhaustion receptors (PD-1 and TIM3) and T cell activation and migration (CD44), as well as quantify the amount of residual cancer cells. IFN-γexpression was quantified through ELISA. To examine the success of potential combinatorial ‘exhaustion treatments’, healthy human T cells were co-cultured with Raji cells for 48 h. One group only received MATCH conjugates, while the other experimental groups received various doses of the combinatorial exhaustion therapy in addition to MATCH conjugates. For this study, PD-1 antibody blockade and IL-10 antibody blockade were assessed as the combinatorial treatments.

Results: PD-1, TIM3, CD44, and IFN-γ expression patterns demonstrated the same general trend during the 72-h antigen rechallenge co-cultures (Figure 2 A-D). At 24 h, significant elevations were observed for all 4 analytes in the MATCH-treated group in comparison to the untreated co-culture. These levels remained elevated or further increased at 48 h. At 72 h, expression of all 4 analytes then decreased close to baseline levels. Combinatorial PD-1 or IL-10 blockade treatment showed similar patterns (Figure 2 E-F). Upon administration of MATCH alone, the residual B cell count at the end of co-culture was significantly reduced in comparison to untreated co-cultures. With increasing doses of antibody blockade (in addition to MATCH), the residual dose was further significantly reduced, with the lowest residual cancer cell amount observed at 10 μg/mL of antibody blockade. Confocal microscopy of co-cultured T cells and Raji cells confirmed the presence of MATCH-induced T cell degranulation events (Figure 3 A). Untreated co-culture of T cells and Raji cells showed nonspecific cell aggregation and a lack of T cell degranulation events. MATCH-treated co-culture of T cells and Raji cells shows several distinct degranulation events at cell interfaces. MATCH-induced T cell degranulation led to cancer cell apoptosis (Figure 3 B).

Conclusion: Based on the expression trends of PD-1 (exhaustion), TIM3 (exhaustion), IFN-γ (T cell function), and CD44 (T cell migration and activation), MATCH induces an activated T cell state that is linked with increasing exhaustion. After exhaustion markers are elevated for multiple days, T cell function decreases. This suggests that exhaustion and activation are correlated and induced by MATCH therapy. Further, even though the same dose of MATCH conjugates and the same quantity of Raji cells were added at all time points, T cells gradually lost their effector functions (determined by IFN-γ release). The residual cancer cell count suppression observed in response to combinatorial blockade treatment demonstrates that both PD-1 blockade and IL-10 blockade functionally reduce T cell exhaustion and improve cancer cell clearance in response to MATCH therapy. Since PD-1 is an inhibitory cell surface receptor and IL-10 is an inhibitory cytokine, this suggests that multiple pathways for reducing T cell exhaustion are possible, and these combinatorial regimens may be further optimized to improve MATCH efficacy. Additionally, this study suggests the possibility of simultaneously administering a checkpoint inhibitor with MATCH therapy to more effectively treat complex or late-stage B cell malignancies.

References: 1. M.T. Gambles, S. Li, I. Kendell, J. Li, D. Sborov, P. Shami, J. Yang, J. Kopeček, Multi-Antigen T Cell Hybridizers. In preparation.
2. T.-W. Chu, R. Zhang, J. Yang, M.P. Chao, P.J. Shami, J. Kopeček, A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab. Theranostics 5, 834-846 (2015).
3. C.U. Blank, W.N. Haining, W. Held, P.G. Hogan, A. Kallies, E. Lugli, R.C. Lynn, M. Philip, A. Rao, N.P. Restifo, A. Schietinger, T.N. Schumacher, P.L. Schwartzberg, A.H. Sharpe, D.E. Speiser, E.J. Wherry, B.Y. Youngblood, D. Zehn, Defining T Cell Exhaustion. Nat. Rev. Immunol. 19, 665-674 (2019).

Acknowledgements: The research was supported by NIH grant R01 CA246716 (to JK) from the National Cancer Institute and by University of Utah UROP award (to IK).


Figure 1. Synthesis and characterization of Fab’-MORF. A generalized synthesis schematic for a single Fab’-MORF conjugate is demonstrated (A). FPLC data from pepsin digestion of whole antibody (B) and intermediates in the synthesis process (C) is presented as well.


Figure 2. MATCH therapy initially induces T cell exhaustion. Expression of exhaustion markers PD-1 (A) and TIM3 (B) on T cells increases in response to MATCH therapy for 48 h after treatment. Fab’CD3-MORF2 does not induce a similar response. At 72 h, expression of PD-1 declines. Concurrent with these exhaustion trends, CD44 expression on T cells (C) increases for the first 48 h of treatment, followed by a return to baseline levels. IFN-γ expression in the supernatant (D) markedly increases over the first 48 h (35-fold), followed by a notable decrease at 72 h. Combinatorial PD-1 blockade (E) and IL-10 blockade (F) improve cancer clearance. PD-1 blockade attenuates exhaustion and improves rates of B cell ablation. MATCH treatment alone reduces the residual B cell population from nearly 75% to 13% in this trial. Increasing doses of PD-1 blockade further suppress that residual value to 5%. Similarly, IL-10 blockade also attenuates exhaustion and improves rates of B cell ablation. MATCH treatment alone reduces the residual B cell population from 70% to 40% in this trial. Increasing doses of IL-10 blockade further suppress that residual value to 20%. Data are represented as mean ± SD (n=3).


Figure 3. MATCH therapy induces degranulation of T cells. Red color indicates CD10, a general B cell marker and green (LysoTracker) indicates T cell granules, in this instance. Untreated co-culture of T cells and Raji cells shows non-specific aggregation and an absence of clear T cell degranulation events. Following MATCH treatment, co-culture of T cells and Raji cells demonstrates several degranulation events at T cell-Raji cell interfaces (A). Mitochondrial depolarization, caspase activation, and generation of reactive oxygen species in target Raji cells are increased with MATCH treatment in co-cultures of T cells and Raji cells, as measured by flow cytometry (B).