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, 8 (3), 4520-4529

Potent CD4+ T Cell-Associated Antitumor Memory Responses Induced by Trifunctional Bispecific Antibodies in Combination With Immune Checkpoint Inhibition


Potent CD4+ T Cell-Associated Antitumor Memory Responses Induced by Trifunctional Bispecific Antibodies in Combination With Immune Checkpoint Inhibition

Nina Deppisch et al. Oncotarget.


Combinatorial approaches of immunotherapy hold great promise for the treatment of malignant disease. Here, we examined the potential of combining an immune checkpoint inhibitor and trifunctional bispecific antibodies (trAbs) in a preclinical melanoma mouse model using surrogate antibodies of Ipilimumab and Catumaxomab, both of which have already been approved for clinical use. The specific binding arms of trAbs redirect T cells to tumor cells and trigger direct cytotoxicity, while the Fc region activates accessory cells eventually giving rise to a long-lasting immunologic memory. We show here that T cells redirected to tumor cells by trAbs strongly upregulate CTLA-4 expression in vitro and in vivo. This suggested that blocking of CTLA-4 in combination with trAb treatment enhances T-cell activation in a tumor-selective manner. However, when mice were challenged with melanoma cells and subsequently treated with antibodies, there was only a moderate beneficial effect of the combinatorial approach in vivo with regard to direct tumor destruction in comparison to trAb therapy alone. By contrast, a significantly improved vaccination effect was obtained by CTLA-4 blocking during trAb-dependent immunization. This resulted in enhanced rejection of melanoma cells given after pre-immunization. The improved immunologic memory induced by the combinatorial approach correlated with an increased humoral antitumor response as measured in the sera and an expansion of CD4+ memory T cells found in the spleens.

Keywords: CTLA-4; T-cell activation; cancer immunotherapy; melanoma; tumor antigen.

Conflict of interest statement


H.L. is the CEO of Trion Research and the inventor or co-inventor of several trifunctional antibody patents. P.R. is an employee of Trion Research. The other authors disclose no potential conflicts of interest.


Figure 1
Figure 1. CD69 and CTLA-4 induction on T cells activated with trAbs in vitro
T cells were cultivated with DCs, irradiated B78-D14 or B16-EpCAM cells and trAb Surek or BiLu, respectively, as outlined in Materials and Methods. At different time points, surface expression of CD69 and CTLA-4 on CD4+ and CD8+ T cells was determined by FACS analyses. Mean values and standard deviations from 3 independent experiments are shown.
Figure 2
Figure 2. Counter-regulatory mechanisms induced by trAb treatment in vivo
Mice received irradiated B78-D14 cells with or without 10 μg of trAb Surek i.p. After 2 and 4 days, spleens were isolated and CD4+ T cells were phenotypically characterized. (A) Expression of CTLA-4 on CD4+ T cells. (B) Percentages of CD4+FoxP3+ cells. (C) Ki67 as a proliferation marker was stained in CD4+FoxP3+ T cells. (D) CTLA-4 upregulation is mainly restricted to the Treg population. Gating was done for CD4+ cells. At least 3 animals were included in each group. Columns indicate means and SEM. Statistics was done using the Mann-Whitney test.
Figure 3
Figure 3. Direct trAb-mediated tumor destruction is moderately improved by combining trAb and anti-CTLA-4 therapy
Antibody treatment of mice started 2 days after challenge with 105 B78-D14 or B16-EpCAM cells. In the experiments shown, 5 to 10 mice were included. (A) Blocking of CTLA-4 alone by HB304 has only a marginal effect on tumor killing. (B) Survival of mice after therapy with Surek alone or with Surek simultaneously delivered with HB304. (C) Moderate survival benefit of mice treated with trAb BiLu and HB304 in comparison to monotherapy in the B16-EpCAM model. Significances were determined using the logrank test.
Figure 4
Figure 4. Immunologic memory induced by trAb Surek with or without CTLA-4 blocking
Immunization was done as outlined in Materials and Methods using the indicated immunogens. Irradiated B78-D14 cells as an antigen source were included in each setting. (A) Th1/Th2 cytokines in sera of mice on day 21 after starting immunization. Up to 8 mice were included in each group. For all cytokines tested, the difference between the Surek and the Surek/HB304 group was not significant, while these two groups were different from all other groups with P < 0.005 (Mann-Whitney). (B) Tumor-directed antibodies in sera of mice vaccinated with different immunogens. Binding of serum IgG1 and IgG2a antibodies to melanoma cells was measured by flow cytometry. Mean fluorescence intensities (MFI) are shown as a quantitative measure for antibody coating of tumor cells. Up to 5 animals were included in each group. The columns show means and SEM. Significances (Mann-Whitney) are indicated. The differences between IgG1 titers are not significant. (C) On day 21, mice were challenged with a lethal dose of melanoma cells. The significance is denoted (logrank).
Figure 5
Figure 5. Characterization of memory T cells after immunization with Surek alone or in combination with CTLA-4 blocking
T cells from spleens of differentially vaccinated mice were stimulated in vitro with splenocytes that were loaded with melanoma-derived peptides [17]. After 7 days, T cells were analyzed for expression of CD4, CD8, CD44 and CD62L. At least 4 mice were included in each group. (A) Representative diagram delineating the differentiation between different memory and effector T-cell compartments. (B) Quantitation of CD4+ T-cell subpopulations from differentially vaccinated mice. (C) Expression of CD137 on CD4+ T cells after in vivo immunization and restimulation with melanoma peptides. CD137+ cells were exclusively identified as CD44+ memory cells (not shown). (D) In the CD8+ T-cell population, an expansion of the memory compartment or a significant upregulation of CD137 was not induced by the combination approach. In all panels, columns represent mean values and SEM. Significances are also shown (Mann-Whitney).

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