Combination of a Bispecific Antibody and Costimulatory Antibody-Ligand Fusion Proteins for Targeted Cancer Immunotherapy

Abstract

Initiation of a tumor-directed immune response and appropriate modulation of its progress are key issues in cancer immunotherapy. Combinatorial strategies addressing both aspects might therefore be especially suitable. Here, we report a targeted approach combining a bispecific antibody with 2 costimulatory antibody-ligand fusion proteins. According to the concept, the bispecific antibody (scDbFAP×CD3) retargets T cells in a MHC-independent manner to tumor cells, providing an artificial first signal that allows the costimulatory antibody-ligand fusion proteins (B7.2-Db and scFv-4-1BBL) likewise targeted to the tumor cells to modulate the T-cell response. In our model system, the target cells coexpress the fibroblast activation protein (FAP) and endoglin as antigens. ScDbFAPCD3 and B7.2-Db are targeted to FAP although by different antibody moieties, whereas scFv-4-1BBL is directed against endoglin. ScDbFAPCD3-induced T-cell stimulation could be enhanced by the addition of either B7.2-Db or scFv-4-1BBL and even further by the combination of both as shown in terms of cytokine release (interleukin-2/interferon γ), proliferation and activation marker expression (CD25). By combined costimulation, overall T-cell population strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype. ScFv-4-1BBL-mediated costimulation of naive CD8+ T cells promoted the expansion and development of cytotoxic T cells with strong effector potential. Thus, combining a bispecific antibody with antibody-ligand fusion protein-mediated CD28 and 4-1BB costimulation in a targeted approach shows great potential to generate and shape an immune response at the tumor site. Therefore, the adaptation of this approach to other immune modulatory ligands and tumor-relevant targets seems to be promising.

Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans

“…31 Also in form of co-stimulatory ligands, co-application of tumor-directed antibody-fusion proteins with 4-1BBL and OX40L have shown to be effective in enhancing T cell stimulation in terms of proliferation and cytotoxic potential. 16 …”

“…in combination with T-cell retargeting bispecific antibodies. 16–20 However, although this approach incorporates a targeted delivery and accumulation in the tumor tissue, activation is restricted to a single receptor type.…”

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

“…43,44 The combination of bispecific antibodies with antibody-ligand fusion proteins targeting these receptors has been shown to significantly increase the efficacy of TCBs both in vitro and in murine tumor models. 45,46 However, the translation of such combinatorial approaches into the clinic requires an in-depth understanding of the effects elicited by potent T-cell stimulation as severe unexpected reactions such as massive cytokine release have been observed in a clinical phase I trial after superagonistic anti-CD28 treatment. 47 Another promising avenue to improve the therapeutic efficacy of TCBs lies in the blockade of inhibitory signals on T-cells.…”

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

“…Fusion proteins at 200 nM in 50% human plasma were incubated at 37 C for 1 d, 3 d and 7 d. Samples were frozen at ¡20 C immediately after preparation (0 d) or after the respective incubation period. The level of intact protein was determined in ELISA via binding of 4-1BBL or OX40L to coated 4-1BB-Fc or OX40-Fc (150 ng/well) and detection was conducted via HRP-conjugated anti-FLAG antibody.…”