Dual antibody therapy to harness the innate anti-tumor immune response to enhance antibody targeting of tumors

The authors have declared that no conflict of interest exists.International audienceCancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing

Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity

Studies of sequential anti-CD137/anti-CD20 therapy have previously shown that the efficacy of anti-CD20 was heavily reliant upon anti-CD137; however, the exact mechanism of the anti-B-cell lymphoma efficacy, and whether this correlates with enhanced adverse effects or toxicity, had not been elucidated. Here, we observed that sequential anti-CD137 administration with anti-CD20 resulted in a synergistic therapy, largely dependent upon Fc receptors (FcR), to prolong survival in an experimental B-cell lymphoma therapy model. Tumor suppression was accompanied by B cell depletion, which was not dependent on one activating FcR. Surprisingly, the B-cell activating factor (BAFF) was elevated in the plasma of mice receiving anti-CD137 alone or in combination with anti-CD20, while a selective increase in some plasma cytokines was also noted and triggered by anti-CD137. These effects were independent of activating FcR. Sustained treatment of advanced lymphoma revealed increased lymphocyte infiltrates into the liver and a significant decrease in the metabolic capability of the liver in mice receiving anti-CD137. Importantly, these effects were not exacerbated in mice receiving the anti-CD20/CD137 combination, and elevations in classical liver damage markers such as alanine aminotransferase (ALT) were less than that caused by the lymphoma itself. Thus, combined anti-CD20/anti-CD137 treatment increases the therapeutic index of anti-CD20 or anti-CD137 alone. These mouse data were corroborated by ongoing clinical development studies to assess safety, tolerability and pharmacodynamic activity of human patients treated by this approach. Together, these data support the use of this sequential antibody therapeutic strategy to improve the efficacy of rituximab in B-cell lymphoma patients

Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus

<div><p>Oncolytic viruses (OV) have recently emerged as a promising therapeutic modality in cancer treatment. OV selectively infect and kill tumor cells, while sparing untransformed cells. The direct cytotoxic effects combined with the capacity to trigger an immune response make OV an appealing combination partner in the burgeoning field of cancer immunotherapy. One of the leading OV therapeutic candidates is the double-stranded RNA virus reovirus. In order to improve the oncolytic activity of reovirus and allow for systemic administration despite the prevalence of neutralizing antibodies, cytokine-induced killer (CIK) cells were explored as cell carriers for reovirus delivery. In this study, CIK cells were successfully loaded with reovirus <i>ex vivo</i>, and viral replication was limited in CIK cells. Confocal microscopy and flow cytometry demonstrated that CIK cells retained reovirus on the surface. Moreover, CIK cells could promote reovirus infection of tumor cells in the presence of neutralizing antibodies; meanwhile, cytotoxicity of CIK cells was increased after loading with reovirus. These findings support further investigation of reovirus and CIK combination for antitumor therapy.</p></div

Cytotoxicity of reovirus-loaded CIK cells toward tumor cell lines.

<p>CIK cells were loaded with 1 pfu/cell reovirus at 4°C for 2 h. DLD-1, PC-3 and NCI-H460 tumor cells were used as target cells. Cytotoxic activity was measured at 6 h by LDH release assay performed on target cells at an effector-to-target ratio of 20:1. The killing assay was performed with CIK cells only (filled black) and reovirus-loaded CIK cells(filled grey) with 7.5% human AB serum. Data are expressed as mean percentage (and SD) of 3 independent experiments. *<i>P</i><0.05, ***<i>P</i><0.01.</p

Reovirus loading onto CIK cells.

<p>(A) Confocal microscopy detected reovirus loading on CIK cell surface. CIK cells were infected with 1 pfu/cell reovirus at 4°C for 2 hours. The inoculum was removed, and CIK cells were incubated in complete growth medium for another 12 hours. The uncoated viruses were washed off; after DAPI (blue) and reovirus (green) staining, imaging was performed by confocal microscopy. Scale bars,10μm. (B) Optimizing the conditions for reovirus loading onto CIK cells. CIK cells were infected with 1 pfu/cell reovirus at 4°C or 37°C for 2 hours or 4 hours, respectively. The unbound viruses were washed off, followed by staining with anti-reovirus–σ3 primary antibody and FITC-goat anti-mouse IgG secondary antibody; reovirus binding on CIK cells was assessed by flow cytometry. Black filled and grey filled histograms represent isotype and untreated control staining, respectively, whereas open-black line histograms represent cells stained with specific FITC-conjugated mAb. (C) Flow cytometry analysis of CIK cell phenotypes before and after reovirus infection. CIK cells were infected with or without 1 pfu/cell reovirus at 4°C for 2 hours, and stained with anti-CD3-APC, anti-CD8-PE or anti-CD56-PE for flow cytometry. (D) Assessment of reovirus binding to CD8⁺CTL and CD3⁺CD56⁺NKT cells. CIK cells were infected with 1pfu/cell reovirus at 4°C for 2 hours, washed twice, and stained with anti-reovirus–σ3 primary antibody at 4°C overnight, cells were than washed and stained with FITC-goat anti-mouse IgG secondary antibody, anti-CD3-APC, anti-CD8-PE or anti-CD56-PE antibodies, before acquisition using flow cytometry.</p

Primers used in qRT-PCR.

<p>Gene short names, Genbank accession numbers, PCR primer sequences and amplicon length are shown.</p

CIK cells deliver reovirus to tumor cells even in the presence of human AB serum.

<p>CIK cells were pre-incubated with 1 pfu/cell live or UV-inactivated reovirus at 4°C for 2 hours, washed twice, and added to PC-3 cells (8×10⁴) at 10:1 ratio in the presence of 7.5% human AB serum. After 2 hours of incubation, CIK cells were washed off with fresh medium, and adherent tumor cells were incubated for additional 24-, 48- and 72-hours, respectively. Cell viability was assessed by the CCK-8 assay at the indicated time points. Representative experiments from three replicates are shown. Error bars represent standard deviation. (* <i>P</i><0.05, ***<i>P</i><0.01)</p