Background Cytokine-induced killer cells (CIKs) are a heterogeneous population of CD3 and CD56 expressing T lymphocytes, expanded and activated ex vivo by peripheral blood mononuclear cells (PBMCs) or umbilical cord blood, with the addition of interferon recombinant human-γ (rhIFN-γ), anti-CD3 monoclonal antibody (mAb), and recombinant human interleukin-2 (rhIL-2). CIK cells are gaining considerable clinical relevance, due to the low risk of acute graft-versus-host disease (GvHD) in both autologous and allogeneic settings, their feasibility and limited production costs.1 2 In this study we combined CIK cells with either Trastuzumab (TRS) mAb, due to CD16a expression, or Trastuzumab (TRS) engineered mAb V90Lec13, which carries two amino acid substitutions (S239D/I332E) and lacks Fc fucosylation, or with the bispecific single-chain fragment variable (bsscFv) Her2xCD3 (figure 1, 2).
Methods CIK cells were obtained from PBMCs from both healthy donors and Her2+ breast cancer patients, under serum-free and a GMP expansion protocol. Effector cell cytotoxicity and dose-dependent activity of immunotools were evaluated by real-time cell assay (xCELLigence) against Her2+ breast cancer cell lines. The concentration of cytokines released was evaluated with a multiplex assay. To evaluated in vivo therapeutic efficacy, NSG mice were injected fat pad with Her2+ cell lines or with Her2+ patient derived xenograft and left untreated or treated with TRS, HerxCD3, 10x106 CIK+Isotype, CIK+TRS or CIK+Her2xCD3. The mice were monitored for tumor growth and survival.
Results CIK cells from patients cultured in GMP condition are able to expand in clinically relevant number. The combination of CIK cells with all immunotools, significantly enhances Her-2+ tumor cell killing (figure 3, 4). In particular at a very low effector/target (E/T) ratio, such as 0.1:1 E/T ratio, CIK cells combined with HER2xCD3 had a remarkable and fast cytotoxicity, that completely kill target cells (figure 5). Interestingly, TRS-resistant tumor cell lines showed to be sensitive to HER2xCD3-armed CIK cell lytic activity. Moreover, bsAb resulted to be effective also at very low concentrations, and the cytokines released from CIK cells matched with a proinflammatory profile, with no significant concentration of cytokines correlated with Cytokines Release Syndrome. In vivo experiments demonstrated that the combination of CIK cells with both TRS or Her2xCD3 delay significantly the tumor growth in vivo and prolong the survival (figure 6).
Conclusions Taken together, these results highlight the potentiality of using new perspectives for the treatment of Her-2+ breast cancer with the adoptive cell therapy by combining non-antigen-specific CIK effector cells with already clinically approved tumor-antigen-specific antibodies or recombinant molecules.3 4
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Palmerini P, Dalla Pietà A, Sommaggio R, Ventura A, Astori G, Chieregato K, Tisi MC, Visco C, Perbellini O, Ruggeri M, Cappuzzello E, Rosato A. A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks. Cytotherapy. 2020 Sep;22(9):511–518. doi: 10.1016/j.jcyt.2020.05.003. Epub 2020 Jul 4. PMID: 32631696.
Dalla Pietà A, Cappuzzello E, Palmerini P, Ventura A, Visentin A, Astori G, Chieregato K, Mozzo V, Perbellini O, Tisi MC, Trentin L, Visco C, Ruggeri M, Sommaggio R, Rosato A. Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells. J Immunother Cancer. 2021 Jul;9(7):e002475. doi: 10.1136/jitc-2021-002475. PMID: 34272306; PMCID: PMC8287629.
Sommaggio R, Cappuzzello E, Dalla Pietà A, Tosi A, Palmerini P, Carpanese D, Nicolè L, Rosato A. Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells. Oncoimmunology. 2020 Jun 11;9(1):1777046. doi: 10.1080/2162402X.2020.1777046. PMID: 32923140; PMCID: PMC7458615.
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