Novel CAR T cells targeting mesothelin (MSLN) expressed on pancreatic cancer cells were developed to overcome the limit of the clinical efficacy of CAR T cell therapy for pancreatic cancer patients. ...Optimal single-chain variable fragments (scFv) binding to MSLN were selected based on the binding activity and the functional effectiveness of various scFv containing CAR-expressing T cells. Engineered MSLN CAR T cells showed successful anti-tumor activity specific to MSLN expression level. Furthermore, MSLN CAR T cells were evaluated for the anti-cancer efficacy in orthotopic mouse models bearing pancreatic cancer cells, MIA Paca-2, MSLN-overexpressed MIA Paca-2 or endogenously MSLN-expressing AsPC-1. Mice were randomized into control, mock treated, MS501 BBz treated, MS501 28z treated or MS501 28BBz treated group. Mice were monitored by weekly IVIS imaging and tumors were harvested and analyzed by immunohistochemical analyses. MSLN CAR T cells produced the therapeutic effect in orthotopic animal models with complete remission in significant number of mice. Histopathological analysis indicated that CD4+ and CD8+ MSLN CAR T cells infiltrated pancreatic tumor tissue and led to cancer cell eradication. Our results demonstrated the anti-tumor efficacy of MSLN CAR T cell therapy against pancreatic cancer, suggesting its therapeutic potential.
BackgroundWe developed tumor microenvironment-targeting immunocytokine or TMEkine™ utilizing strong anti-tumoral effect of interleukin 12 (IL-12). In this effort, we created a bi-specific 1+1 ...antibody fusion with conventional knob-in-hole technology where anti-CD20 was paired with IL-12 fc fusion arm. A couple of IL-12 muteins were used in our therapeutic molecules to reduce systemic toxicity. IL-12 has been known for a key orchestrator in immune response. The main actions of IL-12 include the induction of CD4+ Th0 cells toward Th1 type and enhancement of IFN-γ production, stimulation of cytotoxicity and growth of natural killer (NK) cells and CD8+ T cells. For these reasons, IL-12 has long been considered as a potential therapeutic molecule for treating cancers by enhancing immune activity toward tumor cells. However, systemic administration of IL-12 showed poor efficacy and severe adverse effects. With our therapeutic approach of tumor targeting and attenuated IL-12 mutein, we expect that our IL12-based TMEkine™ holds great promise for the future of cancer immunotherapy.In this study, we targeted CD-20 expressing cancers such as B-cell lymphoma with our anti-CD20/IL-12 mutein TMEkine. We evaluated the biological activity of our molecules with in vitro and in vivo efficacy and safety.MethodsThe target specific binding to CD20 and IL-12 receptor was analyzed by FACS and ELISA. Biological activities as signaling transduction and T cell activation were confirmed in vitro using HEKblue IL12 cell line, primary human T cells and NK cells. The anti-tumor efficacy of TMEkine (CD20-IL-12) was assessed in A20 lymphoma syngeneic mouse model. To demonstrate long term protection to A20, the cured five mice after TMEkine administration were re-challenged with A20 and 4T1 cells.ResultsFirst, we analyzed the specific binding of our TMEkine molecules to CD20 expressing B-cell lymphoma cell lines (such as Raji). We showed that TMEkine (CD20-IL-12) binds to Raji and Ramos, which express CD20, but not to Jurkat, which does not express CD20. We also showed that TMEkine molecules bind to IL-12 receptor in a dose-dependent manner. pSTAT4 alphaLISA assay revealed that TMEkine (CD20-IL-12) transduces STAT4 signaling. In our IL-12 mutein, key residues for heparin binding were mutated. The biological activity of our mutein molecule was attenuated due to this change in human PBMC. In addition, our TMEkine molecules significantly induced IFN-γ secretion from primary human T cells and NK cells. An A20 B-cell lymphoma syngeneic mouse model was utilized to investigate the anti-tumor activity of TMEkine (CD20-IL-12). TMEkine molecules were injected three times with Q3D intraperitoneally. Tumor growth was substantially reduced and no cytotoxicity was observed. To further investigate the underlying mechanism, we analyzed tumor infiltrating lymphocytes (TIL) and as expected, we observed the increase in the number of CD8+ T cells in TIL, compared to control group. Interestingly, our tumor re-challenge result demonstrates that TMEkine (CD20-IL-12) protected animals from tumor recurrence implying that immunologic memory response was generated upon our TMEkine (CD20-IL-12) treatment.ConclusionsAltogether, our data suggest that TMEkine (CD20-IL-12) as an efficacious tumor targeting cytokine opening up a new avenue for the treatment of B-cell lymphoma.
•IL-12 faces limitations in systemic administration.•IL-12 mutant exhibits reduced activity.•CD20-IL-12mut integrates IL-12 mutations and CD20 targeting antibody.•CD20-IL-12mut maximizes therapy ...while minimizing off-target effects.•CD20-IL-12mut shows potent antitumor activities and specific immunological memory.
Immunotherapy has emerged as a promising approach to cancer treatment that utilizes the potential of the immune system to precisely identify and eradicate cancerous cells. Despite significant progress in immunotherapy, innovative approaches are required to enhance the effectiveness and safety of these treatments. Interleukin-12 (IL-12), widely recognized for its essential function in immune responses, has been explored as a potential candidate for treating cancer. However, early attempts involving the systemic administration of IL-12 were ineffective, with significant adverse effects, thus underscoring the need for innovation. To address these challenges, we developed a therapeutic molecule that utilizes a single-chain IL-12 mutant (IL-12mut) linked to a tumor-targeting arm. Here, we describe the development of a highly effective IL-12-based TMEkine™ platform by employing a B-cell lymphoma model (termed CD20–IL-12mut). CD20–IL-12mut combined the attenuated activities of IL-12 with targeted delivery to the tumor, thereby maximizing therapeutic potential while minimizing off-target effects. Our results revealed that CD20–IL-12mut exhibited potent anticancer activity by inducing complete regression and generating immunological memory for tumor antigens. Collectively, our data provide a basis for additional research on CD20–IL-12mut as a potential treatment choice for patients with B-cell lymphomas such as non-Hodgkin’s lymphoma.
Background Cytokines are well-known immunomodulators. Thanks to recent success of immune checkpoint inhibitors there is a renewed interest in cytokines as a promising cancer immunotherapy option. ...Several inflammatory cytokines including IL-12 showed potent anti-tumor activities but severe immune adverse events when administered systemically greatly hindered using them as anti-tumor agents. Methods Previously (SITC 2020) we showed that IL-12 activity was reduced by our introduced mutation (termed as mut1) if measured by pSTAT4 AlphaLISA assay. But when it was treated in human immune cells IFNg production was not reduced as expected. Thus, we further attenuated IL-12 activities by protein engineering and created our candidate molecule KNP-101. Results We showed that KNP-101 maintained potent anti-tumor activities in vivo but gained greatly improved toxicity profiles. When we measured pSTAT4 signals, KNP-101 showed about 30-fold attenuation in IL-12 activities compared with rIL-12. IFNg production from human PBMC was also reduced. Although the IL-12 activity was weakened in order to reduce its systemic toxicity, our KNP-101 mouse surrogate still maintained good anti-tumor potency in various mouse syngeneic models with a single intravenous injection as low as 2 ug/head. In combination with anti-PD-L1, KNP-101 surrogate showed a synergistic anti-tumor effect and further FACS analysis of tumor infiltrated lymphocytes demonstrated that the effects were mediated by immune cell infiltration. Importantly, in CD1 naïve mouse toxicity test, KNP-101 surrogate was tolerable up to 50 ug/head and no survival issue was observed. However, that was not the case with the control group, non-tumor-targeting null/IL-12 showing survival issues with all tested dose levels. Compared with the control, KNP-101 surrogate also showed much safer profiles in terms of organ weight and serum chemistry such as ALT level. We also performed similar toxicity study in tumor-bearing mice. KNP-101 surrogate again showed a very safe profiles being tolerable up to 500 ug/head and no survival issue. Noticeably, when serum IFNg was measured in tumor-bearing mice, our KNP-101 surrogate induced far less IFNg in serum compared to null/IL-12 suggesting that systemic toxicity was greatly reduced. Conclusions Together, we demonstrated that systemic toxicity of IL-12 cytokine therapy can be overcome by tumor-targeting and IL-12 attenuation. Our KNP-101 has a widen therapeutic window by maintaining potent anti-tumor activities and showing much improved safety profiles. We hope that KNP-101 can benefit patients in the future who suffer from primary and acquired resistance of the current anti-PD-1/PD-L1 treatments.
BackgroundAlthough cancer immunotherapy showed promising results in hematological malignancies, it has come up with relatively low tumor response for many solid tumors partly due to ...immune-suppressive tumor microenvironment (TME). Because of the immune-suppressive nature of TME, TME has been an active area of research and therapeutic target for restoring immune system and subsequent tumor growth inhibition. Among the many components in TME, cancer-associated fibroblasts (CAFs) are one of the key cell components of TME where one of the promising solid-tumor TME marker, fibroblast-activating protein (FAP) is highly expressed. Here we have developed an antibody-cytokine fusion protein from our TMEkine™ platform containing anti-FAP and IL-12. Our TMEkine™ (anti-FAP-IL-12) molecule induced strong anti-cancer effects in preclinical solid tumor models by immune-modulation.MethodsIL-12 cytokine was mutated in TMEkine™ (anti-FAP-IL-12) to reduce systemic toxicity and its binding affinity was tested to FAP and IL-12 receptor. The anti-tumor activity of anti-FAP-IL-12 was investigated on CT26 (murine colorectal cancer) syngeneic mouse models with/without NIH-3T3 (murine fibroblast). Additionally, mice showing complete response after anti-FAP-IL-12 administration were re-injected CT26 with/without 4T1 cells for re-challenge study to monitor long-term durable response generated from the initial immune activation.ResultsWe showed that TMEkine™ (anti-FAP-IL-12) interacts with FAP and IL-12 receptor. IL-12 activity was attenuated by our IL-12 mutants. We also showed that TMEkine™ (anti-FAP-IL-12) induced IFN-γ from primary human T cells and NK cells. TMEkine™ (anti-FAP-IL-12) administration resulted in significant reduction of the tumor burden in both CT26+NIH-3T3/FAP+ and CT26/FAP+ models. In the re-challenge experiments, CT26 tumor growth was inhibited significantly compared to 4T1 tumor suggesting memory immune response was generated in TMEkine™ (anti-FAP-IL-12) treated mice.ConclusionsThese findings provide evidences that the treatment of anti-FAP/IL-12 TMEkine™ induced anti-cancer effects without serious adverse effects. Anti-FAP/IL-12 has a strong potential to provide a therapeutic option for cancer-specific immunomodulator and cancer cell eradication.
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