The Antitumor Activity Of Tumor Targeting Antibody-IFNβFusion Protein And Its Mechanism Of Action

The first generation of antibodies (Abs) targeting tumor-associated receptors,especially oncogenic receptors, becomes increasingly used for cancer therapy but relapseor Ab-resistance arises despite initial tumor regression. Most publications and currentstrategies to deal with Ab-resistance have been investigating and targeting theresistance-related signaling pathway within the tumor cell. Currently, the secondgeneration of therapeutic antibodies fused with cytotoxic compounds or toxins to directlytarget the cytotoxicity to the tumor cells. Some of these antibodies, e.g. Herceptin-DM1,have achieved better anti-tumor effect with few side effects, while others have limitedefficacy. Despite this strategy, Ab-resistance still emerged even for Herceptin-DM1.Therefore, next generation of antibody based treatment is urgently needed. We haverecently developed the third generation of therapeutic antibodies based on fusion withimmunomodulators to modulate the tumor microenvironment, changing the mileux fromsuppressive/anergic to stimulatory/energic, in order to re-activate both innate and adaptiveimmune responses against the tumor. We have observed that targeting oncogenic signalingcan trigger the production of type I interferons for tumor regression. It is clear thattargeting tumor tissues with next generation antibody therapies based on fusion proteins,namely the Ab-IFNβ, could re-activate innate and adaptive immune cells which greatlyimprove the therapeutic effect of antibody therapies, even for Ab-resistant tumors.In contrast to previous strategies targeting one of various tumor-intrinsic resistancemechanisms, we have approached resistance from a new angle, impaired innate andadaptive immune responses, which can boardly overcome Ab resistance independent ofvarious tumor-intrinsic mechanisms. We have revealed that the Ab-IFNβ fusion proteintargeted the IFNAR on host DCs but not T cells for better cross-presentation and CTLresponses against the tumor, which supply a novel important immunotherapy target thancurrent widely used inhibitory signal on T cells. Our data also indicated that re-activatedimmune cells by our third generation antibody is a better strategy than direct cytotoxicityby the second generation Ab-toxin treatment for better tumor control with the followingadvantage: reduced treatment period, less direct cytotoxicity to normal cells and preventingrelapse and resistance due to multiple targeting mutated tumor antigens.Furthermore, this study also indicated another important protective mechanism forhost so called treatment induced-adaptive resistance. We revealed that induced expression of PDL-1by Ab-IFNβ is the key molecule for the induced adatpive resistance. BlockingPDL-1could maximize the therapeutic effect of Ab-IFNβ, overcome treatment-acquiredresistance and completely eradicate Ab-resistant tumor. This clearly demonstratedtargeting the specifically induced inhibitory pathway by immune activation should be animportant direction to improve the cancer immune therapy.The superiority of Ab-immunomodulator fusion proteins over the previousgenerations of antibodies will open a new avenue to properly arm various tumor-targetingantibodies (missiles) with a panel of smart bombs. The fusion protein is not a new idea, butmost fusion proteins use bombs that directly kill tumor cells and surrounding immune cells.We propose a new missile that delivers immunomodulators rather than non-specificdestruction, in order to mobilize the host immune responses while mitigating collateraldamage. Since each tumor tissue could have distinct microenvironments, proper screeningof tumor tissues for proper targeting of receptors on tumors and immunomodulators onimmune cells could dictate tumor eradication. Therefore, this study establishes a new andpersonalized strategy to modulate the tumor to a microenvironment that promotes theinnate and adaptive immune responses against the tumor cells.Part One: Ab in vivo responding is dependent on type I interferons.1. Generation of Ab responding, partial resistant and complete resistant tumormodel.2. Ab treatment could induce type I interferon production.3. Blockade of type I interferon signaling impaired the therapeutic effect of Ab.4. Targeting tumor directly by type I interferon is sufficient to control tumor growth.Conclusion: Targeting tumor with type I interferons maybe sufficient to overcometumor immune evasion and is a potential immunotherapeutic target, even for Ab-resistanttumor.Part Two: Targeting delivery of IFNβ enhanced the therapeutic effect of Ab.1. Construction, expression and purification of anti-EGFR-IFNβ fusion protein.2. The binding function of anti-EGFR-IFNβ fusion protein in vitro.3. The biodistribution of anti-EGFR-IFNβ fusion protein in vivo.4. The therapeutic effect of anti-EGFR-IFNβ fusion protein in vivo.Conclusion: Ab-IFNβ fusion protein therapy used at low dose and for short duration isfar superior than first-generation Ab therapy for controlling tumors, even in Ab resistanttumor models and tolerized hosts. Part Three: The anti-tumor effects and mechanisms of anti-EGFR-IFNβ fusionprotein.1. The therapeutic effect of anti-EGFR-IFNβ fusion protein depends on adaptiveimmunity.2. The therapeutic effect of anti-EGFR-IFNβ requires IFNAR expression on hosthematopoietic cells.3. DCs activation contributes to the anti-tumor effect of anti-EGFR-IFNβ.4. Anti-EGFR-IFNβ directly targets DCs to reverse the tolerized tumormicroenvironment for better anti-tumor effect.Conclusion: Anti-EGFR-IFNβ treatment can increase T cell priming, whichcontributes to the anti-tumor effect of anti-EGFR-IFNβ; mediated its anti-tumor effect notby directly inhibiting tumor-cell growth, but by activating host hematopoietic cells thatthen changes the tumor microenvironment; increased DC cross-presentation leading toimproved CD8+CTL priming and function might the major mechanism underlying thetherapeutic effect of anti-EGFR-IFNβ; direct activation of IFNAR-expressing DCs plays amajor role in the anti-EGFR-IFNβ–mediated therapeutic effect, which can be furtherenhanced by engaging IFNAR expressed on T cells.Part Four：Antagonising PDL-1expression induced by anti-EGFR-IFNβ achievedtumor free outcome.1. Anti-EGFR-IFNβ greatly increases the expression of PDL-1.2. Combination of anti-PDL-1and anti-EGFR-IFNβ could further enhance theanti-tumor effect of anti-EGFR-IFNβ.3. No synergic effect of anti-EGFR-IFNβ with anti-CTLA4or anti-BTLA.4. Combination of anti-PDL-1and anti-EGFR-IFNβ could further enhance theanti-tumor specific T cell response.Conclusion: Antagonizing anti-EGFR-IFNβ–induced PDL-1expression on tumorcells can maximize the anti-tumor effect of anti-EGFR-IFNβ and achieve an impressivetumor-free outcome, even for Ab-resistant tumors. This combination-based strategy willlikely increase the overall response and cure rates of Ab-resistant hosts, even in hostsfailing to respond to anti-PD-1antibodies that directly block T cell-expressed PD-1.