MC38 Model Research Articles

Abstract 2451: A novel single domain antibody targeting TIGIT for cancer use in combination therapies

Abstract T-cell immunoglobulin and ITIM domains (TIGIT) has emerged as another popular checkpoint receptor that negatively regulates T-cell functions. TIGIT is upregulated by immune cells, such as activated T cells, natural killer cells, and regulatory T cells. TIGIT binds to two ligands, CD155 (PVR) and CD112 (PVRL2, nectin-2), that are expressed by tumor cells and antigen-presenting cells in the tumor microenvironment. Beyond CTLA4 and PD1, TIGIT therapies recently demonstrated great combo potential in the clinic along with well-tolerated toxicity profile. Here we present HLX53, a novel single domain antibody (sdAb) targeting TIGIT with differentiated format, binding epitope, and functional activity. HLX53 binds to TIGIT and blocks CD155 and CD112 with sub-nanomolar affinity. In mixed lymphocyte reaction assay (MLR), HLX53 significantly increased IL2 secretion compared to Tiragolumab and Vibostolimab analogues. HLX53's IL2 induction seems to require an adequate bridging effect between T-cell and antigen-presenting cell via Fc-FcγR engagement, further promoting the maturation of antigen-specific T-cell. In vivo, HLX53 exhibits a better tumor inhibition than Tiragolumab analogue and shows synergy in combination with anti-PD-L1 antibody in MC38 model. In addition to its unique epitope/MOA, HLX53 being a sdAb has a shorter half-life and thus potentially a better tumor penetration and a differentiated PK/PD profile. Understanding pharmacokinetic and pharmacodynamic relationships enables a dosing regimen providing adequate target coverage and superior tumor eradication. Citation Format: Botong Hua, Ming Yang, Jie Xue, Chen Dong, Yi-Ting Mao, Ou Li, Eric Cheung, Hassan Issafras, Wenfeng Xu, Weidong Jiang. A novel single domain antibody targeting TIGIT for cancer use in combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2451.

Abstract 1562: Syngeneic MC38 mouse colorectal tumor model: Immune profile and response to immunomodulatory agents and radiation

Abstract With 53,200 deaths expected in 2020, colorectal cancer (CRC) is the second most common cause of cancer deaths in the US. Metastatic lesions are common, and CRC remains incurable for many patients, leading to over 900,000 deaths per year worldwide. There is a need to develop safe and effective treatments since not all patients or tumors respond to current therapies and the use of appropriate animal models remains vital. One such model is the MC38 murine CRC cell line, derived from a grade-III adenocarcinoma chemically induced in a female C57BL/6 mouse. The aim of this study was to analyze the immune infiltration in subcutaneous MC38 tumors and track responses to various therapeutics. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. As shown by flow cytometry, naïve tumors in the log phase of growth exhibited low infiltration of CD45+ cells. Infiltration of lymphoid cells, represented by CD8+ T cells and CD4+ T helper cells, was also minimal, characteristic of a cold immune-suppressive landscape. The myeloid population represented ~50% of the CD45+ cells and was characterized predominantly by M1-TAMs, M-MDSCs, M2-TAMs and G-MDSCs, also implicated in immune-suppressive environments. To evaluate the effect of immunotherapies, mice bearing established tumors were administered various checkpoint inhibitors or costimulatory antibodies. The costimulatory antibodies anti-GITR, anti-CD137 and anti-OX40 showed no evident anti-tumor effect. In contrast, anti-PD-L1 and anti-PD-1 treatments produced clear anti-tumor activities with 40 and 50% putative responders and 35 and 37% increase in time to progression, respectively, as compared to controls. Tumor volumes in anti-PD-L1 and anti-PD-1 treated mice were also smaller than those in isotype controls starting day 22 post-tumor inoculation (509+103 and 504+94mm3 vs 899+124mm3, respectively, on day 22). Because radiotherapy is thought to increase the immunogenicity of tumors, we investigated the effects of focal radiation (RT) treatment (10Gy), delivered by the Small Animal Radiation Research Platform (Xstrahl), alone or in combination with checkpoint inhibition. Treatment with RT alone resulted in tumor growth delay of 12 days and an increase in time to progression of 119 %, as compared to controls, and 20% partial regression. Treatment with RT combined with anti-PD-1 potentiated these effects, with tumor growth delay of 14 days, an increase in time to progression of 134%, 30% complete regression, 10% partial regression, and 30% tumor free survivors. These results demonstrate that RT can convert an immune-suppressive milieu as seen in the MC38 model, into a warmer environment, and underscore the value of such relevant mouse models in developing clinically relevant therapeutic multimodality strategies. Citation Format: Sylvie Kossodo, Sheri Barnes, David Draper, Derrick Germain, Scott Wise, Maryland Rosenfeld Franklin. Syngeneic MC38 mouse colorectal tumor model: Immune profile and response to immunomodulatory agents and radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1562.

Abstract 2786: A FcgR IIB tumor enriching anti-CD137 novel antibody to avoid liver Immuno-toxicity

Abstract CD137 (4-1BB/TNFRSF9) is a costimulatory T-cell receptor belonging to the TNF receptor superfamily which is transiently expressed on T cell and NK cells. Targeting CD137 or its natural ligand 4-1BB ligand (4-1BBL) has important implications in many clinical conditions, including cancer. Anti-CD137 agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. In depth analysis revealed that 4-1BB-mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes and high amounts of cytokines, including IFN-γ. However, this approach has suffered significant setback in the clinic due to either dose-dependent severe liver toxicity (e.g. Urelumab), or lack of single agent activity in the case of Utomilumab. Multiple therapeutics that attempt to restrict 4-1BB agonism to the tumor microenvironment and minimize systemic exposure have emerged. Here we present a novel antibody targeting 4-1BB receptor, named HLX25 with engineered Fc region to enhance the binding to FcγR IIB. In vitro, HLX25 induces comparable or superior CD137 activation and IL-2 release compared to Urelumab and Utomilumab, suggesting potentially a better safety profile. We showed that HLX25 binds to unique non-ligand competing epitope, which is different from both Urelumab and Utomilumab. In vivo, HLX25 shows potent dose-dependent single agent anti-tumor activity as low as 0.3mg/kg in MC38 model without sign of liver toxicity up to 20 mg/kg, suggesting superior efficacy and better safety profile. Citation Format: Jie Xue, Jen-Kuan Chang, Chen Dong, Yi-Ting Mao, Pallavi Raghavendra, Ou Li, Hassan Issafras, Wenfeng Xu, Weidong Jiang. A FcgR IIB tumor enriching anti-CD137 novel antibody to avoid liver Immuno-toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2786.

Abstract 1732: Investigation of the best therapeutic approach to target CCR8 expressed on tumor regulatory T cells to boost anti-tumor immune responses

Abstract The goal of this study was to investigate the therapeutic potential of targeting regulatory T cells (Treg) through CC motif chemokine receptor 8 (CCR8) and to determine the optimal mode of action for CCR8 targeting molecules to elicit anti-tumor immune response. Infiltration of high levels of Treg cells in tumors is associated with a poor survival prognosis suggesting that the latter cells restrain effector T cell and overall immune activity in the tumor microenvironment. Modulation and inhibition of Treg cells in the tumor is expected to lead to a reinvigorated immune response against the tumor cells. Since Treg cells are also essential for controlling autoimmunity, modulating regulatory T cell activity is preferably restricted to the tumor microenvironment. CCR8 has been identified as a chemokine receptor expressed specifically on tumor-infiltrating, but not on peripheral Treg cells. Surrogate molecules targeting mouse CCR8 having distinct modes of action were generated and were tested in different syngeneic tumor mouse models. In the CT26 and MC38 models, anti-CCR8 monotherapy showed a reduction in tumor growth, prolonged survival and tumor stasis in the majority of the mice. Tumor regression was observed in 20-40% of the mice. Combining anti-CCR8 with anti-PD-1 treatment resulted in both models in complete tumor regression in the majority of the mice. Immunophenotyping analysis showed that anti-CCR8 treatment resulted in strongly decreased Treg cell levels in the tumor. In addition, anti-CCR8 monotherapy and anti-PD1 combination therapy lead to an increase of intratumoral CD8 T cell levels resulting in a favorable CD8/Treg cell ratio. Molecules lacking Fc mediated effector function and only blocking the CCL1-CCR8 signaling showed limited or no efficacy in these tumor models, suggesting that the observed anti-tumor effect is due to ADCC/ADCP - mediated Treg cell depletion and not to blocking of CCR8. Moreover, re-challenge of the complete responders (in both MC38 and CT26) resulted in full tumor rejection in the majority of the mice, indicative of a strong immunological memory. Next to the surrogate molecules, a large set of molecules binding to human CCR8 were identified. Based on human CCR8 binding, CCL1 blocking and in vitro ADCC cell based assay data a final panel of molecules binding to a diverse set of epitopes on human CCR8 have been selected. Final leads are available and are progressing into preclinical development. Our results demonstrate that targeting CCR8 represents a very attractive and promising method to unleash anti-tumor immune responses in patients. Citation Format: Heleen Roose, Elizabeth Allen, Helena Van Damme, Bruno Dombrecht, Rosa Martín-Pérez, Damya Laoui, Jo A. Van Ginderachter, Pascal Merchiers. Investigation of the best therapeutic approach to target CCR8 expressed on tumor regulatory T cells to boost anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1732.

Abstract 1787: YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor

Abstract Background: The indoleamine 2,3-dioxygenase (IDO), is an enzyme responsible for catabolizing tryptophan (Trp) to kynurenine (Kyn). The depletion of tryptophan and the increase in kynurenine exert important immunosuppressive functions by activating T regulatory cells and myeloid-derived suppressor cells. Therefore, overexpression of IDO is associated with poor clinical outcome. In this study, we evaluated pharmacokinetic and pharmacodynamic profiles and anti-cancer immune responses of YH29407, a novel IDO1 inhibitor. Methods: MC38 murine tumor models were established for pharmacokinetic and pharmacodynamic profiling. The kynurenine levels in plasma and tumor of YH29407, epacadostat and BMS-986205 following 3 day treatment were detected. The antitumor effects of YH29407 100 mg/kg QD/BID, epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD for 10 day administration were evaluated in MC38 models. Various immunologic profiles were evaluated after 5 day treatment of individual inhibitors. Results: The AUC of kynurenine in plasma was decreased by 81.4% with YH29407 100 mg/kg BID, 62.7% with epacadostat 100 mg/kg BID, and 19.8% with BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of kynurenine was also reduced by 99.4%, 89.9% and 1.1% with 3 day treatment of YH29407 100 mg/kg BID, epacadostat 100 mg/kg BID, and BMS-986205 100 mg/kg QD treatment, respectively, compared to control group. The intratumoral concentration of YH29407 BID, epacadostat BID and BMS-986205 QD at 24 hours after 3 day treatment was 9.697 µg/g, 5.478 µg/g and 0.025 µg/g, respectively. These results indicate favorable pharmacokinetic property of YH29407. Compared to epacadostat and BMS-986205, YH29407 100 mg/kg BID treatment exhibited significant proliferative response of splenocytes (P <0.05). IFN-γ secretion levels were also significantly increased by YH29407 treatment, compared to epacadostat and BMS-986205 treatment (P <0.05). Profiling of serum cytokine levels revealed significant increase of Granzyme B, IFN-γ, IL-12, and MMP2 and decrease of FASL, compared to the vehicle group. Upregulation of IFN-γ in CD3+ tumor-infiltrating lymphocytes was significantly induced in the YH29407 treated group compared to epacadostat and BMS-986205 treated groups (P <0.05). Treatment with YH29407 100 mg/kg BID induced significant tumor growth inhibition, compared to epacadostat 100 mg/kg BID and BMS-986205 100 mg/kg QD. Conclusion: The novel IDO1 inhibitor, YH29407, with improved pharmacokinetic and pharmacodynamic profiles, showed antitumor inflammatory reprogramming leading to enhanced antitumor efficacy. Citation Format: Kyoung-Ho Pyo, Ho-Woong Kang, Sun Min Lim, Do Hee Kim, Dong Kwon Kim, Gyu-Jin Lee, Jong-Suk Park, Se-Woong Oh, Byoung Chul Cho. YH29407, a novel IDO1 inhibitor, enhances the anti-tumor effects through increased tumor-reactive T cell functions in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1787.

Abstract 1444: TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models

Abstract Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

Abstract 1735: The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity

Abstract The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1735.

Abstract 606: Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models

Abstract Pyruvate kinase is a crucial enzyme responsible for the last step of glycolysis. Cancer cells can use the M2 isoform of pyruvate kinase (PKM2), to better balance respiration and biosynthesis due to allosteric switching between the less active dimeric and fully active tetrameric forms. Additionally, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells' ability to grow and evade immune detection. Inducing tetramerization presents an opportunity to target PKM2 resulting in the metabolic reprogramming of tumor-immune microenvironment (TME). TP-1454 is a potent PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and multiple cell types (AC < 50 nM), tolerated in mice, rats and dogs after repeat doses as high as 1000 mg/kg/day and has recently entered a Phase I clinical trial (NCT04328740). We hypothesize that PKM2 activation may reverse the immune-suppressive TME. To test this hypothesis, we examined the activity of TP-1454 combination with immunotherapy (I/O) in multiple mouse syngeneic tumor models. TP-1454 and anti-PD-1 combination therapy in colorectal cancer models resulted in tumor growth inhibition versus vehicle (53% in CT26; 99% in MC38, P < 0.001). We observed decreases in multiple glycolytic intermediates in TP-1454-treated tumors versus vehicle. We conducted immunophenotyping of the TME in multiple models to identify targets of PKM2 activation. TP-1454 treatment reduced the CD4+ Foxp3+ T-regulatory (Treg) population in MC38, 4T1, RENCA models. Further, we assayed TP-1454 induced PKM2 activation in different immune cell types. To confirm the effect of PKM2 activation on Treg cells we conducted an in vitro assay to explore TP-1454 treatment response on polarization of Tregs and/or toxicity and proliferation. We further utilized LCMS to explore metabolic intermediates that play a critical role in Treg regulation, including regulation of the O-linked β-N-acetylglucosamine (O-GlcNac) post-translational modification, which is reported to stabilize Foxp3 in CD4+ cells. We are currently exploring the effect of TP-1454 treatment on O-GlcNac of Foxp3 and its stability in HEK293 cells, to support the link between PKM2 activation and stabilization of Foxp3. TP-1454 effects on tumor-specific immunity were validated using tumor rechallenge studies. The results of a tumor rechallenge study will be presented using murine MC38 or RENCA xenograft models that are treated with TP-1454 and I/O combination therapies that exhibited a complete response (CR) and were re-implanted. These preclinical studies indicate a unique mechanism modulating tumor metabolism and the TME to improve the response of cancer patients to immunotherapy. Citation Format: Salah Sommakia, Satya Pathi, Yuta Matsumura, Curtis Allred, Ethika Tyagi, Matthew Lalonde, Jason Foulks, Adam Siddiqui, Clifford Whatcott, David Bearss, Steven Warner. Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 606.

Abstract 1563: DRP-104, a broad acting glutamine antagonist, synergizes with immune checkpoint blockade in vivo

Abstract We previously reported that DRP-104, a novel broad acting glutamine antagonist, has significant therapeutic potential in cancer by directly targeting tumor metabolism and simultaneously inducing a potent antitumor immune response. We also reported the immunomodulatory effect of DRP-104 on tumor growth inhibition (TGI) as a single agent and in combination with PD-1/PD-L1 checkpoint inhibitors in the MC38 and CT26 colon cancer syngeneic models(1). DRP-104 mediated TGI is associated with increased tumor-infiltrating leukocytes (TIL) including T, NKT, and NK cells; M1-polarized tumor associated macrophages; and decreased immune-suppressive cells such as MDSCs.Here we further elucidate the immunomodulatory effect of DRP-104 on tumor growth inhibition. In the MC38 mouse model, CD8+, CD4+, or NK cells were depleted by anti-CD8+Ab, anti-CD4+Ab, or anti-asialoGM1 Ab respectively to assess the contribution of these immune cells towards DRP-104 efficacy. CD8+T cell depletion significantly reduced anti-tumor efficacy for DRP-104 (73.9% TGI in CD8+T depletion setting vs 120% TGI with 57.8% regression in undepleted animals). NK cell depletion delayed early anti-tumor response to DRP-104 treatment, while in undepleted mice DRP-104 treatment showed TGI as early as 2 days after dose initiation. Interestingly, CD4+depleted mice showed enhanced efficacy to DRP-104 with TGI of 213% (regressions) and 6/8 tumor free mice 29 days after the end of treatment vs 1/8 mice tumor free in the CD4+ undepleted group with DRP-104 treatment. As such, we tested the combination of DRP-104 with anti-TIGIT antibody which has been reported to deplete Treg cells in the TME(2). In MC38 (anti-TIGIT insensitive) and CT26 (anti-TIGIT sensitive), DRP-104 showed significant single agent TGI. Furthermore, combination of DRP-104 with anti-TIGIT Ab demonstrated enhanced tumor growth inhibition and resulted in improvement in survival. In summary, immune cell depletion experiments confirmed DRP-104's immuno-oncology mechanism of action. CD8+ or NK cell depletion reduced DRP-104 efficacy while CD4+ cell depletion enhanced efficacy. Furthermore, combination therapy of DRP-104 with anti-PD-1 or anti-TIGIT Ab achieved significantly enhanced anti-tumor efficacy even in checkpoint inhibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in combination with immune checkpoint inhibitors. The first in human clinical study of DRP-104 is ongoing.

Abstract 1791: LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 model

Abstract Objective: Blocking PD-1 with monoclonal antibodies is a highly promising onco-immune therapy and has elicited durable antitumor responses and long-term remissions with a broad spectrum of cancers, However, the response to anti-PD-1 therapy is restricted to only a subset of patients with around 25% response rate in cancer patients. TGF-β, a growth factor, plays a pivotal role in tumor development and progression, by inducing tumor immune evasion, activating cancer associated fibroblasts with induction of migration and invasiveness of tumor cells, and promoting angiogenesis in tumor microenvironment. Therefore, a tetravalent bispecific fusion protein (LBL-015), targeting both PD-1/PD-L1 axis and TGF-β signaling pathway was developed to achieve maximal anti-tumor immunity, while blocking the factors implicated in the tumor microenvironment and overcoming the resistance of tumor cells to anti-PD-1 therapy. Methods: The bispecific fusion protein was designed as 2:2 antibody-cytokine fusion format, using our unique Mab fusion platform. The binding affinity of LBL-015 to human PD-1 and TGF-β1 was determined by Fortebio. The activity of LBL-015 was determined using several in vitro assays, including PD-1/NFAT, TGFβ1/SBE-Luc reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-015 was investigated in the human PD-1 knock-in mouse model (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-015 was measured as 5.24 nM to human PD-1 and 0.14 nM to human TGF-β1, respectively. In vitro LBL-015 could block PD-1-PD-L1 interaction, recovering NFAT reporter gene signal, with EC50 of 0.1565 nM, which was much more potent than M7824 (EC50 of 0.5412 nM). In TGF-β1/SBE-Luc reporter gene assay, LBL-015 inhibited SMAD signaling induced by TGF-β1, with IC50 of 0.00576 nM, which was comparable to M7824. In pre-activated human PBMCs, the release of IFN-γ by PBMCs was completely suppressed with TGF-β1 addition. LBL-015 could relieve the suppression of IFN-γ release induced by PD-1 or TGF-β1, in a dose dependent manner. Surprisingly, M7824 didn't show any effect in this assay system. In a model of huPD-1 knock-in mice (Shanghai Model Organisms) implanted with MC38-OVA cells. LBL-015 was shown significant tumor growth inhibition (TGI) of 84.36% and 55.27% at the dose of 20 mg/kg and 10 mg/kg respectively (p<0.001 compared with vehicle treated mice), indicating its potent anti-tumor activity of this bi-functional fusion protein. Conclusions: LBL-015, an anti-PD-1 and TGF-β bispecific fusion protein was shown a great synergetic anti-tumor efficacy in vitro to antagonize both tumor immune evasion and aberrant microenvironment induced by PD-1 and TGF-β1, also in a mouse tumor model, LBL-015 strongly inhibited tumor growth in a dose dependent manner, hence a promising bispecific fusion protein for further clinical development. Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-015, a novel anti-PD-1 fused with TGF-βRII, shows a great anti-tumor activity in a mouse MC38 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1791.

Lymphatic PD-L1 Expression Restricts Tumor-Specific CD8+ T-cell Responses.

Lymph node (LN)-resident lymphatic endothelial cells (LEC) mediate peripheral tolerance by self-antigen presentation on MHC-I and constitutive expression of T-cell inhibitory molecules, including PD-L1 (CD274). Tumor-associated LECs also upregulate PD-L1, but the specific role of lymphatic PD-L1 in tumor immunity is not well understood. In this study, we generated a mouse model lacking lymphatic PD-L1 expression and challenged these mice with two orthotopic tumor models, B16F10 melanoma and MC38 colorectal carcinoma. Lymphatic PD-L1 deficiency resulted in consistent expansion of tumor-specific CD8+ T cells in tumor-draining LNs in both tumor models, reduced primary tumor growth in the MC38 model, and increased efficacy of adoptive T-cell therapy in the B16F10 model. Strikingly, lymphatic PD-L1 acted primarily by inducing apoptosis in tumor-specific CD8+ central memory T cells. Overall, these findings demonstrate that LECs restrain tumor-specific immunity via PD-L1, which may explain why some patients with cancer without PD-L1 expression in the tumor microenvironment still respond to PD-L1/PD-1-targeted immunotherapy. SIGNIFICANCE: A new lymphatic-specific PD-L1 knockout mouse model reveals that lymphatic endothelial PD-L1 expression reduces tumor immunity, inducing apoptosis in tumor-specific CD8+ central memory cells in tumor-draining lymph nodes.

Tumor-derived GDF-15 to suppress t-lymphocyte recruitment to the tumor microenvironment resulting in resistance to ANTI-PD-1 treatment.

e14532 Background: Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily with low to absent expression in healthy tissue. GDF-15 has been linked to feto-maternal immune tolerance, to prevention of excessive immune cell infiltration during tissue damage, and to anorexia. Various major tumor types secrete high levels of GDF-15. In cancer patients, elevated GDF-15 serum levels correlate with poor prognosis and reduced overall survival (OS). Methods: Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking was analyzed by whole blood adhesion assays, a HV18-MK melanoma-bearing humanized mouse model and a GDF-15-transgenic MC38 model. Additionally, patient GDF-15 serum levels were correlated with clinical response and overall survival in oropharyngeal squamous cell carcinoma (OPSCC) and melanoma brain metastases. Results: In whole blood cell adhesion assays GDF-15 impairs adhesion of T and NK cells to activated endothelial cells. Neutralization of GDF-15 by CTL-002 rescued T cell adhesion. In HV18-MK-bearing humanized mice CTL-002 induced a strong increase in TIL numbers. Subset analysis revealed an overproportional enrichment of T cells, in particular CD8+ T cells. As immune cell exclusion is detrimental for checkpoint inhibitor (CPI) therapy, a GDF-15-transgenic MC38 model was tested for anti-PD-1 therapy efficacy. In GDF-15 overexpressing MC38 tumors response to anti PD-1 therapy was reduced by 90% compared to wtMC38 tumors. Combining aPD-1 with CTL-002 resulted in 50% of the mice rejecting their GDF-15 overexpressing tumors. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for HPV+ OPSCC and for melanoma brain metastases. GDF-15 serum levels were significantly higher in HPV- than in HPV+ OPSCC patient (p < 0.0001). Low GDF-15 levels corresponded to longer OS in both HPV- and HPV+ OPSCC. In two independent melanoma patient cohorts treated with nivolumab or pembrolizumab low baseline serum GDF-15 levels were predictive for clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 independently predicts poor survival in aPD-1 treated melanoma patients. Conclusions: Taken together our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of T cells to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. In melanoma, patients with higher GDF-15 levels have significantly shorter survival and are less likely to respond to anti-PD1 therapy. GDF-15 may thus serve as a new predictive biomarker for anti-PD1 response, but most importantly also represents a novel target for cancer immunotherapy to improve tumor immune cell infiltration and response to anti-PD1 therapy.

Pharmacologic ascorbate enhances the therapeutic index of ATM-inhibitor based chemoradiation for colorectal cancer.

3609 Background: Ataxia telangectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury. Methods: Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57blj/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors. Results: Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated P-ATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT. Conclusions: P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

Assessment of cancer-specific microbiome signature of response to immune checkpoint inhibitors.

2574 Background: Clinical and preclinical experiments suggest that the gut microbiome can affect outcome in cancer patients treated with immune checkpoint inhibitors (ICI). Most data to date has been in melanoma, so the relationship of the gut microbiome with treatment outcome in other cancers is poorly understood. Here, we evaluated the microbiome composition in correlation to ICI response in patients with metastatic lung, urothelial, or renal cancer, as well as metastatic melanoma. Methods: Fecal microbiome samples were obtained from patients with metastatic melanoma, lung, urothelial, or renal cancer immediately before ICI therapy was initiated. Bacterial genomic DNA was isolated and profiled by whole metagenome sequencing. Sequence data were analyzed using a custom implementation of MetaPhlAn2. Response to ICI was defined as partial or complete response or remaining on therapy for more than 6 months. Results: Samples were prospectively collected from 94 patients, including metastatic melanoma (n = 17), lung (n = 44), urothelial (n = 23), or renal cancer (n = 10). Treatment included anti-PD(L)1 monotherapy (n = 51), anti-PD1 + anti-CTLA4 combination therapy (n = 17), or a combination of anti-PD1 and chemotherapy (n = 26). Clinical response was observed in 58% of patients, including partial or complete response (45%) and on treatment for more than 6 months (55%, with 31% on treatment for more than 1 year). Although the variance in the composition of pretreatment microbiome samples did not explain response alone (R vs NR, PERMANOVA, p = 0.273), a significant portion of the variance in microbiome composition was explained by the interaction of cancer type and outcome (PERMANOVA, p = 0.014), suggesting a cancer-specific microbiome relationship. Notably, there was some similarity in the signature of NR across three cancer types (lung, urothelial and melanoma). One sample in this NR cluster was from a patient whose metastatic NSCLC was nonresponsive to pembrolizumab and carboplatin/pemetrexed. This microbiome sample was evaluated in vivo using subcutaneous MC38 and CT26 tumor models in germ-free mice. In contrast to mice colonized with stool from a healthy donor, mice colonized with stool from this patient yielded a nonresponsive result upon treatment with anti-PD1 or anti-PD-L1 in combination with anti-CTLA4. Conclusions: Analysis of the fecal microbiome composition from patients with metastatic lung, urothelial, renal cancer, and melanoma identified a cancer-specific signature of R and NR to ICI. Across three cancer types, a consistent signature of NR was identified and corroborated experimentally in preclinical models.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets.

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/β sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.

Abstract PO-088: Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076

Abstract Ionizing radiation therapy (RT) is a standard of care for many cancer indications. RT promotes the activation of transforming growth factor (TGF)-β, a pleotropic cytokine that mediates RT-induced fibrosis and suppresses anti-tumor immunity. Additionally, RT induces the expression of the immunosuppressive checkpoint PD-L1 on tumor cells. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, functioning as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1. Bintrafusp alfa enhances anti-tumor immunity elicited by RT and reverses RT-induced fibrosis in preclinical models. Inhibition of ataxia telangiectasia mutated (ATM) increases the cancer cell's sensitivity to radiotherapy by blocking cellular pathways which facilitate the response to, and repair of, RT induced DNA double strand breaks. We hypothesize that a combination therapy consisting of bintrafusp alfa, RT and the ATM kinase inhibitor M4076 will show greater anti-tumor efficacy than the respective mono- or dual-combination therapies in immune competent preclinical solid tumor models. The anti-tumor efficacy of bintrafusp alfa, RT, and M4076 triple combination therapy was explored in the immune therapy resistant 4T1 murine syngeneic breast carcinoma model. This triple combination therapy significantly increased anti-tumor efficacy and prolonged overall survival relative to the bintrafusp alfa + RT or M4076 + RT dual combination therapies. The triple combination was further evaluated in the more immunogenic MC38 colon carcinoma model. MC38 tumor bearing mice treated with the triple combination therapy showed a significantly greater tumor growth inhibition and long term tumor free survival when compared with the bintrafusp alfa + RT or M4076 + RT treatment groups. Finally, the bintrafusp alfa + RT + M4076 combination therapy also showed significantly greater tumor growth inhibition and long term tumor free survival when compared to the combination of anti-PD-L1 + RT + M4076. The bintrafusp alfa triple combination resulted in 8 of 10 mice showing long term tumor free survival, whereas the anti-PD-L1 triple combination failed to elicit tumor free survival (0 of 10 mice tumor free). These studies demonstrate the superior anti-tumor efficacy of the Bintrafusp alfa + RT + M4076 triple combination therapy over that of the dual combination therapies of RT + bintrafusp alfa or RT + M4076. These data in the MC38 model also suggest that bintrafusp alfa may promote a superior anti-tumor immune response relative to that of anti-PD-L1 when in combination with RT and M4076. Together, these data support the rational to develop a bintrafusp alfa + RT + M4076 combination therapy for the treatment of solid tumors. Citation Format: Adam Lazorchak, Huakui Yu, Guozhong Qin, Jin Qi, Bo Marelli, Yan Lan. Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-088.

A global live cell barcoding approach for multiplexed mass cytometry profiling of mouse tumors.

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

Single-Domain Antibody Nuclear Imaging Allows Noninvasive Quantification of LAG-3 Expression by Tumor-Infiltrating Leukocytes and Predicts Response of Immune Checkpoint Blockade.

Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte-associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. Methods: SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3-expressing immune cells using a γ-counter and flow cytometry respectively. PD-1/LAG-3-blocking antibodies were injected in MC38-bearing mice. Results: The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1-blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. Conclusion: These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.

Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12.

The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen-specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.