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4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Supplementary figures and methods

Alligator’s Neo-X-Prime platform aims to enable antigen presenting cells to efficiently enhance priming of tumor neoantigen-specific T cells. We have demonstrated that binding of a CD40 x TAA bispecific antibody (bsAb) to CD40 on dendritic cells (DCs) and a tumor-associated antigen (TAA) on tumor exosomes or tumor debris leads to activation of the DC, uptake of the tumor material, cross-presentation of a tumor-derived antigen and priming of tumor antigen-specific T cells. This has the potential to result in an increased quantity and/or quality of the tumor-targeting T cell pool and enhanced anti-tumor activity. Herein, we present ATOR-4066, a Neo-X-Prime bsAb targeting CD40 and CEA (CEACAM5), a TAA highly expressed in several cancers. Using in vitro models, we have demonstrated CEA-dependent activation of CD40-expressing cells, and an ability to mediate co-localization of CEA-expressing tumor debris and CD40 expressing antigen presenting cells. In addition, ATOR-4066 was shown to activate CD40-expressing cells in the presence of primary human tumor cells from CEA+ colorectal and gastric cancer patients. Furthermore, in vitro treatment of primary colorectal and gastric tumor-derived cell cultures and tumoroids with ATOR-4066 induced activation of tumor-infiltrating immune cells. In vivo studies using human CD40 transgenic mice bearing CEA-transfected MC38 tumors showed superior anti-tumor effects of ATOR-4066 compared to a CD40 mAb, demonstrating the ability of ATOR-4066 to efficiently induce a tumor-targeting immune response. Overall, these data show the ability of ATOR-4066 to remodel the immune microenvironment and activate tumor-infiltrating immune cells in primary human tumors expressing CEA, demonstrating the promise of this new candidate drug for further clinical development. Citation Format: Karin Hägerbrand, Anette Sundstedt, Mattias Levin, Yago Pico de Coaña, Laura Varas, Lill Ljung, Anneli Nilsson, Mona Celander, David Gomez Jimenez, Hampus Andersson, Malin Lindstedt, Peter Ellmark. ATOR-4066, a Neo-X-Prime bispecific antibody targeting CD40 and CEA, activates myeloid cells in primary human tumors in vitro and induces anti-tumor immunity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2939.

Mitazalimab is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on myeloid cells, such as dendritic cells and tumor infiltrating macrophages, leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. Mitazalimab is a FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and manageable safety profile. Immunologically cold and immune-excluded tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, effector CD8+ T cells are excluded from the tumor by the desmoplastic tumor stroma, which surrounds the tumor and hosts immunosuppressive macrophages that dampen the immune response in the tumor microenvironment. By activating and re-directing tumor infiltrating myeloid cells, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective anti-tumor response by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor thereby enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy, inducing long-term survival. The combined treatment improved activation of antigen presenting cells in the circulation, intratumoral T cell responses as well as reduced the amount of intratumoral immunosuppressive M2 macrophages. By converting the MB49 tumor cell line resistant to FOLFIRINOX treatment, we could further demonstrate that the combination of mitazalimab and FOLFIRINOX induced a strong anti-tumor activity also in a chemoresistant variant of the MB49 cell line. In conclusion, mitazalimab synergizes effectively with chemotherapy, leading to induction of long-term survival in a preclinical tumor model by reducing immunosuppressive M2 macrophages and improving T cell responses intratumorally. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099), where mitazalimab was well tolerated up to 1200 μg/kg with a manageable safety profile, support the ongoing clinical phase 2 study OPTIMIZE-1 (NCT04888312) of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Karin Enell Smith, Mia Thagesson, Anneli Nilsson, Doreen Werchau, Peter Ellmark. Mitazalimab, a potent CD40 agonist in combination with chemotherapy redirects and activates tumor infiltrating myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4155.

Mitazalimab (ADC-1013, JNJ-64457107) is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on antigen-presenting cells (APC), leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. There are several CD40 agonists in clinical development with different characteristics, affecting both efficacy and tolerability. Mitazalimab is an FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and safety. Immunologically “cold” tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, infiltration of effector CD8+ T cells is blocked by the desmoplastic tumor stroma, which surrounds the tumor and hosts immune-suppressive macrophages that dampen the immune response in the tumor microenvironment (TME). By activating and re-directing the immunosuppressive M2 macrophages into tumoricidal M1 macrophages in the TME, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective immune reaction by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy and induced a long-term survival. Further, it was also demonstrated that mitazalimab induced a concentration-dependent activation of tumor-associated macrophages (TAMs), purified from human tumor samples, into a more tumoricidal M1 like phenotype, by upregulation of cell surface markers such as CD83.In conclusion, mitazalimab re-directs human macrophages into a M1 like tumoricidal, less immunosuppressive phenotype, and synergizes effectively with chemotherapy, leading to induction of a long-term survival in a preclinical tumor mouse model. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099) where mitazalimab was well tolerated up to 1200 μg/kg with manageable side effects, support further clinical development of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Adnan Deronic, Mia Thagesson, Anneli Nilsson, Peter Ellmark, Anette Fält, Charlotte Fält, Karin Enell Smith. Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy [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 1593.

The ability to induce potent anti-tumor activity by stimulating 4-1BB (CD137), a key co-stimulatory receptor, makes 4-1BB an attractive immunotherapeutic target. However, a clinically tested, 4-1BB targeting monospecific antibody has been hampered by dose-limiting hepatic toxicities. To improve safety of 4-1BB targeting therapies we have developed a 4-1BB x 5T4 bispecific antibody designed to direct tumor-specific T cell responses to the tumor by stimulating 4-1BB only when co-engaged with 5T4, a tumor-associated antigen. The preclinical dataset presented here provides an overview of the mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic. ALG.APV-527 was built using the ADAPTIR™ platform with binding domains from the ALLIGATOR-GOLD® human scFv library. Its 5T4-dependent agonistic function was assessed using primary CD8+ T cells or NK cells in the presence of 5T4-expressing cells. Secretion of IFN-γ or granzyme B was measured at 72 hrs using ELISA. To measure proliferation, PBMCs were labelled with Cell Trace™ and gated CD8+ T cells were analyzed using flow cytometry. For tumor inhibition studies, the human 5T4-expressing colon carcinoma HCT116 xenograft model was used. 5T4 expression was evaluated in normal human tissues and different human tumors by immunohistochemistry. The preclinical safety profile of ALG.APV-527 was evaluated in a single and repeated dose, dose-range finding toxicology study in non-human primates (NHP). The study design included all the standard repeated dose toxicity parameters and in addition, pharmacokinetics, immunogenicity, and pharmacodynamic end-points. ALG.APV-527 induces a 5T4-dependent increase in IFN-γ and granzyme B production and enhances proliferation of T cells and NK cells. Furthermore, ALG.APV-527 inhibits tumor growth in a human 5T4-expressing colon carcinoma xenograft model. 5T4 is overexpressed in multiple solid tumors, potentially directing the activity of 4-1BB induced by ALG.APV-527 to 5T4-expressing tumors, improving the risk/benefit profile. Four doses (administered once weekly) did not cause any adverse events in the NHP toxicity study. In conclusion, ALG.APV-527 induces potent CD8+ T cell and NK co-stimulation but only in the presence of 5T4. Based on its efficacy and preclinical safety profile, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of multiple 5T4-expressing solid tumors. Citation Format: Anna Dahlman, Michelle Nelson, Jeannette Bannink, Starrla Johnson, Doreen Werchau, Anneli Nilsson, Lill Ljung, Gabriele Blahnik-Fagan, Robert Bader, Adnan Deronic, Peter Ellmark, Maria Askmyr, Gabriela Hernandez-Hoyos, Cathy McMahan, Sara Fritzell. Preclinical safety and efficacy of a tumor-directed T cell activating 4-1BB x 5T4 ADAPTIR™ bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2380.