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PDF file - 278K, Figure 1. Local low-dose anti-CD40 therapy generates distant anti-tumor effects and inhibits B16 tumor growth.

PDF file - 195K, Figure 3. In vivo biodistribution of target organs 48h and 72h post injection of 125I-CD40-specific and 131I-rat IgG2a control antibody.

Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic. Cancer Immunol Res; 2(1); 80–90. ©2013 AACR.

PDF file - 68K, Table 3: In vivo biodistribution 48h (also see Supplementary Fig. S3A). Statistical differences calculated between anti-CD40 and rat IgG2a and the two administration routes (n=4, Student?s paired t-test).

PDF file - 68K, Table 1: In vivo biodistribution 4h (also see Fig. 4A). Statistical differences calculated between anti-CD40 and rat IgG2a and the two administration routes (n=4, Student?s paired t-test).

PDF file - 67K, Table 2: In vivo biodistribution 24h (also see Fig. 4B). Statistical differences calculated between anti-CD40 and rat IgG2a and the two administration routes (n=4, Student?s paired t-test).

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.

PDF file - 404K, Figure 2. Retained CD40 specificity after radiolabeling and modulation of CD40 expression as well as immune cell distribution after repeated injections of FGK4.5 in vivo.

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Supplementary figures and methods

Purpose: Local administration of immune-activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here, we report the development and affinity maturation of a fully human agonistic CD40 antibody (IgG1), ADC-1013.Experimental Design: We have used molecular engineering to generate an agonistic antibody with high affinity for CD40. The functional activity of ADC-1013 was investigated in human and murine in vitro models. The in vivo effect was investigated in two separate bladder cancer models, both using human xenograft tumors in immune deficient NSG mice and using a syngeneic bladder cancer model in a novel human CD40 transgenic mouse.Results: Activation of dendritic cells (DC) by ADC-1013 results in upregulation of the costimulatory molecules CD80 and CD86, and secretion of IL12. ADC-1013 also activates DCs from human CD40 transgenic mice, and peptide-pulsed and ADC-1013–stimulated DCs induce antigen-specific T-cell proliferation in vitro. In vivo, treatment with ADC-1013 in a syngeneic bladder cancer model, negative for hCD40, induces significant antitumor effects and long-term tumor-specific immunity. Furthermore, ADC-1013 demonstrates significant antitumor effects in a human bladder cancer transplanted into immunodeficient NSG mice.Conclusions: Our data demonstrate that ADC-1013 induces long-lasting antitumor responses and immunologic memory mediated by CD40 stimulation. To the best of our knowledge, ADC-1013 represents the first immunomodulatory antibody developed for local immunotherapy of cancer. Clin Cancer Res; 21(5); 1115–26. ©2014 AACR.See related commentary by Dronca and Dong, p. 944

Supplementary Figure 3. Anti-tumor effect on MB49 tumors. ADC-1013 generates a significant anti-tumor effect on CD40 negative MB49 cells in hCD40tg mice. Treatment with ADC-1013 or isotype control was performed on day 7 and 10 post tumor inoculation. Increasing doses of ADC-1013 were administred peritumorally in 100 ul PBS and the individual tumor growth curves are displayed.

Supplementary Figure 2. Microphotographs from cryo sections showing representative images of A) Spleen, B) Liver and C) Brain (one slide representing data from one female and one male mice) from hCD40+ mice stained with an anti-human CD40 antibody or anti mouse CD40 antibody.

Supplementary Figure 1. A) Annexin V positive moDC after ADC-1013 stimulation and B) the effect of cross-linking on percentage of Annexin stained cells (n=6).

Supplementary Figure 6. Splenocytes from complete responders (CR) from ADC-1013 treatment of MB40 tumors were transplanted to naive mice. The receiving naïve mice were then challenged with MB49 tumors and the tumor volume followed over time until the first mice was sacrificed (**p

Supplementary Figure 5. hCD40tg mice with one tumor on each flank were treated peritumorally in the tumor on the right flank. Tumor volumes of individual untreated tumors (red) and treated tumors (black) are displayed.

Supplementary Methods

Supplementary Table 1. Selection strategy Supplementary Table 2. Kinetic parameters for ADC-1013 and B44 measured by Biacore Supplementary Table 3. In vitro potency (EC50) of ADC-1013 in DC and B cell assays Supplementary table 4. Phenotypic profiling of hCD40tg mice

Supplementary Figure 4. Anti-tumor effect on MB49 tumors. ADC-1013 generates a significant anti-tumor effect on CD40 negative MB49 cells in hCD40tg mice. Treatment with ADC-1013 or isotype control was performed on day 7 and 10 post tumor inoculation. Increasing doses of ADC-1013 were administred peritumorally in 100 ul PBS and the individual tumor growth curves are displayed.

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.

Non-responders to checkpoint inhibitors generally have low tumor T cell infiltration and could benefit from immunotherapy that activates dendritic cells, with priming of tumor-reactive T cells as a result. Such therapies may be augmented by providing tumor antigen in the form of cancer vaccines. Our aim was to study the effects of mitazalimab (ADC-1013; JNJ-64457107), a human anti-CD40 agonist IgG1 antibody, on activation of antigen-presenting cells, and how this influences the priming and anti-tumor potential of antigen-specific T cells, in mice transgenic for human CD40. Mitazalimab activated splenic CD11c+ MHCII+ dendritic cells and CD19+ MHCII+ B cells within 6 h, with a return to baseline within 1 week. This was associated with a dose-dependent release of proinflammatory cytokines in the blood, including IP-10, MIP-1α and TNF-α. Mitazalimab administered at different dose regimens with ovalbumin protein showed that repeated dosing expanded ovalbumin peptide (SIINFEKL)-specific CD8+ T cells and increased the frequency of activated ICOS+ T cells and CD44hi CD62L− effector memory T cells in the spleen. Mitazalimab prolonged survival of mice bearing MB49 bladder carcinoma tumors and increased the frequency of activated granzyme B+ CD8+ T cells in the tumor. In the ovalbumin-transfected tumor E.G7-OVA lymphoma, mitazalimab administered with either ovalbumin protein or SIINFEKL peptide prolonged the survival of E.G7-OVA tumor-bearing mice, as prophylactic and therapeutic treatment. Thus, mitazalimab activates antigen-presenting cells, which improves expansion and activation of antigen-specific T cells and enhances the anti-tumor efficacy of a model cancer vaccine.

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.

BackgroundAlligator's Neo-X-Prime platform aims to enable antigen presenting cells to efficiently enhance priming of tumor neoantigen-specific T cells with the goal of overcoming PD-1 resistance in certain tumor types. We hypothesize 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 (i) activation of the DC, (ii) uptake of the tumor material, (iii) cross-presentation of tumor-derived neoantigen (present in exosomes or debris) and, iv) priming of tumor neoantigen-specific T cells, resulting in an increased quantity and/or quality of the tumor-targeting T cell pool.MethodsFunctionality was evaluated in vitro using CD40 reporter cells and monocyte-derived DCs, co-cultured with cells expressing TAA. Further, co-localization of TAA-expressing cellular debris with a CD40-expressing human B cell line in the presence of bsAbs was assessed using live cell imaging. In vivo, anti-tumor efficacy and immunological memory were assessed in human CD40 transgenic (hCD40tg) mice bearing MB49 bladder carcinoma tumors transfected with human TAA or controls. T cells isolated from OVA-specific TCR-transgenic mice were used to evaluate the effect of Neo-X-Prime bsAbs on antigen-specific T cell expansion in the presence of hCD40tg DCs and exosomes from MB49 tumors transfected with both human TAA and OVA using flow cytometry.ResultsUsing CEA as a highly expressed TAA, we have developed lead Neo-X-Prime CD40-CEA bsAbs engineered to achieve an optimal profile. Further, using Neo-X-Prime concept molecules targeting EpCAM, we have demonstrated the ability to mediate co-localization of tumor debris and CD40 expressing antigen presenting cells that is dependent on the receptor density of the TAA. We have further shown that addition of Neo-X-Prime bsAbs to a co-culture of murine DCs, T cells and tumor-derived exosomes induces increased expansion of model neoantigen-specific T cells. In vivo, Neo-X-Prime bsAbs display a potent, TAA-dependent anti-tumor effect that is superior to CD40 mAbs. Cured mice develop a broad immunological memory that is not dependent on expression of the TAA. The tumor-localizing property of Neo-X-Prime bsAbs also shows potential for improved safety compared to CD40 monospecific antibodies.ConclusionsNeo-X-Prime bsAbs have the potential to tumor-selectively target CD40-expressing antigen-presenting cells to mediate an expansion of the tumor-specific T cell repertoire, resulting in increased T cell infiltration and potent anti-tumor effects.Ethics ApprovalAll experiments were performed after approval from the Malmö/Lund Animal Ethics Committee.

Background4-1BB (CD137) is an activation-induced co-stimulatory receptor that regulates immune responses of activated CD8+ T cells and NK cells. Leveraging the therapeutic benefit of 1st generation 4-1BB monospecifics has been challenging due to dose limiting hepatotoxicity. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel 4-1BB x 5T4 bispecific antibody that stimulates 4-1BB function only when co-engaged with 5T4, a highly selective tumor-associated antigen. The combined preclinical dataset presented here provides an overview of the potential indication landscape, mechanism of action and the efficacy and safety profile of ALG.APV-527, supporting its advancement into the clinic.MethodsGenevestigator Software was used to analyze curated transcriptomic data from bulk tumor mRNA-sequencing data libraries and from single cell RNA-seq libraries for the expression profiles of CD8, 4-1BB and 5T4 across selected human solid tumor datasets. ADCC and ADCP reporter bioassays were utilized to assess Fc engagement by ALG.APV-527. For in vitro tumor lysis studies, human T cells were co-cultured with labelled tumor cells and sub-optimally activated with anti-CD3. Cytotoxicity of tumor cells were continually assessed using a Live-Cell Analysis System.ResultsDual expression of CD8 and 5T4 occurred in many tumor types and correlated well with indications that are pursued in the clinical development of ALG.APV-527. 4-1BB expression was observed in tumor-derived lymphoid subpopulations, especially in those with an exhausted phenotype. Since ALG.APV-527 is designed with a non-Fcγ receptor binding Fc, minimal ADCC & ADCP was induced in vitro. Additionally, ALG.APV-527 enhanced primary immune cell-mediated killing of 5T4-expressing tumor cells when compared to anti-CD3 alone, demonstrating the potential benefit of 4-1BB agonism for enhancing cytotoxic anti-tumor responses in the clinic.ConclusionsALG.APV-527 is designed to elicit safe and efficacious 4-1BB-mediated antitumor activity in a range of 5T4-expressing tumor indications. Transcriptional profiling of patient tumor samples demonstrates 4-1BB expression in multiple tumor-infiltrating lymphocyte subsets and identifies potential indications with 5T4 expression and CD8+ T cell infiltration. The unique design of the molecule minimizes systemic immune activation and hepatotoxicity, allowing for highly efficacious tumor-specific responses as demonstrated by potent activity in in vitro models. Based on these preclinical data, ALG.APV-527 is a promising anti-cancer therapeutic for the treatment of a variety of 5T4-expressing solid tumors and is progressing towards a phase I clinical trial in 2021.

Mitazalimab is a human CD40 agonistic IgG1antibody being developed as cancer immunotherapy. Targeting CD40 kickstarts the cancer immunity cycle by licensing DCs leading to tumor-specific T cell priming and activation. Furthermore, in PDAC, CD40 agonism activates myeloid cells and promotes the degradation of the desmoplastic tumor stroma, improving the influx of T cells and chemotherapeutic agents into the tumor. Mitazalimab has shown to be safe and well tolerated (at doses up to 1200 μg/kg) with signs of clinical activity in solid tumors in a Phase I study (NCT02829099). Most drug related adverse events (AE) were grade 1 or 2. OPTIMIZE-1 (NCT04888312) is a phase 1b/2, open-label, multicenter study designed to evaluate safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX in adults diagnosed with previously untreated mPDAC. The objective of the first (phase 1b) part of the study was to determine the RP2D of mitazalimab + mFOLFIRINOX. Mitazalimab was escalated from 450 µg/kg to 900 µg/kg following a Bayesian optimal interval design with at least 3 patients enrolled per dose level. In the first 21-day treatment cycle (Dose Limiting Toxicity assessment period), mitazalimab is administered intravenously on day 1 and 10 and mFOLFIRINOX starts on day 8. In the second and subsequent cycles, treatment follows a 14-day cycle schedule where mitazalimab is administered 2 days after mFOLFIRINOX. In part 2 of the study (phase 2), mitazalimab at the RP2D will be administered in combination with mFOLFIRINOX. Primary endpoint is RECIST-defined overall response rate. Progression-free survival and overall survival will be assessed as secondary endpoints. We report data from phase 1b (dose escalation) part of this study. As of March 9, 2022, 11 patients were treated with mitazalimab: 5 at 450 µg/kg and 6 at 900 µg/kg mitazalimab doses. One patient in the 900 µg/kg dose cohort withdrew from the trial for administrative reasons after the first mitazalimab infusion, prior to receiving mFOLFIRINOX and was not included in RP2D determination. Key baseline characteristics included: 7 female, 4 male; median age 63 (range 57-70); ECOG 0-1; median time since mPDAC diagnosis, 25 days. Mitazalimab related AEs were reported in 9/11 patients. Treatment related AEs occurring in >1 patient were fever (60%), muscle pain (50%) and fatigue (20%). At the 450 µg/kg dose, all mitazalimab related AEs were grade 1-2. At the 900 µg/kg dose, 4 patients (67%) experienced grade 1-2 mitazalimab related AEs. One patient in the 900 µg/kg dose experienced mitazalimab related grade 3 fatigue and grade 3 headache that led to treatment discontinuation after the first cycle. There were no mitazalimab related grade 4 or 5 AEs. 1/10 patients required mFOLFIRINOX dose reduction and, at the cutoff date, the range of treatment length was 1-14 weeks. Mitazalimab combined with mFOLFIRINOX is safe and well tolerated. The 900 µg/kg dose of mitazalimab was selected as the RP2D. Phase 2 of the OPTIMIZE-1 trial is currently enrolling patients. Citation Format: Jean-Luc Van Laethem, Ivan Borbath, Hans Prenen, Aurélien Lambert, Karen Geboes, Jean-Frédéric Blanc, Yago Pico de Coaña, Karin Enell-Smith, Lena Schultz, Karin Nordbladh, Peter Ellmark, Sumeet Ambarkhane, Malin Carlsson, Philippe Cassier. Mitazalimab (CD40 agonist) in combination with mFOLFIRINOX in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Safety data and recommended dose for phase 2 (RP2D) from OPTIMIZE-1, a phase 1b/2 study [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A018.

BackgroundIndications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation, generate new tumor-specific T cells, and activate tumor-infiltrating myeloid cells to remodel the tumor microenvironment, represent a promising opportunity to meet this need. In this study, we present the first in vivo data supporting a role for tumor-associated antigen (TAA)-mediated uptake and cross-presentation of tumor antigens to enhance tumor-specific T-cell priming using CD40×TAA bispecific antibodies, a concept we named Neo-X-Prime.MethodsBispecific antibodies targeting CD40 and either of two cell-surface expressed TAA, carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) or epithelial cell adhesion molecule (EpCAM), were developed in a tetravalent format. TAA-conditional CD40 agonism, activation of tumor-infiltrating immune cells, antitumor efficacy and the role of delivery of tumor-derived material such as extracellular vesicles, tumor debris and exosomes by the CD40×TAA bispecific antibodies were demonstrated in vitro using primary human and murine cells and in vivo using human CD40 transgenic mice with different tumor models.ResultsThe results showed that the CD40×TAA bispecific antibodies induced TAA-conditional CD40 activation both in vitro and in vivo. Further, it was demonstrated in vitro that they induced clustering of tumor debris and CD40-expressing cells in a dose-dependent manner and superior T-cell priming when added to dendritic cells (DC), ovalbumin (OVA)-specific T cells and OVA-containing tumor debris or exosomes. The antitumor activity of the Neo-X-Prime bispecific antibodies was demonstrated to be significantly superior to the monospecific CD40 antibody, and the resulting T-cell dependent antitumor immunity was directed to tumor antigens other than the TAA used for targeting (EpCAM).ConclusionsThe data presented herein support the hypothesis that CD40×TAA bispecific antibodies can engage tumor-derived vesicles containing tumor neoantigens to myeloid cells such as DCs resulting in an improved DC-mediated cross-priming of tumor-specific CD8+T cells. Thus, this principle may offer therapeutics strategies to enhance tumor-specific T-cell immunity and associated clinical benefit in indications characterized by poor T-cell infiltration or deficiencies in T-cell priming.

Agonistic CD40 antibodies activate dendritic cells and can expand and activate tumor-specific T cells. Our purpose was to assess the CD40 agonistic antibody ADC-1013 in the clinical setting including intratumoral administration since preclinical studies have indicated that intratumoral is better than intravenous administration. A Phase I, open label, multicenter study was conducted in patients with advanced solid tumors who had received established treatments. A modified 3 + 3 dose-escalation was applied (every other week dosing). Twenty-three patients were treated with ADC-1013 intratumorally (dosing from 22.5 μg/kg up to 400 μg/kg) or intravenously (dosing at 75 μg/kg). The pharmacodynamic effects observed in the patients were further verified in an hCD40tg mouse model. Adverse events were mostly Common Terminology Criteria for Adverse Events (CTCAE) Grades 1 or 2 and transient. The serum concentration ADC-1013 and cytokine release (MCP-1, TNFα and IL-6) were more pronounced in patients receiving injections in deep metastases compared to patients receiving injections in superficial metastases. Treatment with ADC-1013 resulted in a marked decrease in B cell levels in peripheral blood after 24 h while remaining B cells significantly increased their expression of the cell surface activation marker CD86. Activation of antigen-presenting cells and subsequent activation of T cells were demonstrated in hCD40tg mice. Moreover, ADC-1013 treatment in this mouse model acted synergistically with a PD-1 inhibitor. The results from the first-in-human study of ADC-1013 indicate that intratumoral administration of ADC-1013 into superficial lesions is well tolerated at clinically relevant doses and associated with pharmacodynamic responses.

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.

e16237 Background: Mitazalimab is a human CD40 agonistic IgG1antibody being developed for cancer immunotherapy. Targeting CD40 kickstarts the cancer immunity cycle by licensing DCs leading to tumor-specific T cell priming and activation. Furthermore, in PDAC CD40 agonists on myeloid cells promote degradation of the desmoplastic tumor stroma, improving influx of T cells and chemotherapeutic agents into the tumor. Mitazalimab has shown to be safe and well tolerated (at doses up to 1200 μg/kg), with signs of clinical activity in solid tumors in a Phase I study (NCT02829099). Most drug related adverse events (AE) were grade 1 or 2. Methods: OPTIMIZE-1 (NCT04888312) is a Phase 1b/2, open-label, multicenter study designed to evaluate safety, tolerability, and efficacy of mitazalimab in combination with mFOLFIRINOX in adults diagnosed with previously untreated metastatic PDAC. In the first 21-day treatment cycle (Dose Limiting Toxicity (DLT) assessment period), mitazalimab is administered intravenously on day 1 and 10 and mFOLFIRINOX infusion starts on day 8. In the second and subsequent cycles, treatment follows a 14-day cycle schedule where mitazalimab is administered 2 days after mFOLFIRINOX. The primary objective of the first part of the study (Phase 1b) is to determine the recommended Phase 2 Dose (RP2D) of mitazalimab in combination with mFOLFIRINOX. Mitazalimab will be escalated from 450 µg/kg to 900 µg/kg following a Bayesian optimal interval design with at least 3 patients enrolled per dose level. A minimum of 6 patients will be evaluated at the RP2D. In Part 2 of the study, mitazalimab at the RP2D will be administered in combination with mFOLFIRINOX. The primary endpoint is RECIST-defined overall response rate. Progression-free survival and overall survival will be assessed as secondary endpoints. Here we report data from the Phase 1b (dose escalation) part of this study. Results: Five patients with histologically confirmed, previously untreated metastatic PDAC were treated at the 450 µg/kg dose level. Key baseline characteristics included: 2 female, 3 male; median age 65 (range 60-68); ECOG score of 0 or 1; median time since primary diagnosis 13 days (range 7-82). Treatment related AEs were only grade 1 or 2 (reported in 4 of 5 patients). Treatment related AEs occurring in > 1 patient were fever, muscle pain and nausea. No DLTs were reported. No patients required dose interruption/reduction with mitazalimab or mFOLFIRINOX. Two patients discontinued treatment due to disease progression and three remain on study. The study continues and patients are currently being enrolled at the 900 µg/kg mitazalimab dose in combination with mFOLFIRINOX. Conclusions: Mitazalimab at 450 µg/kg dose combined with mFOLFIRINOX is safe and well tolerated. Enrollment at the 900 µg/kg dose is ongoing for this Phase 1b/2 study. Updated data from part 1 of the study will be presented. Clinical trial information: NCT04888312.

Introduction: CD40 signaling activates dendritic cells leading to improved T cell priming against tumor antigens. CD40 agonism expands the tumor-specific T cell repertoire and has the potential to increase the fraction of patients that respond to established immunotherapies. Areas covered: This article reviews current as well as emerging CD40 agonist therapies with a focus on antibody-based therapies, including next generation bispecific CD40 agonists. The scientific rationale for different design criteria, binding epitopes, and formats are discussed. Expert opinion: The ability of CD40 agonists to activate dendritic cells and enhance antigen cross-presentation to CD8+ T cells provides an opportunity to elevate response rates of cancer immunotherapies. While there are many challenges left to address, including optimal dose regimen, CD40 agonist profile, combination partners and indications, we are confident that CD40 agonists will play an important role in the challenging task of reprogramming the immune system to fight cancer. (Less)

Background Bladder cancer is categorized as non-muscle invasive (NMIBC) or muscle invasive (MIBC). NIMBC makes up around 70% of the cases and although it is less aggressive, the recurrence rate is 50-70%, thus requiring extensive monitoring. Additionally, there is a risk of progression into MIBC with a 5-year survival of only 50% even when treated with radical cystectomy. Immune checkpoint inhibitors have shown promising results for treatment of bladder cancer; however, only around 30% of patients have a therapeutic effect and novel therapies are thus required. With the aim of pinpointing novel targets for T-cell based therapy, we have performed transcriptomic profiling of specific T cell populations in MIBC and NMIBC, as well as in control bladder tissue. Methods Muscle-invasive (n=7) as well as non-muscle invasive (n=13) bladder tumor biopsies were obtained from untreated patients and control bladder tissue (n=7). Upon digestion, cells were stained with an antibody panel to enable sorting of CD8+ cytotoxic T-cells (CD8T), CD4+ T-helper cells (Th) and regulatory T-cells (Treg) using fluorescence activated cell sorting. RNA was extracted and subject to sequencing. Differential gene expression analysis was performed, using DESeq2 (genes with padj Results Principal component analysis demonstrated that CD8T, unlike Th and Tregs, cluster according to the invasiveness of the disease. Accordingly, many genes were significantly differentially expressed between CD8T in MIBC and NMIBC compared to control, and also between CD8T in MIBC compared to NMIBC. Several genes associated with CD8 T-cell exhaustion were significantly upregulated in MIBC compared to both NMIBC and control. Further, GSEA results indicated biological differences of the CD8T compartment between different tumor stages. Conclusions The gene expression profiles of CD8 T-cells were significantly different in NMIBC, MIBC and control. The transcriptional profiles give clues on biological differences and disease progression and can be relevant for development of novel treatment strategies. Ethics Approval The study was approved by the Regional Ethics Committee (EPN - Regionala Etikprovningsnamnden i Lund), approval number 2017/34. Consent Written informed consent was obtained from all patients included in the study.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans., Costimulation has been shown to be required for optimal activation of T cells and it could be delivered either in trans with respect to the source of CD3-TCR ligation or in cis on the same cell. Here the authors show that CD137 costimulation is more effective when delivered in cis to enhance T cell proliferation and activation.