DataSheet_1_Interferon gamma immunoPET imaging to evaluate response to immune checkpoint inhibitors.pdf

IntroductionWe previously developed a 89Zr-labeled antibody-based immuno-positron emission tomography (immunoPET) tracer targeting interferon gamma (IFNγ), a cytokine produced predominantly by activated T and natural killer (NK) cells during pathogen clearance, anti-tumor immunity, and various inflammatory and autoimmune conditions. The current study investigated [89Zr]Zr-DFO-anti-IFNγ PET as a method to monitor response to immune checkpoint inhibitors (ICIs).MethodsBALB/c mice bearing CT26 colorectal tumors were treated with combined ICI (anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death 1 (PD-1)). The [89Zr]Zr-DFO-anti-IFNγ PET tracer, generated with antibody clone AN18, was administered on the day of the second ICI treatment, with PET imaging 72 hours later. Tumor mRNA was analyzed by quantitative reverse-transcribed PCR (qRT-PCR).ResultsWe detected significantly higher intratumoral localization of [89Zr]Zr-DFO-anti-IFNγ in ICI-treated mice compared to untreated controls, while uptake of an isotype control tracer remained similar between treated and untreated mice. Interestingly, [89Zr]Zr-DFO-anti-IFNγ uptake was also elevated relative to the isotype control in untreated mice, suggesting that the IFNγ-specific tracer might be able to detect underlying immune activity in situ in this immunogenic model. In an efficacy experiment, a significant inverse correlation between tracer uptake and tumor burden was also observed. Because antibodies to cytokines often exhibit neutralizing effects which might alter cellular communication within the tumor microenvironment, we also evaluated the impact of AN18 on downstream IFNγ signaling and ICI outcomes. Tumor transcript analysis using interferon regulatory factor 1 (IRF1) expression as a readout of IFNγ signaling suggested there may be a marginal disruption of this pathway. However, compared to a 250 µg dose known to neutralize IFNγ, which diminished ICI efficacy, a tracer-equivalent 50 µg dose did not reduce ICI response rates.DiscussionThese results support the use of IFNγ PET as a method to monitor immune activity in situ after ICI, which may also extend to additional T cell-activating immunotherapies.</p

Supplementary Data from Immune Function Abnormalities in Peripheral Blood Mononuclear Cell Cytokine Expression Differentiates Stages of Cutaneous T-Cell Lymphoma/Mycosis Fungoides

Supplementary Data from Immune Function Abnormalities in Peripheral Blood Mononuclear Cell Cytokine Expression Differentiates Stages of Cutaneous T-Cell Lymphoma/Mycosis Fungoide

DataSheet_1_Evaluation of an ImmunoPET Tracer for IL-12 in a Preclinical Model of Inflammatory Immune Responses.pdf

The immune cytokine interleukin-12 (IL-12) is involved in cancer initiation and progression, autoimmunity, as well as graft versus host disease. The ability to monitor IL-12 via imaging may provide insight into various immune processes, including levels of antitumor immunity, inflammation, and infection due to its functions in immune signaling. Here, we report the development and preclinical evaluation of an antibody-based IL-12-specific positron emission tomography (PET) tracer. To mimic localized infection and stimulate IL-12 production, BALB/c mice were administered lipopolysaccharide (LPS) intramuscularly. [89Zr]Zr-DFO-αIL12 tracer was given one hour post LPS administration and PET images were taken after 5, 24, 48, and 72 hours. We observed significantly higher uptake in LPS-treated mice as compared to controls. Biodistribution of the tracer was evaluated in a separate cohort of mice, where tracer uptake was elevated in muscle, spleen, lymph nodes, and intestines after LPS administration. To evaluate the utility of [89Zr]Zr-DFO-αIL12 as an indicator of antigen presenting cell activation after cancer immunotherapy, we compared PET imaging with and without intratumoral delivery of oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (Adv/GM-CSF), which we have shown promotes anti-tumor immunity. BALB/c mice were inoculated orthotopically with the mouse mammary carcinoma line TUBO. Once TUBO tumors reached a volume of ~50 mm3, mice were treated with either three intratumoral injections of 108 PFU Adv/GM-CSF or vehicle control, given every other day. Upon the last dose, [89Zr]Zr-DFO-αIL12 was injected intravenously and 72 hours later all mice were imaged via PET. Tumor-specific uptake of [89Zr]Zr-DFO-αIL12 was higher in Adv/GM-CSF treated mice versus controls. Tissues were harvested after imaging, and elevated levels of macrophages and CD8+ Tc cells were detected in Adv/GM-CSF treated tumors by immunohistochemistry. We validated that IL-12 expression was induced after Adv/GM-CSF by qRT-PCR. Importantly, expression of genes activated by IL-12 (IFNγ, TNFα, and IL-18) were unaffected after IL-12 imaging relative to mice receiving an IgG control tracer, suggesting the tracer antibody does not significantly disrupt signaling. Our results indicate that targeting soluble cytokines such as IL-12 by PET imaging with antibody tracers may serve as a noninvasive method to evaluate the function of the immune milieu in situ.</p

Supplemental Figure 5 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Specificity control of mouse immune serum.</p

Supplemental Figure 1 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Authentication of the cat mammary tumor lines K248 and K12 used in this study via short tandem repeat (STR) analysis at 4 loci.</p

Supplemental Figure 2 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

RTK analysis of FMC primary tissue and cell lines.</p

Supplemental Table 1 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Feline mammary carcinoma patients.</p

Supplemental Figure 3 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Sequence alignment of HER2 extracellular and transmembrane regions (ECTM) from 5 mammalian species.</p

Supplemental Figure 4 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Q141K substitution in feline HER2.</p

Supplemental Table 2 from Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination

Cloning primers.</p