Figure S27. Experimental design and dosing schematic for in vivo combination therapy studies. A schematic timeline illustrating the therapeutic protocol utilized for subcutaneous prostate cancer xenograft models (22Rv1).

Figure S25. PET/CT imaging and biodistribution with [89Zr]DFO-YS5-MMAE in DU145 tumor-bearing mice. (A) Axial, coronal, and MIP PET/CT images of mice (M1–M3) at 24, 48, 96, and 168 hours post-injection show sustained tumor uptake of [89Zr]DFO*-YS5-MMAE. Color scale indicates %ID/g. (B) Organ distribution of [89Zr]DFO*-YS5-MMAE in DU145 bearing mice, at 168 h post-injections.

Figure S13: A saturation binding assay was performed on DU145 cells, showing Kd values of 3.2 and 3.0 nM for YS5-MMAE and Macropa-PEG4-YS5-MMAE.

Supplementary Table S5. Region of interest analysis data reported as %ID/cc of the [89Zr]PEG-DFB1-TLZ3 on on different subcutaneous tumors from Day 1-4 post injection. (n = 4)

Supplementary Figure S13. Autoradiographic images and H&E staining images of subcutaneous CT26 tumor sections from day 1 to day 4 post injection of 89Zr labeled nanocarriers. *Indicate the presence of necrosis.

Supplementary Figure S4. Coronal μPET/CT fusion and MIP images obtained at 18 h, 48 h, 72 h, and 96 h following administration of 150-170 μCi of 89Zr labeled nanocarriers in nude mice bearing 22rv1 subcutaneous tumors over left flanks.

Figure S9: Experimental design and dosing schematic for in vivo combination therapy studies shown in Figure 2. A schematic timeline illustrating the therapeutic protocol utilized for subcutaneous prostate cancer xenograft models (22Rv1).

Supplementary Figure S2. Pilot PET/CT studies in prostate, pancreatic, and colorectal cancer models to measure the enhanced permeability retention effect using [89Zr]PEG-DFB1-TLZ3.

Infectious diseases are a major cause of morbidity and mortality worldwide. With the increasing frequency of antibiotic resistance, efficient and noninvasive diagnostic methods are more important than ever. In this report, we interrogate the use of several intravenously administered, bacteria-specific, 13C-enriched metabolites whose conversion to [13C]-CO2 can be detected via a portable and inexpensive method, namely nondispersive infrared (NDIR) spectroscopy. The enriched metabolites [U-13C]-maltose, [U-13C]-maltotriose, d-[U-13C]-mannitol, and l-[U-13C]-arabinose were metabolized to [13C]-CO2 by several pathogens in vitro, while showing minimal [13C]-CO2 production in uninfected mice. We further demonstrated that myositis, bacteremia, pneumonia, and osteomyelitis could be detected in vivo using one or more 13C-enriched metabolites. Additionally, in a model of Escherichia coli myositis, [13C]-CO2 production correlated with bacterial burden following ceftriaxone therapy, showing that exhaled [13C]-CO2 could be employed to monitor antimicrobial efficacy. Finally, [13C]-CO2 production by Staphylococcus aureus clinical isolates treated with [U-13C]-maltose was correlated with the performance of its cognate PET tracer [2-18F]-maltose, suggesting that [13C]-CO2 breath testing could predict the performance of pathogen-targeted positron emission tomography (PET) tracers in vivo. [13C]-CO2 breath testing using an expanded metabolite toolbox and on-site detection tools represents a unique and complementary method to identify bacterial infection in clinical practice.

Figure S17. Biodistribution of [225Ac]Macropa-PEG4-YS5-MMAE in 22Rv1 tumor-bearing mice at day 4 post-injection comparing low (0.1 kBq/µg) and high (6.3 kBq/µg) specific activity formulations (n=4).

Supplementary Table S4. Region of interest analysis data reported as %ID/cc of the [89Zr]PEG-DFB4 on different subcutaneous tumors from Day 1-4 post injection. (n = 4)

Supplementary Figure S23. Organ biodistribution presented in (left) %ID/g, and (right) %ID/organ of 89Zr labeled nanocarriers in nude mice inoculated with 22rv1 cells via intracardiac injection at 72 h postinjection. (n = 4, mean ± SD)

Supplementary Figure S21. Autoradiographic images and H&E staining images of metastatic tumor, and kedney sections from day 1 to day 4 post injection of [89Zr]PEG-DFB1-TLZ3 in nude mice inoculated with 22rv1 cells in the left kidney capsule. (NA = Data not available)

Figure S2: ZIP synergy analysis of the combination of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE in DU145 and LNCaP cells. (A) ZIP synergy score heatmap for DU145 cells treated with matrix of YS5-MMAE and [225Ac]Macropa-PEG4-YS5 to study the synergistic effect. The heatmap indicates regions of synergistic interaction (red regions) and antagonistic interaction (green regions). (B) ZIP synergy score heatmap for C4-2B cells treated with varying dose of [225Ac]Macropa-PEG4-YS5 and YS5-MMAE, and their combinations. The heatmap demonstrates differential synergy and antagonism compared to DU145 cells, highlighting cell-line-specific responses to the combination therapy.

Figure S19. iTLC showing radiolabeling of [134Ce]Macropa-PEG4-YS5-MMAE (A), and purity following the PD10 purification process (B).

Supplementary Table S3. Organ biodistribution data in %ID/Organ tissue for [89Zr]PEG-DFB1-TLZ3 at 72 h in mice model bearing different subcutaneous tumors. (n = 4)

Supplementary Table S1. Region of interest analysis data reported as %ID/cc of the [89Zr]PEG-DFB1-TLZ3 on on different subcutaneous tumors on 24 h and 72 h post injection. (n = 4)

Figure S22. Biodistribution of [134Ce]Macropa-PEG4-YS5 and [134Ce]Macropa-PEG4-YS5-MMAE in 22Rv1 tumor-bearing mice at 168 hours post-injection (n=4). %IA/g values are shown for major organs, including the liver, kidneys, spleen, and tumor.

Figure S1: MALDI mass spectrometry analysis of YS5-MMAE and Macropa-PEG4-YS5-MMAE revealed an average conjugation of 0.91 Macropa-PEG4 moieties per YS5-MMAE molecule. The disulfide bond reduction process used for MMAE conjugation led to the appearance of multiple peaks in the YS5-MMAE spectrum. For quantifying Macropa-PEG4 conjugation, the doubly charged peak at 75 kDa was used. The difference in peak intensity between YS5-MMAE and Macropa-PEG4-YS5-MMAE was divided by the molecular weight of Macropa-PEG4 to estimate the degree of conjugation.

Supplementary Table S14. Comparison of influx and efflux rates of [89Zr]PEG-DFB4 and [89Zr]PEG-DFB1-TLZ3 conjugates in different subcutaneous tumor models (n = 4, mean ± SD). *Prior published data.6