Abstract Non-small cell lung cancer (NSCLC) is the primary cause of cancer-related mortality worldwide, necessitating novel interventions to improve survival rates. Current treatment modalities for locally advanced NSCLC involve cytotoxic chemotherapy with concurrent external beam radiation therapy (XRT), followed by immunotherapy. However, the impact of XRT on the complement system, a crucial component of innate immunity, remains unclear. The complement regulatory proteins (CRPs), including CD46, CD55, and CD59, tightly control complement activation mediated by the three pathways. In this study, we investigated the surface expression of CRPs in NSCLC cell lines in vitro and within the tumor microenvironment (TME) in vivo. We assessed the activation of the three complement pathways in the TME and monitored tumor growth and survival in C57BL/6 wild-type, C3-/-, and C3AR-/- mice following XRT. Following XRT, NSCLC cell lines A549 and H460 exhibited a significant increase in the cell surface expression of CD46, CD59, and CD55 compared to controls (p<0.05), whereas normal lung epithelial cells (MRC5) did not display such upregulation. Irradiated Lewis lung carcinoma (LLC) tumors in wild-type mice demonstrated elevated levels of C3 and C5 mRNA and protein (p=0.0013). Also, C3AR and C5AR were significantly upregulated in the irradiated LLC tumor as compared to the sham. Additionally, the alternative complement pathway components, factor B and factor D, were significantly upregulated (p<0.001) in the LLC TME following XRT. However, the membrane attack complex (MAC) did not form in XRT-treated LLC tumors compared to sham. Deficiency of the central C3 and C3AR complement components in C3-/- and C3AR-/- mice delays LLC tumor growth in combination with XRT. Our findings suggest that XRT increases the expression of CRPs in NSCLC, potentially facilitating immune evasion by tumor cells. We also found activation of the alternative pathway of the complement system. Targeting C3 and C3AR effectively delayed tumor growth, highlighting the potential of complement pathway modulation to enhance the efficacy of XRT in NSCLC treatment. These results underscore the importance of elucidating the intricate interplay between radiation-induced complement activation and tumor survival for devising novel therapeutic strategies in NSCLC. Citation Format: Harendra Shah, Minakshi Saikia, Xiaobo Wu, John P. Atkinson, Abhay K. Singh, Vaishali Kapoor. Radiation-mediated complement cascade activation within the lung tumor microenvironment diminishes the efficacy of radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7377.

Tumor-associated factors released by cancer cells can promote immune suppression and tolerance to radiation or immune therapy. Squamous cell carcinoma antigen (SCCA) is a tumor-associated antigen released by cancer cells. In cervical cancer, elevated pre-treatment SCCA in the serum is seen in 28-88% of patients and these patients are more likely to experience treatment resistance and recurrence. The poor prognosis for patients with elevated SCCA shows the need for improve treatment strategies. Here, we aim to understand the mechanism of action of circulating SCCA to tumor progression and response to radiotherapy. Using sequencing analysis of paired pre- versus on-treatment biopsies, we found that patients with elevated serum SCCA had increased macrophages expressing immunosuppressive features and T-cell immune checkpoints, inversely correlated with inflammatory signaling and radiation-induced interferon-stimulated gene expression. The supplement of SCCA protein (rSCCA) to the media during in vitro macrophage differentiation promotes anti-inflammatory cytokines and gene expression. Peritoneal macrophages isolated from mice treated with intra-peritoneal rSCCA expressed high M2-associated markers and functional inhibition of T cell activation. Moreover, we found that mouse tumors engineered to express SCCA with a signal peptide promoting extracellular secretion displayed increased CSF1R+ TAM infiltration and decreased T cell activation, associated with reduced response to combined radiation and anti-PDL1 treatment. Giving the findings, we hypothesized that targeting TAMs in tumors with elevated SCCA is imperative. We further demonstrated that tumor-bearing mice showed improved response to radiation and immune checkpoint therapy when receiving concurrent CSF1R blockade, abolishing the protective effect of high circulating SCCA. Our results suggest that blockade of CSF1R signaling or other myeloid-targeting therapeutics will optimize response to radiation and PD-L1 blockade in cervical cancer, particularly those with high circulating SCCA. Liyun Chen, Eric Liu, Victoria Shi, Marlene L. Campos Guerrero, Jasmin Yang, Abhay Singh, Matthew Inkman, Jin Zhang, Julie Schwarz, Stephanie Markovina. Targeting CSF1R+ macrophages to synergize immune checkpoint therapy in SCCA-mediated radioresistant cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4696.

Supplementary Figure 3. A. Representative chromatogram showing protein A purification profile of the L111 antibody. B. Representative size exclusion chromatogram in S200 10/300 GL columns of the purified L111 antibody.

Over the last two decades, Electron Energy Loss Spectroscopy (EELS) imaging with a scanning transmission electron microscope has emerged as a technique of choice for visualizing complex chemical, electronic, plasmonic, and phononic phenomena in complex materials and structures. The availability of the EELS data necessitates the development of methods to analyze multidimensional data sets with complex spatial and energy structures. Traditionally, the analysis of these data sets has been based on analysis of individual spectra, one at a time, whereas the spatial structure and correlations between individual spatial pixels containing the relevant information of the physics of underpinning processes have generally been ignored and analyzed only via the visualization as 2D maps. Here, we develop a machine learning-based approach and workflows for the analysis of spatial structures in 3D EELS data sets using a combination of dimensionality reduction and multichannel rotationally invariant variational autoencoders. This approach is illustrated for the analysis of both the plasmonic phenomena in a system of nanowires and in the core excitations in functional oxides using low loss and core-loss EELS, respectively. The code developed in this manuscript is open sourced and freely available and provided as a Jupyter notebook for the interested reader.

Supplementary Figure 11. Specific uptake of the [89Zr]Zr-DFO-L111 in tumors in vivo. Bar graph showing the uptake of the [89Zr]Zr-DFO-Isotype or [89Zr]Zr-DFO-L111 at days 2 and 5 in nude mice bearing A549 tumors in the hindlimb (n=4). Tumors were irradiated with three doses of 3Gy over 24h, followed by injection with radiolabeled antibodies. Cold anti-TIP1 Ab was injected before injecting the radiolabeled for the blocking study. Bar graphs represent %ID/gm. *P<0.05, **P<0.01

Supplementary Table 3. Rank ordering of the DFO conjugated L111 antibodies based on their KD values

Supplementary Figure 1. Strategy for WashU II phage display library creation. The human antibody repertoires were amplified by PCR from the cDNA of mixed human peripheral blood mononuclear cells and cloned randomly into a phagemid vector. This vector encodes for a 16 aa linker (G4S)3T between the VH and VL domain of the scFv and adds an additional C-terminal 6xHis and Flag tag. First, the VH and VL repertoires were amplified in the first set of PCRs and completed by SfiI/XhoI (VH) or SalI/NotI (VL) restriction sites in the second set of PCRs. The VH repertories were cloned first by electroporation in E. coli TG1 (Lucigen, #60502), followed by cloning of the VLkappa or VL-lambda repertories.

Supplementary Figure 13. A. Schematic representation of the tumor implantation, irradiation, antibody injection, PET imaging and post-PET biodistribution schedule. B and C. The tumor SUV max in H460 tumors (B) and A549 tumors (C) at days 2 and 5 post-injection (n=4).

Supplementary Figure 9. A. HPLC-SEC analysis of L111 and L111-DFO on an Agilent 1200 series HPLC system using a Zorbax GF-250 9.4 mm ID x 25 cm column (Agilent). DFO conjugation did not change the retention time of the antibody, and no aggregation was observed. B. iTLC showing the radiochemical purity of the radiolabeled L111 antibody. C. Radio-HPLC confirming the radiolabeling of the L111 antibody with [89Zr]Zr.

Supplementary Figure 6. Flow cytometry evaluation of the purified anti-TIP1 antibodies on H460 (A) and A549 (B) cells. The percentage of anti-TIP1+ vs. antibody concentration was plotted in GraphPad Prism software using One Site-specific binding model. The table below represents the Kd values. C. Immunohistochemistry shows intense TIP1 staining in the human patient-derived xenograft tissue.

Supplementary Figure 10. A. In vitro stability of [89Zr]Zr-DFO-L111 antibodies that were either freshly conjugated with DFO or thawed from -80°C after DFO conjugation. The stability was assessed in human serum by instant thin-layer chromatography. B. In vitro serum stability of [89Zr]Zr-DFO-L111 antibodies in four different buffers. The stability was assessed in human serum by instant thin layer chromatography.

Supplementary Figure 12. In vitro characterization of [89Zr]Zr-DFO-L111. A. SDS-PAGE under non-reducing and reducing conditions for the [89Zr]Zr-DFO-L111 developed by autoradiography. Four two-fold dilutions were loaded on the SDS-PAGE gel and developed by autoradiography. B. ELISA assay showing binding affinity of the [89Zr]Zr-DFO-L111 to recombinant TIP1 protein.

Supplementary Table 2. The analysis of the DFO-to-antibody ratio for the D10-L111 by native mass spectrometry

Supplementary Figure 8. DFO-to-L111 ratio (DAR) analysis by native MS. The deconvoluted mass spectra (deconvoluted using Intact Mass, Protein Metrics Inc) show the increasing numbers of DFO (+1 to +8). The respective DARs are shown in red.

AbstractPurpose: Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. Experimental Design: A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors’ blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). Results: We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. Conclusions: L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

Supplementary Figure 2. A. Colony PCR showing the completeness of the WashU II library. B. Western blots for the WashU II phages at different dilutions were developed with an anti-pIII antibody showing the fused scFv with the pIII coat protein of the M13 phage. C. Western blots for the WashU II phages at different dilutions were developed with an anti-Flag antibody showing the full-length scFv on the M13 phage.

Supplementary Figure 7. Biophysical characterization of the purified L111 antibody. A. Analysis of purified L111 antibody integrity by non-reduced (top) and reduced (bottom) capillary electrophoresis-SDS (CE-SDS) on a Perkin Elmer LabChip GXII Touch HT. The table shows the size and migration times of the non-reduced and reduced antibody samples. B. Size-exclusion chromatography high-performance liquid chromatography (SEC-HPLC) analysis of the purified L111 antibody. The table shows the percentage of monomers and high molecular weight species (HMWS). C. Cation-exchange chromatography of the purified L111 antibody. D. Deconvoluted mass spectrometry spectra of the purified L111 antibody. E and F. Analysis of Fc N-Glycan profiles and major glycans distribution by Hydrophilic interaction liquid chromatography (HILIC)-in combination with fluorescence detection (FLD). F. Table shows the percentage of each N-Glycan in the L111 antibody.

Supplementary Figure 5. ELISA shows the affinity of purified human anti-TIP1 full-length IgG1 to TIP1 protein. Recombinant TIP1 protein was coated on ELISA plates. Three-fold serial dilutions (starting at 20 nM) of the purified human anti-TIP1 full-length IgG1 antibodies were incubated with the protein. Anti-human HRP conjugated antibody was used as the detection antibody along with TMB substrate. Absorbance at 450nm vs concentration is plotted in the graph. The data were fitted using the A. “One-site specific” and B. log(agonist) vs. response -- Variable slope (four parameters) model in GraphPad Prism software. The Kd and EC50 values are shown in the table below each graph.

Supplementary Table 1. Rank ordering of the anti-TIP1 antibodies based on their KD values

Supplementary Figure 4. SDS-PAGE of eight unique purified human anti-TIP1 full-length IgGs under non-reducing (A) and reducing conditions (B). A. Intact full-length IgGs are observed above 150 kDa. B. In the reducing condition, heavy chains and light chains are observed at 50 kDa and 25 kDa, respectively.