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.

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.

Supplementary Figure from Targeting a Radiosensitizing Antibody–Drug Conjugate to a Radiation-Inducible Antigen

Abstract Lung cancer is the leading cause of cancer death worldwide. There is an unmet need for therapies to improve lung cancer outcomes without causing additional toxicities. Tax-interacting protein 1 (TIP1) is a radiation-inducible antigen that plays a role in lung cancer progression and resistance to therapy. TIP1 is a targetable cell surface protein. We developed human antibodies (Ab) that bind specifically to lung cancer. This study aimed to evaluate our lead anti-TIP1 human antibody targeting lung cancer in vitro and in vivo. Anti-TIP1 IgG1 Ab was developed by biopanning phage display human antibody library. The purified Ab was characterized by size-exclusion chromatography-HPLC (SEC-HPLC), mass spectrometry, ELISA, BIAcore and flow cytometry. Ab was labeled with 89Zr using desferrioxamine chelator. The surface binding of 89Zr-Ab was evaluated in the A549 and H460 lung cancer cells. Stability of the 89Zr-Ab was assessed in human serum. Mice bearing 3 patient-derived xenografts (PDX) lung tumors or A549 tumors were injected with 50 µCi of 89Zr-Ab. Small animal PET imaging and biodistribution were conducted over 7 days. Specific tumor uptake was evaluated in biodistribution studies on day 5 by injecting cold Ab before 89Zr-Ab. We obtained 95% pure anti-TIP1 Ab by SEC-HPLC. Mass spectrometry confirmed the intact mass and glycosylation pattern of the anti-TIP1 IgG1. The Ab is bound to recombinant TIP1 protein and cancer cell surface with high affinity. The radiochemical purity of the 89Zr-Ab was 99.9%, and the molar activity was 11.54 MBq/nmol with a radiolabeling yield of 91.4%. Both A549 and H460 cell lines demonstrated specific binding as >70% inhibition was observed when the cold Ab was added. The ratio of SUV tumor to SUV muscle increased from 5.8 at 2 days to 6.3 at 7 days in PDX. In biodistribution studies, we found significantly higher (p<0.05) tumor uptake of the anti-TIP1 Ab (9.15±3.13 %ID/gm) vs. isotype control (5.72±1.02 %ID/gm). Cold anti-TIP1 Ab significantly blocked (p<0.0001) binding of the 89Zr-Ab in A549 tumors. The anti-TIP1 Ab targets lung cancer cells in vitro and in vivo. The Ab retained antigen-binding potential following labeling with 89Zr. This Ab can detect TIP1 positive tumors for therapy when conjugated with therapeutic radionuclides. IND enabling studies with the lead anti-TIP1 Ab are underway. Citation Format: Abhay Kumar Singh, Calvin D. Lewis, Cristian AWV Boas, Philipp Diebolder, Prashant N. Jethva, Aaron Rhee, Jong Hee Song, Young Ah Goo, Shunqiang Li, Yongjian Liu, Buck Rogers, Vaishali Kapoor, Dennis E. Hallahan. Preclinical development of a 89Zr-labeled human antibody as a novel immuno-PET agent for noninvasive cancer detection and monitoring response to treatment. [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 5035.

Therapeutic antibodies used to treat cancer are effective in patients with advanced-stage disease. For example, antibodies that activate T-lymphocytes improve survival in many cancer subtypes. In addition, antibody–drug conjugates effectively target cytotoxic agents that are specific to cancer. This review discusses radiation-inducible antigens, which are stress-regulated proteins that are over-expressed in cancer. These inducible cell surface proteins become accessible to antibody binding during the cellular response to genotoxic stress. The lead antigens are induced in all histologic subtypes and nearly all advanced-stage cancers, but show little to no expression in normal tissues. Inducible antigens are exploited by using therapeutic antibodies that bind specifically to these stress-regulated proteins. Antibodies that bind to the inducible antigens GRP78 and TIP1 enhance the efficacy of radiotherapy in preclinical cancer models. The conjugation of cytotoxic drugs to the antibodies further improves cancer response. This review focuses on the use of radiotherapy to control the cancer-specific binding of therapeutic antibodies and antibody–drug conjugates.