Human ox40 imaging agents for detecting t cell activation in vivo

Abstract

In one aspect, the disclosure relates to immunoPET agents for detecting activated T cells expressing human OX40, methods of making same, pharmaceutical compositions comprising same, and methods of monitoring OX40 positive cells in a subject using same. In an aspect, the immunoPET agents can include at least an antibody or an antibody fragment, wherein the antibody or antibody fragment is labeled with a radioisotope. In a further aspect, the antibody can be ivuxolimab or a fragment thereof, while the radioisotope can be Zr-89 or other related positron emission tomography or single photon emission tomography or theranostic isotope.

Description

ATTORNEY DOCKET NO. 221910-2400HUMAN 0X40 IMAGING AGENTS FOR DETECTING T CELL ACTIVATION IN VIVOCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 733,575 filed on December 13, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under contract CA286998 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Immunotherapy (IOT) has fast established itself as the fourth pillar of cancer treatment alongside surgery, radiation therapy, and chemotherapy. Its success, which relies on stimulation of the host immune system for effective cancer cell killing, has revolutionized the treatment of patients with advanced and metastatic disease, achieving extended survival and even cures. The observed variable clinical efficacy, however, combined with growing investment in developing new candidates and optimizing existing ones, highlights the urgent unmet need for technologies that permit non-invasive, longitudinal and whole-body monitoring of IOT response. Current standard of care techniques to assess IOT efficacy face limitations in specificity and capturing spatial, global, and / or longitudinal information. Tumor biopsies are invasive and fail to capture tissue heterogeneity while peripheral blood sampling for multiparametric analysis of circulating immune cells and cytokines lacks spatial information and does not accurately reflect the tumor microenvironment. Morphological changes measured using anatomical imaging typically manifest over an extended period (8-12 weeks) and struggle to distinguish disease progression and pseudo progression, an initial increase in lesion size and frequency caused by a usually favorable lOT-induced immune cell influx.

[0004] Central to the success of cancer immunotherapy is the ability of an immunotherapeutic agent to prime a patient’s immune system to induce T cell activation, an early and critical indicator of downstream expansion and infiltration into tumors for enhanced cell killing. Molecular imaging technologies that can interrogate T cell responses in patients within the tumor microenvironment and beyond represent a powerful approach for immune-monitoring. While generally beneficial in the context of immuno-oncology, activated T cells can also be a key driver and hallmark of immune-driven diseases including auto-immune disorders such as multiple sclerosis andATTORNEY DOCKET NO. 221910-2400 rheumatoid arthritis, chronic inflammatory diseases like inflammatory bowel disease, and transplant complications such as Graft versus Host Disease and solid organ transplant rejection.

[0005] Positron emission tomography (PET) imaging is a highly sensitive and quantitative clinical molecular imaging modality perfectly poised to provide non-invasive, whole-body mechanistic insights into T cell responses. Widely used metabolic tracers such as18F-fluorodeoxyglucose (18F- FDG) often exhibit non-specificity, as targeted metabolic pathways can also be upregulated in tumor-infiltrating lymphocytes (TILs), other activated immune cells, and cancer cells. To overcome this limitation, cluster of differentiation (CD) markers specific to immune subsets have increasingly garnered interest as potentially valuable imaging biomarkers. ImmunoPET, which combines the superior sensitivity of PET with the ultra-high specificity of monoclonal antibodies (mAbs) or antibody fragments, is a promising approach to visualize cell surface markers. Thus far, the most clinically advanced T cell immunoPET tracers targeted to cytotoxic CD8+TILs, have shown great potential for delineating anti-cancer responses. However, this approach overlooks CD4+T cells, which are also crucial for a robust T cell response. Furthermore, imaging these constitutively expressed lineage markers may fail to report on potential T cell dysfunction caused by the immunosuppressive tumor microenvironment. Imaging T cell function, specifically activation, could enable earlier, more accurate prediction of IOT outcomes.

[0006] Despite advances in molecular imaging, there is still a scarcity of high affinity probes that are selective for imaging activated T cells in order to monitor the progress of IOT while addressing deficiencies in spatial, global, and / or longitudinal information associated with known methods, and for other purposes. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0007] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to immunoPET agents for detecting activated T cells expressing the human 0X40 receptor (CD134), methods of making same, pharmaceutical compositions comprising same, and methods of monitoring 0X40 positive cells in a subject using same. In an aspect, the immunoPET agents can include at least an antibody or an antibody fragment, wherein the antibody or antibody fragment is labeled with a radioisotope. In a further aspect, the antibody can be ivuxolimab or a fragment thereof, while the radioisotope can be Zr-89.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings andATTORNEY DOCKET NO. 221910-2400 detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIGs. 1A-1C show production of human 0X40 PET tracer89Zr-lvuxolimab. FIG. 1A: Scheme summarizing optimized conditions for radiolabeling of deferoxamine (DFO)-lvuxolimab with89Zr. FIG. 1 B: Radio-iTLC results post-labeling and column-purification. FIG. 1C: Radio-and UV chromatograms of the final tracer product.

[0011] FIGs. 2A-2D show cell binding assays for in vitro validation of89Zr-lvuxolimab binding and specificity. FIG. 2A:89Zr-lvuxolimab binding to resting and activated primary human T cells, and activated T cells blocked by co-incubation with 25-fold higher mass dose of non-radioactive Ivuxolimab. FIG. 2B: Tracer binding to parental HEK293 cells, OX40+HEK293 cells and OX40+HEK293 cells was blocked by co-incubation with 25-fold higher mass dose of non-radioactive Ivuxolimab. Flow cytometry confirms increased 0X40 expression on (FIG. 2C) human activated compared to resting T cells and on (FIG. 2D) human OX40+HEK293 cells compared to parental HEK293 cells. Values represent mean ± SD, n=5-6 replicates / group from two independent experiments, **p<0.01 , ****p<0.0001 by 1-way ANOVA. T cell data are based on cells isolated from two healthy donors.

[0012] FIGs. 3A-3D show (FIG. 3A) Representative89Zr-lvuxolimab PET / CT whole body maximum intensity projection (MIP) coronal images of mice bearing HEK293 or human OX40+HEK293 tumors at 72 h and 120 h post tracer injection. White dashed lines indicate tumors (T); H: heart; L: liver; F: femur. FIG. 3B: Quantification of89Zr-lvuxolimab PET signal at 72 h (clear bars) and 120 h p.i (checked bars) in HEK293 and OX40+HEK293 tumor bearing mice. TissueATTORNEY DOCKET NO. 221910-2400 associated radioactivity is expressed as percentage injected dose / gram (%ID / g). FIG. 3C: Quantification of tumor: muscle ratios derived from PET images of the OX40+HEK293 tumor bearing (grey bars) versus the HEK293 tumor bearing (white bars) mice. FIG. 3D: Biodistribution analysis of89Zr-lvuxolimab using ex vivo gamma counting of tissues post necropsy at 72 h (clear bars) and 120 h p.i (checked bars). Tissue associated radioactivity is expressed as percentage injected dose / gram (%l D / g). Data presented as mean ± SD, results pooled from two independent experiments, n=4-6 mice per group, *p<0.05, p<0.001 , ***p<0.001). S.lnt: small intestine; L. Int: large intestine.

[0013] FIGs. 4A-4C show (FIG. 4A)89Zr-lvuxolimab PET allows detection of pathogenic activated T cells in early Graft versus Host Disease (GvHD), prior to overt clinical symptoms. PET / CT whole body MIP coronal and sagittal images of control total body irradiation (TBI) and bone marrow mice, shown alongside GvHD mice. FIG. 4B: Quantification of89Zr-lvuxolimab PET signal at 48 h p.i. in TBI, BM, and GvHD groups. FIG. 4C: Biodistribution analysis of89Zr-lvuxolimab using ex vivo gamma counting of tissues post necroscopy at 48 h p.i.

[0014] FIGs. 5A-5B show (FIG. 5A) High-resolution visualization of89Zr-lvuxolimab in lymphoid and gastrointestinal tissues via ex vivo autoradiography (ARG). FIG. 5B: IHC of 4iim thick tissue sections confirms presence of 0X40+ cells in intestinal tissue and mesenteric lymph node of GvHD mice, not observed in control TBI and BM mice. Corresponding H&E-stained images of adjacent tissue sections (bottom panel). Images were acquired at 40x magnification. L. Int: large intestine; S.lnt: small intestine; M. LN: mesenteric lymph node.

[0015] FIGs. 6A-6D show bioconjugation of ivuxolimab, a human 0X40 mAb (huQX40mAb) with DFO chelate and its subsequent characterization. FIG. 6A: Scheme summarizing optimized conditions for DFO conjugation to ivuxolimab. FIG. 6B: Electrospray ionization-mass spectrometry data and (FIG. 6C) Size-exclusion chromatography (SEC)-HPLC chromatogram of conjugate. FIG. 6D: Immunoreactivity test; binding of ivuxolimab and its DFO-modified version to OX40+HEK293 cells.

[0016] FIGs. 7A-7C show quality control to monitor radiolabeling reactions. Radio-TLC (left ) and radio-HPLC (right) profiles for (FIG. 7A)89Zr-oxalate in reaction buffer, (FIG. 7B) an incomplete radiolabeling reaction showing89Zr as free metal and a fraction bound by DFO-ivuxolimab and (FIG. 7C) a complete reaction in which89Zr is fully incorporated by the DFO-lvuxolimab to form89Zr-lvuxolimab.ATTORNEY DOCKET NO. 221910-2400

[0017] FIG. 8 shows representative flow cytometry plots showing 0X40 expression on CD4+ and CD8+ T cells from a healthy donor. T cells isolated from healthy donors were either rested or activated using anti-CD3 / anti-CD28 coated Dynabeads for 48 hours. Cells were gated on the CD4+ and CD8+ populations. While resting human CD4+ and CD8+ T cells show negligible 0X40 pression, it is markedly upregulated on both activated CD4+ and CD8+ T cells.

[0018] FIGs. 9A-9B show initial estimates of surface expression levels / OX40 antigen density on human T cells activated in vitro, yielded between 15,684 to 19,469 0X40 molecules / Dynabead- activated cell.

[0019] FIGs. 10A-10B shows ex vivo stability of89Zr-lvuxolimab assessed by (FIG. 10A) radio- iTLC and (FIG. 10B) radio-HPLC analysis of plasma collected at defined timepoints.

[0020] FIG. 11 shows longitudinal PET / CT imaging with89Zr-lvuxolimab enables visualization of 0X40 in vivo. Axial 20 min static PET / CT images of89Zr-lvuxolimab (48 hr, 72 hr and 120 hr p.i.) in representative NSG tumor bearing mice (tumors delineated with dotted white lines).

[0021] FIGs. 12A-12C show (FIG. 12A) Ex vivo macroscopic analysis of89Zr-lvuxolimab distribution in OX40+HEK293 and HEK293 tumortissue slices using autoradiography (ARG). The ARG of a representative human OX40+HEK293 tumor and HEK293 tumor taken 120 h p.i are shown overlaid with the H&E-stained images, alongside ARG of muscle taken from corresponding mice (top panel). ARG images of 40|im thick tissue sections (middle panel) and H&E-stained images of 12|im thick adjacent tissue sections (bottom panel) are also shown separately. Images were acquired at x40 magnification. FIG. 12B: ARG quantification; regions-of-interest were drawn around the tissues to calculate the mean pixel intensities (MPI) and derive tumor to muscle ratios. Data are presented as mean ± SD and are pooled from two independent experiments (n=4-15 mice per group), ***p<0.001. FIG. 12C: Immunohistochemical staining of the 0X40 receptor (CD134) in representative OX40+HEK293 and HEK293 tumors.

[0022] FIG. 13A shows SEC-HPLC showed a 0.6 min longer retention time for the F(ab’)2fragment (retention time, Rt = 10. 1 min), consistent with its smaller size compared to full-length Ivuxolimab (Rt = 9. 5 min). FIG. 13B shows ESI-MS confirmed the size difference between Ivuxolimab and its F(ab’)2fragment, with masses of 146,823 and 96,388 Da, respectively. FIG. 13C shows an SDS-PAGE of full-length Ivuxolimab (IgG), unpurified and purified lvux-F(ab’)2stained with Coomassie-blue, confirms successful generation and purification of the F(ab’)2fragment.ATTORNEY DOCKET NO. 221910-2400

[0023] FIGs. 14A-14D show synthesis and quality control of DFO-lvux-F(ab’)2 and89Zr-DFO- lvux-F(ab’)2radiotracer. FIG. 14A: Size exclusion chromatography revealed >92% pure F(ab’)2fragment generated from Ivuxolimab (retention time, Rt= 10.14 min, top). The smaller peak at Rt ~12 minutes is likely an impurity with lower MW (<8% abundance). DFO conjugated F(ab’)2fragment chromatogram largely coincides with unmodified F(ab’)2protein (Rt= 10.16 min bottom). FIG. 14B: Mass spectrometry confirming the molecular weight of the lvux-F(ab’)2fragment: 96388 (top). The mass spec profile of DFO-lvux-F(ab’)2indicates the presence of unmodified F(ab’)2(96389), and conjugates modified at a ratio of F(ab’)2:DFO of 1 :1 (97142), 1 :2 (97895), and 1 :3 (98692). FIG. 14C: Schema showing production of the new lvux-F(ab’)2PET tracer requiring two steps — 1) DFO conjugation of lvux-F(ab’)2in PBS buffer at pH ~9.0 followed by 60 mins agitation at 37 °C, and 2) radiolabeling of the DFO-lvux-F(ab’)2precursor with89Zr-oxalate in 0.5 M Hepes buffer for 45 min at 37 °C with agitation. D) Post-radiolabeling, radio-iTLC indicates pure89Zr- DFO-lvux-F(ab’)2radiotracer, with high radiochemical purity, suitable for further preclinical evaluation.

[0024] FIG. 15 shows89Zr-DFO-lvux-F(ab’)2maximum intensity projection (MIP) PET / CT images. PET / CT images of a representative HEK293 tumor bearing mouse (left) and a human 0X40+ HEK293 tumor bearing mouse (right) at 72 h post injection (p.i.) of tracer. Coronal, sagittal and axial views shown for each mouse with white dashed lines indicating the tumors (T). L=liver, K=kidney.

[0025] FIG. 16 shows post-necropsy, biodistribution analysis of89Zr-DFO-lvux- F(ab’)2using ex vivo gamma counting of tissues from HEK293 tumor bearing (white bars) and 0X40+ HEK293 tumor bearing mice (black bars), 72 h p.i.. Data presented as mean SD. Results pooled from a single experiment (n=4-5 per group), Mann-Whitney U-test, **P = 0.001), bone; femur, I. Intestine; large intesting, s. intestine; small intestine.

[0026] FIG. 17 shows ex vivo macroscopic analysis of89Zr-DFO-lvux-F(ab’)2distribution in huOX40+ HEK293 and parental HEK293 tumors. Autoradiography of representative huOX40+ HEK293 tumor and HEK293 tumor sections taken 72 h p.i, alongside autoradiography of muscle taken from corresponding mice (40 pm thick tissue sections). Images were acquired at x40 magnification.

[0027] FIGs. 18A-18B show a bead-based immunoreactivity assay for89Zr-lvuxolimab at end-of- synthesis (EOS) and at 66 h post-EOS. High was observed at end-of-synthesis (EOS) and at 66h post-EOS in unblocked 0X40 antigen coated Dynabeads (green bars). Minimal nonspecificATTORNEY DOCKET NO. 221910-2400 binding (NSB) was observed when using control beads (lacking antigen coating). Immunoreactivity was also significantly reduced when89Zr-lvuxolimab and antigen coated-beads were co-incubated with excess Ivuxolimab (n=3 tubes / condition).

[0028] FIG. 19 shows calculation of binding parameters (Bmax and Kd) for89Zr-lvuxolimab using a radioactive cell binding assay. Total binding was determined by incubating huOX40+ HEK293 cells with increasing concentrations of89Zr-lvuxolimab. Antigen negative HEK293 cells were used to determine non-specific binding of89Zr-lvuxolimab. Specific binding of89Zr-lvuxolimab to huOX40+ HEK293 cells was determined by subtracting non-specific binding from total binding. Values represent mean ± SD taken (n=4 replicates / concentration).

[0029] FIG. 20 shows in vitro serum stability of89Zr-lvuxolimab. Minimal demetallation was observed during incubation in human and mouse serum over 7 days at 37°C (ascertained by radio-TLC). Values represent mean ± SD taken from two independent experiments (n=4 replicates / timepoint in each group).

[0030] FIG. 21 shows longitudinal89Zr-lvuxolimab PET / CT imaging for preclinical dosimetry studies. Whole body maximum intensity projection (MIP) coronal images of a representative female and male C57BL / 6N huOX40tg mice, acquired as 20-minute scans, over several days. %l D / g, percent injected dose per gram of tissue.

[0031] FIG. 22 shows biodistribution analysis of89Zr-lvuxolimab in female (grey bars) and male (black bars) C57BL / 6N huOX40tg mice at 160 h p.i.. Tissue associated radioactivity is expressed as percentage injected dose per gram (%l D / g). Data presented as mean ± SD, n=4-5 mice per group, *p<0.05, **p<0.001). S.lnt: small intestine; L. Int: large intestine.

[0032] FIGs. 23A-23B show optimization of immunohistochemical (IHC) staining of the human 0X40 receptor (CD134) in representative (FIG. 23A) 0X40 negative (HEK293) and (FIG. 23B) 0X40 positive (huOX40+ HEK293) tumor samples derived from tumor-bearing NSG mice. N=3 individual tumors / group. Enlarged view of antigen-positive tumor (ID: M8) shows strongmembrane associated 0X40 expression (bottom panel).

[0033] FIG. 24 shows baseline 0X40 expression in healthy human lymphoid tissues.

[0034] FIG. 25 shows elevated and heterogeneous 0X40 expression in Human Papilloma Virus (HPV) positive and negative Head and Neck Squamous Cell Carcinoma biopsy samples (n=three de-identified patients, + indicates higher levels of 0X40 expression).ATTORNEY DOCKET NO. 221910-2400

[0035] FIG. 26 shows heterogeneous 0X40 expression observed in a Head and Neck Squamous Cell Carcinoma tumor microarray (TMA). Comprising 180 tumor samples (left panel), the TMA enables high throughput analysis of 0X40 expression across several malignant and benign samples in parallel. Magnified view of sample ID: I3 (right), a lymph node with metastasized Squamous Cell carcinoma exhibits elevated 0X40 expression.

[0036] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0037] The 0X40 receptor (CD134), a costimulatory molecule on the surface of T cells that can potentiate T cell receptor signaling, has been identified as a promising marker of T cell activation. While absent on resting naive T cells, 0X40 is highly expressed on activated T cells following antigen-recognition. Its expression is largely restricted to the T cell population (and can be found on both CD4+and CD8+murine and human T cells); this is in stark contrast to other surface activation markers previously evaluated with immunoPET (e.g. CD25 and CD44), which are found on a wide repertoire of immune, endothelial, and cancer cells. In one aspect, the clinical relevance of 0X40 in immunopathology and oncology is well documented, providing rationale for its evaluation as a therapeutic target. In a further aspect, its prognostic value has also been reported in several cancers: high 0X40 expression levels in the tumor immune infiltrate taken from cutaneous melanoma, colorectal cancer, and non-small cell lung cancer patients were associated with positive immune contexture and improved survival.

[0038] In one aspect, although several ongoing clinical investigations are ongoing, including most notably18F-labeled ArabinoFuranosylGuanine ([18F]F-AraG) and18F-Clofarabine (18F-CFA), no gold-standard tracers in the clinic to date are able to report T cell activation with high specificity. Given both the clinical relevance of 0X40 and promising prognostic value of murine 0X40 PET imaging, in one aspect, disclosed herein is a human OX40-specific immunoPET tracer. Herein is reported the development of a new OX40-immunoPET tracer based on the human 0X40 therapeutic ivuxolimab (PF-04518600), developed by Pfizer Inc., and its evaluation in vitro and in vivo. Ivuxolimab is a fully human lgG2 agonist clone that has been evaluated in Phase l / ll trials.ATTORNEY DOCKET NO. 221910-2400In a further aspect, the tracer is well tolerated in patients with locally advanced or metastatic cancers (NCT03217747), ivuxolimab also showed on-target immune activation at clinically relevant therapeutic doses. In a further aspect, it is hypothesized that the human 0X40- immunoPET tracer will be a highly specific imaging agent for whole-body visualization of 0X40 and T cell activation, and a candidate for future clinical translation with the potential to enable early and accurate prediction of IOT response.

[0039] In one aspect, disclosed herein is a new PET tracer for human 0X40 (89Zr-lvuxolimab). In a further aspect, 0X40 is a co-stimulatory molecule specifically upregulated predominantly by T cells. In one aspect, the expression of 0X40 is particularly upregulated at high levels on activated T cells, making 0X40 a promising imaging target for probing activated T cells. Also disclosed are imaging and / or diagnostic agents containing89Zr-lvuxolimab.

[0040] In one aspect, in an initial preclinical evaluation,89Zr-lvuxolimab showed selective binding to activated primary human T cells versus resting naive cells, demonstrated favorable in vivo stability, and enabled high-contrast visualization of human 0X40 expression on activated T cells in vivo. In a further aspect, these results support89Zr-ivuxolimab as a strong candidate for clinical translation. Therefore, herein is described further translational development of89Zr-ivuxolimab, encompassing Good Manufacturing Practice (GMP) production and preclinical dosimetry studies in rodents to generate estimated human dose equivalents, which are components required for investigational new drug (IND) application for first-in-human imaging agents. In parallel, disclosed herein is an assessment of 0X40 expression in human tissue specimens with particular focus on head and neck cancer squamous cell carcinoma, to reinforce the biological rationale for imaging activated T cells within this population. In an aspect, these studies provide the preclinical and chemistry and manufacturing controls (CMC) foundations necessary for IND approval, in addition to translational biomarker validation to support the clinical translation of89Zr-ivuxolimab for first- in-human studies.

[0041] In one aspect, F(ab')2antibody fragments can be obtained by cleavage of a whole IgG antibody. In a further aspect, the fragments lack the Fc region but retain the hinge region, which holds the two antigen binding sites together through disulfide bonds. In a still further aspect, this makes F(ab')2fragments bivalent and about two thirds the size of whole IgG antibodies (~100 kDa). In one aspect, several potential advantages can result from using an lvux-F(ab')2fragment for 0X40 imaging:ATTORNEY DOCKET NO. 221910-2400

[0042] In one aspect, use of a fragment can result in faster clearance: The F(ab')2 fragment is smaller (~100 kDa) than a whole antibody (~150 kDa) which generally enables faster clearance from the bloodstream and improved tissue penetration. This could allow for earlier / same day imaging and potentially a more favorable radiation dose for the subject

[0043] In another aspect, use of a fragment can result in improved contrast: More rapid clearance has the potential to achieve a higher contrast between the target tissue (e.g., a lymph node / tumor) and the surrounding normal tissue.

[0044] In still another aspect, use of a fragment can result in improved theranostic potential: F(ab')2 fragments, like whole antibodies, can be labeled with isotopes suitable for both PET imaging and targeted radionuclide therapy.

[0045] Disclosed herein is immunoPET agent for detecting activated T cells expressing human 0X40, the immunoPET agent including at least an antibody or an antibody fragment labeled with a radioisotope. In some aspects, when the immunoPET agent is an antibody, the radioisotope can be Zr-89, Cu-64, Cu-61, Cu-67, 1-131 , In-111 , Lu-177, Ac-225, Pb-212, Bi-212, Ra-223, Re- 188, Th-227, F-18, Ga-68, Sc-44, C-11 , or any combination thereof. In one aspect, the antibody can be ivuxolimab. In another aspect, when the immunoPET agent is or includes an antibody fragment, the antibody fragment can be selected from a nanobody, a bivalent minibody, a cys- diabody, an F(ab)2 fragment, a bispecific T-cell engager (BITE), or any combination thereof. In a further aspect, when the immunoPET agent is an antibody fragment, the radioisotope can be Zr- 89, Cu-64, Cu-61 , Cu-67, 1-131 , In-111 , Lu-177, Ac-225, Pb-212, Bi-212, Ra-223, Re-188, Th- 227, F-18, Ga-68, Sc-44, C-11 , or any combination thereof. In an aspect, and without wishing to be bound by theory, shorter-lived radioisotopes can, in some cases, be used with antibody fragments that may not be possible to use with a complete antibody. Moreover, pre-targeting approaches can be used with this antibody or fragment, which involves a two-step strategy in which ivuxolimab (for example) is modified with a TCO tag first and then administered to a living subject. This agent then localizes to the target tissue, followed later by administration of a small, rapidly clearing18F-labeled tetrazine molecule that selectively “clicks” to the antibody in vivo, enabling high-contrast imaging with minimal background signal. In some aspects, the immunoPET agent can include a chelator such as, for example, deferoxamine (DFO), 1,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), 1 ,4,8,11-tetraazacyclotetradecane-1 ,4,8,11-tetraacetic acid (TETA), diethylenetriaminepentaacetic acid (DTPA), 6-hydrazinonicotinic acid (HYNIC), 2,2'-(1,10-ATTORNEY DOCKET NO. 221910-2400 diazacyclooctadecane-1 ,10-diyl)diacetic acid (Macropa), a Macropa derivative selected from Macropa-PEG, Macropa-TATE, and Macropa- BP, a hydroxypyridinone (HOPO) chelator selected from 3-HOPO, 4-HOPO, and 1 ,2-HOPO, a DOTA-HOPO hybrid, or any combination thereof.

[0046] Also disclosed herein are pharmaceutical composition including the disclosed immunoPET agents. Furthermore, disclosed herein is a method for quantifying and tracking 0X40 positive cells in a subject, the method including at least the step of administering the immunoPET agent or the pharmaceutical composition to the subject and monitoring a signal produced by the immunoPET agent. In one aspect, the subject is a human.

[0047] In some aspects, the disclosed antibodies and antibody fragments can be labeled with radioisotopes useful for single-photon emission computed tomography (SPECT) imaging or can be labeled with fluorophores for use in optical methods such as, for example, optical surgical navigation.

[0048] In any of these aspects, the subject can have a cancer such as, for example, head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), malignant melanoma (MEL), renal cell carcinoma (RCC), bladder cancer, breast cancer, gastric cancer, cervical cancer, non-small cell lung cancer (NSCLC), lymphomas, or any combination thereof. In another aspect, the disclosed compositions and methods can be used to detect the presence of OX40-presenting cells, monitor the progress of a cancer treatment, or a combination thereof. In an aspect, treatments that can be monitored by the disclosed methods include, but are not limited to, radiation therapy or an immunotherapy such as, for example, a cancer vaccine with or without adjuvant, immune checkpoint therapy, or adoptive T cell therapies. In still another aspect, the disclosed compounds and methods can be used in conjunction with theranostic agents used to ablate activated T cells in immune driven diseases.

[0049] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.ATTORNEY DOCKET NO. 221910-2400

[0050] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0051] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0052] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0053] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0054] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0055] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of theATTORNEY DOCKET NO. 221910-2400 specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0056] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0057] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0058] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a PET tracer,” “an antibody fragment,” or “an excipient,” includes, but is not limited to, mixtures or combinations of two or more such PET tracers, antibody fragments, or excipients, and the like.

[0059] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0060] When a range is expressed, a further aspect includes from the one particular value and / orATTORNEY DOCKET NO. 221910-2400 to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0061] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0062] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.ATTORNEY DOCKET NO. 221910-2400

[0063] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0064] In accordance with the present disclosure, “a detectably effective amount” of the labeled probe of the present disclosure is defined as an amount sufficient to yield an acceptable image using equipment that is available for clinical use. A detectably effective amount of the labeled probe of the present disclosure may be administered in more than one injection. The detectably effective amount of the labeled probe of the present disclosure can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the like. Detectably effective amounts of the probe of the present disclosure can also vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art.

[0065] As used herein, the term “subject” includes vertebrates such as humans and mammals (e.g., cats, dogs, horses, etc.). Typical subjects to which embodiments of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, horses, primates, and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use, such as mammalian (particularly primate such as human) blood, urine, or tissue samples, or blood, urine, or tissue samples of the animals mentioned for veterinary applications. In some embodiments, a system includes a sample and a subject. The term “living subject” refers to a subject noted above that is alive and is not dead. The term “living subject” refers to the entire subject and not just a part excised (e.g., a liver or other organ) from the living subject.

[0066] The term “detectable” refers to the ability to detect a signal over the background signal.

[0067] The term “detectable signal” is a signal derived from non-invasive imaging techniques such as, but not limited to, positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The detectable signal is detectable and distinguishable from other background signals that may be generated from the subject. In other words, there is aATTORNEY DOCKET NO. 221910-2400 measurable and statistically significant difference (e.g., a statistically significant difference is enough of a difference to distinguish among the detectable signal and the background, such as about 0.1%, 1 %, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or more difference between the detectable signal and the background) between the detectable signal and the background. Standards and / or calibration curves can be used to determine the relative intensity of the detectable signal and / or the background.

[0068] The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a probe of the disclosure is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the probes of the disclosure and pharmaceutically acceptable carriers preferably should be sterile. Water is a useful carrier when the probe of the disclosure is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.

[0069] The disclosure encompasses compositions and dosage forms of the compositions of the disclosure that can include one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. In addition, pharmaceutical compositions or dosage forms of the disclosure may contain one or more solubility modulators, such as sodium chloride, sodium sulfate, sodium or potassium phosphate, or organic acids. An exemplary solubility modulator is tartaric acid.

[0070] "Pharmaceutically acceptable salt" refers to those salts that retain the biological effectiveness and properties of the free bases and that are obtained by reaction with inorganic orATTORNEY DOCKET NO. 221910-2400 organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.

[0071] Embodiments of the present disclosure include pharmaceutical compositions that include the labeled probe, pharmaceutically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of labeled probe to a subject (e.g., human).

[0072] Embodiments of the present disclosure may be salts and these salts are within the scope of the present disclosure. Reference to a compound of any of the formulas herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, when an embodiment of the present disclosure contains both a basic moiety and an acidic moiety, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds may be formed, for example, by reacting an active compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0073] Embodiments of the present disclosure that contain a basic moiety may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3- phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.ATTORNEY DOCKET NO. 221910-2400

[0074] Embodiments of the present disclosure that contain an acidic moiety may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t butyl amines, and salts with amino acids such as arginine, lysine, and the like.

[0075] Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0076] Solvates of the compounds of the disclosure are also contemplated herein. Solvates of the compounds are preferably hydrates.

[0077] The amounts and a specific type of active ingredient (e.g., a labeled probe) in a dosage form may differ depending on various factors. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician or other attending professional within the scope of sound medical judgment. The specific effective dose level for any particular subject will depend upon a variety of factors, including for example, the activity of the specific composition employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; the existence of other drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.

[0078] The term “positron emission tomography” as used herein refers to a nuclear medicine imaging technique that produces a three-dimensional image or map of functional / molecular processes in the body. The system detects pairs of gamma rays emitted indirectly by a positronemitting radioisotope, which is introduced into the body via a molecule specific for a target / process of interest. Images of the target / process of interest in space are then reconstructed by computerATTORNEY DOCKET NO. 221910-2400 analysis. Using statistics collected from tens-of-thousands of coincidence events, a set of simultaneous equations for the total activity of each parcel of tissue can be solved by a number of techniques, and a map of radioactivities as a function of location for parcels or bits of tissue may be constructed and plotted. The resulting map shows the tissues in which the molecular probe has become concentrated. PET technology can be used to trace the biologic pathway of any compound in living humans (and many other species as well), provided it can be radiolabeled with a PET isotope. The half-life of zirconium-89 is long enough such that imaging agents labeled with this radioisotope can be manufactured commercially at an offsite location. In some aspects, PET and / or SPECT isotopes with shorter half-lives can be used for antibody fragments.

[0079] The term “single-photon emission computed tomography” or “SPECT” as used herein refers to a nuclear medicine imaging scan that uses gamma rays to produce 3D images of a subject, typically as a series of cross-sectional slices. In a SPECT scan, a gamma-emitting radioisotope is administered to the subject, such as by injection of a ligand (e.g., antibody, antibody fragment, or the like) conjugated to the radioisotope. In a further aspect, SPECT scans can monitor biological activity in addition to producing images of anatomical structures.

[0080] The term “label” as used herein refers to any moiety that may be linked (e.g. bonded or otherwise associated with) to the agent (e.g., 0X40) of the present disclosure and which may be used to provide a detectable image including, but not limited to, a radiolabel such as a PET or SPECT probe. Alternatively the label may be a fluorophore for use in optical surgical navigation.

[0081] The term "in vivo imaging" as used herein refers to methods or processes in which the structural, functional, molecular, or physiological state of a living being is examinable without the need for a life-ending sacrifice.

[0082] The term "non-invasive in vivo imaging" as used herein refers to methods or processes in which the structural, functional, molecular, or physiological state of being is examinable by remote physical probing without the need for breaching the physical integrity of the outer (skin) or inner (accessible orifices) surfaces of the body.Dosage Forms

[0083] Embodiments of the present disclosure can be included in one or more of the dosage forms mentioned herein. Unit dosage forms of the pharmaceutical compositions (the “composition” includes at least the labeled probe of the present disclosure, e.g.,89Zr-lvuxolimab imaging probe) of this disclosure may be suitable for oral, mucosal (e.g., nasal, sublingual,ATTORNEY DOCKET NO. 221910-2400 vaginal, buccal, or rectal), parenteral (e.g., intramuscular, intraperitoneal, subcutaneous, intravenous, intra-arterial, or bolus injection), topical, subcutaneous, intravitreal, retro-orbital, intranasal, intrathecal, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0084] The composition, shape, and type of dosage forms of the compositions of the disclosure typically vary depending on their use. For example, a parenteral dosage form may contain smaller amounts of the active ingredient than an oral dosage form used to treat the same condition or disorder. These and other ways in which specific dosage forms encompassed by this disclosure vary from one another will be readily apparent to those skilled in the art (See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990)).

[0085] Typical compositions and dosage forms of the compositions of the disclosure can include one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy or pharmaceutics, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms, such as tablets or capsules, may contain excipients not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. For example, the decomposition of some active ingredients can be accelerated by some excipients, such as lactose, or by exposure to water. Active ingredients that include primary or secondary amines are particularly susceptible to such accelerated decomposition.

[0086] The disclosure encompasses compositions and dosage forms of the compositions of the disclosure that can include one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,”ATTORNEY DOCKET NO. 221910-2400 include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. In addition, pharmaceutical compositions or dosage forms of the disclosure may contain one or more solubility modulators, such as sodium chloride, sodium sulfate, sodium or potassium phosphate, or organic acids. An exemplary solubility modulator is tartaric acid.

[0087] "Pharmaceutically acceptable salt" refers to those salts that retain the biological effectiveness and properties of the free bases and that are obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.

[0088] Embodiments of the present disclosure include pharmaceutical compositions that include the labeled probe (e.g.,89Zr-lvuxolimab), pharmaceutically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of labeled probe (e.g.,89Zr- Ivuxolimab) to a subject (e.g., human).

[0089] Embodiments of the present disclosure may be salts and these salts are within the scope of the present disclosure. Reference to a compound of any of the formulas herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, when an embodiment of the present disclosure contains both a basic moiety and an acidic moiety, zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of an active compound may be formed, for example, by reacting an active compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0090] Embodiments of the present disclosure that contain a basic moiety may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,ATTORNEY DOCKET NO. 221910-2400 hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3- phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.

[0091] Embodiments of the present disclosure that contain an acidic moiety may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N- bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine, and the like.

[0092] Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0093] Solvates of the compounds of the disclosure are also contemplated herein. Solvates of the compounds are preferably hydrates.

[0094] The amounts and a specific type of active ingredient (e.g., a labeled probe such as89Zr- Ivuxolimab) in a dosage form may differ depending on various factors. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician or other attending professional within the scope of sound medical judgment. The specific effective dose level for any particular subject will depend upon a variety of factors, including for example, the activity of the specific composition employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; the existence of other drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the composition at levelsATTORNEY DOCKET NO. 221910-2400 lower than those required to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.

[0095] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).

[0096] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.EXAMPLES

[0097] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Development and Evaluation of a Human OX40-lmmunoPET Tracer for Highly Specific Detection of T Cell ActivationMaterials and MethodsAnimal Studies

[0098] All animal experiments were completed in accordance with the Stanford Administrative Panel on Laboratory Animal Care (APLAC), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC International). Female NOD.Cg-Prkdcscid H2rgtm1 Wjl / SzJ (NSG) (8-10 weeks) and female BALB / cJ mice (8-10 weeks) were purchased from Jackson Laboratories. Female human 0X40 transgenic (hOX40tg) knock- in C57BL / 6 mice (8-10 weeks) were purchased from GenOway. Briefly, in this strain, the extracellular part of the murine 0X40 (mOX40) gene is replaced with the human extracellular 0X40 coding sequence, keeping the mouse transmembrane and intracellular parts intact. All miceATTORNEY DOCKET NO. 221910-2400 were acclimatized for at least 1 week prior to experiments and housed in a temperature-controlled environment under a 12 h light / dark schedule with unrestricted access to food and water.Cell Culture

[0099] All cell culture media, fetal bovine serum (FBS), and penicillin / streptomycin were obtained from Life Technologies (Carlsbad, CA). Human embryonic kidney 293 (HEK293) cells (ATCC) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 100 U / mL penicillin, 100 g / mL streptomycin (1 % penicillin / streptomycin). Recombinant HEK293 cells stably transfected with human 0X40 (OX40+HEK293 cells) were cultured in growth media 1 F as per manufacturer’s instructions (BPS Bioscience). Cell lines were routinely tested for mycoplasma using the MycoStrip™ mycoplasma detection kit (InvivoGen).DFO Conjugation

[0100] DFO was conjugated to anti-human (hu) 0X40 antibody (ivuxolimab, PF-04518600, Pfizer Inc.), using metal-free buffers and previously described procedures. Briefly, 1.2 mg of Ivuxolimab was buffer exchanged with PBS (pH adjusted to 8.8-9.0 using 1M sodium carbonate) and diluted to a final concentration of 1 mg / mL. Subsequently, 20-fold molar excess of 1-(4- isothiocyanatophenyl)-3-[6,17-dihydroxy-7, 10,18,21-tetraoxo-27-(A / -acetylhydroxylamino)-6,11,17, 22-tetraazaheptaeicosine] thiourea chelate (DFO-isothiocyanate or DFO-SCN, B-705; Macrocyclics Inc.) dissolved in DMSO (Thermofisher) was gradually added with gentle vortexing. The reaction was allowed to proceed for 1 h at 37°C, with shaking at 400 rpm. DFO-modified mAb (DFO-lvuxolimab) was subsequently buffer exchanged three times in PBS, pH 7.4, using a 2 mL Vivaspin filter with a 50KDa cut-off (Sartorius) to remove unbound chelate. The conjugate was concentrated and left at 4°C for 16 h, prior to retrieval, for enhanced protein recovery. Final protein concentration was determined using a NanoDrop™ UV-Vis spectrophotometer (Thermo Scientific™). Electrospray ionization (ESI) mass spectrometry was conducted to determine the final mAb:chelate ratios. Immunoreactivity of the final conjugate was compared to that of the parent mAb using OX40+HEK293 cells.Immunoreactivity assessment of DFO-lvuxolimab

[0101] Human (hu) 0X40+ HEK293 cells were seeded in a V-bottomed 96 well plate (4 x 105per well). Serial dilutions of Ivuxolimab (huOX40mAb) or DFO-lvuxolimab were performed in buffer (2% FBS in PBS) and 50mL added to the cells in each well (duplicates for each concentration) and incubated at 4 °C for 60 minutes. Cells were washed twice in buffer. All wells except unstainedATTORNEY DOCKET NO. 221910-2400 controls were subsequently incubated with secondary antibody, goat-anti human IgG Fc Dylight650 antibody (4 mg / ml, abeam) for 30 minutes at room temperature. Cells were spun down (2000 rpm, 2 minutes) and washed twice with buffer, prior to being resuspended in 100 ml buffer and analyzed for 673nm fluorescence intensity on a BD LSRII cytometer.Radiolabeling

[0102] Radiolabeling of DFO-lvuxolimab conjugate with89Zr (half-life: 78.4 h) was performed using varying ratios of radioactivity: conjugate to optimize labeling efficiency and purity. The following optimized conditions were chosen for all subsequent radiolabeling experiments: briefly, 1 mCi (37 MBq)89Zr oxalate (3D Imaging) was added to 0.5 M HEPES buffer (240 pL), to achieve a pH of 6.8-7.2. DFO-lvuxolimab was added to the radiometal at a final concentration of 15- 20pCi / pg of protein (0.56 MBq / pg). The radioactive mixture was briefly centrifuged for 5 seconds at 1000 rpm and incubated at 37°C for 1 h with shaking at 400 rpm. The reaction was monitored by instant thin layer chromatography (iTLC) strips (Biodex). The crude final product was purified through a Zeba spin desalting column with 7000 MWCO cut-off (ThermoFisher). Radio-TLC was developed in a 50 mM EDTA solution, with 2 pL of column-purified reaction added to the TLC strip to quantify the final radiochemical purity of the radiolabeled protein (89Zr-lvuxolimab Rfvalue: 0.2 and free radiometal89Zr Rfvalue: 0.4). Protein concentration and final specific activity were determined by spectrophotometry (Thermo Scientific Nanodrop UV-Vis spectrophotometer). Finally,89Zr-lvuxolimab was analyzed by size exclusion chromatography (SEC)-radioHPLC using a BioSeo™ 5 pm SEC-s3000 290 A (300 x 7.8 mm) column.Cell Binding Studies

[0103] Primary human T cells were isolated from buffy coats and either rested or Dynabead- activated using a previously established protocol for 48h (Supplemental Methods). For the cell binding assay, 0.5x106primary human T cells and immortalized cell lines (HEK293 and human OX40+HEK293) were seeded in a 96 well plate and incubated in 200 pL pre-warmed Hanks balanced salt solution (HBSS) containing89Zr-lvuxolimab (activity: 5 pCi; mass dose: 0.3 pg) at 37°C for 30 to 60 minutes. To assess tracer specificity, additional samples of activated T cells and human OX40+HEK293 cells, were co-incubated with the PET tracer, and 25-fold cold Ivuxolimab. Cells were subsequently washed twice with cold PBS and resuspended in 200 pL of the same buffer; 150 pL was transferred to collection tubes and cell-associated radioactivity was measured using an automated gamma counter (Hidex AMG Automatic Gamma Counter). The remaining sample were used for cell number quantification. Final cell-associated radioactivityATTORNEY DOCKET NO. 221910-2400 values were expressed as a percent of total tracer added, normalized to cell number (%total tracer / 1x105cells). Flow cytometric analysis of primary resting and activated human T cells and immortalized cell lines was performed to confirm 0X40 expression. Fluorescence quantitation was performed to estimate 0X40 antigen density on human T cells, using the Quantum™ Simply Cellular kit anti-mouse IgG (Bangs Laboratories) as per the manufacturer’s instructions and Alexa Fluor 647 anti-human 0X40 (clone Ber-ACT35, Biolegend).Radiotracer Stability

[0104] For ex vivo evaluation of radiotracer stability, female NSG mice were injected with89Zr- Ivuxolimab (292 ± 21.2 pCi, 15.7 pg) intravenously (i.v.) via the tail vein. Blood was collected by cardiac puncture at 24, 48, 72, 120, and 168 h post-tracer injection (p.i.) and the derived plasma fraction was analyzed using radio-TLC to measure free89Zr (to assess89Zr-DFO complex stability) and by radio-HPLC (to analyze89Zr-lvuxolimab protein integrity).Subcutaneous tumor model

[0105] 1x107HEK293 and human OX40+HEK293 cells suspended in 100 mL sterile PBS were mixed 1 :1 with matrigel (BD Biosciences) and subcutaneously injected into the flank of female NSG mice. T umor growth was monitored twice a week using an electronic caliper and the volume calculated using the following equation: volume=(P / 6) x height x width x length). PET imaging was conducted when tumors reached ~150mm3. Briefly, ~ 90 pCi was injected into mice and PET / CT images were acquired over 3 days.Human 0X40 Transgenic (hQX40tq), MHC-Mismatch Murine Model of Acute Graft Versus Host Disease

[0106] To evaluate the utility of89Zr-lvuxolimab for T cell activation in vivo, a modified version of a previously described, well-established MHC-mismatch murine model of acute GvHD was employed. In this model irradiated female BALB / cJ mice receive an infusion of T cell depleted bone marrow (TCD-BM) cells and splenocytes from C57BL / 6 mice. The latter induces GvHD mediated by the donor T cells present in the infused splenocytes, becoming activated. Briefly, this protocol was modified by obtaining splenocytes from female human 0X40 transgenic (hOX40tg) knock-in C57BL / 6 mice, thereby introducing human 0X40 expression on pathogenic donor T cells. Additional control groups included BALB / cJ mice which received total body irradiation (TBI group) and BALB / cJ mice which only received TCD-BM cells (BM group).ATTORNEY DOCKET NO. 221910-2400PET / CT Imaging and Image Analysis in Tumor Bearing Mice and hQX40tq Acute Graft Versus Host Disease Model

[0107] Mice were anesthetized using isoflurane (2-2.5% for induction and 1.5-2.0% for maintenance) delivered by 100% oxygen.89Zr-lvuxolimab radiotracer (60 pCi / 2.2 MBq; volume: 60|JL saline; mass dose: 3.2 pg) was administered i.v. via the tail vein of mice. PET / CT imaging studies were subsequently performed using the GNEXT PET / CT scanner (Sofie Biosciences). The first (20 minute static) PET scans were acquired 24 h p.i. in list mode format and then at 24 h intervals up to 5 days post-tracer injection in NSG tumor bearing mice. Similarly, 20 minute static PET scans were acquired in list mode format for mice in the acute GvHD study at 48 h p.i tracer injection. The PET system can deliver 0.54mm isotropic spatial resolution at the center of the 130mm field of view. Isotropic resolution was achieved using OSEM3D reconstruction algorithms with 24 subsets, 3 iterations, and a matrix size of 240x240x191. All PET scans were followed by a CT scan to provide anatomical reference and for PET attenuation correction. Region of interest (ROI) analysis of the PET images was performed using a 3D volume drawing mode around the heart, tumor, (thigh) muscle, liver, and femur for tumor bearing mice (Inveon Research Workplace). For the hOX40tg acute GvHD model, ROI analysis of the PET images was performed using a 3D volume drawing mode around the heart, liver, spleen, (thigh) muscle, femur, mesenteric lymph node and abdomen. PET data were normalized to injected dose and were expressed as percentage injected dose / g of tissue (%l D / g).

[0108] For further validation of the PET imaging data, tumor bearing mice were euthanized at 72 h or following completion of the terminal scan at 120 h and tissue associated radioactivity verified by ex vivo gamma counting for biodistribution analysis or autoradiography. For the hOX40tg acute GvHD model, ex vivo gamma counting for biodistribution analysis or autoradiography was performed on mice euthanized at 48 h following completion of the terminal scan.Statistical Analysis

[0109] Statistical analyses were performed using Prism 9.0 (GraphPad Software). Data are represented as mean ± SD with individual values plotted on bar graphs. Statistical significance was determined using unpaired, 2-tailed Student’s t test or 1-way analysis of variance (ANOVA) for column and multiple column analyses respectively. A Bonferroni’s post-test was applied when appropriate to correct for multiple comparisons. A p value of less than 0.05 was considered statistically significant.Results and DiscussionATTORNEY DOCKET NO. 221910-2400Human OX4Q-irnrnunoPET Tracer Production

[0110] DFO conjugation of Ivuxolimab (FIG. 6A) was confirmed via ESI-mass spectrometry, which showed peaks corresponding to unconjugated (m / z 146824.8) and conjugated Ivuxolimab with mAb:DFO ratios within the desired range of 1:1 (m / z 147576.8), and 1 :2 (m / z 148328.5) (FIG. 6B), to preserve immunoreactivity. SEC-HPLC analysis of the bioconjugate (DFO-lvuxolimab) confirmed a single protein peak (retention time [Rt]=10.9 mins, FIG. 6C). To ensure DFO- modification did not adversely affect the immunoreactivity of Ivuxolimab, cell-binding studies were performed with (human) OX40+HEK293 cells which confirmed that binding of DFO-lvuxolimab to cells, detected by a fluorescent anti-human secondary antibody, was comparable to that of the parent mAb, with over 94% of binding of the parent mAb retained (FIG. 6D). Subsequent radiolabeling of DFO-lvuxolimab with89Zr oxalate (FIG. 1 A) was highly reproducible, yielding the89Zr-lvuxolimab tracer with mean radiolabeling efficiency (derived from radio-iTLC) of 85.71 ± 2.62 %. Post-Zeba column purification, radio-iTLC confirmed a single-peak corresponding to the89Zr- Ivuxolimab immunoPET tracer (Rf=0.2; final radiochemical purity: 98.27 ± 1.73%; FIG. 1 B) with final specific activity of 18.5 ± 2.91 mCi / mg (684.5 ± 107.3 MBq / mg, Table 1). Radio-SEC-HPLC analysis of the final column-purified tracer shows a single peak (Rt=11.5 min, FIG. 1 C), with a corresponding UV-protein peak (Rt=10.9 min), confirming the identity of89Zr-lvuxolimab. Radio- iTLC and radio-SEC-HPLC were both utilized as part of the quality control to monitor all89Zr labeling reactions (FIGs. 7A-7C).In vitro Validation of Radiotracer Specificity

[0111] In vitro specificity of89Zr-lvuxolimab was evaluated by comparing its binding to isolated primary human T cells that were rested or Dynabead-activated for 48 h (FIG. 2A). Significantly higher binding of89Zr-lvuxolimab to activated versus resting human T cells was observed (10.8- fold higher post 30 minutes co-incubation; 7.2-fold higher post 60 minutes of co-incubation;ATTORNEY DOCKET NO. 221910-2400 p<0.0001; FIG. 2A). Tracer binding to recombinant (human) OX40+HEK293 cells was also assessed and compared to parental HEK293 cells (FIG. 2B). Tracer binding to OX40+HEK293 was significantly higher versus parental HEK293 cells (26.8-fold higher post 30 minutes; 20.9-fold higher post 60 minutes; p<0.0001 ; FIG. 2B). Co-incubation of the tracer with a blocking dose of 25-fold higher cold / non-radioactive Ivuxolimab significantly reduced tracer binding to activated T cells and OX40+HEK293 cells (by 88% and 87% respectively, p<0.0001), further confirming tracer specificity (FIGs. 2A-2B). Flow cytometric analysis confirmed significantly higher 0X40 expression on total activated versus resting human T cells (FIG. 2C, p=0.0079). This increased expression coincided with both CD4+(FIG. 8A) and CD8+subsets (FIG. 8B). Initial estimates of surface expression levels / OX40 antigen density on human T cells activated in vitro, yielded between 15,684 to 19,469 0X40 molecules / Dynabead-activated cell (FIGs. 9A-9B). Higher target expression on OX40+HEK293 cells versus parental cells was also confirmed by flow cytometry (FIG. 2D, p=0.0022).Radiometabolism studies

[0112] In developing a human OX40mAb radiotracer, DFO was chosen as the chelator since it is FDA-approved and known to maintain a stable complex with89Zr in humans. Radiometabolism studies were performed to evaluate overall tracer stability in mice.89Zr- Ivuxolimab was highly stable in vivo: plasma analysis by radio-iTLC and radio-HPLC confirmed minimal demetallation over 5 days (FIGs. 10A-10B). Area under the curve calculations of radio-HPLC profiles indicated 5.4% ± 2.6% free metal at 72 h and 7.1 % ± 1.5% at 120 h post tracer injection. Moreover, smaller radiolabeled fragments were not observed by radio-HPLC, indicating overall preservation of the mAb integrity in vivo.Validation of Radiotracer Specificity in vivo

[0113] Having established radiotracer stability, the in vivo specificity and biodistribution profile of89Zr-lvuxolimab were next interrogated in a subcutaneous tumor model using PET / CT imaging. Longitudinal imaging over 5 days p.i. revealed high contrast images with markedly higher signal in OX40+HEK293 tumors versus HEK293 tumors evident at both 72 h and 120 h p.i. (FIG. 3A). Quantification of the PET signal using region-of-interest analysis confirmed significantly higher tracer accumulation in OX40+HEK293 tumors compared to the negative control HEK293 tumors (11.41 ± 2.81 %ID / g versus 3.90 ± 0.96 %ID / g at 72 h p.i., p<0.0001 ; 15.85 ± 3.90 %ID / g versus 3.71 ± 0.71 %l D / g at 120 h p.i., p=0.0007; FIGs. 3B, 11). The liver and heart also showed notable tracer uptake, consistent with the known biodistribution of an intact mAb. Tumor-to-muscle ratiosATTORNEY DOCKET NO. 221910-2400 derived from PET quantification were significantly higher for the OX40+HEK293 tumors versus the HEK293 group at all imaging timepoints (24 h: p=0.0225; 48 h: p=0.0001 ; 72 h: p=0.0002; 120 h: p=0.0048, FIG. 3C). Notably, this ratio increased longitudinally only in the OX40+HEK293 group, reflecting specifically bound tracer accumulation and clearance of the unbound tracer over time (tumocmuscle ratios at 24 h: 5.95 ± 0.78; 48 h: 10.1 ± 0.99; 72 h: 14.5 ± 1.60; 120 h: 20.43 ± 3.44; FIG. 3C). It was noted that increasing non-specific accumulation of free89Zr was observed in bone and joints at later imaging timepoints since the89Zr-DFO complex is prone to dissociation in mice.

[0114] Biodistribution analysis using ex vivo gamma counting of tissues corroborated PET results with significantly increased tracer binding in OX40+HEK293 tumors versus HEK293 tumors at both 72 h p.i. (16.0 ± 2.91 %ID / g and 4.13 ± 0.61 %ID / g respectively, p= 0.0031) and 120 h p.i. (13.15 ± 2.18 %ID / g and 2.30 ± 0.32% I D / g respectively, p<0.0001 , FIG. 3D). Femur from OX40+HEK293 tumor-bearing mice showed 1.4-fold elevated radioactivity compared to those from HEK293 tumor-bearing mice at the late 120h imaging timepoint (p=0.0198). Autoradiography (ARG) of tumor and muscle at 120 h p.i., enabling high resolution visualization of radiotracer signal in these tissues, visually corroborated PET images and biodistribution analysis (FIG. 12A). Quantification of the tissue-associated signal from ARG images confirmed higher mean tumor-to- muscle ratios for the OX40+HEK293 tumor-bearing mice (5.30 ± 1.46) versus the HEK293 tumorbearing mice (1 .60 ± 0.24, p=0.0005, FIG. 12B). IHC of tumor tissues confirmed high extracellular 0X40 staining in OX40+HEK293 tumors compared to HEK293 tumors (FIG. 12C).

[0115] To evaluate the utility of89Zr-lvuxolimab for detecting T cell activation in vivo, the tracer was subsequently evaluated in a modified murine acute GvHD model. Splenocytes containing T cells from a human 0X40 transgenic (hOX40tg) knockin donor mouse were employed; these, when transplanted into recipient BALBC mice, become activated, initially proliferating in lymphoid tissues before infiltrating in to GvHD target organs such as the intestinal tract and induce GvHD. PET / CT images obtained 48 h p.i. with89Zr-lvuxolimab, showed elevated signal in known compartments of T cell activation and proliferation of the GvHD group compared to control groups (FIG. 4A). These tissues include the spleen and the mesenteric lymph node (ml_N) and intestines in the abdominal region. Quantification of PET signal from these regions (FIG. 4B) and ex vivo gamma counting of these tissues (FIG. 4C) corroborated PET images and confirmed statistically higher PET tracer binding in the spleen, MLN, large and small intestine of GvHD mice compared to TBI and BM control mice. Ex vivo macroscopic analysis of89Zr-lvuxolimab using ARG enabled high resolution visualization of tracer distribution in these key tissues (FIG. 5A). The specificity ofATTORNEY DOCKET NO. 221910-2400 the high ARG signal observed in GvHD group was confirmed with IHC staining (FIGs. 5B-5C), which shows increased 0X40 target expression in intestinal and lymphoid tissue obtained from GvHD mice as a result of infiltration of pathogenic activated T cells.Discussion

[0116] It is challenging to monitor therapeutic responses and predict clinical outcomes following cancer immunotherapy (IOT) administration due to the resulting varying spatiotemporal immune dynamics. ImmunoPET technologies show high potential to become integral tools for IOT monitoring and optimization: mounting clinical evidence indicates their potential to outperform biopsy measurements of biomarkers (using immunohistochemistry and RNA) by capturing tissue heterogeneity and precise whole body molecular information that can more accurately guide patient management.

[0117] Here is reported the reproducible synthesis of a first-in-class immunoPET tracer,89Zr- Ivuxolimab, to image human 0X40, a highly specific biomarker of T cell activation. The tracer exhibits appropriate stability, pharmacokinetics and, above all, specificity for human 0X40, which deem it suitable for high-contrast, longitudinal imaging of the target in vivo. Definitive evidence for tracer specificity is provided using in vitro and in vivo assays employing both primary activated T cells expressing human 0X40 and a human OX40-expressing stable cell line. Both activated T cells and the 0X40+ cell line showed markedly higher tracer binding compared to their respective negative controls. Importantly, herein it is demonstrated that89Zr-lvuxolimab PET can detect activated T cells in vivo using a GvHD model. Similar detection can be used to evaluate89Zr- Ivuxolimab monitoring of therapy response to clinically relevant GvHD pharmacological interventions such as calcineurin-inhibitors and post-transplant cyclophosphamide.

[0118] Specific imaging of T cell functionality is an unmet clinical need that is rapidly gaining momentum. Two such promising approaches currently under clinical evaluation are i) granzyme B imaging, a biomarker of direct cell kill mediated by cytotoxic CD8+T cells and natural killer cells (NCT04169321), and ii) imaging deoxyguanosine kinase expression and activity, which increases in activated T cells (NCT04524195). T cell activation is an early event in the cancer immunity cycle, and a critical determinant of efficacy across different cancer lOTs and overall treatment success. Previously, the utility of murine OX40-immunoPET was demonstrated for detecting and quantifying early T cell activation induced by different lOTs, across solid and liquid tumors. In intratumoral CpG-treated lymphoma tumors, the murine OX40-PET signal correlated with treatment response prior to occurrence of gross morphological changes and predicted therapeuticATTORNEY DOCKET NO. 221910-2400 efficacy with greater accuracy than anatomic or blood-based biomarkers alone. Additionally, the tracer revealed unique insights into the spatiotemporal dynamics of vaccine-induced T cell responses that occurred beyond the tumor microenvironment. These included the requisite immune priming in tumor draining lymph nodes, which was deemed an identifier of a robust response. These findings in mice were key in the motivation to generate a human-specific 0X40- PET tracer with the potential to enable early and accurate prediction of responses to lOTs in the clinic.

[0119] To date, intact mAbs remain the most validated vector format for probing expression of cell surface targets in the clinical setting. Their long circulation time (weeks) compared to small molecule tracers (hours) prolongs the temporal window available for immunoPET tracers to bind their targets. This provides a distinct advantage, typically leading to a higher PET signal and enhanced sensitivity, especially for targets low in abundance (as low as -10,000 surface molecules per cell). Accordingly, the choice of radiometal, zirconium-89 (89Zr; ti / 2=78.4 h), is well- suited for mAbs by matching their long biological half-life. It is believed that the described approach is suitable for gleaning first-in-human insights prior to evaluation of smaller engineered mAb fragments that would afford faster pharmacokinetic profiles for same-day imaging and compatibility with short-lived isotopes. The estimated receptor density of 0X40 on human activated T cells taken from humanized IOT models and patient samples is currently being quantified and will ultimately evaluate the detection sensitivity and utility of the human 0X40 radiotracer for imaging T cell activation in vivo. Ultimately, both receptor density and the concentration of activated T cells will affect the ability to visualize these cells in human subjects; critical studies using IHC and autoradiography-based estimates using human tumor biopsies are being performed to address this. Ongoing antigen density quantitation studies will help to better define the threshold for 0X40 detection by89Zr-lvuxolimab; its applicability to target expression levels on activated T cells in in vivo models and in humans requires careful assessment. IND- enabling studies for the clinical translation of89Zr-lvuxolimab, including radiation dosimetry assessment in humanized 0X40 mice and streamlined and reproducible production of the tracer under Good Manufacturing Process (GMP) conditions are ongoing.

[0120] Generating an 0X40 PET tracer based on a clinically evaluated mAb allows use of the extensive pharmacokinetic and safety data to ensure timely investigational new drug (IND) approval in the future, advancing the end goal of this project for its eventual clinical translation. Minimal safety concerns are anticipated from the low / sub-pharmacological mass doses used for imaging: While agonistic engagement of 0X40 by Ivuxolimab at therapeutic doses has beenATTORNEY DOCKET NO. 221910-2400 shown to result in T cell proliferation, cytokine secretion and preliminary anti-tumor activity, we foresee minimal safety concerns in human subjects due to the low / sub-pharmacological mass doses used for imaging. While Diab et al. reported agonist effects at 0.1 mg / kg in patients, this far exceeds the total mass dose anticipated to be used for imaging. Nevertheless, thorough validation of the radiotracer will be performed to evaluate any potential biological perturbations in humanized cancer IOT models. Advantageously, the clone does not induce Fc-mediated Ab- dependent cellular cytotoxicity (ADCC), thus avoiding T cell depletion. It is anticipated that 0X40- immunoPET imaging may be useful for identifying a subgroup of cancer patients with high basal levels of 0X40 expression within the tumor; these individuals may benefit from OX40-agonist mono- or combination therapy with immune checkpoint blockade. The tracer may also have utility in stratifying responders and non-responders to IOT interventions known to increase 0X40 expression, allowing timely optimization of treatment protocols.Conclusion

[0121] 89Zr-lvuxolimab is a new radiotracer for molecular imaging of human T cell activation. The data reported herein provides definitive evidence of its high specificity for activated primary human T cells and stably transfected human OX40+cells in vitro, as well as its ability to allow sensitive and longitudinal visualization of the target in vivo.89Zr-lvuxolimab is a promising candidate for clinical translation with the potential to guide patient management and improve clinical outcomes by providing early, non-invasive, whole-body, immune-monitoring of IOT responses. Future studies are required to elucidate its sensitivity and utility for visualizing activated T cells in human subjects.Example 2: Development of89Zr-lvux-F(ab’)2- PET agentGeneration of F(ab’)2- fragment derivative of Ivuxolimab

[0122] The F(ab’)2fragment was prepared from Ivuxolimab (an lgG2), using an immobilized IdeS enzyme system (Genovis, FabRICATOR Fab2 Kit Midispin). The IdeS system is an IgG-specific, cysteine protease which enables highly specific digestion at a single amino acid position below the hinge region of the parent mAb, generating a F(ab')2and Fc fragments. The latter is then affinity captured on a Fc capture column resulting in purified F(ab')2.

[0123] Briefly 9 mg Ivuxolimab (in 1.8 ml_ PBS buffer) was incubated with the FabRICATOR column (Genovis) containing immobilized IdeS enzyme, with constant rotation and inversion for 12 hours. The digested mix was eluted in PBS by centrifugation of the column at 100 g for 1ATTORNEY DOCKET NO. 221910-2400 minute, followed by two washes of the column, each with 1 mL PBS, followed by centrifugation of the column at 100g, 1 minute. The F(ab’)2fragment was subsequently isolated from the Fc portion in the eluent using the CaptureSelect™ Fc column (Genovis); briefly the eluent (total 3.7 mL), was incubated with the CaptureSelect™ Fc column for 60 minutes with constant rotation and inversion. The purified F(ab’)2fragment was then eluted by centrifugation of the column at 200 g, 1 minute, followed by two more washes with 1 mL PBS, with centrifugation of the column at 200 g, 1 minute each time. The final yield of the lvux-F(ab’)2fragment was 5.091 mg in 5.3 mL total volume.

[0124] To confirm successful purification of lvux-F(ab’)2size exclusion chromatography was performed; this showed a longer retention time for the lvux-F(ab’)2compared to Ivuxolimab, consistent with a species of smaller MW (FIG. 13A). Additionally, Electrospray ionization (ESI) mass spectrometry further confirmed the MW of the lvux-F(ab’)2of approximately 96kDa (FIG. 13B). Ivuxolimab (a full length IgG) and post-ldeS enzyme digestion samples (both unpurified and purified lvux-F(ab’)2) were also subjected to electrophoresis under reducing conditions through a 4-20% Tris-Hepes SDS-PAGE gel alongside molecular markers (25-250kDa). The gel was stained with Coomassie blue revealing banding patterns consistent with IgG and F(ab’)2profiles. Affinity column purified F(ab’)2 appears as a single band (~25kDa) corresponding to dissociated heavy and light chains (FIG. 13C).DFO conjugation of lvux-F(ab’)2fragment

[0125] A total of 1 .8 mg F(ab’)2fragment was taken (in PBS buffer, pH 7.4) and buffer exchanged with PBS buffer, pH ~9.0 (adjusted with 1 M Na2COs solution), three times using a 2 mL Vivaspin filter with a 10 KDa cut-off. The 1.8 mg was divided into 3 microcentrifuge tubes (1.5 mL) for 3 separate reactions (each reaction: 0.5 mg F(ab’)2in 500 pL PBS buffer, pH 9). Next, F(ab’)2 was incubated with 10-fold (reaction 1), 20-fold (reaction 2), and 30-fold (reaction 3) equivalents of bifunctional chelate SCN-Bz-DFO: F(ab’)2, at 37 °C for 1 hour (Table 2).ATTORNEY DOCKET NO. 221910-2400

[0126] Post-conjugation, the reactions were buffer exchanged three times in PBS, pH 7.4, using a 0.5 mL Vivaspin filter with a 10 KDa cut-off to remove unbound chelate. The conjugates were concentrated in the same Vivaspin filter. Final protein concentration was determined using a NanoDrop™ UV-Vis spectrophotometer (Thermo Scientific™). The typical yield of the DFO- F(ab’)2conjugate retrieval was -80%.

[0127] Post DFO-conjugation, size exclusion chromatography (SEC-HPLC) was used to determine the purity of both lvux-F(ab’)2and DFO-lvux-F(ab’)2constructs (FIG. 14A). Mass spectrometry provided more accurate estimation of the molecular weight of the Ivuxolimab F(ab’)2and DFO conjugated F(ab’)2 sample (representative mass spectometry profile of a 10-fold DFO: F(ab’)2reaction, FIG. 14B, bottom panel).

[0128] Storage: All (DFO-F(ab’)2conjugates are stored at -80 °C in freezer for long storage. A vial is thawed and stored at 4 °C in refrigerator (for at least 24 h) prior to further use.89Zr radiolabeling of DFO-lvux-F(ab’)2fragment

[0129] A total of 170.8 pg of the DFO-lvux-F(ab’)2conjugate was added to a reaction vial with 1.71 mCi of89Zr-oxalate in 0.5 M HEPES buffer (400 pL), pH 6.5-7.0. The reaction tube was vortexed for 5 seconds and agitated in an Eppendorf ThermoMixer® F1.5 shaker for 45 minutes at 37 °C (Table 3, FIG. 14C). Successful radiolabeling (incorporation of the89Zr into the DFO- lvux-F(ab’)2) and high radiochemical purity of the product was confirmed by radio-TLC.

[0130] To further purify the final formulation, a NAP™5 column employed. Briefly, a NAP™5 column was equilibrated with 10 mL of PBS buffer. Saline (1.5 mL) was added and the columnATTORNEY DOCKET NO. 221910-2400 eluent was collected in fractions ranging from 0.2 to 0.5 mL. Radioactivity of each fraction is measured in a dose calibrator. Fractions with significant radioactivity and radiochemical purity > 95% were combined for formulation. The total volume of combined fractions is typically 1.0 ± 0.5 mL. The product was further diluted to 2 mL with 0.9% saline (USP). The radiochemical purity of the final tracer product was checked by radio-TLC (FIG. 14D). The decay-corrected radiochemical yield for the qualification runs was (EOS (decay-uncorrected): 70.17 %.PET / CT evaluation of89Zr-DFO-lvux-F(ab’)2 in vivo

[0131] Subcutaneous tumor model: 1 *107HEK293 and huOX40+ HEK293 cells suspended in 100 pL sterile PBS were mixed 1 :1 with matrigel (BD Biosciences) and subcutaneously injected into the flank of female NSG mice (8-10 weeks). Tumor growth was monitored twice a week using an electronic caliper and the volume calculated. PET / CT imaging was conducted when tumors reached ~70 mm3. Briefly, ~90 pCi was injected into mice and PET / CT images were acquired over 3 days.

[0132] 89Zr-DFO-lvux-F(ab’)2exhibited markedly higher signal in 0X40+ HEK293 tumors versus antigen negative HEK293 tumors (FIG. 15).89Zr-DFO-lvux-F(ab’)2exhibits predominantly hepatic clearance in addition to renal clearance as indicated by high PET signal in liver and kidney respectively. These findings are also confirmed by ex vivo biodistribution analysis (FIG. 16)

[0133] Ex vivo biodistribution analysis. For further validation of the PET imaging data, mice were euthanized following the completion of the terminal PET / CT scans. Mice were deeply anesthetized (2.5 - 3.0 % isoflurane) and blood collected via cardiac puncture. Tissues of interest were collected and wet-weights immediately recorded to allow normalization of tissue-associated radioactivity to weight. Tissues underwent gamma counting (Hidex AMG Automatic Gamma Counter) to determine the tissue-associated radioactivity. Biodistribution data were normalized to injected dose and were expressed as percentage injected dose / g of tissue (%ID / g).

[0134] Biodistribution analysis using ex vivo gamma counting of tissues corroborated PET results, with significantly increased (~5.5-fold higher)89Zr-DFO-lvux-F(ab’)2associated radioactivity in huOX40+ HEK293 tumors versus HEK293 tumors at 72 h after injection (p= 0.007937) (FIG. 16).

[0135] Autoradiography. Tumors and muscle were fresh frozen in optimum cutting temperature compound (O.C.T., Sakura Finetek Inc.) and sectioned at 40 pm and 12 pm using a cryostat (Microm) for autoradiography and histology respectively. Slide-mounted tissue sections wereATTORNEY DOCKET NO. 221910-2400 subsequently exposed to a storage phosphor film (Fujifilm, GE Healthcare) for 2.5 weeks (i.e., 5 half-lives) at -20 °C and scanned using a typhoon phosphorimager (Amersham Biosciences). The autoradiography signal was analyzed with Imaged (image processing software, version 2.0.0) to obtain high resolution images of89Zr-lvuxolimab signal in tissues, expressed as mean-pixel intensity / background (MPI / background).

[0136] Autoradiography of tumor and muscle at 72 h after injection enabled high-resolution visualization of89Zr-DFO-lvux-F(ab’)2 signal in these tissues (FIG. 17). Tissue-associated signal from autoradiography images showed higher signal in the huOX40+ HEK293 tumors versus the HEK293 tumors and background muscle tissue.Example 3: Translational Development of89Zr-lvuxolimab Including GMP Manufacturing, Preclinical Dosimetry, and Biomarker ValidationMaterials and MethodsAnimal studies

[0137] All animal experiments were completed in accordance with the Stanford Administrative Panel on Laboratory Animal Care (APLAC), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC International). Female and male human 0X40 knock-in transgenic (huOX40tg) C57BL / 6 mice (8-10 weeks) were obtained from GenOway (France). Briefly, in this strain, the extracellular part of the murine 0X40 gene is replaced with the human extracellular 0X40 coding sequence, while the mouse transmembrane and intracellular parts remain intact. All mice were acclimatized for at least 1 week prior to experiments and housed in a temperature-controlled environment under a 12 h light / dark schedule with unrestricted access to food and water.Cell culture

[0138] All cell culture media, fetal bovine serum (FBS), and penicillin / streptomycin were obtained from Life Technologies (Carlsbad, CA). Recombinant HEK293 cells stably transfected with human 0X40 (huOX40+HEK293 cells) were cultured in growth media 1 F as per manufacturer’s instructions (BPS Bioscience).Radiosynthesis of89Zr-lvuxolimab

[0139] To optimize89Zr-lvuxolimab manufacturing process, two initial technical runs were conducted (Tables 4 and 5) prior to performing three consecutive clinical validations runs (TableATTORNEY DOCKET NO. 221910-24006). The validation runs were executed under conditions representative of those for intended clinical production and were required to meet all the predefined release criteria (Table 7) to demonstrate GMP-executed manufacturing and process consistency representative in scale, equipment and materials to the intended clinical process.ATTORNEY DOCKET NO. 221910-2400ATTORNEY DOCKET NO. 221910-2400

[0140] For the clinical validation runs the following method was used; a total of 424 ± 15.39 pg of the DFO-lvuxolimab conjugate was added to a reaction vial with 4.24 ± 0.15 mCi of89Zr-oxalate in 0.5 M HEPES buffer (400 pi L) , pH 6.5-7.0. The reaction tube was briefly vortexed for 5 seconds, and then incubated for 45 minutes at 37 °C with agitation, in an Eppendorf ThermoMixer® F1.5 shaker. Radiochemical purity of the product was checked by radio-TLC and radio-HPLC.Post-radiolabeling purification and sterilization of89Zr-lvuxolimab

[0141] A NAPTM5 column was equilibrated with 10 mL of PBS buffer to purify the final formulation and remove any free89Zr. Saline (total volume 1.5 mL was added in increments of 0.2-0.3 mL), and the eluent collected in 0.2 to 0.5 mL fractions. Radioactivity of each fraction was measured in a dose calibrator. Fractions with significant radioactivity (> 300 pCi) and radiochemical purity > 95% were combined for formulation. The total volume of combined fractions ranged from 0.8-1.2 mL.

[0142] The sterile product vial, product needle, vent needle with a 4 mm GV vent filter, and Millex- GV sterile filter were assembled in a sterile laminar flow hood. The combined fractions were diluted to 10 mL with 0.9% saline (USP) and passed through a Millex-GV sterile filter (0.22 pm pore size, 33 mm diameter) into a pre-assembled septum-sealed sterile collection vial (30 mL) for final sterilization. The control information for each batch was assigned a unique batch number forATTORNEY DOCKET NO. 221910-2400 every production run (labeling format: yyyymmdd-n, where y=year, m=month, d=date, n=synthesis number). For example, the first clinical validation production on May 17, 2024 would be labeled 20240517-1). Approximately 1 ml_ of the final product was removed aseptically for further quality control tests. The total time from initiation of synthesis to release of a single unit dose was approximately 3 hours. The final chemical purity of the product was confirmed by radio- TLC and radio-HPLC. The decay-corrected radiochemical yield for the validation runs was decay- corrected for end of synthesis (EOS): 80.09 ± 4.66 % (n=3).Regulatory specifications, procedures and tests

[0143] For a clinical validation run to be deemed successful, the89Zr-lvuxolimab batch at EOS was required to meet the release specifications summarized in Table 7. Associated tests summarized in Table 7 are described below (see methods for visual inspection for particulates, membrane filter integrity test, pH assessment, HPLC, radio-TLC, half-life, gas chromatography, limulus amebocyte lysate (LAL), multi-channel analyzer (MCA) test, and post-release sterility test). All these tests (except membrane filter integrity test) were also repeated at 66 h post EOS (Table 8). All the analytical test procedures were performed using high quality solvents and reagents, which were carefully logged, controlled, and verified in the same manner as the reagents for the manufacturing process. Testing forms were developed to ensure consistency for documentation of the collected test information. These criteria were guided by the United States Pharmacopeia (USP <29>).Visual inspection for particulatesATTORNEY DOCKET NO. 221910-2400

[0144] As part of the release specifications, the chemical purity of theS9Zr-lvuxolimab solution was examined visually to ensure the final drug product in the vial was intact, clear and colorless without any visible particulates, as per USP <823> and USP <631 > Color and Achromicity.Membrane filter integrity test

[0145] In addition to working in an ISO Class 7 environment when preparing solutions used in the manufacture of the89Zr-lvuxolimab and the final product vial, the radiotracer solution was passed through a 0.22 pm sterilizing filter into the final sterile product vial. After the89Zr- Ivuxolimab was collected, the sterilizing filter was tested for filter integrity to give an indication of likelihood of product sterility. Filter integrity was tested with a bubble point procedure, whereby the sterilizing filter was placed on a gas line with a pressure gauge and the outlet of the filter was immersed in water. The gas pressure on the inlet to the filter was increased slowly until a steady stream of bubbles was observed at the filter outlet. The pressure when the bubble stream began was recorded and compared with the manufacturer’s pressure rating for the filter (found in the certificate of quality). If the observed bubble point pressure exceeded the manufacturer’s specifications, the filter integrity test passed. If the bubble point pressure measured lower than the specification, the product was re-sterilized by passing through a second sterilizing filter, which was required to pass the bubble point test. The final89Zr-lvuxolimab drug product was permitted to undergo only a single re-sterilization cycle. This test was required to be passed for the89Zr- Ivuxolimab dose to be released.Assessment of pH

[0146] Given the small volume of the radioactive product, pH test strips were employed as a suitable alternative to a pH meter. The pH test strips were first qualified prior to use by applying pH 5 and pH 7 calibrated commercial pH standards to individual strips. The resulting colors were required to match the corresponding pH values on the manufacturer’s color key. Following verification,89Zr-lvuxolimab was applied to a new test strip and the color was compared with the color key. The observed pH value was documented. For batch release, the measured pH of89Zr- Ivuxolimab was required to fall within the acceptable range of pH 4.5 to 8.0.HPLC to determine chemical and radiochemical purity

[0147] The chemical and radiochemical purities of89Zr-lvuxolimab were confirmed by using reverse phase analytical HPLC. The final radiochemical purity release specification was established at > 90%. In HPLC analysis, no UV peaks (unspecified impurities) were observedATTORNEY DOCKET NO. 221910-2400 before and after the DFO-lvuxolimab peak; only the expected HEPES peak, with retention time=12-13 min was present. Reverse phase analytical HPLC was performed on an Agilent HPLC system (1260 Quat pump VL) plus auto sampler with a BioSep 5 pm SEC-s3000 290 A column and a mobile phase consisting of PBS buffer (pH ~7) at an isocratic flow rate of 1 mL / min. Radioactivity was monitored with a Raytest (GABI) radiodetector. The UV absorbance was monitored at 280 nm using an Agilent 1260 DAD WR system. The DFO-lvuxolimab UV peak was typically observed at 9 to 10 minutes, with the corresponding89Zr-lvuxolimab radioactivity peak appearing ~0.2 minutes later due to the downstream placement of the radioactivity detector.

[0148] Calibration standards were prepared at concentrations that bracketed either the sample or the minimum acceptable mass limit. All standards were required to be baseline resolved (resolution >1 .5), producing cleanly resolved chromatography peaks, for a valid analysis. A linear regression of the UV absorbance peak areas of the DFO-lvuxolimab standards was generated to construct the calibration curve. The UV peak area of the DFO-lvuxolimab drug product was then interpolated on this curve to determine the protein concentration in each sample. The concentration of the DFO-lvuxolimab in the final89Zr-lvuxolimab product was <200 pg / mL and the protein mass was <1 mg per dose. No low or high molecular weight UV-visible impurities were detected in any of the clinical validation runs.Radio-TLC to determine radiochemical purity and identity

[0149] Radiochemical purity and identity were also assessed by radio-TLC, using 50 mM EDTA as the mobile phase. This test effectively allows for detection of any unbound89Zr and89Zr bound to free DFO; if present, both move with the solvent front with an Rfvalue equal to >0.5, whereas the89Zr-lvuxolimab product remains at Rf < 0.5. A radiochemical purity specification of >90% was established based on this criterion. As such, the radio-TLC assay served primarily as a screen for free89Zr and complemented the radiochemical purity assessment obtained by analytical HPLC. Product identity was confirmed by HPLC co-injection of the nonradioactive DFO-protein reference standard with the drug product to verify matching retention times.Immunoreactivity assessment using a bead-based assay

[0150] To test the immunoreactivity of89Zr-lvuxolimab, a well-established magnetic bead-based assay was employed. Briefly, 20 pL of magnetic DynaBeads™ MyOne™ Streptavidin Tl (Thermofisher) were incubated with 1pg biotinylated human 0X40 (CD134, BPS Bioscience). Subsequently, the resulting human OX40-coated Dynabeads were incubated with 1 ng89Zr- Ivuxolimab for 45 minutes at room temperature on a rotating mixer (experiment performed inATTORNEY DOCKET NO. 221910-2400 triplicates). After the incubation period, the 3 tubes were placed on a DynaMag™-2 magnet (Thermofisher) which enables separation of the supernatant containing unbound89Zr-lvuxolimab from the fraction bound to bead-antigen (0X40) complex. Radioactivity (cou nts pe r m i n ute , C P M) of all six microcentrifuge tubes (3 tubes containing beads and 3 tubes with their respective supernatant solutions) were counted using a gamma counter. The immunoreactivity for each tube was calculated using the following equation and reported as: % Immunoreactivity =beads CPM (beads CPM + supernatant CPM). Immunoreactivity for the following controls were performed alongside; i)89Zr-lvuxolimab binding to OX40-coated dynabeads alongside a blocking dose of 7.5 g cold Ivuxolimab was tested to confirm binding specificity of the unblocked samples and ii) Dynabeads lacking bound 0X40 antigen, enabled measurement of non-specific binding (NSB).Bmax and Kd determinationCalculation of binding parameters (Bmax and Kd) for89Zr-lvuxolimab was performed using a radioactive cell binding assay. Briefly, 3 x105huOX40+ HEK293 or HEK293 cells were seeded in 12-well plates, 24 hours preceding the experiment. The cells were then incubated with a starting concentration of 148 pCi / mL of89Zr-lvuxolimab (with subsequent serial dilutions to yield concentrations ranging from 0.2-1 OOnM), prepared in pre-warmed DMEM media. After 60 mins incubation at 37 °C, the tracer containing media was aspirated and cells were washed three times in 1ml_ ice-cold PBS. Cells were subsequently detached with 150 pL trypsin, and subsequently neutralized with 500 pL media; 500 pL of this was transferred to a gamma counting tube to measure radioactivity (Hidex AMG Automatic Gamma Counter) and the remaining sample was used to quantify the cell number. Radioactivity (CPM) and cell count were used to derive CPM / cell values. Total binding was determined from huOX40+ HEK293 cells with increasing concentrations of89Zr-lvuxolimab. The antigen negative HEK293 cells were used to determine non-specific binding of89Zr-lvuxolimab. Specific binding of89Zr-lvuxolimab to huOX40+ HEK293 cells was determined by subtracting non-specific binding from total binding. Values represent mean + / - SD (n=4 replicates / concentration). Saturation curve for specific binding was fitted using nonlinear regression with one site specific binding to obtain Bmax and Kd values.In vitro serum stability assay

[0151] Human and mouse serum (Sigma Aldrich, St Louis, MO) were centrifuged at 4°C for 10 min at 13000 rpm. 500 pL of the supernatant was removed and incubated with column-purified89Zr-lvuxolimab (500 pCi, 18.5MBq). After gentle vortexing, the radiolabeled mixtures wereATTORNEY DOCKET NO. 221910-2400 incubated at 37°C. Instant Thin Layer Chromatography (radio-TLC) analyses were performed at 0, 12, 24 and further 24 hour increments out to 7 days to measure bound versus free89Zr to determine the overall stability of the89Zr-lvuxolimab in serum.PET-CT imaging and dosimetry studies

[0152] For dosimetry studies, [18F]OP-801 was synthesized via the preclinical synthesis protocol described previously. Estimated radiation-absorbed dose for human subjects was calculated based on in vivo imaging of transgenic C57BL / 6N mice expressing human 0X40 (C57BL / 6N C57BL / 6N-Tnfrsf4,m1 1 (TNFRSF4) / costm1 1<ICOS\ Genoway, France). Mice (female n=5; male n=5) were anesthetized using isoflurane in oxygen (2.0-3.5% for induction and 1.0-2.5% for maintenance). Formulated tracer89Zr-lvuxolimab (~82 + 5.8 pCi in saline) was administered intravenously via tail-vein injection. PET / CT imaging studies were subsequently performed using a GNEXT PET / CT scanner (Sofie Biosciences). A 20-min static PET scan was acquired in list mode format at the following timepoints: 1.5, 24, 72, 120 and 168 hours post tracer injection (p.i.). Isotropic resolution was achieved using OSEM3D / MAP reconstruction algorithms with 24 subsets and 3 iterations and a matrix size of 240x240x191. A CT scan was acquired immediately after PET imaging to provide attenuation correction and an anatomic reference for the PET data. Calibration correction factors for Zr-89 for the pPET / CT scanner was obtained at the time of study, using PET standards with known amounts of89Zr-lvuxolimab radioactivity. The weights of the mice were recorded at the time of the study (mean weights: 18.64g and 20.74g for female and male mice respectively).

[0153] Volumes of interest (VOIs) were hand-drawn over regions of interest (i.e. , bladder, bone, brain, heart, kidney, liver, lung, muscle and spleen) using image analysis software (IRW) and CT images for anatomic reference. VOIs were drawn -30% smaller than the organ itself to avoid partial volume effects and signal spillover artifacts. The total percent injected dose per gram (% I D / g) associated with each VOI was calculated and summed over the PET imaging time points (area under curve [AUC]), and converted to %ID / organ. A previously described percent kg / g method for animal-to-human biokinetic extrapolation was then employed. Source organ residence times were calculated using a standard quantitation platform, organ level internal dose assessment (OLINDA), and bi-exponential model without bladder voiding. Lastly, projected human doses were computed for male and female phantoms using these source organ residence times.Ex vivo gamma counting of tissues for tracer biodistribution analysisATTORNEY DOCKET NO. 221910-2400

[0154] For validation of the PET imaging data, mice were euthanized following the terminal PET- CT scans at 168h p.i., and tissue associated radioactivity was verified by ex vivo gamma counting of mouse tissues. Briefly, mice were deeply anesthetized (2.5 - 3.0 % isoflurane) and blood collected via cardiac puncture prior to cervical dislocation. Tissues of interest were collected and wet-weights immediately recorded to allow normalization of tissue-associated radioactivity to weight. Tissues underwent gamma counting (Hidex AMG Automatic Gamma Counter) to determine the tissue-associated radioactivity. Biodistribution data were normalized to injected dose and were expressed as percentage injected dose / g of tissue (%ID / g).Immunohistochemistry0X40 immunohistochemistry was performed manually, using 4 pm thick formalin-fixed paraffin- embedded tissue sections. Briefly, slides were deparaffinized and rehydrated using conventional methods. Heat-induced epitope retrieval was performed at 113°C for 3 minutes (pH 9). Slides were cooled and subsequently placed in 3 % hydrogen peroxide for 15 minutes and then rinsed twice with PBS. Normal horse serum was applied at room temperature for 30 minutes. Primary antibody incubation was performed overnight at 4 °C using anti-CD134 clone ACT-35 murine lgG1 antibody (BD catalog #555836). The following day, sections were rinsed in PBS and a secondary universal antibody was applied. After a final rinse in PBS, the sections were saturated with DAB solution. Slides were thoroughly rinsed in de-ionized water, counterstained with hematoxylin, dehydrated and coverslips applied. Staining optimization was performed using NSG mouse-derived tumor samples generated from HEK293 cells stably transfected with huOX40+(antigen-positive control) and unmodified HEK293 cells (antigen-negative control). After establishing optimal conditions, the protocol was validated in human lymphoid tissues (lymph node and spleen) and ultimately applied to Head and Neck Squamous Cell Carcinoma (HNSCC) biopsy samples (Origene) and an HNSCC tumor microarray provided by the Stanford Department of Pathology to evaluate 0X40 expression in this population.ResultsOptimized radiolabeling and quality control testing of89Zr-lvuxolimab radiotracer consistently met the established clinical dose release criteria

[0155] Two preliminary technical runs were completed prior to the clinical validation runs to determine the optimal89Zr labeling conditions for Ivuxolimab. Various conditions were screened, such as89Zr-oxalate activity (pCi): DFO-lvuxolimab amount (pg) ratios and volume of 0.5 M HepesATTORNEY DOCKET NO. 221910-2400 buffer to achieve >95% radiochemical conversion and purity. (See Tables 4 and 5 for a summary of reaction conditions.) The reaction mixture was briefly centrifuged for 15 seconds at 1000 rpm and incubated at 37°C for 45 minutes with agitation at 400 rpm. A (pCi: j g) ratio of 10 produced >95% pure89Zr-lvuxolimab within 45 minutes of incubation. The progress of the89Zr labeling was monitored via radio-TLC in 0.5 mM EDTA mobile phase (89Zr-lvuxolimab Rfvalue: 0.112 and free radiometal89Zr Rfvalue: 0.5). Successful labeling in the first technical run (>95% radiochemical purity) required 200 pL of 0.5 M HEPES buffer while the second run required 400 pL of 0.5 M HEPES to yield 100% pure tracer. The increased buffer volume in the latter technical run was likely needed to neutralize the larger volume of acidic89Zr-oxalate and achieve the target reaction pH of 6.8-7.2 (15 pL in run 1 vs. 18-20 pL in run 2). Once radio-TLC confirmed >95% purity of89Zr-ivuxolimab in the crude reaction mixture, the product was purified on a NAPTM-5 column and eluted with saline. The eluant was then diluted with USP-grade 0.9% saline to a final volume of 10 mL and sterilized through a 0.22 pm Millex-MP filter (Millipore). Radiochemical purity of the final product was assessed by radio-TLC and by size-exclusion chromatography (SEC) using PBS (pH 7) as the mobile phase.

[0156] Based on these results of the technical runs, three consecutive clinical validation runs were conducted involving the final conditions; 4.17 ± 0.17 mCi89Zr-oxalate and 417.33 ± 17.04 mg Ivuxolimabwere incubated at 37 °C for 45 minutes with agitation at 400 rpm. In the subsequent purification step, the labeled DFO-lvuxolimab was passed through a NAPTM5 column and eluted with saline. A radio-TLC test of the purified89Zr-lvuxolimab showed >95% radiochemical purity (Rf 0.112). Radiochemical purity was further checked by HPLC. HPLC chromatograms did not indicate the presence of additional high or low molecular weight radiation or unspecified UV peaks (except HEPES buffer), indicating that no fragmented or aggregated byproducts of protein were generated during manufacturing.

[0157] These validations resulted in end of synthesis (EOS) activity of 3.31 ± 0.07 mCi pure tracer in 10 mL saline with final specific activity 9.85 ± 2.08 mCi / pg. The results of the three validation runs are summarized in Table 6). The release criteria required for successful completion of clinical validation runs and for future clinical production of89Zr-lvuxolimab are summarized in Table 7.HEPES BufferATTORNEY DOCKET NO. 221910-2400HEPES buffer played critical role in89Zr labeling. The minimum requirement for 0.5 M HEPES buffer volume yielded (7.71 ± 0.74) mg HEPES in 1 mCi dose. The amount of HEPES in the clinical batch was less than 15 mg.Bead-based immunoreactivity assay

[0158] 89Zr-lvuxolimab showed high immunoreactivity at the end of synthesis (EOS), measuring of 92 ± 0.4 % (FIG. 18A). Binding to OX40-coated Dynabeads was effectively blocked by the addition of 7.5 g unlabeled Ivuxolimab, resulting in an 89 % drop in immunoreactivity compared to the unblocked sample, thereby confirming target-specific binding. Dynabeads not coated with 0X40 antigen, were used to assess non-specific binding (NSB), showed <1% binding of89Zr-lvuxolimab. Immunoreactivity remeasured at 66 hours post-EOS, decreased to 70 ± 2.1 % (FIG. 18B) consistent with expected reduction in specific activity over time due to radioactive decay. Based on these observations, the final release criteria for immunoreactivity at EOS was set at >60% accordingly.Radioligand binding assay for Bmax and Kd calculation

[0159] Saturation binding assays performed using HEK293 cells and 0X40+ HEK293 cells incubated with increasing concentrations of89Zr-lvuxolimab, confirmed minimal non-specific binding and an estimated binding affinity constant (Kd) of 4.6 to 8.5 nM, indicating high affinity for human 0X40, respectively (FIG. 19).89Zr-lvuxolimab stability in human and mouse serumRadio-Thin Layer Chromatography (radio-TLC) confirmed that89Zr-lvuxolimab showed high serum stability in vitro, with less than 7% and 9% demetallation over 7 days of incubation at 37°C, in human and mouse serum respectively (FIG. 20). This is consistent with previously reported ex vivo stability studies performed with89Zr- Ivuxolimab in mice.Estimated mouse-to-human dosimetry analysis

[0160] Human radiation dose estimates were generated using data from both male and female mice, analyzed with a widely used and validated image-based dosimetry approach for generating reliable absorbed dose calculations. Image-based dosimetry permits longitudinal evaluation in individual animals although precise segmentation of small organs remains challenging. Since these small volumes of interest account for only a minor fraction of the total absorbed dose and have no meaningful effect on dose limits in mice or humans, they were not included in the imagebased analysis. Based on murine imaging data focusing on organs of major uptake such as theATTORNEY DOCKET NO. 221910-2400 heart, liver, lungs and spleen, absorbed doses were quantified for 24 organs of interest and used to predict human dosimetry (Table 9).

[0161] The highest dose was detected in the liver, which was thus predicted to be the critical or dose-limiting organ in humans, with an average of 0.366 and 0.271 mSv / MBq for females and males, respectively. Other organs that receive highest dose equivalents include the spleen and heart wall. The effective dose calculated was 0.0495 mSv / MBq and 0.0358 mSv / MBq for females and males, respectively. Thus the effective dose from a 37 M Bq (1mCi) administration of89Zr-ATTORNEY DOCKET NO. 221910-2400Ivuxolimab, typical of a clinical dose for89Zr-labeled antibody, is estimated to be 1.83 mSv for females and 1.32 mSv for males. These values are comparable to the estimated dosimetry profile reported for89Zr-Rituximab and fora89Zr-labeled PDLIantibody. The average yearly background effective dose in the United States is 3 mSv. For context, the average annual background radiation exposure or effective dose in the United States is ~3 mSv. Thus, the additional radiation from a 37 MBq (1 mCi) dose in the planned first-in-human studies would remain below typical yearly background exposure. Overall, the dosimetry profile of89Zr-lvuxolimab supports its use in clinical PET imaging studies

[0162] Ex vivo gamma counting of tissues collected after terminal dosimetry scans acquired at 168 h p.i. corroborated PET-derived biodistribution findings the highest uptake observed in liver, spleen, lung and bone (FIG. 22). Female mice exhibited modestly higher radiotracer retention in several tissues, with statistically significant differences observed in the brain, heart, kidney and muscle.

[0163] 0X40 immunohistochemistry (IHC) was first optimized successfully in NSG mouse- derived tumor samples (FIGs. 23A-23B) generated from HEK293 cells stably transfected with huOX40+(antigen-positive control) and unmodified HEK293 cells (antigen-negative control). 0X40 IHC was further validated in healthy human lymphoid tissues which showed overall low levels of baseline 0X40 expression as expected (FIG. 24). In contrast HNSCC biopsy samples showed higher and heterogenous levels of 0X40 expression, irrespective of Human Papilloma Virus (HPV) status (FIG. 25). HNSCC tumor microarrays further facilitate high-throughput visualization of 0X40 expression across multiple resected samples, encompassing both malignant and benign tissues, in parallel (FIG. 26). Future studies will aim to further characterize OX40-expressing cells within the immune infiltrate in HNSCC and other cancers through costaining with T-cell markers such as CD3, CD4, and CD8, using conventional IHC and multiplexed spatial biology approaches.

[0164] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCESATTORNEY DOCKET NO. 221910-2400 Alam IS, et al. Imaging activated T cells predicts response to cancer vaccines. J Clin Invest. 2018;128:2569-2580. Alam IS, et al. Visualization of activated T cells by OX40-immunoPET as a strategy for diagnosis of acute Graft-versus-Host-Disease. Cancer Res. 2020;80:4780-4790. Alam IS. Nuclear Imaging of Endogenous Markers of Lymphocyte Response. In: Harsini S, Alavi, A., Rezaei, N., ed. 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Claims

ATTORNEY DOCKET NO. 221910-2400CLAIMSWhat is claimed is:

1. An immunoPET agent for detecting activated T cells expressing human 0X40, the immunoPET agent comprising an antibody or an antibody fragment labeled with a radioisotope.

2. The immunoPET agent of claim 1, further comprising a chelator.

3. The immunoPET agent of claim 2, wherein the chelator comprises deferoxamine (DFO), 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), 1 ,4,7-triazacyclononane-1 , 4, 7-tri acetic acid (NOTA), 1 ,4,8,11-tetraazacyclotetradecane-1 ,4,8,11-tetraacetic acid (TETA), diethylenetriaminepentaacetic acid (DTPA), 6-hydrazinonicotinic acid (HYNIC), 2,2'-(1 , 10- diazacyclooctadecane-1 ,10-diyl)diacetic acid (Macropa), a Macropa derivative selected from Macropa-PEG, Macropa-TATE, and Macropa- BP, a hydroxypyridinone (HOPO) chelator selected from 3-HOPO, 4-HOPO, and 1 ,2-HOPO, a DOTA-HOPO hybrid, or any combination thereof.

4. The immunoPET agent of claim 2, wherein the chelator conjugates the antibody or antibody fragment to the radioisotope.

5. The immunoPET agent of claim 2, wherein the immunoPET agent comprises an antibody and the radioisotope comprises Zr-89, Cu-64, Cu-61 , Cu-67, 1-131 , In-111 , Lu-177, Ac-225, Pb-212, Bi-212, Ra-223, Re-188, Th-227, F-18, Ga-68, Sc-44, C-11 , or any combination thereof.

6. The immunoPET agent of claim 2, wherein the immunoPET agent comprises an antibody fragment and the radioisotope comprises Zr-89, Cu-64, Cu-61, Cu-67, 1-131 , In-111, Lu-177, Ac-225, Pb-212, Bi-212, Ra-223, Re-188, Th-227, F-18, Ga-68, Sc-44, C-11, or any combination thereof.

7. The immunoPET agent of claim 5, wherein the antibody comprises ivuxolimab.

8. The immunoPET agent of claim 7, wherein the immunoPET agent comprises89Zr-ivuxolimab.

9. The immunoPET agent of claim 6, wherein the antibody fragment comprises a nanobody, a bivalent minibody, a cys-diabody, an F(ab)2fragment, a bispecific T-cell engager (BITE), or any combination thereof.

10. The immunoPET agent of claim 9, wherein the antibody fragment is an F(ab)2fragment produced by enzymatic digestion with IdeS.

11. The immunoPET agent of claim 9, wherein the antibody fragment comprises lvux-F(ab)2andATTORNEY DOCKET NO. 221910-2400 has a molecular weight of about 96 kDa.

12. A pharmaceutical composition comprising the immunoPET agent of any one of claims 1-11.

13. A method for quantifying and tracking 0X40 positive T cells in a subject, the method comprising administering the pharmaceutical composition of claim 12 to the subject and monitoring a signal produced by the immunoPET agent.

14. The method of claim 13, wherein the signal comprises a positron emission tomography (PET) signal or a single-photon emission computed tomography (SPECT) signal.

15. The method of claim 13, wherein presence of the signal produced by the immunoPET agent above a background threshold indicates presence of 0X40 positive T cells in the subject.

16. The method of claim 13, wherein the subject is a human.

17. The method of claim 13, wherein the subject has or is suspected of having cancer, an autoimmune disorder, a chronic inflammatory disease, or a transplant complication.

18. The method of claim 17, wherein the cancer comprises cutaneous melanoma, colorectal cancer, non-small cell lung cancer, head and neck cancer, hepatocellular carcinoma (HCC), malignant melanoma (MEL), renal cell carcinoma (RCC), bladder cancer, breast cancer, gastric cancer, cervical cancer, lymphomas, or any combination thereof.

19. The method of claim 17, wherein the auto-immune disorder comprises multiple sclerosis or rheumatoid arthritis.

20. The method of claim 17, wherein the chronic inflammatory disease comprises inflammatory bowel disease, type 1 diabetes, psoriasis, psoriatic arthritis, Crohn's disease, autoimmune hepatitis, autoimmune uveitis, Systemic Lupus Erythematosus (SLE), atopic dermatitis, or any combination thereof.

21. The method of claim 17, wherein the transplant complication comprises graft versus host disease or solid organ transplant rejection.

22. A method for monitoring progress of treatment for a disease in which 0X40 positive T cells are involved, the method comprising administering the immunoPET agent the pharmaceutical composition of claim 12 to the subject a first time, wherein the first time is prior to beginning the treatment and monitoring a first signal produced by the immunoPET agent; administering the immunoPET agent to the subject a second time, wherein the second time is following performing the treatment and monitoring a second signal produced by the immunoPET agent; and comparing the first signal to the second signal.ATTORNEY DOCKET NO. 221910-240023. The method of claim 22, wherein a successful treatment is indicated when the second signal has a lower intensity than the first signal.

24. The method of claim 13, wherein the immunoPET agent is administered through intravenous, intramuscular, subcutaneous, intraperitoneal, intravitreal, retro-orbital, topical, intranasal, or intrathecal administration.

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