Multimodal imaging for the detection and characterization of liver lesions in a mouse model of neuroendocrine tumor

Background

The aim of this study was to compare in vivo magnetic resonance imaging (MRI) and ex vivo autoradiography with histopathological results for the detection and characterization of liver lesions in an experimental model of human neuroendocrine tumors.

Material and methods

Intestinal STC-1 endocrine tumor cells were injected into 30 nude mice to achieve hepatic dissemination. Seven to 30 days after injection, T2-weighted in vivo images covering the entire liver were acquired with a 7-T system. Autoradiographs were also obtained in 28 mice after injection of fluorodeoxyglucose (¹⁸F-FDG). The autoradiographic liver samples were then stained with an antichromogranin antibody before histological analysis. Tumor size and the hepatic tumor fraction were measured using the three imaging modalities.

Results

Metastatic tumors visualized on the histological liver sections ranged in size from 50 μm (day 7) to 3 mm (day 30). The hepatic tumor fraction increased with time, reaching 30% of the hepatic surface area on day 30. Visual analysis revealed variable tumor distribution and type (solid and/or cystic). On MRI, lesions were identified from day 12 (about 100 μm in diameter) and the hepatic tumor fraction was up to 48% at day 30. The smallest lesions (350 μm in diameter) were also detected at day 12 on the autoradiographs. There was good correlation between tumor fractions determined from autoradiographic and histological data.

Conclusion

In vivo, MRI appears to be well suited to the follow-up of liver lesions in a mouse model of neuroendocrine tumor. Preliminary results using ¹⁸F-FDG in this animal model are promising, showing differences in FDG uptake.     

Abundant in vitro expression of the oncofetal ED-B-containing fibronectin translates into selective pharmacodelivery of 131I-L19SIP in a prostate cancer patient

Purpose

The extradomain B of fibronectin (ED-B) is a promising vascular target for selective pharmacodelivery in cancer patients. We analyzed a large series of prostatectomies from patients with prostate cancer, hyperplastic prostate disease, and normal prostates to study extent and tumor-selectivity of ED-B expression.

Methods

Using immunohistology, 68 adenocarcinomas of the prostate or prostate cancer-inflicted lymph nodes, 4 samples of benign prostatic hyperplasia, and 6 normal prostate glands were studied for ED-B expressing newly formed blood vessels. Further, we treated an advanced prostate cancer patient with the anti-ED-B antibody ¹³¹I-L19SIP to study in vivo target accessibility.

Results

ED-B-positive blood vessels were found significantly more frequent in prostate cancers as compared with peritumoral prostate tissues or normal prostate glands, independent of tumor differentiation. The ED-B-positive blood vessels’ density was 97 (±23), 65 (±9), and 59 (±9)/mm² in G3, G2, and G1 prostate cancers, respectively, and 7 (±5)/mm² in normal prostate glands. In high-grade (G3) prostate cancers, also the peritumoral tissue showed a higher density of ED-B vessels than normal prostate glands. Similar results were obtained when ED-B-positive vessel density was expressed as a fraction of CD34-positive vessel density. Finally, selective uptake of ED-B-binding ¹³¹I-L19SIP to tumor lesions was found in an advanced prostate cancer patient by whole-body planar scintigraphy.

Conclusions

ED-B-positive blood vessels were found to a large extent in prostate cancer tissues, but only rarely in normal prostates or benign prostatic hyperplasia. Whole-body planar scintigraphy in a prostate cancer patient confirmed selective uptake of ¹³¹I-L19SIP in the prostate cancer tissues, qualifying ED-B as a promising target for selective pharmacodelivery of anticancer agents in prostate cancer.     

Improved experimental radioimmunotherapy of colon xenografts by combining131I-CC49 and Interferon-γ

PURPOSE: This study was designed to determine whether the ability of interferon- to upregulate the expression of a human tumor antigen improved the therapeutic efficacy of a radionuclide-conjugated monoclonal antibody. METHODS: Tumor xenografts of the moderately differentiated human colon tumor cell line HT-29 were grown in athymic mice. Constitutive levels of the human tumor antigen, tumor-associated glycoprotein-72, were measured before and after treatment with interferon-. Antitumor effects of an¹³¹I-labeled antitumor-associated glycoprotein-72 monoclonal antibody, CC49, were determined by measuring changes in tumor volumes in the respective groups of athymic mice. RESULTS: Interferon- induced a time-dependent and dose-dependent increase in tumor-associated glycoprotein-72 expression in the HT-29 tumors. Immunohistochemical staining revealed a more homogeneous tumor-associated glycoprotein-72-positive tumor cell population in tumors isolated from mice treated for eight days with interferon-, which accounted for the enhanced tumor localization of¹³¹I-CC49 in mice. That experimental model was used to examine the antitumor effects of combining interferon- with¹³¹I-CC49. Administration of 300 Ci of¹³¹I-CC49 to mice bearing HT-29 tumors induced a transient suppression of tumor growth. Conversely, a long-term, sustained HT-29 tumor growth suppression was achieved in mice given 300Ci of¹³¹I-CC49 and interferon-. In fact, the cytokine/radioimmunoconjugate combination eradicated any evidence of tumor in approximately 30 percent of the mice. CONCLUSION: The ability of interferon- to enhance tumor-associated glycoprotein-72 expression substantially augmented the antitumor effects of the radioimmunoconjugate. Those observations provide additional argument for use of a radioimmunoconjugate in combination with a cytokine to improve tumor diagnosis and therapy.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

Summary It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the prliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [³H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE₂. The underlayer of tumor macrophages P 388 D₁ resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [³H] thymidine uptake by EL-4 cells.These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.Supported by a grant from the Consiglio Nazionale delle Ricerche (CNR). Progetto Finalizzato Control of neoplastic growth. Contract n^o 8001591.     

Prediction of tumor biological characteristics in different colorectal cancer liver metastasis animal models using 18F-FDG and 18F-FLT

Background: Positron emission tomography(PET) is a noninvasive method to characterize different metabolic activities of tumors, providing information for staging, prognosis, and therapeutic response of patients with cancer. The aim of this study was to evaluate the feasibility of~(18)F-fludeoxyglucose(~(18)F-FDG) and 3'-deoxy-3'-~(18)F-fluorothymidine(~(18)F-FLT) PET in predicting tumor biological characteristics of colorectal cancer liver metastasis.Methods: The uptake rate of~(18)F-FDG and~(18)F-FLT in SW480 and SW620 cells was measured via an in vitro cell uptake assay. The region of interest was drawn over the tumor and liver to calculate the maximum standardized uptake value ratio(tumor/liver) from PET images in liver metastasis model. The correlation between tracer uptake in liver metastases and VEGF, Ki67 and CD44 expression was evaluated by linear regression.Results: Compared to SW620 tumor-bearing mice, SW480 tumor-bearing mice presented a higher rate of liver metastases. The uptake rate of~(18)F-FDG in SW480 and SW620 cells was 6.07% ± 1.19% and2.82% ± 0.15%, respectively(t = 4.69, P = 0.04); that of~(18)F-FLT was 24.81% ± 0.45% and 15.57% ± 0.66%, respectively(t = 19.99, P 0.001). Micro-PET scan showed that all parameters of FLT were significantly higher in SW480 tumors than those in SW620 tumors. A moderate relationship was detected between metastases in the liver and~(18)F-FLT uptake in primary tumors(r = 0.73, P = 0.0019).~(18)F-FLT uptake was also positively correlated with the expression of CD44 in liver metastases(r = 0.81, P = 0.0049).Conclusions: The uptake of~(18)F-FLT in metastatic tumor reflects different biological behaviors of colon cancer cells.~(18)F-FLT can be used to evaluate the metastatic potential of colorectal cancer in nude mice.     

An attempt to enhance chemosensitivity of quiescent cell populations in solid tumors by combined treatment with nicotinamide and carbogen

cis-Diamminedichloroplatinum(II) (cisplatin) was intraperitoneally injected into mice bearing SCC VII or EMT6/KU tumors after ten administrations of 5-bromo-2-deoxyuridine (BrdU) to label all the proliferating tumor cells. The tumors were excised 1 h after the cisplatin injection, minced, and trypsinized. The tumor cell suspensions were then incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus frequency was determined, using immunofluorescence staining for BrdU. Cells that were not labeled with BrdU were regarded as quiescent. The micronucleus frequency in the total number of tumor cells was determined in tumors that had not been pretreated with BrdU. To modify the sensitivity to cisplatin, nicotinamide was intraperitoneally injected before the administration of cisplatin or mice were placed in a circulating carbogen (95% O₂, 5% CO₂) chamber for 30 min after cisplatin administration. In both tumor systems, the micronucleus frequency in quiescent cells was lower than that in the total cells. Nicotinamide pretreatment increased the micronucleus frequency in total and in quiescent cells in both tumor systems, and to a higher extent in total cells. The combination of nicotinamide and carbogen increased the micronucleus frequency more markedly than treatment with either nicotinamide or carbogen alone. In total cells of both tumors, the nicotinamide injection increased the uptake of [^195mPt]cisplatin. The combined treatment raised the uptake more markedly than did treatment with either agent alone. In total cells of the SCC VII tumor, these increases in micronucleus frequency and the [^195mPt]cisplatin uptake following nicotinamide or combined pretreatment were significant. In both tumors, carbogen breathing also elevated the micronucleus frequency to some degree in total and quiescent cells and the [^195mPt]cisplatin uptake in total cells. The combined nicotinamide and carbogen treatment was considered to be useful for sensitizing tumor cells to chemotherapy with cisplatin in vivo.Abbreviations Q cells quiescent cells - P cells proliferating cells     

The effect of antibody against vascular endothelial growth factor on tumor growth and metastasis

Vascular endothelial growth factor (VEGF), a very important in the process of tumor angiogenesis, was chosen as a target in a study to determine whether manipulation of angiogenesis with antibody against VEGF may interrupt tumor growth and metastasis. Anti-VEGF antibody was obtained from immunized rabbits, purified on an affinity column, and identified as neutralized antibody by Mile's assay. IVTA₂MA891, a murine spontaneous breast cancer with a high rate of metastasis in lung in TA₂ × 615 F1 mice, was chosen as an animal model in this study, because of the high expression of VEGF in the primary tumor as well as in the lung metastatic tumor. The anti-VEGF antibody could inhibit growth of S180 sarcoma in a dose-dependent manner, and the inhibition rate could reach 41.0% with a dose of 200 μg mouse⁻¹ day⁻¹. Anti-VEGF antibody could inhibit tumor growth by 76.2% in nude mice bearing human gastric cancer (MGC 803). When anti-VEGF antibody was combined with ¹³¹I-3H11, a murine monoclonal antibody conjugated with ¹³¹I, only one of five nude mice developed tumor and 84.0% more inhibition of tumor growth was obtained in comparison with treatment by ¹³¹I-3H11 alone. The growth of the primary tumor was inhibited by 44.0% and the number and size of the metastatic foci in the lungs were reduced by 73.0% and 83.7% respectively in the animal model, with a high rate of metastasis in lung. The anti-VEGF antibody may be potentially useful for clinical treatment of cancer and metastasis. Received: 2 January 1998 / Accepted: 17 March 1998     

Improved experimental radioimmunotherapy of colon xenografts by combining<Superscript>131</Superscript>I-CC49 and Interferon-γ

PURPOSE: This study was designed to determine whether the ability of interferon-γ to upregulate the expression of a human tumor antigen improved the therapeutic efficacy of a radionuclide-conjugated monoclonal antibody. METHODS: Tumor xenografts of the moderately differentiated human colon tumor cell line HT-29 were grown in athymic mice. Constitutive levels of the human tumor antigen, tumor-associated glycoprotein-72, were measured before and after treatment with interferon-γ. Antitumor effects of an¹³¹I-labeled antitumor-associated glycoprotein-72 monoclonal antibody, CC49, were determined by measuring changes in tumor volumes in the respective groups of athymic mice. RESULTS: Interferon-γ induced a time-dependent and dose-dependent increase in tumor-associated glycoprotein-72 expression in the HT-29 tumors. Immunohistochemical staining revealed a more homogeneous tumor-associated glycoprotein-72-positive tumor cell population in tumors isolated from mice treated for eight days with interferon-γ, which accounted for the enhanced tumor localization of¹³¹I-CC49 in mice. That experimental model was used to examine the antitumor effects of combining interferon-γ with¹³¹I-CC49. Administration of 300 μCi of¹³¹I-CC49 to mice bearing HT-29 tumors induced a transient suppression of tumor growth. Conversely, a long-term, sustained HT-29 tumor growth suppression was achieved in mice given 300μCi of¹³¹I-CC49 and interferon-γ. In fact, the cytokine/radioimmunoconjugate combination eradicated any evidence of tumor in approximately 30 percent of the mice. CONCLUSION: The ability of interferon-γ to enhance tumor-associated glycoprotein-72 expression substantially augmented the antitumor effects of the radioimmunoconjugate. Those observations provide additional argument for use of a radioimmunoconjugate in combination with a cytokine to improve tumor diagnosis and therapy.     

Scintigraphic detection of neural-cell-derived small-cell lung cancer using glioma-specific antibody

Radiolabeled GA-17, a murine monoclonal antibody that reacts specifically with glioma cells, bound to a small-cell lung cancer (SCLC) cell line NCI-H69 derived from neural cells, both in vitro and in vivo. The affinity constant of GA-17 F (ab)₂ fragment binding to NCI-H69 was 1.02×10⁸/M while that to the glioma cell line U87MG was 1.22×10⁸/M. Iodine-125-labeled GA-17 F (ab)₂ fragments injected i.v. localized well in NCI-H69 cells xenografted in nude mice. The percentage of the injected dose per gram accumulated in the xenografted tumor was 6.87±1.34%g^–1 (mean±SD,n=5) 24 h after injection. On the other hand, control monoclonal F (ab)₂ fragments accumulated in the xenografted tumor at 0.75±0.30%g^–1. The tumor-to-blood ratio was 1.8 for NCI-H69, while that of control F (ab)₂ was 0.60. In conclusion, the radiolabeled GA-17 F (ab)₂ fragment is expected to be useful clinically to visualize the small-cell lung cancer and in radioimmunotherapy.Abbreviations SCLC small-cell lung cancer - mAb monoclonal antibody     

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.During metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed “tumor clumps”) are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5–7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of epithelial tumors, a process termed “collective invasion” (9, 10). In breast cancer, collective invasion is facilitated by invasive leader cells, a subpopulation of tumor cells that highly express keratin 14 (K14) and other basal epithelial markers (11). K14⁺ cells are migratory, protrusive, and lead trailing K14⁻ cells, while maintaining cell–cell cohesion and E-cadherin–based cell contacts.In this study, we sought to understand how these K14⁺ cells exit collective invasion strands in the primary tumor and travel to distant organs (12). One hypothesis is that collective invasion is an intermediate step toward eventual single-cell dissemination and monoclonal metastasis. However, tumor cell clusters are detected in circulation (5) and primary human breast tumors can disseminate collectively into the surrounding extracellular matrix in ex vivo assays (13–15). These data prompted an alternative hypothesis, that collectively invading K14⁺ cancer cells could initiate and complete the metastatic process as a cohesive multicellular unit. Here we define the clonal nature of metastases in a spontaneous mouse model of metastasis to the lungs (16, 17), in which the predominant invasive form in the primary tumor is collective invasion strands led by K14⁺ cells (11). We establish that the majority of metastases arise from polyclonal seeds, and show that disseminated tumor cell clusters are predominantly composed of K14⁺ cells. We propose a mechanism for polyclonal metastasis via the collective invasion, dissemination, and colonization of clusters of K14⁺ cancer cells.     

Noninvasive brain cancer imaging with a bispecific antibody fragment,generated via click chemistry

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)₂] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal “click” ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105^+/+) tumors with ⁶⁴Cu-labeled Bs-F(ab)₂ revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of ⁶⁴Cu-NOTA-Bs-F(ab)₂. ⁶⁴Cu-NOTA-Bs-F(ab)₂ was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1–labeled Bs-F(ab)₂ for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.Despite advances in diagnostic procedures and clinical patient management, early detection and diagnosis of cancers remains the most important endeavor for reducing cancer morbidity and mortality (1). Although ultrasonography, computed tomography (CT), and magnetic resonance imaging are essential to clinical oncology, tumor detection using these technologies is based primarily on anatomical characteristics, providing limited information about the molecular profile during tumor progression (2). On the other hand, noninvasive molecular imaging techniques, which can be designed to specifically detect alterations in gene amplification or mutations that occur early during cancer progression, have the potential to visualize carcinogenesis at earlier stages (3). Given its excellent sensitivity (picomolar range), adequate spatial resolution, and the ability to accurately quantify the biodistribution of a radiotracer, PET imaging is becoming the modality of choice to noninvasively study the biochemistry of human tumors in situ (4). PET imaging with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), which allows clinicians to scrutinize glucose metabolism in vivo, has largely dominated the clinical diagnostic oncology setting. However, a common disadvantage of the use of ¹⁸F-FDG as an imaging tracer has been its limited sensitivity and specificity, which can lead to confounding diagnosis (5); other pathological processes including inflammation and infection also present high glucose metabolism. Additionally, ¹⁸F-FDG PET often fails at detecting small malignant lesions (<5 mm in diameter) (6). Therefore, there is a pressing need for the implementation of molecular imaging probes that specifically target cancer-associated biological pathways and that can detect earlier such processes at the molecular level (7).Antibodies are of high interest as molecular imaging agents, particularly in oncology, because of their excellent antigen specificity and binding affinity. ImmunoPET probes can be designed to seek and target tumor cell-specific surface epitopes in vivo while maintaining low off-target effects (8). This enables the acquisition of high-quality PET images, which is highly desirable for cancer diagnosis, staging, and therapy response assessment. Compared with ¹⁸F-FDG and several other small-molecule PET tracers, antibodies provide greater specificity and phenotypic information on primary and metastatic diseases that can guide treatment decisions (3). However, the implementation of antibody-based imaging has been limited by practical complications related to long circulation half-lives, slow tumor penetration, immunogenicity, and regulatory hurdles. Fortunately, various protein engineering technologies can alleviate many of these issues. For example, humanized and fully human antibodies are available that minimized the risk of eliciting host immune responses. Furthermore, antibody fragments can exhibit significantly improved pharmacokinetic profiles compared with the intact antibody while retaining excellent antigen-binding affinity. A myriad of such immunoderivatives have been used for immunoPET imaging including monovalent fragments, diabodies, triabodies, minibodies, and single-domain antibodies (9). However, although PET imaging with antibody fragments offers several advantages in terms of radiation exposure, time to image, and multiple/repeated imaging, the fragments typically display significantly reduced tumor uptake and a much higher renal accumulation (10, 11).Given the inherent complexity of cancer, which involves a sophisticated cross-talk and promiscuity between multiple disease-mediating pathways and growth-promoting factors, targeting an isolated process usually fails to provide a satisfactory diagnosis and treatment efficacy (12). On the other hand, bispecific antibody fragments simultaneously targeting two antigens make for a promising alternative to enhance tumor uptake as well as specificity (13, 14). Although the value of bispecific antibodies for combination therapies has been proposed (15, 16), their potential as molecular imaging agents for cancer detection remains largely unexplored.Herein, we developed a bispecific construct, Bs-F(ab)₂—via conjugation of two antibody Fab fragments targeting epidermal growth factor receptor (EGFR) and CD105, respectively—for radiolabeling with ⁶⁴Cu and noninvasive PET imaging. The antibody fragments were obtained by enzymatic digestion of cetuximab (CET), an anti-human EGFR chimeric mAb, and TRC105, a mAb that recognizes both human and murine CD105. To conjugate the two Fab fragments, we exploited the fast reaction kinetics and selectivity of the inverse electron-demand Diels–Alder reaction between electron-deficient tetrazine (Tz) and strained transcyclooctene (TCO) derivatives (17). EGFR has been extensively studied as a target for anticancer therapy, and its activation stimulates tumor proliferation and angiogenesis (18). Similarly, CD105 (also called endoglin) is abundantly expressed on activated endothelial cells, and such overexpression is a negative prognostic factor in many malignant tumor types (19, 20). To date, simultaneous targeting of EGFR and CD105 has not been investigated. We hypothesized that our bispecific Bs-F(ab)₂ will harness the targeting capabilities of CET-Fab and TRC105-Fab and display a synergistic effect via dual targeting of EGFR and CD105. To test our hypothesis, we determined the advantages of dual EGFR/CD105 targeting in terms of tumor-binding affinity and specificity of Bs-F(ab)₂ in a glioblastoma multiforme (GBM) xenograft model, which expresses high levels of both EGFR and CD105 (^+/+). We presented here a generalizable rationale that could be potentially applied to produce bispecific imaging probes from other disease-targeting antibody fragments.     

T helper 17 cells play a critical pathogenic role in lung cancer

Lung cancer development is associated with extensive pulmonary inflammation. In addition, the linkage between chronic obstructive pulmonary disease (COPD) and lung cancer has been demonstrated in population-based studies. IL-17–producing CD4 helper T cells (Th17 cells) play a critical role in promoting chronic tissue inflammation. Although Th17 cells are found in human COPD and lung cancer, their role is not understood. We have thus used a mouse model of lung cancer, in which an oncogenic form of K-ras (K-ras^G12D), frequently found in human lung cancer, is restrictedly expressed in lung epithelial cells [via Clara cell secretory protein (CCSP^cre)]. In this model, Th17 and Treg but not Th1 cells were found enriched at the tumor tissues. When CCSP^cre/K-ras^G12D mice were weekly challenged with a lysate of nontypeable Haemophilus influenza (NTHi), which induces COPD-type inflammation and accelerates the tumor growth, they showed greatly enhanced Th17 cell infiltration in the lung tissues. Lack of IL-17, but not IL-17F, resulted in a significant reduction in lung tumor numbers in CCSP^cre/K-ras^G12D mice and also those treated with NTHi. Absence of IL-17 not only resulted in reduction of tumor cell proliferation and angiogenesis, but also decreased the expression of proinflammatory mediators and reduced recruitment of myeloid cells. Depletion of Gr-1⁺CD11b⁺ myeloid cells in CCSP^cre/K-ras^G12D mice suppressed tumor growth in lung, indicating Gr-1⁺CD11b⁺ myeloid cells recruited by IL-17 play a protumor role. Taken together, our data demonstrate a critical role for Th17 cell-mediated inflammation in lung tumorigenesis and suggest a novel way for prevention and treatment of this disease.Inflammation plays an important role in tumor development (1, 2). Although targeting inflammation and tumor microenvironment has been considered as a new direction of cancer therapy, the mechanisms underlying cancer-associated inflammation have not been well understood. Lung cancer is a leading cause of death in the world. Accumulating evidence has shown that inflammation is associated with pathogenesis of lung cancer, especially those induced by cigarette smoke (3). The primary risk factor among smokers to develop lung cancer is the presence of chronic obstructive pulmonary disease (COPD) (4), which is characterized by chronic pulmonary inflammation, airway remodeling and destruction of lung parenchyma. Human lung cancers are inflicted with alterations in various subsets of lymphocytes and myeloid cells (5, 6), reminiscent of immune activation during chronic inflammation. Several studies have shown NFκB signaling as a mechanistic link between inflammation and lung cancer using a mouse model of lung adenocarcinoma (7, 8). However, the specific inflammatory cell types or molecules potentiating lung cancer are not understood clearly.We and others have identified a novel subset of CD4 helper T cells that produce IL-17 and are referred as Th17 cells (9, 10). Th17 cells have been associated with inflammatory diseases such as rheumatoid arthritis, asthma, lupus, and allograft rejection. An important function of IL-17 is to promote tissue inflammation through the up-regulation of proinflammatory cytokines and chemokines (11). Consistently, we have shown that transgenic overexpression of IL-17 in the lungs resulted in chemokine up-regulation and tissue infiltration by leukocytes, although mice treated with neutralizing IL-17–specific antibody were also found to be resistant to the induction of experimental autoimmune encephalomyelitis (9). These and other studies collectively demonstrated that IL-17 and Th17 cells play nonredundant function in promoting inflammation.Increased frequencies of IL-17 and Th17 cells have been reported in patients with different types of tumors (12), including lung adenocarcinoma (13). The density of intratumoral IL-17–positive cells in primary human nonsmall cell lung cancer was inversely correlated with patient outcome and correlated with smoking status of the patients (14). Th17 cells specific for a common tumor antigen were found in lung cancer patients as part of their spontaneous immune response to the autologous tumor (15). However, the function of Th17 cells and IL-17 in the development of lung cancer remains to be shown. Animal model studies have revealed contrasting roles of IL-17 in various tumors (16). Tumor-promoting effect of IL-17 was shown in some models such as colon cancer (17–20), whereas in others, IL-17 supported anti-tumor immunity, including in B16 melanoma model (21–24). Thus, the role of IL-17 could be complex and tumor-specific.To properly evaluate the role of IL-17 in inflammation-associated lung cancer, we used a model of oncogenic K-ras mutation expressed only in the lung. Mice expressing K-ras mutation in Clara cells (CCSP^cre/K-ras^G12D mice) spontaneously develop lung adenocarcinoma (25). In addition, we induced COPD-type lung inflammation by challenging mice with lysates of nontypeable Haemophilus influenza (NTHi). Inflammation driven by NTHi can promote tumor growth in CCSP^cre/K-ras^G12D mice (25). These experiments collectively indicate a tumorigenic role of IL-17–mediated inflammation in the development of lung cancer.     

A novel therapeutic strategy with anti-CD9 antibody in gastric cancers

Background

CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as motility, cell signaling, proliferation, adhesion, and metastasis. However, very little is known about the involvement of CD9 in the process of development of primary tumors. In the present study, we investigated whether anti-CD9 monoclonal antibody (ALB6) has antitumor effects in human gastric cancer cell xenografts.

Methods

Human gastric cancer cell lines (MKN-28) (5 × 10⁶ cells/animal) were inoculated subcutaneously into the dorsal region of SCID mice (five mice in each group). After a tumor was visualized, animals were assigned to either the ALB6 treatment group or the control IgG treatment group (100 μg/body/time, intravenous, three times per week. Day 1, 4, and 7 of first week). Then tumor volumes were monitored every day. Proliferation of tumor was analyzed by 5-bromo-2′-deoxyuridine (BrdU) immunostaining, apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) methods, and angiogenesis was assessed by counting the number of CD34-positive endothelial cells.

Results

Tumor volume was significantly suppressed (1,682 ± 683 mm³ versus 4,507 ± 1,012 mm³; P = 0.049), the BrdU labeling indexes were significantly decreased (10.9 ± 1.1% versus 17.2 ± 1.4%; P = 0.009), the apoptotic indexes were significantly increased (1.98 ± 0.48% versus 0.72 ± 0.09%; P = 0.034), and tumor microvessel densities were significantly suppressed (671,922 ± 34,505 pixels/mm² versus 1,135,043 ± 36,086 pixels/mm²; P = 0.037) in the ALB6 treatment group compared with the control IgG treatment group.

Conclusions

These results suggest that administration of anti-CD9 antibody to mice bearing human gastric cancer cells successfully inhibits tumor progression via antiproliferative, proapoptotic, and antiangiogenetic effects.     

High FDG uptake in PET/CT predicts worse prognosis in patients with metastatic gastric adenocarcinoma

Purpose

We evaluated the role of FDG-PET/CT in patients with metastatic gastric adenocarcinoma before palliative chemotherapy to predict prognosis and chemotherapeutic response.

Methods

The study included 35 consecutive newly diagnosed patients with metastatic gastric adenocarcinoma who underwent FDG-PET/CT before palliative chemotherapy. Maximum standardized uptake value (SUVmax) of the primary tumor was assessed to evaluate survival and chemotherapeutic response. Survival analysis was performed for time to progression and overall survival using the Kaplan–Meier method. Cox proportional hazard models were used to determine independent prognostic factors.

Results

All primary tumors were visualized using FDG-PET/CT (mean SUVmax = 8.1 ± 4.5, range 2.5–22.1). Sensitivity, specificity, and accuracy of FDG-PET/CT in detection of solid organ metastasis were 95.2% (20/21), 100% (14/14), and 97.1% (34/35), respectively. No significant difference of primary tumor SUVmax was found among the chemotherapeutic response groups. Univariate survival analysis demonstrated ECOG performance status (≥2), presence of solid organ metastasis, number of organs involved in distant metastasis (≥2), and SUVmax of the primary tumor (>8) as significant predictors for poor overall survival. Multivariate survival analysis showed SUVmax of the primary tumor (P = 0.048), presence of solid organ metastasis (P = 0.015), and ECOG performance status (P = 0.002) as significant independent prognostic predictors for overall survival.

Conclusions

High FDG uptake of the primary tumor in patients with metastatic gastric adenocarcinoma is associated with poor overall survival. Assessment of tumor FDG uptake has limited value for prediction of chemotherapeutic response, but provides useful information regarding prognosis.     

Simultaneously targeting CD45 significantly increases cytotoxicity of the anti-CD33 immunoconjugate, gemtuzumab ozogamicin, against acute myeloid leukemia (AML) cells and improves survival of mice bearing human AML xenografts

Targeting CD33 or CD45 is currently exploited for immunotherapy of acute myeloid leukemia (AML). Gemtuzumab ozogamicin (GO), an immunoconjugate of an anti-CD33 antibody that facilitates cellular uptake of a toxic calicheamicin-₁ derivative, induces complete remissions in a subset of patients with AML. We herein tested whether simultaneous targeting of CD45 could improve GO cytotoxicity against AML cell lines and primary AML cells. We found that the anti-CD45 antibody, BC8, dose-dependently increased cytotoxicity induced by GO, and, to a lesser degree, free calicheamicin-₁. BC8 promoted CD33 endocytosis, suggesting that its effect on GO cytotoxicity may be, at least partly, due to increased uptake and intracellular GO availability. Finally, compared with either agent alone, BC8 combined with GO resulted in marked tumor growth inhibition and superior survival rates of mice bearing human AML xenografts. These data suggest that further study of this antibody combination for clinical use in AML is warranted.     

B72.3 Immunoreactivity in benign abdominal lymph nodes associated with gastrointestinal disease

PURPOSE: Immunolocalization of the tumor-associated glycoprotein 72 antigen with the monoclonal antibody B72.3 has been used as a cancer marker in radioimmunoscintigraphy and radioimmunoguided surgery (RIGS). Radioimmunoscintigraphy and RIGS have been used to detect occult metastatic deposits from colorectal adenocarcinoma. It has been suggested that RIGS is superior to histologic examination in detecting lymph node metastases from colorectal cancer. To determine the specificity of immunodetection of the tumor-associated glycoprotein -72 antigen as a marker for metastatic adenocarcinoma, we studied benign intraabdominal lymph nodes with B72.3 and an immunohistochemical technique. METHODS: Formaldehyde-fixed, paraffin-embedded sections of 276 benign abdominal lymph nodes, resected with 35 cases of colonic adenocarcinoma and 33 cases of benign gastrointestinal disorders, were evaluated for B72.3 immunoreactivity using an avidin-biotin complex immunohistochemical technique. Lymph nodes from cases of colonic carcinoma were also studied with cytokeratin immunostaining to help eliminate occult micrometastases. RESULTS: B72.3 immunoreactivity was seen in the germinal centers of benign lymph nodes associated with 49 percent of the cases of colonic adenocarcinoma and 12 percent of the cases of benign gastrointestinal disease. CONCLUSIONS: B72.3 immunoreactivity can be seen in benign abdominal lymph nodes associated with gastrointestinal disease. We advise caution in the use of diagnostic techniques that equate B72.3 immunoreactivity with the presence of adenocarcinoma.Read at the meeting of the United States and Canadian Academy of Pathology, Toronto, Ontario, Canada, March 11 to 17, 1995.     

A <Emphasis Type="Italic">Tbc1d1</Emphasis><Superscript>Ser231Ala</Superscript>-knockin mutation partially impairs AICAR- but not exercise-induced muscle glucose uptake in mice

Aims/hypothesis

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser²³¹, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis.

Methods

Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 ^Ser231Ala-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 ^Ser231Ala-knockin mice.

Results

Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 ^Ser231Ala-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 ^Ser231Ala-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 ^Ser231Ala-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice.

Conclusions/interpretation

TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.

     

Radioiodinated anti-hepatocellular carcinoma (HCC) ferritin Targeting therapy,tumor imaging and anti-antibody response in HCC patients with hepatic arterial infusion

Summary Radioimmunoimaging and radioimmunotherapy with radioiodinated anti-(hepatocellular carcinoma ferritin) antibody (¹³¹I-or¹²⁵I-FtAb) have been applied in patients with primary liver cancer. A total of 41 patients with surgically unresectable hepatocellular carcinoma (HCC) and receiving hepatic artery ligation and cannulation during exploratory laparotomy were treated with this regimen by intrahepatic arterial infusion. Compared with the control group, a decline of serum -fetoprotein (65.7% versus 42.9%) and shrinkage of tumor (68.3% versus 33.9%) were observed in the treated group, and a higher second-look resection rate (31.7% versus 5.1%) and longer survival (1-year: 61.0% versus 37.3%, 3-year: 25.0% versus 6.9%) resulted. The administration of antibody through a hepatic arterial catheter (n=16) was compared with intravenous injection (n-17) in terms of the tumor-imaging sensitivity in 33 patients with liver cancer. The results indicated that hepatic arterial infusion was superior to intravenous injection. The sensitivity 7 days after the administration was 100% in the i.a. group and 76.5% in the i.v. group, the uptake ratio of tumor to liver being 1.74±0.57 in the former and 1.34±0.29 in the latter. Furthermore, intrahepatic arterial infusion revealed a lower anti-antibody detection rate than intravenous injection (0/14 versus 4/11).Abbreviations AFP -tetoprotein - FtAb ferritin antibody     

Purification and characterization of α-L-fucosidase from human primary hepatocarcinoma tissue

AIM: To purify and characterize alpha-L-fucosidase from human liver cancer tissue and to detect the localization of alpha-L-fucosidase in tumor tissue. METHODS: Cation exchange chromatography on CM-52 and ultrafiltration were used to separate alpha-L-fucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-alpha-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues. RESULTS: Human alpha-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue. CONCLUSION: The purified alpha-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties from alpha-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.     

Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo

The noninvasive detection and quantification of CD8⁺ T cells in vivo are important for both the detection and staging of CD8⁺ lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-C_H3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8⁺ T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8⁺ T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for ⁶⁴Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both ⁶⁴Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.The rapid increase of therapeutic antibodies approved by the US Food and Drug Administration (FDA) and those currently in phase I–III clinical trials for oncological, autoimmune, and inflammatory diseases, among other conditions, has benefited from advances in antibody engineering, protein conjugation chemistry, and biomarker identification (1–3). Concurrently, immuno-PET imaging agents based on intact antibodies have shown promise both preclinically and clinically for the detection of cancer in vivo (4). Noninvasive detection of specific biomarkers of disease can provide crucial information for diagnosis, prognosis, response to therapy, dosage for radioimmunotherapy, and targeted therapy selection.Although much progress has been made in the immuno-PET detection of oncological markers (4), the noninvasive monitoring of immune cells in the fields of oncology, autoimmunity, and infection remains challenging. Practiced methods for lymphocyte detection include isolation of cells from the peripheral blood or, less commonly, the tissue of interest. However, the invasive tissue sampling methods are prone to error and do not provide dynamic information that reflects the number, location, and movement of lymphoid cells. Therefore, problems still exist for the evaluation of immunotherapy protocols due to the lack of effective methods to monitor the extent and duration of the therapy.Current methods to monitor immune cells noninvasively using emission tomography include direct cell labeling, reporter genes, small-molecule PET tracers, and radiolabeled intact antibodies. The ex vivo direct labeling of immune cells with PET or single-photon emission computed tomography probes before subsequent reinjection and imaging has enabled in vivo trafficking of lymphocytes (5, 6). However, this method has inherent limitations, such as radioisotope t_1/2 and cell division in vivo that lead to probe dilution. Reporter gene imaging, whereby cells are transfected with a PET reporter gene that encodes a protein specifically targeted via a radiolabeled reporter probe (7, 8), has been used to image adoptive cell transfer of transduced T-cell receptor-engineered lymphocytes (9). Reporter gene imaging allows for longitudinal tracking of cells but relies on the ex vivo transfection of cells and, for clinical translation, the development of nonimmunogenic PET reporter proteins (8).In another approach, small-molecule PET probes targeting metabolic pathways, including [¹⁸F]-fluorodeoxyglucose ([¹⁸F]FDG), [¹⁸F]-fluorothymidine ([¹⁸F]FLT), and [¹⁸F]-1-(2-doexy-2-fluoro-arabinofuranosyl)-cytosine ([¹⁸F]FAC), all have the potential to monitor diverse cell types of both innate and adaptive immunity noninvasively (reviewed in ref. 10). Clinically, [¹⁸F]FDG-PET has been used to evaluate inflammation in a range of diseases. However, in the context of immune cell detection in oncology, false-positive signals can arise from the utilization of glycolysis in both cancerous and immune cells, both innate and adaptive, in the tumor itself or in the draining lymph nodes (11, 12). [¹⁸F]FLT-PET accumulates in highly proliferative tissues, and most research has been focused on cancer detection. [¹⁸F]FLT-PET suffers from high uptake in proliferating bone marrow, therefore limiting detection of lesions in bone. However, [¹⁸F]FLT-PET was used clinically to detect cytotoxic T-lymphocyte antigen-4 blockade-induced cell replication in the spleens of patients with melanoma (13) and, more recently, to detect antigen-specific immune responses in patients with melanoma who have lymph node metastases using dendritic cell therapy (14). [¹⁸F]FAC-PET can distinguish between innate and adaptive immune cells due to the up-regulation of deoxycytidine kinase in proliferating T cells, but the uptake in a Moloney murine sarcoma virus/murine leukemia virus complex-induced sarcoma model was limited to proliferating T cells in the draining lymph nodes and spleen (15). Therefore, like the other metabolic tracers FDG and FLT, FAC uptake due to activation-induced T-cell proliferation is restricted to the draining lymph nodes and was unable to image tumor T-cell infiltration (15). This makes immuno-PET imaging using antibody fragments targeting specific immune cell antigens (i.e., CD8 expressed on cytotoxic T cells and CD4 expressed on helper T cells) potentially critical for immunotherapeutic diagnosis, because the expression of CD8 is present on all cytotoxic T cells and binding is not proliferation-dependent.Intact antibodies have relatively long serum t_1/2s (1–3 wk) compared with their engineered counterparts, such as the diabody and minibody (Mb), which have terminal t_1/2s that range from 2 to 5 h and from 5 to 12 h, respectively (16). Although decreasing the total uptake in tumors, the rapid clearance of engineered antibody fragments allows for higher tumor-to-background images at earlier times postinjection (p.i.). This not only allows for the potential of same-day imaging but reduces the overall radiation dose. Furthermore, engineered diabody and Mb fragments are biologically inert because they lack Fc effector functions. Intact antibodies have been used previously to image T cells in patients with a range of disorders (reviewed in ref. 17). The majority of these studies use planar gamma imaging using therapeutic intact mouse anti-human antibodies. The expanding knowledge of the importance of immune cell subtypes in diseases and the improvements in antibody-based immuno-PET imaging indicate that antibody-based imaging of immune cells in vivo should be revisited.In this report, we develop two anti-murine CD8 Mbs (Fig. 1A) for the detection of CD8 expression. Murine CD8 is a cell surface glycoprotein expressed mainly on a subset of T cells known as cytotoxic T cells and a subset of dendritic cells. Functional CD8 is expressed as either the homodimer CD8αα or the heterodimer CD8αβ of the two isoforms of CD8, α and β. Mice have two alleles for CD8α, Lyt2.1 and Lyt2.2, which are restricted to certain mouse strains. Lyt2.1, for example, is expressed in the mouse strains CBA, AKR, C3H, and DBA, whereas Lyt2.2 is expressed in the mouse strains BALB/c and C57BL/6 (B/6). The difference between Lyt2.1 and Lyt2.2 is a methionine (Lyt2.2)-to-valine (Lyt2.1) substitution at residue 78 of the mature CD8α (Fig. 1B).Open in a separate windowFig. 1.Mb construction and epitope specificity are shown. (A) Anti-CD8 2.43 and YTS169 Mbs contain the rat V_H-V_L separated by an 18-aa linker, followed by the murine IgG2a hinge (h), murine C_H3, and a C-terminal hexahistidine (HisTag). (B) Murine CD8α (Lyt2) is expressed as two isoforms, Lyt2.1 and Lyt2.2, that differ in a single amino acid, and it is restricted to specific mouse strains. The 2.43 Mb binds CD8α only in Lyt2.2⁺ mouse strains, whereas the YTS169 Mb binds CD8α in all mouse strains.In this study, the parental antibodies from the hybridomas YTS 169.4.2.1 (YTS169) and 2.43 were engineered into Mb fragments (Fig. 1A). Both the YTS169 and 2.43 antibodies bind mCD8α (Lyt2). However, they differ in that the YTS169 antibody binds both Lyt2.1 and Lyt2.2, whereas the 2.43 antibody binds an epitope that is Lyt2.2-specific (Fig. 1B). These newly engineered Mbs retained their antigen specificity, as shown by flow cytometry and ⁶⁴Cu immuno-PET imaging. Most importantly, both the 2.43 and YTS169 Mbs produce high-contrast immuno-PET images of CD8⁺ lymphoid organs at only 4 h p.i. This report details successful antibody fragment-based immuno-PET detection of CD8 expression in vivo.