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Targeted α-therapy (TAT) delivers high-linear-transfer-energy α-particles to tumors with the potential to generate tumor immune responses that may be augmented by antigen-targeted immunotherapy. Methods: This concept was evaluated in immunocompetent carcinoembryonic antigen (CEA) transgenic mice bearing CEA-positive mammary or colon tumors. Tumors were targeted with humanized anti-CEA antibody M5A labeled with (225)Ac for its 10-d half-life and emission of 4 α-particles, as well as being targeted with the immunocytokine M5A–interleukin 2. Results: A dose response (3.7, 7.4, and 11.1 kBq) to TAT only, for orthotopic CEA-positive mammary tumors, was observed, with a tumor growth delay of 30 d and an increase in median survival from 20 to 36 d at the highest dose. Immunocytokine (4 times daily) monotherapy gave a tumor growth delay of 20 d that was not improved by addition of 7.4 kBq of TAT 5 d after the start of immunocytokine. However, TAT (7.4 kBq) followed by immunocytokine 10 d later led to a tumor growth delay of 38 d, with an increase in median survival to 45 d. Similar results were seen for TAT followed by immunocytokine at 5 versus 10 d. When a similar study was performed with subcutaneously implanted CEA-positive MC38 colon tumors, TAT (7.4 kBq) monotherapy gave an increase in median survival from 29 to 42 d. The addition of immunocytokine 10 d after 7.4 kBq of TAT increased median survival to 57 d. Immunophenotyping showed increased tumor-infiltrating interferon-γ–positive, CD8-positive T cells and an increased ratio of these cells to Foxp3-positive, CD4-positive regulatory T cells with sequential therapy. Immunohistochemistry confirmed there was an increase in tumor-infiltrating CD8-positive T cells in the sequential therapy group, strongly suggesting that immunocytokine augmented TAT can lead to an immune response that improves tumor therapy. Conclusion: Low-dose (7.4 kBq) TAT followed by a 4-dose immunocytokine regimen 5 or 10 d later gave superior tumor reductions and survival curves compared with either monotherapy in breast and colon cancer tumor models. Reversing the order of therapy to immunocytokine followed by TAT 5 d later was equivalent to either monotherapy in the breast cancer model.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled

Background Tumor-associated glycoprotein (TAG)–72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72–specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. Methods Anti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49–IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. Results Subcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. Conclusions Anti-huCC49–IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Introduction Ovarian cancer has only a 17% 5‐year survival rate in patients diagnosed with late stage disease. Tumor‐associated glycoprotein‐72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody‐targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium. Methods Using the clinically relevant anti‐TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS‐MMAE), bromoacetamido (Br‐MMAE), and maleimido (mal‐MMAE). Results The drug antibody ratios (DARs) of the three ADCs were 2.3 for VS‐MMAE, 10 for Br‐MMAE, and 9.5 for mal‐MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49‐mal‐MMAE (3.3%ID/g) was low compared to CC49‐Br‐MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49‐VS‐MMAE and CC49‐Br‐MMAE, and 7% for CC49‐mal‐MMAE. CC49‐VS‐MMAE was not further studied due to its low DAR, while CC49‐mal‐MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49‐Br‐MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49‐Br‐MMAE. Conclusion We conclude that CC49‐Br‐MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model., Antibody drug conjugates with three different linker chemistries were evaluated for targeting TAG72 positive xenografts in an ovarian cancer model. An ADC with ten bromoacetamido linked monomethylauristatin drugs per antibody performed best in terms of tumor targeting and therapy.

Background/aim Fluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models. Materials and methods Humanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated. Results Intra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76). Conclusion Anti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

Pretargeted positron emission tomography is a macromolecule driven nuclear medicine technique that involves targeting a pre-administered antigen target-bound macromolecule with a radioligand in vivo, aiming to minimize the overall radiation dose. This study investigates the use of antibody based host-guest chemistry methodology for pretargeted positron emission tomography. We hypothesize that the novel pretargeting approach reported here overcomes the challenges the current pretargeting platforms have with the in vivo stability and modularity of the pretargeting components. A cucurbit[7]uril host molecule modified, anti-carcinoembryonic antigen antibody (M5A; CB7-M5A) and a (68)Ga-radiolabeled ferrocene guest radioligand ([(68)Ga]Ga-NOTA-PEG(3)-NMe(2)-Fc) were studied as potential host-guest chemistry pretargeting agents for positron emission tomography in BxPC3 xenografted nude mice. The viability of the platform was studied via in vivo biodistribution and positron emission tomography. Tumor uptake of [(68)Ga]Ga-NOTA-PEG(3)-NMe(2)-Fc was significantly higher in mice which received CB7-M5A prior to the radioligand injection (pretargeted), (3.3 ± 0.7 %ID/g) compared to mice which only received the radioligand (non-pretargeted), (0.2 ± 0.1 %ID/g).

Radioimmunotherapy, an approach using radiolabeled antibodies, has had minimal success in the clinic with several β-emitting radionuclides for the treatment of ovarian cancer. Alternatively, radioimmunotherapy with α-emitters offers the advantage of depositing much higher energy over shorter distances but was thought to be inappropriate for the treatment of solid tumors, for which antibody penetration is limited to a few cell diameters around the vascular system. However, the deposition of high-energy α-emitters to tumor markers adjacent to a typical leaky tumor vascular system may have large antitumor effects at the tumor vascular level, and their reduced penetration in normal tissue would be expected to lower off-target toxicity. Methods: To evaluate this concept, DOTAylated-huCC49 was labeled with the α-emitter (225)Ac to target tumor-associated glycoprotein 72–positive xenografts in a murine model of ovarian cancer. Results: (225)Ac-labeled DOTAylated-huCC49 radioimmunotherapy significantly reduced tumor growth in a dose-dependent manner (1.85, 3.7, and 7.4 kBq), with the 7.4-kBq dose extending survival by more than 3-fold compared with the untreated control. Additionally, a multitreatment regime (1.85 kBq followed by 5 weekly doses of 0.70 kBq for a total of 5.4 kBq) extended survival almost 3-fold compared with the untreated control group, without significant off-target toxicity. Conclusion: These results establish the potential for antibody-targeted α-radionuclide therapy for ovarian cancer, which may be generalized to α-radioimmunotherapy in other solid tumors.

BackgroundPhotoimmunotherapy (PIT) employs the use of a near-infrared (NIR) laser to activate an antibody conjugated to a NIR-activatable dye to induce cancer cell death. PIT has shown to be effective in a number of studies, however, there are no data on its use in colorectal cancer in an orthotopic model.MethodsHumanized anti-CEA antibody (M5A) was conjugated to NIR-activatable IRDye700DX (M5A-700). PIT was validated in vitro with a colon cancer cell-line, using a laser intensity of either 4 J/cm2, 8 J/cm2, or 16 J/cm2. Orthotopic colon cancer mouse models were established by surgical implantation of LS174T tumor fragments onto the cecum. M5A-700 was administered and PIT was performed 24 hours later using a 690 nm laser. Repeat PIT was performed after 7 days in one group. Control mice received laser treatment only.ResultsIn vitro PIT demonstrated tumor cell death in a laser intensity dose-dependent fashion. In orthotopic models, control mice demonstrated persistent tumor growth. Mice that underwent PIT one time had tumor growth arrested for one week, after which re-growth occurred. The group that received repeated PIT exposure had persistent inhibition of tumor growth.ConclusionPIT arrests tumor growth in colon cancer orthotopic nude-mouse models. Repeated PIT arrests colon cancer growth for a longer period of time. PIT may be a useful therapy in the future as an adjunct to surgical resection or as primary therapy to suppress tumor progression.

While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

Development of a humanized fluorophore-conjugated antibody that can improve contrast for fluorescence-guided oncologic surgeries. BxPC-3-GFP pancreatic cancer cells were injected into flanks of nude mice. Fragments of subcutaneous tumors were grafted onto the pancreatic tail of recipient mice to create orthotopic xenograft models of pancreatic cancer. After tumors developed for 4 weeks, a humanized anti-carcinoembryonic antigen antibody conjugated to an 800 nm near-infrared fluorescent dye (hM5A-IR800) was injected intravenously. Mice were imaged at 6, 12, 24, 48, and 72 h after injection. Fluorescence imaging showed that hM5A-IR800 specifically localized to BxPC-3 human pancreatic cancer cells. The fluorescent probe localized to cell surfaces in vitro and specifically co-localized with green fluorescent protein-labeled tumors in an orthotopic pancreatic xenograft model in vivo. Serial imaging at specific time points showed peak signal intensity of the orthotopic pancreatic tumor at 48 h; this time point corresponded with a maximal tumor-to-background ratio (TBR) of 16.6 at 48 h. hM5A-IR800 was successfully able to specifically label orthotopic pancreatic tumors in situ. The longer wavelength allowed deeper tissue penetration, particularly in tumor areas covered by normal pancreatic parenchyma. The probe had expected kinetics for an antibody-fluorophore conjugate, with the peak signal intensity reached at 48 h. A clear tumor signal was observed with a TBR > 5 at all time points, with high contrast (TBR of 16.6) at 48 h. hM5A-IR800 demonstrated excellent tumor localization and a very bright signal. It is a promising agent for future clinical fluorescence-guided surgery applications.

Introduction Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in knowledge and implications for patient care Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to an LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery.

Purpose: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia, which results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific noninvasive imaging method to detect disease, including in extramedullary organs, representing an unmet clinical need. About 85% to 90% of human myeloid leukemia cells express CD33 cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET. Experimental Design: We evaluated whether [64Cu]Cu-DOTA-anti-CD33 murine mAb can be used for immunoPET imaging of AML in a preclinical model. MicroCT was adjusted to detect spatial/anatomical details of PET activity. For translational purposes, a humanized anti-CD33 antibody was produced; we confirmed its ability to detect disease and its distribution. We reconfirmed/validated CD33 antibody-specific targeting with an antibody–drug conjugate (ADC) and radioimmunotherapy (RIT). Results: [64Cu]Cu-DOTA-anti-CD33–based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). The CD33+ PET activity was significantly higher in specific skeletal niches [femur (P < 0.00001), tibia (P = 0.0001), humerus (P = 0.0014), and lumber spine (P < 0.00001)] in AML-bearing mice (over nonleukemic control mice). Interestingly, the hybrid PET-CT imaging showed high disease activity in the epiphysis/metaphysis of the femur, indicating regional spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (P = 0.02) and [225Ac]Ac-anti-CD33-RIT (P < 0.00001) significantly reduced disease burden over that of respective controls. Conclusions: We have successfully developed a novel anti-CD33 immunoPET-CT–based noninvasive modality for AML and its spatial distribution, indicating a preferential skeletal niche.

Background Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Methods An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. Results The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. Conclusion dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells. Electronic supplementary material The online version of this article (10.1186/s12885-019-6056-8) contains supplementary material, which is available to authorized users.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (

BackgroundThe success of a curative surgery for cancer is dependent on the complete removal of all cancer cells. Tumor visualization by the surgeon can be enhanced through fluorescent-antibody targeting. To further develop such technology, we selected humanized anti-carcinoembryonic antigen (CEA) conjugated to a near-infrared dye to target orthotopically-implanted human colon cancer in nude mice.Materials and methodsThe HT-29 human colon cancer cell line was grown in culture and subcutaneously injected in mice. After 3wk of growth, tumors were resected and cut into 2mm3 fragments that were sutured to the cecum of five additional nude mice for orthotopic implantation. The tumors were allowed to grow for 4wk at which point 3 had successful orthotopic tumor growth and were selected for injection of the humanized anti-CEA antibody conjugated to the near-infrared dye IRDye800CW (anti-CEA-IRDye800CW). The antibody-dye conjugate (75μg) was administered via tail vein injection. Images were obtained with the Pearl Trilogy Small Animal Imaging System with both 700 and 800nm channels and evaluated using Image Studio.ResultsLaparotomy was performed 24h after labeling the tumors. When imaged through the 800nm channel, the tumors were observed to be strongly labeled with anti-CEA-IRDye800. At 48h, laparotomy was repeated which again demonstrated strong labeling of the tumors through the 800nm channel, but with a lower absolute intensity (in relative units), than at 24h.ConclusionsHumanized anti-CEA-IRDye800CW can rapidly and effectively label CEA-expressing human colon cancer in an orthotopic nude mouse model. Given the ability of this technology to target and label tumors with great specificity, the anti-CEA-IRDye800CW is currently being developed for clinical use in fluorescence-guided surgery.

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer.A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient-based partial-volume correction, and the results were compared with ex vivo biodistribution.Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing (124)I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing (89)Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P0.0001 for each), but the (124)I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor-to-soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 (124)I-A11 and 4.87 ± 0.52 (89)Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides.Both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the (124)I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor-to-soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both (124)I- and (89)Zr-labeled A11 minibody.

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.

Diabodies are noncovalent dimers of single-chain antibody fragments that retain the avidity of intact IgG but have more favorable blood clearance than intact IgG. Radiometals offer a wide range of half-lives and emissions for matching imaging and therapy requirements and provide facile labeling of chelate-antibody conjugates. However, because of their high retention and metabolism in the kidney, the use of radiometal-labeled diabodies can be problematic for both imaging and therapy. Methods: Having previously shown that 111In-DOTA-polyethylene glycol (PEG)3400-anti–carcinoembryonic antigen diabody has less than half the kidney uptake and retention of non-PEGylated diabody and that the two have similarly high tumor uptake and retention, we synthesized a similar derivative for an anti–tumor-associated glycoprotein 72 diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1,321 and 617, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with 125I, 111In, or 64Cu in tumor-bearing athymic mice. Results: The addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 percentage injected dose/g) comparable to that obtained with radiometal-labeled intact IgG. The PEG27 and PEG12 diabody conjugates also demonstrated low kidney uptake without reduction of tumor uptake or tumor-to-blood ratios. When radiolabeled with 64Cu, the DOTA-PEG12 and -PEG27 diabody conjugates gave high-contrast PET images of colon cancer xenografts in athymic mice. Conclusion: PEGylated diabodies may be a valuable platform for delivery of radionuclides and other agents to tumors.

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-CH3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 ± 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 ± 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 ± 4.0% ID/g). A minibody derived from a second anti-p185HER2 antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 ± 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-CH2-CH3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 ± 2.4% ID/g) and reduced kidney uptake (13.1 ± 1.5% ID/g). Thus, by manipulating the size and format of anti-p185HER2 antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.

Purpose: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (VL-linker-VH-CH3; 80 kDa) that demonstrates bivalent and high affinity (4 × 1010 m−1) binding to carcinoembryonic antigen (CEA). The variable regions (VL and VH) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the CH3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab′)2 fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether 123I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer.Experimental Design: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5–10 mCi (1 mg) of 123I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48–72 h. Surgery was performed immediately after the last nuclear scan.Results: Tumor imaging was observed with 123I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. 123I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9–65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed.Conclusion: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as 111In.

Single-step iodination is often performed in preference to use of a radiometal label for initial animal biodistributions. Yet loss of iodine occurs in vivo so that it is important to measure uptake (%ID/g) differences between radiolabels.Murine biodistributions of four radioiodinated and (111)In-labeled cognate anti-carcinoembryonic antigen antibodies were compared. Uptakes were obtained in athymic mice out to 96 hours for diabody, minibody, scFv-Fc, and intact humanized (M5A) versions of the T84.66 antibody. Tissues included liver, spleen, kidneys, lungs, and human LS174T colorectal xenografts. Ratios (R) of iodine uptake to indium uptake were calculated.For all cognates, no significant differences were found in the blood uptakes between the two labels. In normal solid organs, iodine was generally cleared more rapidly with decreasing molecular weight (MW). In liver and spleen by 24 hours, the R value was 5% for diabody and minibody, whereas a value of 50% was seen for the intact mAb. Renal differences were even more marked for the two lower MW species. Tumor losses, however, were found to be essentially independent of MW and were modest; 50% by 48 hours. To test the generality of the results, comparisons were then made for normal organs and tumors of the R values found above for M5A and MN-14 intact antibody literature results. Good agreement, within estimated errors in R, was seen between these two cases, although (111)In and (88)Y were the respective radiometals.Loss of iodine from labeled antibodies can be marked in normal organs and that correction (by 1/R) of the iodine biodistribution to estimate the associated radiometal result may be possible. Explicit differences between the two types of biodistribution also imply that separate uptake results need to be considered when evaluating the impact on imaging and therapy using various possible radiolabels.

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers1–5. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition6–10. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors. Mol Cancer Ther; 10(6); 1036–45. ©2011 AACR.

Retention mapping of chimeric monoclonal IgG(1), Fc, Fab, F(ab')(2), and aggregated antibody was conducted on hydroxyapatite (HA) by systematically varying phosphate and chloride concentrations during gradient elution in order to characterize the interactions of each solute with calcium and phosphate residues on the solid phase. Lysozyme was used as a control to model cation exchange-dominant interactions. Bovine serum albumin was used as a control for calcium affinity-dominant interactions. Calcium affinity and phosphoryl cation exchange were positively cooperative for IgG-related species. Fc retention was dominated by calcium affinity, while retention of Fab was dominated by cation exchange. F(ab')(2) exhibited a curve shape similar to Fab, but stronger retention. The retention curve for intact IgG incorporated the distinctive elements of its fragments but stronger retention than that predicted by their addition to one another. Aggregate retention paralleled the curve for non-aggregated antibody, with stronger retention by both binding mechanisms. Experimental data revealed evidence of charge repulsion between IgG carboxyls and HA phosphate at low conductivity values. Electrostatic repulsion of amino residues and attraction of carboxyls by HA calcium appeared to be blocked by strong complexation of calcium with mobile phase phosphate.

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.

Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents.

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti–carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2β, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2β, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy. [Cancer Res 2007;67(2):718–26]

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti–carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using 125I- and 131I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was ∼12 days. Additionally, 124I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.

Chimeric T84.66 (cT84.66) is a monoclonal antibody (mAb) of high specificity and affinity for the tumor-associated carcinoembryonic antigen (CEA). Radiolabeled cT84.66 has demonstrated utility in the clinic as a reagent for the radioimmunoscintigraphy and radioimmunotherapy of CEA-positive colorectal and breast malignancies. To extend the therapeutic efficacy of T84.66, humanization by complementary determining region (CDR) grafting was employed. CDR grafting is a well-established technique, though often a series of framework back-mutations is required to restore high affinity. Recently, the crystal structure of the T84.66 diabody (scFv dimer) derived from the murine T84.66 mAb was determined, facilitating the humanization process by the availability of crystal structure data for both the graft donor and graft acceptor. A search of the Protein Data Bank revealed close structural similarity (r.m.s.d. of 1.07 A) between the Fv of T84.66 and the Fv of 4D5v8, a humanized anti-p185HER2 antibody marketed as Herceptin (Trastuzumab). This resulted in two humanized versions of the T84.66 M5A and M5B mAbs that differed only in the number of murine residues present in the C-terminal half of CDR-H2. Biochemical analysis and animal biodistribution studies were conducted to evaluate the humanized mAbs. The M5A, M5B and cT84.66 mAbs showed sub-nanomolar affinity for CEA and as radiolabeled mAbs exhibited specific tumor localization in tumor bearing mice. The T84.66 M5A mAb was selected for clinical development due to a slightly higher tumor uptake and a larger content of human residues, and was renamed hT84.66. A limited-scale production and animal imaging study have demonstrated hT84.66's ability to support clinical trials. Planned clinical trials will determine the effective utilization of this structure-based approach in the development of a promising new therapeutic.

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.

An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.

Three analytic indicators were used to compare five members of a monoclonal antibody (Mab) family. The cognates consisted of the genetically engineered intact chimeric IgGI (cT84.66) and related engineered fragments [scFv, diabody, minibody, F(ab')2] reactive against the same epitope of carcinoembryonic antigen (CEA). All analyses were based on radioiodinated Mabs targeting to colorectal xenografts of LS174T tumors in nude mice. Affinity constants were evaluated initially. A second indicator was the imaging figure of merit (IFOM) which determines how rapidly a statistically significant tumor image can be acquired. Finally, deconvolution was used to determine tumor temporal response to an arterial bolus. This last analysis gave the possible tumor accumulation in the absence of normal tissue sequestration. Affinities were all in excess of 10(8) M-1 and were highest for the divalent Mabs. Using the IFOM criterion, an 131I label was best suited as a radiolabel for the intact (IgG) T84.66, while an 123I label indicated optimal imaging with either minibody or F(ab')2. Deconvolution analyses showed that divalent members behaved similarly while the univalent member (scFv) had a tumor residence time smaller by an order of magnitude. The diabody had the largest impulse response function, but renal uptake may limit its present usefulness.

Targeting of monoclonal antibody (Mab) to solid tumor sites is a function of the blood curve of activity versus time. It has been suggested that the blood curve be artificially reduced to approach zero so that the contrast between tumor and blood uptake is maximized. We analyzed tumor uptake as a function of the time t c of blood curve truncation. By using a convolution approach, we were able to find the optimal times for setting the blood curve to zero in either diagnostic or therapeutic animal examples. Two iodinated cT84.66 anti-CEA engineered fragments, diabody and minibody, were considered using previous data from nude mouse studies involving the LS174T colorectal tumor model. Figures of merit (FOMs) were used to compare ordinary and truncated blood curves and their associated tumor accumulations. Using a 123 I label, it was seen that the appropriate time for diagnostic truncation occurred when tumor uptake, as measured, was a maximum. The corresponding point for therapy (with 131 I as a label) was at infinite time. We also demonstrated that the use of traditional indices led to ambiguities in the choice of truncation times. The traditional therapy index, the ratio of the integral of the tumor uptake to the integral of the blood uptake, was found to be a numerical constant independent of t c . This ratio was proved to be the integral of the tumor impulse response function. Use of such convolution techniques to assess truncation of the perfused material is probably also applicable to multistep processes as well as to lesion targeting with other tumor-specific pharmaceuticals.

Background Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors. Methods and findings As proof-of-concept, we selected Herceptin (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice. Conclusions Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.

Calsequestrin was isolated from chicken fast-twitch skeletal muscle, and partial amino terminal sequence was determined. The sequence (NH2) EEGLNFPTYDGKDRVIDLNE shows high identity with known mammalian calsequestrins contained in the Protein Identification Resource data bank (1). Most importantly, this 20 amino acid sequence shares complete identity with the amino terminus of aspartactin, a putative laminin-binding protein of the extracellular matrix (2, 3). The possible relationship of aspartactin to calsequestrin is discussed.

Inhibition of rat liver lysosomal phospholipases is one of the main events that leads to accumulation of tissue phospholipids during drug-induced phospholipidosis. Drug inhibition of lysosomal phospholipase A may occur by direct effects of drugs on the enzyme (or substrate) or by drug-induced increases in intralysosomal pH. Although beta-adrenoceptor blockers have not been reported to cause lipid storage, they do inhibit lysosomal phospholipase A. To investigate the structural requirements for drug inhibition, we studied the effects of six beta-adrenoceptor blockers on purified rat liver lysosomal phospholipase A1. The agents studied include: propranolol, timolol, metoprolol, practolol, atenolol and the combined alpha and beta adrenoceptor blocking agent, labetalol. The drugs varied by two logs in their abilities to inhibit phospholipase A1 activity. The relative inhibitory potencies were propranolol greater than labetalol much greater than timolol greater than metoprolol much greater than practolol greater than atenolol. Our studies identify drug hydrophobicity as a key determinant for phospholipase A1 inhibition. A strong negative correlation was noted between the octanol/water partition coefficients and IC50 for phospholipase inhibition (r = -0.91). The ability of propranolol to inhibit phospholipase A1 was identical for the d, l and the d and l stereoisomers.

Amiodarone is effective in the treatment of ventricular and supraventricular arrhythmias. In man its clinical use is associated with the accumulation of phospholipid-rich multilamellar inclusions in various tissues including lung, liver and others. This report presents evidence showing that amiodarone is a potent inhibitor of lysosomal phospholipase A from rat alveolar macrophages, J-744 macrophages and rat liver. When compared with other cationic amphiphilic agents which are known to produce phospholipidosis, amiodarone is one of the most potent inhibitors yet discovered. The subcellular localization of amiodarone has been determined in lung and its distribution was consistent with a lysosomal localization. It is hypothesized that amiodarone causes cellular phospholipidosis by concentrating in lysosomes and inhibiting phospholipid catabolism.

The uptake and intracellular distribution of amantadine were examined to better define the site and mechanism of action of this drug. Amantadine is rapidly concentrated in lysosomes and in the cytosol (postmicrosomal supernatant) of Madin Darby canine kidney (MDCK) cells. A considerable portion of the cell-associated amantadine resists elution and remains in the lysosomes and the cytosol when the cells are subsequently washed and incubated in amantadine-free medium. In contrast, the antiviral activity of amantadine is rapidly lost when the drug is removed from the extracellular medium. These results indicate that most of the cell-bound amantadine does not contribute to the antiviral action of the drug. That portion of the cell-associated amantadine which does account for its antiviral activity is rapidly exchanged with the extracellular medium, and in fact, may actually be the drug in the extracellular medium.