Paul J. Yazaki

Radioiodinated versus Radiometal-Labeled Anti–Carcinoembryonic Antigen Single-Chain Fv-Fc Antibody Fragments: Optimal Pharmacokinetics for Therapy

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 (T1/2beta, 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 (T1/2beta, 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.

Pilot Trial Evaluating an 123I-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (cT84.66 Minibody) in Patients with Colorectal Cancer

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.

Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications.

Genetically engineered radiolabeled antibody fragments have shown great promise for the radioimmunoscintigraphy of cancer. Retaining the exquisite specificity of monoclonal antibodies yet smaller in molecular size, antibody fragments display rapid tumor targeting and blood clearance, a more uniform distribution in the tumor, and present a lower potential to elicit an immune response. However, one of the factors that has limited clinical evaluation of these antibody-derived proteins has been the difficulty in expressing and purifying the quantities necessary for clinical trials. This study outlines the capability of mammalian expression for the production of recombinant antibody fragments intended for clinical use. Two anti-carcinoembryonic antigen antibody fragments, the T84.66/212 Flex minibody (scFv-C(H)3) and the T84.66 diabody (scFv dimer) have been previously expressed and have shown excellent radioimaging properties in tumor bearing animals. To proceed toward human studies, these high affinity recombinant fragments and a second minibody version, the T84.66/GS18 Flex minibody, were expressed using a high-level mammalian expression system. Production of all three antibody fragments in a small-scale hollow fiber bioreactor resulted in 137-307 mg of crude antibody harvest. A purification protocol that employed ceramic hydroxyapatite and anion exchange chromatography resulted in 50-150 mg of purified T84.66 diabody and T84.66 minibody. The development of this level of research grade material established conditions for clinical production as well as provided material to complete pre-clinical studies and undertake protein crystallization studies. Scale-up for clinical studies produced 3.4 g of the T84.66 minibody in the harvest. A portion of this material was purified yielding 180 mg of highly purified T84.66 minibody intended for pilot radioimmunoscintigraphy studies of carcinoembryonic antigen (CEA) positive disease.

The Crystal Structure of an Anti-CEA scFv Diabody Assembled from T84.66 scFvs in VL-to-VH Orientation: Implications for Diabody Flexibility

Diabodies (scFv dimers) are small, bivalent antibody mimetics of approximately 55kDa in size that possess rapid in vivo targeting pharmacokinetics compared to the intact parent antibody, and may prove highly suitable for imaging and therapeutic applications. Here, we describe T84.66Di, the first diabody crystal structure in which the scFvs comprise V domains linked in the V(L)-to-V(H) orientation. The structure was determined by X-ray diffraction analysis to 2.6 A resolution. The T84.66Di scFv was constructed from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66 variable domains connected by an eight residue peptide linker to provide flexibility between Fv modules and promote dimer formation with bivalent affinity to the cell-surface target, CEA. Therefore, it was surprising to observe a close association of some Fv module complementarity-determining regions in the T84.66 diabody crystal, especially compared to other diabody structures all of which are linked in the opposite V(H)-to-V(L) orientation. The differences between the arrangement of Fv modules in the T84.66Di V(L)-to-V(H) linked diabody structure compared to the crystal structure of L5MK16 and other proposed V(H)-to-V(L) linked diabodies has been investigated and their potential for flexibility discussed. The comparison between V(H)-to-V(L) and V(L)-to-V(H) linked diabodies revealed in this study represents a limited repertoire of possible diabody Fv orientations, but one that reveals the potential flexibility of these molecules. This analysis therefore provides some signposts that may impact on future molecular designs for these therapeutic molecules with respect to diabody flexibility and avidity.

Neural stem cells as a novel platform for tumor-specific delivery of therapeutic antibodies.

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.

Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody

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 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods: A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-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. Results: Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I- and 89Zr-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 124I-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 89Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the 124I-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 124I-A11 and 4.87 ± 0.52 89Zr-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. Conclusion: Both 124I- and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-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 124I- and 89Zr-labeled A11 minibody.

CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

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.

The bispecific immunoligand ULBP2-aCEA redirects natural killer cells to tumor cells and reveals potent anti-tumor activity against colon carcinoma

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody‐derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re‐directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell‐mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti‐tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti‐tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.

Site-specific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing peg size on kidney clearance and tumor uptake with improved 64-copper PET imaging.

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.

A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance

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.