Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

Improved immunoscintigraphy by subcutaneous injection of 99mTc or 111In labelled F(ab′)2 fragments of an anti-melanoma monoclonal antibody

Technetium-99m and/or ¹¹¹In labelled F(ab′)₂ fragments of a melanoma associated MoAb 225.28S were injected i.v. in 80 patients affected by stage I to IV malignant melanoma. Seventy five percent of metastatic lesions already documented by other methods were detected by immunoscintigraphy, which was also capable of detecting a certain number of unknown metastases. However, we observed a lower percentage of positive scans in liver, lung and skin because of the poor tumour to background ratio. In some patients, subcutaneous (s.c.) injection allowed us to visualize documented metastases undetected by i.v. administration. An equal amount of non-specific F(ab′)₂ fragments (MoAb 4C4) injected s.c. as a negative control showed no positive scans. Clinical studies and Chromatographic patterns of patient serum samples suggest that the s.c. route of administration offers, with respect to the i.v. route, the advantage of reducing vascular background and aspecific accumulation in liver, probably because of retention of possible contaminants by the lymphatic system.     

Breakage of the T cell receptor α chain locus in non malignant clones from patients with ataxia telangiectasia

Summary Patients with ataxia telangiectasia (A-T) develop specific chromosome translocations, which may confer a proliferative advantage, resulting in the appearance of large clones in the peripheral blood lymphocytes. These lymphocytes are not malignant. Using in situ hybridisation techniques we have investigated a consistent 14q11 translocation break-point observed in a t(X;14)(q28;q11) translocation clone from each of two different patients and a t(14;14)(q11;q32) clone from a third patient. In all cases the chromosome translocation involved breakage within the chain locus of the T cell receptor (TCR), between the variable and constant regions, at 14q11. Chromosome rearrangement involving breakage within TCR can therefore precede the development of malignancy. Further chromosomal rearrangement may be required in these patients, for progression to the leukaemic state.     

Rapid and potent induction of cell death and loss of NK cell cytotoxicity against oral tumors by F(ab′)2 fragment of anti-CD16 antibody

Freshly isolated untreated NK cells undergo rapid apoptosis and lose their cytotoxic function upon the addition of F(ab′)₂ fragment of anti-CD16 antibodies. Loss of NK cell cytotoxic function after treatment with F(ab′)₂ fragment of anti-CD16 antibody can be seen against K562 and UCLA-2 oral tumor cells when either added immediately in the co-cultures of NK cells with the tumor cells or after pre-treatment of NK cells with the antibody before their addition to the tumor cells. Addition of Interleukin-2 (IL-2) in combination with anti-CD16 antibody to NK cells delayed the induction of DNA fragmentation in NK cells, and even though decreased cytotoxicity could still be observed against K562 and UCLA-2 oral tumors when compared to IL-2 alone treated NK cells, the cytotoxicity levels remained relatively higher and approached those obtained by untreated NK cells in the absence of antibody treatment. No increases in IFN-γ, Granzymes A and B, Perforin and TRAIL genes could be seen in NK cells treated with anti-CD16 antibody. Neither secretion of IFN-γ nor increased expression of CD69 activation antigen could be observed after the treatment of NK cells with anti-CD16 antibody. Furthermore, IL-2 mediated increase in CD69 surface antigens was down-modulated by anti-CD16 antibody. Finally, the addition of anti-CD16 antibody to co-cultures of NK cells with tumor target cells was not inhibitory for the secretion of VEGF by oral tumor cells, unlike those co-cultured with untreated or IL-2 treated NK cells. Thus, binding and triggering of CD16 receptor on NK cells may enhance oral tumor survival and growth by decreased ability of NK cells to suppress VEGF secretion or induce tumor cell death during the interaction of NK cells with oral tumor cells. This work was supported by RO1-DE18830 from NIH.     

High efficiency reduction capability for the formation of Fab׳ antibody fragments from F(ab)2 units

Antibodies have widespread applications in areas ranging from therapeutics to chromatography and protein microarrays. Certain applications require only the fragment antigen-binding (Fab) units of the protein. This study compares the cleavage efficacy of dithiothreitol (DTT), mercaptoethylamine (MEA), and dithiobutylamine (DTBA) – a relatively new reducing agent synthesized in 2012. Pseudo-first order kinetic analyses show DTBA to be ~213 times faster than DTT and ~71 times faster than MEA in the formation of Fab? antibody fragments from polyclonal rabbit antibodies. Monoclonal mouse antibodies were also used to show the feasibility of the reduction process on antibodies from a different species and with a different clonality. DTBA cleaved the monoclonal mouse F(ab)₂ units most efficiently, ~2 times faster than DTT ~10 times faster than MEA. Due to the extremely quick reactivity of all the reducing agents in the first five minutes of monoclonal antibody reductions as well as for the DTBA reductions of the polyclonal rabbit antibodies, the pseudo-first order kinetic analyses should be interpreted qualitatively for these results. Nucleophilic sulfides on Fab? fragments are preserved in the DTBA reduction process, demonstrated by their reactivity with Ellman?s reagent. Degradation of the Fab? fragments was observed with the monoclonal mouse antibodies after reduction with DTBA or DTT. In conclusion, DTBA is the more efficient reducing agent compared to DTT and MEA, however, the reduction process should be optimized as degradation of the Fab? fragments is possible.     

Radioimmunolocalization of pelvic recurrences from rectosigmoid cancer employing 111In anti-CEA F(ab′)2

Thirty-two immunoscintigraphic studies have been performed in 26 patients previously operated upon for rectosigmoid cancer, for an early detection of pelvic recurrence, employing ¹¹¹In anti-CEA, F(ab′)₂. The sensitivity was 1, specificity 0.76 and accuracy 0.84, leading to a positive predictive value of 0.69 and a negative value of 1. The combined use of a non-imaging surgical probe in 6 of the 10 patients who underwent second look surgery, detecting the ¹¹¹In radioactivity used for RIS, improved diagnostic accuracy, ruling out the presence of tumors in 3 patients with false positive RIS.     

Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state

CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF?) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF?-coated 96-well plates and cultured under na?ve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF?-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF?-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.     

Identification of prognostic factors for γδT cell immunotherapy in patients with solid tumor

Background aimsActivated γδT cells have been shown to exhibit cytotoxicity against tumor cells. However, the efficacy of γδT cell immunotherapy for a large number of patients with solid tumors remains unclear. In this study, we examined the efficacy of γδT cell immunotherapy using in vitro-activated γδT lymphocytes in combination with standard therapies in terms of the survival of patients with solid tumors, and determined prognostic factor for γδT cell immunotherapy.Methods131 patients enrolled in this study received γδT cell immunotherapy with or without standard therapies. Their overall survival was analyzed by the Kaplan-Meier with log-rank test and Cox regression methods. Immunological analysis was performed by flow cytometry (FCM) before and after six cycles of γδT cell immunotherapy.ResultsMultivariable analysis revealed that patients who showed stable disease (SD) and partial response (PR) to γδT cell immunotherapy showed better prognosis than those with a progressive disease (PD) (P = 0.0269, hazard ratio [HR], 0.410, 95% confidence interval [CI], 0.190–0.901). Furthermore, when immunological parameters were examined by FCM, the high Vγ9/γδT ratio (i.e., the high purity of the Vγ9 cells within the adoptively transferred γδT cells) before treatment was found to be a good prognostic factor for γδT cell immunotherapy (P = 0.0142, HR, 0.328, 95% CI, 0.125–0.801). No serious adverse events were reported during γδT cell immunotherapy.ConclusionThus, γδT cell immunotherapy might extend the survival of patients with solid tumors.     

Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.     

Differences in biodistribution of the anti-(carcinoembryonic antigen) murine monoclonal antibody CE-25, its F(ab′)2 fragment and its intact mainly human chimeric form CE 4-8-13

The effect of the size of the tumour and the amount of antibody injected on the biodistribution of a family of radioiodinated antibodies was studied. The intact mouse anti-(carcinoembryonic antigen) (anti-CEA) monoclonal antibody CE-25, its F(ab)₂ fragment and the intact human-mouse chimeric from CE 4-8-13 were evaluated in a model system using the human CEA-producing colon xenograft T 380 grown in nude mice. The relative retention (the percentage of the injected dose per gram of tissue), of mouse mAb and F(ab)₂ in tumour and most normal tissues 1 day after injection was independent of the antibody dose; after 4 days the mAb values increased with increasing antibody dose. The relative retention of chimeric mAb increased with increasing antibody dose 1 day after injection and also slightly after 4 days. The relative retention in tumour tissue was lower in bigger xenografts for all antibodies. The relative retention of mouse mAb in small tumours increased from day 1 to day 4; for chimeric mAb this value decreased. In normal tissues the relative retention of mouse mAb decreased from day 1 to day 4, but the relative retention of chimeric mAb in normal tissue dropped rapidly and changed little afterwards. Thus the biokinetics of antibodies is species-dependent: foreign, mainly human, chimeric antibody clears faster from normal mouse tissue than mouse antibody and reaches lower concentrations.     

Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT

We previously reported that microSPECT/CT imaging with ¹¹¹In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab′)₂ of pertuzumab modified with NOTA chelators for complexing ⁶⁴Cu. The effect of the administered mass (5–200 µg) of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to ¹¹¹In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with ⁶⁴Cu-NOTA-pertuzumab F(ab′)₂ detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to ¹¹¹In-labeled pertuzumab.     

Induction of parturition in the mare with prostaglandin F2α

Thirty-one mares of Quarter Horse and Thoroughbred breeding were utilized in two experiments to evaluate the efficacy of prostaglandin F₂α (PGF₂α)_for induction of equine parturition and to monitor the effects of this treatment on viability of the resulting foals.Three of five mares given 5 mg PGF₂α (im) on day 338 of gestation foaled 19.6 ± 8.2 hr postinjection. In the second experiment immediately following 3 daily injections of 10 mg estradiol cypionate (ECP) given on days 326, 327 and 328 of gestation, seven mares were infused (iv) with PGF₂α at the rate of 1.3 mg/hr for 24 hr or until parturition occurred. Four of the seven mares foaled in 8.8 ± 1.8 hr after the start of infusion. Side effects including sweating, hypothermia, increased respiration rate and diarrhea were evident in both injected and infused mares, but effects were transient. Neither the injection, nor infusion route of administration of PGF₁α adversely affected the viability of foals. However, some mares induced to foal 12 days prior to expected parturition had foals with slightly weaker pasterns than those of control mares.     

Interaction of the α2A domain of integrin with small collagen fragments

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.     

A genetic variant of immunoglobulin γ2 is strongly associated with immunity to mucin 1 in patients with breast cancer

High levels of antibodies to mucin 1 (MUC1), a membrane-bound glycoprotein that is overexpressed in adenocarcinomas, are associated with good prognosis in patients with breast cancer. The aim of the present investigation was to determine whether GM and KM allotypes—genetic markers of IgG heavy chains and κ-type light chains, respectively—contribute to the magnitude of natural antibody responsiveness to MUC1 in patients with breast cancer. A total of 153 Caucasian subjects with breast cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. Anti-MUC1 IgG antibody levels in subjects who were carriers of the immunoglobulin γ2 allele GM 23 were significantly higher than in those who were noncarriers (P = 0.003). These results could potentially divide the population into high or low responders to MUC1, which has important implications for MUC1-based immunotherapeutic interventions in breast cancer.     

Induction of specific T cell immunity in patients with prostate cancer by vaccination with PSA146–154 peptide

T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4⁻CD8⁺ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.