The potential for enhanced tumour localisation by poly(ethylene glycol) modification of anti-CEA antibody (original) (raw)
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Bioconjugate Chemistry, 1992
Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridy1dithio)propionate or 4-@-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-Sg) and two different S-C conjugates (5 x 3 and S-Cg) were prepared using 3-and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95 % of lllIn-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with ll1In, the conjugates had a specific radioactivity of 90-120 pCi/pg. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-Sg was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). 5 x 3 and S-Cg produced practically the same blood clearances (much slower than that of S-Sg) and significant tumor uptake (9-1074 ID/g at 24 h). S-C, gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for 5 x 3 and S-Cg was rather high (45% ID/g at 24 h), but much lower than for S-Sg.
Protein Engineering Design and Selection, 2013
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 onehalf 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 pretargeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.
International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology, 1991
Tumour-to-normal tissue ratios of i.p. injected 125I-labelled monoclonal antibodies (MAbs), reacting with CEA were determined in nude rats xenografted with human colonic cancer cells (LS 174 T). Two MAbs, I-38S1 and II-16, reactive with the GOLD 1-epitope on CEA were tested. MAb I-38S1 was also tested after additional purification using anion exchange chromatography (thereafter named AEC 38). In the external activity measurements, MAb AEC 38 showed significantly better tumour-to-liver ratios than did MAb II-16 on all 4 days after injection. MAb I-38S1 gave intermediate ratios but was significantly better than II-16 only on day 3. The mean tumour-to-blood ratios were 3.0, 2.6 and 1.5 and the mean tumour-to-liver ratios were 6.6, 4.8 and 3.5 for MAbs AEC 38, I-38S1 and II-16 respectively. Gamma camera registrations in 3 animals on 4 days showed good imaging properties for all three MAbs and the patterns of tissue uptake were consistent with those seen in the external measurements. Fur...
Scientific reports, 2017
Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT). We focus here on mAb modifications by grafting an increase amount of trans-cyclooctene (TCO) derivatives (0 to 30 equivalents with respect to mAb) bearing different polyethylene glycol (PEG) linkers between mAb and TCO (i.e. PEG (1), PEG (2) and PEG (3)) and assessing their functionality. We used colorectal xenograft (HT29/Ts29.2) and peritoneal carcinomatosis (A431-CEA-Luc/35A7) as tumor cells/mAbs models and fluorescent tetrazines (TZ). MALDI-TOF MS shows that grafting with 2,3 increases significantly the number of TCO per mAb compared with no PEG. In vitro immunofluorescence showed that Ts29.2 and 35A7 labeling intensity is correlated with the number of TCO when using 1,3 while signals reach a maximum at 10 equivalents when using 2. Under 10 equivalents conditions, the capacity of resulting mAbs-1-3 for antigen recognition is similar when reported per grafted TCO and comparable to ...
Bioconjugate Chemistry, 2000
The aim of this study was to localize 99m Tc and 188 Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 × 734 (Fab′ × Fab′), anti-carcinoembryonic antigen [CEA] × anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N′-o-Phenylenedimaleimide was used to cross-link the Fab′ fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)-DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH 2 {TscG ) 3-thiosemicarbazonylglyoxyl}], with two indium-DTPAs and a chelate for selectively binding 99m Tc or 188 Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with 99m Tc (94-99%) at up to 1836 Ci/mmol and with 188 Re (97%) at 459-945 Ci/mmol of peptide. [ 99m Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 × murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) × 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 × m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 × m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 µg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 × 734, respectively), [ 188 Re]IMP-192 (7 µCi) or [ 99m Tc]IMP-192 (10 µCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [ 99m -Tc] or [ 188 Re]IMP-192 was 12.6 ( 5.2 and 16.9 ( 5.5% ID/g at 3 h postinjection, respectively. Tumor/ nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with 99m Tc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when 188 Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.
PLoS ONE, 2013
Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N9-N99-N999-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA) 1 -decalysine, (DOTA) 3 -decalysine or (DOTA) 5 -decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with 177 Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.664.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine] 2 , 30.6612.0% ID/g at 24 h for chCE7agl-[(DOTA) 3 -decalysine] 2 and 49.963.1% ID/g at 48 h for chCE7agl-[(DOTA) 5 -decalysine)] 2 . The rapid elimination from the blood of chCE7agl-[(DOTA)decalysine] 2 (1.060.1% ID/g at 24 h) is associated with a high liver accumulation (23.264.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.161.0 (DOTA) 3 versus 11.761.4% ID/g (DOTA) 5 , p,0.005 at 24 h) and lower radioactivity levels in the liver (21.463.4 (DOTA) 3 versus 5.860.7 (DOTA) 5 , p,0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA) 5 -decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).
Cancer research, 1990
Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and nonsmall cell lung cancer as well as weak or nondetectable reactivity to the majority of normal adult tissues, with the exception of secretory endometrium. Radiolabeled B72.3 has demonstrated MAb localization of carcinoma in approximately 70% of several hundred colorectal and ovarian carcinoma patients. The B72.3-reactive antigen, tumor-associated glycoprotein 72, has been purified from a human colon cancer xenograft and used as an immunogen to generate second generation MAbs. Twenty-eight of these MAbs, designated CC (colon cancer), were shown to be reactive with tumor-associated glycoprotein 72; direct-binding radioimmunoassays, Western blotting, live cell surface binding assays, liquid competition radioimmunoassays, ...
International Journal of Cancer, 1989
The in vivo uptake, therapeutic potential, and host toxicity were evaluated for both the intact IgG and F(ab'), fragment of 2 murine monoclonal antibodies (MAbs) directed against either carcino-embryonic antigen (CEA), called NP-4, or colon-specific antigen-p (CSAp), called Mu-9, in the GW-39 human colorectal carcinoma grown in the hamster cheek pouch. Mu-9 IgG and F(ab'), were retained longer by the tumor than was the NP-4 IgG or F(ab'),, respectively. Localization of the two antibodies by micro-autoradiography revealed two distinct patterns. Mu-9 was seen densely around whole acini of tumor cells, whereas NP-4 was found around each individual cell, albeit less densely. The anti-tumor effects of '3'l-labelled Mu-9 and NP-4 IgG were equal, but the therapeutic effectiveness of Mu-9 F(ab'), was significantly higher than NP-4 F(ab'),, as measured by change in tumor size. A dosedependent increase in host toxicity, as measured by change in body weight and change in peripheral white blood cells (p. WBCs), was observed with 0.5 to 3.0 mCi doses of '3'l-lgG regardless of the MAb used. A 10-2296 loss in body weight lasting 3-7 weeks and a 5040% loss in pWBCs lasting 68 weeks were observed in these animals. In contrast, 3 X 2 mCi doses of "'I-NP-4 or Mu-9 F(ab'), given at 3-day intervals, a schedule which was equally therapeutic to a single 2-mCi dose of '3'l-lgG, resulted in only a 9% loss in body weight and a 50% loss in pWBCs that lasted only I week. This was followed by a complete recovery over the next 2-3 weeks. These data suggest that multiple doses of F(ab'), can be as tumoricidal as a single dose of an intact MAb IgG, but significantly less toxic to the host. MATERIAL AND METHODS Antibody purijication and labelling All MAbs were purified from mouse ascites using Protein A and ion exchange chromatography over S-Sepharose (Pharmacia, Piscataway, NJ). NP-4 anti-CEA MAb has been described previously (Sharkey et al., 1987). Mu-9 was developed by immunization of mice with an extract from GW-39 (Nocera et al., 1987). Unlike the other Mu-series MAbs, Mu-9 was found to identify an epitope that had properties similar to those reported earlier for CSAp (Shochat et al., 1982), namely, sensitivity to destruction by reducing agents or heat. The antigen is not found in serum or in normal tissues with the exception of normal colon (Pant et al., 1977). An irrelevant IgG of the same IgG, isotype, anti-AFP MAb, designated AFP-7-31, was obtained from Immunomedics, Newark, NJ. F(ab'), fragments of each antibody were prepared by pepsin digestion in 0.1 M sodium citrate buffer, PH 3.5, at 37°C for 45-90 min. The reaction was stopped by elevating the PH to 7.0. The preparation was desalted on a column of fractogel TSK HW-5OF (EM Science, Gibbstown, NJ) and fractionated by ion-exchange chromatography on Q-Sepharose equilibrated with TrislHCl buffer, PH 7.5, so that intact IgG, peptides of the Fc region, and pepsin bind to the gel. The F(ab'), was collected and concentrated by YM-30 ultrafiltration (Amicon, Danvers, MA) and dialyzed against PBS (40 m sodium phosphate), PH 7.4. IgG and F(ab'), were radio-iodinated by the chloramine-T method (McConahey and Dixon, 1966). Free radio-iodine was separated from antibody-bound iodine by passage over a PD-10
Nuclear medicine and biology, 2014
This paper proposes liposomes as a potential new tool for radioimmunotherapy in solid tumours with a two step targeting system. Tumour pretargeting is obtained by using a monoclonal bispecific antibody (BsmAb, anti CEA x anti-DTPA-In) and pegylated liposomes containing lipid-hapten (DSPE-DTPA-In or DSPE-PEG-DTPA-In). To optimise at the same time in vivo behaviour and specific targeting, the study focuses on the liposome formulation in order to determine more precisely the role of pegylation on both the blood half-life and the specific recognition with the BsmAb. Different liposome formulations containing two PEG length (1000 and 2000) in varying amount (1.5-6 mol%) were prepared with DTPA directly coupled to DSPE or at the end of the PEG chain (DSPE-DTPA or DSPE-PEG-DTPA). Liposomes were immobilized on an L1 chip to measure by SPR (Surface Plasmon Resonance) the effect of pegylation on the BsmAb recognition of the DTPA-In hapten. Pharmacokinetic studies were performed in mice. Tumou...
Journal of Immunotherapy, 2009
Carcinoembryonic antigen (CEA, CD66e) is a wellcharacterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/ CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 + T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/ CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach. FIGURE 4. The effect of various E:T ratios on redirected lysis of transfected Chinese hamster ovary cells expressing carcinoembryonic antigen by bscAb A11 in a fluorescence-activated cell sorting-based cytotoxicity assay. Unstimulated human CD3 + T cells from 2 different donors (A, #322 and B, #806) were used as effector cells at the indicated E:T ratios. Duration of the assay was 72 hours. Points, mean (n = 4); bars, SD. bscAb indicates bispecific single-chain antibodies, E:T, effector-to-target.