ABSTRACT
INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.
ABSTRACT
BACKGROUND: Paclitaxel has recently been reported by this laboratory to potentiate the high-LET radiation therapeutic (212)Pb-TCMC-trastuzumab, which targets HER2. To elucidate mechanisms associated with this therapy, targeted α-particle radiation therapeutic (212)Pb-TCMC-trastuzumab together with paclitaxel was investigated for the treatment of disseminated peritoneal cancers. METHODS: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pre-treated with paclitaxel, followed by treatment with (212)Pb-TCMC-trastuzumab and compared with groups treated with paclitaxel alone, (212)Pb-TCMC-HuIgG, (212)Pb-TCMC-trastuzumab and (212)Pb-TCMC-HuIgG after paclitaxel pre-treatment. RESULTS: (212)Pb-TCMC-trastuzumab with paclitaxel given 24 h earlier induced increased mitotic catastrophe and apoptosis. The combined modality of paclitaxel and (212)Pb-TCMC-trastuzumab markedly reduced DNA content in the S-phase of the cell cycle with a concomitant increase observed in the G2/M-phase. This treatment regimen also diminished phosphorylation of histone H3, accompanied by an increase in multi-micronuclei, or mitotic catastrophe in nuclear profiles and positively stained γH2AX foci. The data suggests, possible effects on the mitotic spindle checkpoint by the paclitaxel and (212)Pb-TCMC-trastuzumab treatment. Consistent with this hypothesis, (212)Pb-TCMC-trastuzumab treatment in response to paclitaxel reduced expression and phosphorylation of BubR1, which is likely attributable to disruption of a functional Aurora B, leading to impairment of the mitotic spindle checkpoint. In addition, the reduction of BubR1 expression may be mediated by the association of a repressive transcription factor, E2F4, on the promoter region of BubR1 gene. CONCLUSION: These findings suggest that the sensitisation to therapy of (212)Pb-TCMC-trastuzumab by paclitaxel may be associated with perturbation of the mitotic spindle checkpoint, leading to increased mitotic catastrophe and cell death.
Subject(s)
Colonic Neoplasms/drug therapy , Colonic Neoplasms/radiotherapy , Lead Radioisotopes/therapeutic use , M Phase Cell Cycle Checkpoints/drug effects , Paclitaxel/therapeutic use , Radioimmunotherapy , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Death/drug effects , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Combined Modality Therapy , Female , Humans , Mice , Mice, Nude , Paclitaxel/pharmacology , Radioimmunotherapy/methods , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND AND PURPOSE: The studies described here are the first to evaluate the in vitro and in vivo properties of (111)In-CHX-A''-panitumumab for radioimmunotherapy (alpha- and beta(-)-emitters) and radioimmunoimaging (single photon emission computed tomography and positron emission tomography). EXPERIMENTAL APPROACH: Twenty-seven human carcinoma cell lines were analysed for expression of epidermal growth factor receptors by flow cytometry. Panitumumab was conjugated with CHX-A''-DTPA (diethylenetriamine-pentaacetic acid) and radiolabelled with (111)In. Immunoreactivity of the CHX-A''-DTPA-panitumumab and (111)In-CHX-A''-DTPA-panitumumab was evaluated by radioimmunoassays. Tumour targeting was determined in vivo by direct quantitation of tumour and normal tissues and by gamma-scintigraphy. KEY RESULTS: For 26 of 27 human tumour cell lines, 95% of the cells expressed epidermal growth factor receptors over a range of intensity. Immunoreactivity of panitumumab was retained after modification with CHX-A''-DTPA. Radiolabelling of the immunoconjugate with (111)In was efficient with a specific activity of 19.5 +/- 8.9 mCi.mg(-1) obtained. Immunoreactivity and specificity of binding of the (111)In-panitumumab was shown with A431 cells. Tumour targeting by (111)In-panitumumab was demonstrated in athymic mice bearing A431, HT-29, LS-174T, SHAW or SKOV-3 s.c. xenografts with little uptake observed in normal tissues. The (111)In-panitumumab was also evaluated in non-tumour-bearing mice. Pharmacokinetic studies compared the plasma retention time of the (111)In-panitumumab in both non-tumour-bearing and A431 tumour-bearing mice. Tumour targeting was also visualized by gamma-scintigraphy. CONCLUSIONS AND IMPLICATIONS: Panitumumab can be efficiently radiolabelled with (111)In with high labelling yields. Based on the efficiency in tumour targeting and low normal tissue uptake, panitumumab may be an effective targeting component for radioimmunodiagnostic and radioimmunotherapeutic applications.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , ErbB Receptors/metabolism , Immunoconjugates/pharmacology , Isothiocyanates/chemistry , Pentetic Acid/analogs & derivatives , Radiopharmaceuticals/pharmacology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Immunoconjugates/chemistry , Indium Radioisotopes , Mice , Mice, Nude , Panitumumab , Pentetic Acid/chemistry , Positron-Emission Tomography , Radioimmunotherapy , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/therapeutic use , Transplantation, HeterologousABSTRACT
Better tumor markers are needed for early diagnosis and staging of prostate cancer, and for monitoring therapeutic response than the currently used prostate specific antigen (PSA). Prostate specific membrane antigen (PSMA) is highly expressed on the surface of prostatic epithelial cells making it a good target for prostate cancer. In this study, mAb 3C6, specific for the extracellular epitope of PSMA, was evaluated both in vitro and in vivo for PSMA-targeting. Immunoreactivity and specificity of mAb 3C6 was evaluated by flow cytometry using prostate cell lines expressing PSMA such as LNCaP and 22Rv1 and a cell line, DU145, that expresses very little PSMA. 3C6 was conjugated with the acyclic CHX-A" DTPA chelate, radiolabeled with (111)In, and its in vitro and in vivo properties were assessed. The biodistribution of the radioimmunoconjugate evaluated in athymic mice bearing xenografts of three human prostate carcinoma cell lines shows high uptake after 72 hr in LNCaP tumors (%ID/g 22.93 +/- 6.32) and 22Rv1 (%ID/g 10.44 +/- 2.32) in contrast to low uptake by the DU145 tumors (%ID/g 4.27 +/- 0.37). Planar gamma-scintigraphic images obtained for xenografted tumor bearing mice demonstrated targeting for PSMA positive tumors suggesting possible applications in imaging and for targeted radiation therapy.
ABSTRACT
Detailed synthesis of the bifunctional chelating agents 2-methyl-6-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (1B4M-DOTA) and 2-(p-isothiocyanatobenzyl)-5, 6-cyclohexano-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetate (CHX-DOTA) are reported. These chelating agents were compared to 2-(p-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (C-DOTA) and 1, 4, 7, 10-Tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N', N", N"'-tris(acetic acid) cyclododecane (PA-DOTA) as their (177)Lu radiolabeled conjugates with Herceptin. In vitro stability of the immunoconjugates radiolabeled with (177)Lu was assessed by serum stability studies. The in vivo stability of the radiolabeled immunoconjugates and their targeting characteristics were determined by biodistribution studies in LS-174T xenograft tumor-bearing mice. Relative radiolabeling rates and efficiencies were determined for all four immunoconjugates. Insertion of the 1B4M moiety into the DOTA backbone increases radiometal chelation rate and provides complex stability comparable to C-DOTA and PA-DOTA while the CHX-DOTA appears to not form as stable a (177)Lu complex while exhibiting a substantial increase in formation rate. The 1B4M-DOTAmay have potential for radioimmunotherapy applications.
Subject(s)
Chelating Agents/pharmacokinetics , Colonic Neoplasms/metabolism , Heterocyclic Compounds, 1-Ring/pharmacokinetics , Isotope Labeling/methods , Lutetium/pharmacokinetics , Radioimmunoassay , Animals , Antibodies, Monoclonal , Cell Line, Tumor , Chelating Agents/chemical synthesis , Colonic Neoplasms/diagnostic imaging , Female , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Metabolic Clearance Rate , Mice , Organ Specificity , Radioisotopes/pharmacokinetics , Radionuclide Imaging , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Tissue DistributionABSTRACT
Among the radionuclides considered for radioimmunotherapy, alpha-emitters such as the bismuth isotopes, 212Bi and 213Bi, are of particular interest. The macrocyclic ligand, DOTA, has been shown to form stable complexes with bismuth isotopes. The kinetics of the complexation of bismuth with the DOTA chelate, however, are slow and impractical for use with 212Bi and 213Bi that have half-lives of 60.6 and 45.6 min. The study described herein compares six DTPA derived bifunctional chelates with the goal of identifying an alternative to the DOTA ligand for radiolabeling with bismuth. Radioimmunoconjugates comprised of MAb B72.3, each of the six DTPA chelates, and radiolabeled with 206Bi, which facilitated the evaluation due to its readily detectable gamma-emission. In vitro studies showed that each of the radioimmunoconjugates retained immunoreactivity that was comparable to its 125I-labeled counterpart. The 206Bi- and 125I-labeled immunoconjugates were then co-injected i.p. into normal athymic mice. Injection of Afree@ 206Bi demonstrated that the kidneys were the critical organ to evaluate for retention of bismuth in the chelate complex. Major differences were identified among the six preparations. The CHX-A and -B immunoconjugates were found to have 1) the lowest %ID/gm in the kidney; 2) a level of 206Bi in the kidney that was comparable to that of 125I-B72.3; and 3) no significant uptake of 206Bi evident in other organs such as bone, lung and spleen. The results described herein suggest that either of the cyclohexyl derivatives of DTPA may be suitable candidates for the labeling of immunoconjugates with alpha-emitting bismuth isotopes for radioimmunotherapeutic applications.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antibodies, Neoplasm/metabolism , Antineoplastic Agents/pharmacokinetics , Bismuth/pharmacokinetics , Pentetic Acid/pharmacokinetics , Radioisotopes/pharmacokinetics , Animals , Chelating Agents , Female , Immunoconjugates , Kidney/metabolism , Mice , Mice, Nude , Radioimmunoassay , Radioimmunotherapy , Spleen/metabolism , Tissue DistributionABSTRACT
Monoclonal antibody (MAb) CC49 reacts with a pancarcinoma antigen, tumor associated glycoprotein (TAG)-72. To circumvent human anti-murine antibody (HAMA) responses in patients, we earlier developed a humanized CC49 (HuCC49) by grafting the complementarity-determining regions (CDRs) of MAb CC49 onto variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28'CL, respectively. With the aim of minimizing its immunogenicity further, we have now generated a variant HuCC49 MAb by grafting the specificity-determining residues (SDRs) of MAb CC49 onto the frameworks of the human MAbs. Based on the evaluation of its binding affinity for TAG-72 and its reactivity with anti-idiotypic antibodies present in sera from patients who have been treated with murine CC49, this variant retains its antigen-binding activity and shows minimal reactivity with anti-idiotypic antibodies in patients' sera. Development of this variant, which is a potentially useful clinical reagent for diagnosis and therapy of human carcinomas, demonstrates that for humanization of a xenogeneic antibody grafting of the potential SDRs should be sufficient to retain its antigen-binding properties.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Animals , Antibodies, Heterophile/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Antibodies, Neoplasm/therapeutic use , Antineoplastic Agents/immunology , Antineoplastic Agents/therapeutic use , Complementarity Determining Regions/analysis , Complementarity Determining Regions/genetics , Drug Design , Genetic Engineering , Genetic Variation , HumansABSTRACT
Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T(1/2) = 52.02 h) has application potential in RII, with a gamma-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T(1/2) = 10 h) is a source of highly cytotoxic alpha-particles via its decay to its 212Bi (T(1/2) = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb.
Subject(s)
Chelating Agents/chemical synthesis , Heterocyclic Compounds/chemical synthesis , Isothiocyanates/chemical synthesis , Lead Radioisotopes , Chelating Agents/chemistry , Electrophoresis, Polyacrylamide Gel , Heterocyclic Compounds/blood , Heterocyclic Compounds/chemistry , Humans , Hydrogen-Ion Concentration , Isothiocyanates/blood , Isothiocyanates/chemistry , Molecular StructureABSTRACT
The evolution of monoclonal antibody forms for radioimmunotherapy and other antibody-based applications has been driven by a series of problems that each new form has introduced. Ehrlich was the first to present the concept that antibodies could be exploited in such a manner. Four decades were required before technological advances allowed the exploration of the potential of antibodies for radioimaging and radioimmunotherapeutic applications. Advances in DNA technology have led to the ability to tailor and manipulate the immunoglobulin molecule for specific functions and in vivo properties. This article discusses the use of monoclonal antibodies for radiotherapy with an emphasis on the problems that have been encountered and the subsequent solutions.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Neoplasms/radiotherapy , Radioimmunotherapy , Animals , Antibodies, Monoclonal/chemistry , Drug Design , Humans , Peptides/therapeutic useABSTRACT
Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.
Subject(s)
Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/chemistry , Antibodies, Neoplasm/immunology , Antibody Specificity , Carcinoma/immunology , Immunoglobulin Variable Region/immunology , Amino Acid Sequence , Animals , Antibodies, Anti-Idiotypic/blood , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/blood , Antibodies, Neoplasm/administration & dosage , Antibodies, Neoplasm/blood , Antibody Affinity/genetics , Antibody Specificity/genetics , Antigens, Neoplasm/blood , Antigens, Neoplasm/immunology , Base Sequence , Binding Sites, Antibody/genetics , Carcinoma/blood , Electrophoresis, Polyacrylamide Gel , Genes, Synthetic/immunology , Humans , Immunoglobulin Heavy Chains/blood , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/blood , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/blood , Immunoglobulin Variable Region/genetics , Injections, Intravenous , Ligands , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , Organ Specificity/immunology , Protein Engineering/methods , Spodoptera/genetics , Spodoptera/immunology , Transplantation, HeterologousABSTRACT
Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Neoplasms, Experimental/radiotherapy , Radioimmunotherapy , Animals , Antibodies, Monoclonal/pharmacokinetics , Copper Radioisotopes/pharmacokinetics , Humans , Isotope Labeling , Mice , Mice, Inbred BALB C , Mice, Nude , Octreotide/pharmacokinetics , Tissue Distribution , Tumor Cells, CulturedABSTRACT
One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Monoclonal/immunology , Antibody Affinity , Antigen-Antibody Reactions , Baculoviridae/genetics , Binding Sites/genetics , Binding, Competitive , Cell Line , Humans , Immunoglobulin Variable Region/immunology , Kinetics , Mice , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SpodopteraABSTRACT
Monoclonal antibodies (MAbs) labeled with radiometallonuclides via metal chelators are being investigated in the laboratory for use in clinical trials. The biodistribution of 111In- and 88Y-labeled antibody (MAb B72.3) using two isomeric forms (CHX-A and CHX-B) of the 2-(p-isothiocyanatobenzyl)-cyclohexyl-DTPA was compared in athymic mice bearing LS-174T tumors, human colon carcinoma xenografts. CHX-(A or B)-125I-DTPA-B72.3 was co-injected in all athymic mice to assess if the chelate conjugation altered the properties of MAb B72.3. In vitro studies demonstrated maintenance of integrity and immunoreactivity for both radioimmunoconjugates. The in vivo analysis, however, indicated major differences between the two isomer forms. In fact, the 88Y-CHX-A-DTPA radioimmunoconjugate demonstrated over the 7-day study period, a more efficient and stable tumor localization as well as a slower blood clearance rate than the CHX-B-DTPA chelate conjugate, suggesting a greater in vivo stability. Differences were also evident in critical normal organ uptake: no significant increase in liver- and spleen- or bone-to-blood ratios was observed when the CHX-A-DTPA chelate was labeled with indium or yttrium. The results described here demonstrate that the CHX-A-DTPA chelate conjugate can be considered more suitable than the CHX-B-DTPA isomer form when radiometallonuclides are coupled to an MAb.
Subject(s)
Colonic Neoplasms/radiotherapy , Indium Radioisotopes/pharmacokinetics , Isothiocyanates , Pentetic Acid/analogs & derivatives , Radioimmunotherapy , Radiopharmaceuticals/pharmacokinetics , Yttrium Radioisotopes/pharmacokinetics , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/therapeutic use , Humans , Indium Radioisotopes/therapeutic use , Isomerism , Ligands , Mice , Mice, Nude , Radiopharmaceuticals/therapeutic use , Tissue Distribution , Transplantation, Heterologous , Yttrium Radioisotopes/therapeutic useABSTRACT
The aim of this study was to assess the utility of a radioimmunoconjugate containing a lead radionuclide for therapy and scintigraphy applications. The radioimmunoconjugate evaluated consisted of a bifunctional DOTA ligand and monoclonal antibody (MAb) B72.3 using athymic mice bearing LS-174T tumors, human colon carcinoma xenografts. In the studies reported here, the lead-203-DOTA complex itself was first demonstrated to have in vivo stability. MAb B72.3 was then conjugated with the DOTA ligand and labeled with 203Pb, and the immunoreactivity of B72.3 was maintained. The localization of the radioimmunoconjugate to tumor tissue and other select organs paralleled that of DOTA-125I-B72.3, suggesting a similar metabolic pattern of the two radioimmunoconjugates. Thus, the DOTA-metal complex does not alter the behavior of the radioimmunoconjugate. Tumor localization of the 203Pb-DOTA-B72.3 conjugate was demonstrated with biodistribution studies as well as immunoscintigraphy studies. Such data highlight the stability of a lead radionuclide in the DOTA ligand. The suitability of this chelation chemistry for labeling radioimmunoconjugates with a lead radionuclide now makes its application in nuclear medicine a feasible proposition.
Subject(s)
Antibodies, Monoclonal , Heterocyclic Compounds , Immunoconjugates/therapeutic use , Lead Radioisotopes/therapeutic use , Radioimmunodetection , Radioimmunotherapy , Animals , Antibodies, Monoclonal/therapeutic use , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/radiotherapy , Heterocyclic Compounds/therapeutic use , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Tissue Distribution , Transplantation, HeterologousABSTRACT
While in vivo gene inoculation is being increasingly exploited to express genes of choice and elicit specific immune responses in animal models, the utility of this method has not been explored extensively for the expression of antibody genes. The primary constraint of this method is the need to deliver to, and express in, a single cell two functional genes, i.e., those encoding heavy and light chains of an antibody molecule. Several single-gene constructs encoding variants of the monoclonal antibody (MAb) CC49 have been developed, MAb CC49 recognizes a tumor-associated glycoprotein, TAG-72. SP2/O myeloma cells, transfected with the CC49 single gene, express a single-chain protein which is secreted by the transfectoma as a homodimer. Following intramuscular injection of mice with the expression plasmids of the single-gene constructs, the encoded CC49 antibody (AB1) was detected in the plasma of the host. In addition, cellular and humoral immune responses to AB1 have been demonstrated. Antibodies (AB2) to the in vivo-produced variable region of AB1 have been detected and persisted for at least 70 days post-inoculation of the recombinant plasmid. Thus, in vivo gene inoculation of single-chain immunoglobulins may be an alternative or complimentary approach to the induction of anti-idiotypic responses in immunotherapy protocols.
Subject(s)
Antibodies, Neoplasm/genetics , Gene Transfer Techniques , Genetic Therapy , Immunoglobulins/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Gene Expression , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
While several murine monoclonal antibodies (MAbs) directed against carcinoma associated antigens have shown excellent tumor targeting properties in clinical trials, the use of radiolabeled MAbs for both diagnostic and therapeutic applications has been hindered by two factors: (a) the induction of host anti-immunoglobulin (Ig) responses and (b) slow plasma clearance of unbound radiolabeled MAb, resulting in bone marrow toxicity for therapeutic application, and long intervals between MAb administration and tumor detection for diagnostic applications. This report describes the development of the first recombinant Ig with properties designed to reduce or eliminate both of the above problems: a complementarity determining region (CDR)-grafted humanized (Hu) MAb with a CH2 domain deletion (delta CH2). The MAb chosen for engineering was CC49, which is directed against a pancarcinoma antigen designated TAG-72 that is expressed on the majority of colorectal, gastric, breast, ovarian, prostate, pancreatic and lung carcinomas. When characterized for antigen binding in solid phase competition radioimmunoassays, the HuCC49 delta CH2 MAb completely inhibited the binding of murine (mu) CC49 and HuCC49 for TAG-72. The relative affinity constants (Ka) of MAbs HuCC49 delta CH2, HuCC49 and muCC49 were 5.1 x 10(-9), 2.1 x 10(-9) and 2.3 x 10(-9), respectively. The plasma clearance of 131I-HuCC49 delta CH2 was significantly faster than that of intact 125I-HuCC49 after either i.v. or i.p. administration in athymic mice (p(2)0.05). Biodistribution studies in athymic mice bearing human colon carcinoma xenografts after i.v. or i.p. administration of 131I-HuCC49 delta CH2 and 125I-HuCC49 demonstrated the efficient tumor localization and substantially lower percent of the injected dose (%ID/g) of the HuCC49 delta CH2 in normal tissues. This is reflected in the significantly higher radiolocalization indices (%ID/g in tumor divided by %ID/g in normal tissue) observed with the HuCC49 delta CH2 for most normal tissues tested (p(2)0.05). The differential between the rate of plasma clearance of HuCC49 delta CH2 and HuCC49 was even more pronounced in SCID mice, which have been shown to be an appropriate model to study the metabolism of human IgG. These studies thus describe the development of a recombinant Ig molecule which, for the first time, combines 1) the properties of more rapid blood clearance than an intact humanized Ig molecule--without loss of antigen binding affinity--and 2) reduced potential for eliciting a human anti-murine antibody (HAMA) response in patients. These studies also demonstrate the potential utility of HuCC49 delta CH2 for i.p. as well as i.v. radioimmunodiagnosis and radioimmunotherapy in patients with TAG-72 positive tumors.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antigens, Neoplasm/immunology , Glycoproteins/immunology , Radioimmunotherapy , Animals , Antibodies, Monoclonal/therapeutic use , Humans , Mice , Mice, Nude , Mice, SCID , Radioimmunoassay , Recombinant Proteins/pharmacokinetics , Tissue Distribution , Tumor Cells, CulturedABSTRACT
CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72. In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas. We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells. As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000. The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49. Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49. Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly. However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49. Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v. administration of the delta CH2 cMAb versus the 72 h required for cCC49. Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue). 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49. Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs. However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49. These results were similar when the cMAbs were labeled with either iodine or lutetium. In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49. The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings. However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.
Subject(s)
Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/immunology , Animals , Antibodies, Neoplasm/metabolism , Carcinoma/diagnostic imaging , Colonic Neoplasms/diagnostic imaging , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/genetics , Gene Deletion , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Iodine Radioisotopes , Macaca mulatta , Mice , Mice, Nude , Radioimmunodetection , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Transfection , Tumor Cells, CulturedABSTRACT
CC49, a murine monoclonal antibody that recognizes the tumor-associated glycoprotein 72, was conjugated to the chemical chelate 1,4,7,10-tetraaza-1-(1-carboxy-3-(4-aminophenyl) propyl)-tris-4,7,10- ((carboxy)methyl)cyclododecane that had been labeled with a beta emitter, Lu. Preclinical studies had shown that Lu-labeled CC49 caused regression of human colon adenocarcinoma xenografts in nude mice. Patients with advanced adenocarcinoma who had failed standard treatment and whose tumors expressed the tumor-associated glycoprotein 72 antigen were eligible for treatment to determine the maximum tolerated dose of Lu-labeled CC49. The starting dose of Lu was 10 mCi/m2 given i.v. with the dose of CC49 held constant at 20 mg. Pharmacokinetic sampling and immunoscintigraphy were performed over the ensuing 3 weeks. The dose of radioactive Lu was escalated by 15 mCi/m2 for each successive dose level. Unexpected bone marrow toxicity developed in patients treated at the second dose level with 25 mCi/m2 Lu; two patients developed grade 4 thrombocytopenia, while the third patient developed grade 3 thrombocytopenia. Pharmacokinetic studies showed that the plasma half-life of the immunoconjugate was 67 h; whole-body retention, however, was prolonged with a biological half-life of 258 h. Serial gamma camera imaging localized known tumor in all patients, and also demonstrated prolonged Lu retention in the reticuloendothelial system (RES). Bone marrow dosimetry estimates ranged from 4 to 5 REMS/mCi Lu based on imaging and biopsy data. Analysis of bone marrow biopsies demonstrated that most of the Lu was localized in the cellular compartment and not in the bone. No antitumor responses were observed. Intravenous administration of 15 mCi/m2 Lu-labeled CC49 to previously treated advanced cancer patients was associated with acceptable hematological toxicity and was the maximum tolerated dose. However, prolonged retention of Lu in the RES, including the bone marrow, was observed and limited the dose of Lu that could be given. Additional studies are indicated to reduce RES uptake and retention of this immunoconjugate.
Subject(s)
Adenocarcinoma/radiotherapy , Antibodies, Neoplasm/administration & dosage , Antineoplastic Agents/administration & dosage , Lutetium/administration & dosage , Neoplasms/radiotherapy , Radioisotopes/administration & dosage , Adenocarcinoma/pathology , Adult , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Antibodies, Neoplasm/adverse effects , Antineoplastic Agents/adverse effects , Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Colonic Neoplasms/pathology , Colonic Neoplasms/radiotherapy , Female , Humans , Lung Neoplasms/pathology , Lung Neoplasms/radiotherapy , Lutetium/adverse effects , Male , Middle Aged , Neoplasms/pathology , Radioimmunotherapy/methods , Radioisotopes/adverse effects , Radiotherapy DosageABSTRACT
Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Neoplasm/biosynthesis , Antibodies, Neoplasm/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Neoplasm/genetics , Antigens, Neoplasm/immunology , Base Sequence , Binding, Competitive/immunology , Female , Genes, Immunoglobulin , Genetic Vectors , Glycoproteins/immunology , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mice , Mice, Nude , Molecular Sequence Data , Radioimmunoassay , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacokinetics , Tumor Cells, CulturedABSTRACT
Previous studies have shown that (a) single-chain antibody binding proteins, or sFvs, localize experimental tumor xenografts (D.E. Milenic et al, Cancer Res., 51: 6363-6371, 1991) and (b) the administration of gamma-interferon (IFN-gamma) increases the expression of a high molecular weight glycoprotein, tumor-associated glycoprotein 72 (TAG-72), which improves mAb-based tumor targeting as well as radioimmunotherapy (J. W. Greiner et al., Cancer Res., 53: 600-608, 1993). The present experimental study was designed to determine whether exploiting those two observations in combination could augment tumor detection. Initial results revealed significant localization of a single-chain antibody binding protein of CC49 (i.e., CC49 sFv), a second generation anti-TAG-72 mAb, to human colon tumor xenografts (HT-29), which express low constitutive TAG-72 levels. IFN-gamma treatment of mice bearing HT-29 tumors significantly increased TAG-72 levels in the tumor xenografts. Increased TAG-72 expression was accompanied by a 2-4-fold augmentation of CC49 sFv localized to the HT-29 tumors, measured by direct quantitation of 125I-labeled CC49 sFv tumor deposition as well as tumor:normal tissue ratios. Enhanced CC49 sFv tumor localization improved HT-29 tumor visualization by external scintigraphy as well as when using a hand-held gamma-detecting probe to discriminate between normal (i.e., heart, hind leg) and tumor tissue. The gamma-detecting probe was the same as that used intraoperatively with 125I-labeled CC49 IgG to identify occult tumors in patients. The present experimental findings indicate that the efficiency by which 125I-labeled CC49 sFv localizes tumor in vivo can be enhanced with IFN-gamma. Results of the present study suggest that (a) the incorporation of an IFN-gamma treatment schema prior to radioimmunscintigraphy may increase the signal from the tumor site(s), thus providing a better discrimination between tumor and background, and (b) combining 125I-labeled CC49 sFv with IFN-gamma will not only reduce the time interval between antibody injection and surgery, but will also increase the efficiency of tumor localization using the intraoperative gamma-detecting probe.