Doxorubicin immunoconjugates containing bivalent,lysosomally-cleavable dipeptide linkages

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.     

Significance of antigen, drug, and tumor cell targets in the preclinical evaluation of doxorubicin, daunorubicin, methotrexate, and mitomycin-C monoclonal antibody immunoconjugates

We tested drug monoclonal antibody immunoconjugates in vitro in 72 h 3H-thymidine assays and in vivo in athymic mice bearing human tumor xenografts of the same target cells. Experimental arms included control, monoclonal antibody, drug, drug + antibody, the test immunoconjugate, and a negative control immunoconjugate with an equivalent molar amount of drug for in vitro experiments, and the amount of drug conjugated to 500 micrograms of antibody in the animal experiments. Monoclonal antibodies included T101, an IgG2a that reacts with a rapidly modulating antigen, 9.2.27, an IgG2a that reacts with a slowly modulating antigen, and ME7, an IgG1 that reacts with a slowly modulating antigen. Cells used in testing included MOLT-4 (T lymphoma), 8392 (B lymphoma), and M21 (melanoma). Drugs tested were doxorubicin, daunorubicin, methotrexate, and mitomycin-C. M21 cells were resistant to daunorubicin in vitro but were inhibited by the 9.2.27 daunorubicin immunoconjugate. T101, 9.2.27, and ME7 cis-aconitate anthracycline immunoconjugates and mitomycin-C-glutarate immunoconjugates were specifically cytotoxic only for antigen positive cells in vitro and were superior to free drug in vivo. These results confirm that antigen specific-cytotoxic drug immunoconjugates can be produced that are superior to the same dose of free drug. However, each monoclonal antibody drug target system is unique and must be well-characterized for appropriate interpretation of data.     

Determination of immunoreactivity of doxorubicin antibody immunoconjugates by a Le(y) competitive RIA.

Many monoclonal antibody-drug immunoconjugates have been evaluated for their ability to deliver cytotoxic drugs to tumors. It is essential to establish that the ability of the conjugates to bind antigen, i.e. their immunoreactivity, is not adversely affected by the drug conjugation procedure. We have described herein a measurement of the immunoreactivity of BR96-DOX, a conjugate comprised of BR96, a chimeric monoclonal antibody specific for the Le(y) tetrasaccharide commonly expressed on human carcinomas, and doxorubicin, an anticancer agent in widespread clinical use. We have employed a competitive RIA, in which microtiter wells were coated with synthetic Le(y) conjugated to human serum albumin and then incubated with 125I-labeled antibody BR96 in the presence of test conjugate or intact BR96 mAb. The test conjugates were found to compete as effectively as unconjugated BR96. This assay is highly applicable to QC processes with the intra-assay CV = 2.0% and the interassay CV = 4.3%.     

Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay

Capillary electrophoresis coupled with laser-induced fluorescence was used for the characterization of quantum dots and their conjugates to biological molecules. The CE-LIF was laboratory-built and capable of injection (hydrodynamic and electrokinetic) from sample volumes as low as 4 μL via the use of a modified micro-fluidic chip platform. Commercially available quantum dots were bioconjugated to proteins and immunoglobulins through the use of established techniques (non-selective and selective). Non-selective techniques involved the use of EDCHCl/sulfo-NHS for the conjugation of BSA and myoglobin to carboxylic acid-functionalized quantum dots. Selective techniques involved 1) the use of heterobifunctional crosslinker, sulfo-SMCC, for the conjugation of partially reduced IgG to amine-functionalized quantum dots, and 2) the conjugation of periodate-oxidized IgGs to hydrazide-functionalized quantum dots. The migration times of these conjugates were determined in comparison to their non-conjugated QD relatives based upon their charge-to-size ratio values. The performance of capillary electrophoresis in characterizing immunoconjugates of quantum dot-labeled IgGs was also evaluated. Together, both QDs and CE-LIF can be applied as a sensitive technique for the detection of biological molecules. This work will contribute to the advancements in applying nanotechnology for molecular diagnosis in medical field.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Design and construction of novel molecular conjugates for signal amplification (II): use of multivalent polystyrene microparticles and lysine peptide chains to generate immunoglobulin-horseradish peroxidase conjugates

Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody–enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and horseradish peroxidase (HRP) were combined in a 1:5 ratio and attached to 0.44 μm streptavidin microparticles or N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 μm amino microparticles with highly reactive free sulfhydryl groups on their surface. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with streptavidin or sulfhydryl groups-containing microparticles. The antibody–poly-HRP immunoconjugates contained an estimated number of 10⁵ HRP/streptavidin microparticle and 10⁶ HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV-1 antibodies that had been captured by HIV antigens on 5 μm carboxyl magnetic microparticles and, upon reaction with orthophenyldiamine substrate, produced a detection signal with 5–8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG. The signal amplification technique by microparticle immunoconjugates may provide potentially novel tools for the development of highly sensitive diagnostic systems.     

Preclinical investigation of the antitumour effects of anti-CD19-idarubicin immunoconjugates

Idarubicin (Ida), an analogue of daunomycin, was linked to a mouse monoclonal antibody against the B cell differentiation antigen CD19. Determination of the activity of both the antibody and drug moieties was carried out in vitro using a pre-B cell human acute lymphoblastic leukaemia cell line (NALM-6). A 23% loss in antibody binding and a 20-fold loss in drug activity were observed upon conjugation. Selective cytotoxicity for CD19⁺ cells, however, was obtained. Measurement of the cytotoxicity, antibody activity and release of the breakdown product, 14-OH-Ida, showed that the conjugates remained stable for more than 100 days after lyophilization and storage at –20° C. In vivo studies were performed in irradiatednu/nu mice bearing NALM-6 tumours. Initial dose response studies with free idarubicin demonstrated that three i.p. doses (every other day) of 10 g resulted in little antitumour activity, but the death of all the animals by day 23. The same treatment regime using 15 g Ida-anti-CD19 conjugate caused the disappearance of four out of five tumours with three complete cures and no evidence of toxicity as assessed by weight loss. Administration of a conjugate of idarubicin with an irrelevant antibody at this dose led to no significant antitumour response. The administration of free drug at a dose of 6 g resulted in a minor antitumour response but high toxicity, whereas injection of Ida-anti-CD19 conjugate at this dose caused no toxicity and a substantial antitumour effect with eradication of two out of five tumours. These results clearly demonstrate that the administration of Ida-anti-CD19 conjugates can result in complete tumour regression in an experimental model. Idarubicin-containing immunoconjugates should be useful for the treatment of patients with non-Hodgkin's lymphoma.     

Carbohydrate-directed conjugation of cobra venom factor to antibody by selective derivatization of the terminal galactose residues

Cobra venom factor (CVF) can cause cell death by complement-mediated bystander cell lysis. Several studies have investigated CVF for application in cancer therapy by conjugating CVF to antibodies against tumor cell surface-specific antigens via the side-chain amino acid residues. In most cases, the activity of CVF was markedly impaired, presumably by modification of the factor B binding domain due to random derivatization. Since CVF is a glycoprotein and its oligosaccharide chains are distal to the factor B binding domain, coupling of CVF to antibodies through its oligosaccharide chains is expected to yield immunoconjugates with retention of CVF activity and elimination of the immunoreactivity of the terminal alpha-galactosyl residues. In this study, we investigated the carbohydrate site-directed conjugation of CVF to a monoclonal IgG specific to a cell-surface antigen of human ovarian cancer cells. The terminal galactosyl residues of CVF were selectively modified at C-6 by treatment with galactose oxidase, and the generated aldehyde groups were derivatized in situ with hydrazides containing either protected thiol or maleimide functional groups. The CVF derivatives were allowed to react with thiol groups introduced to the antibody by derivatization with 2-iminothiolane to yield carbohydrate site-directed CVF-antibody conjugates. In both cases, 30-40% of the antibody cross-linked to CVF to yield predominantly monovalent CVF-antibody conjugates. The purified immunoconjugates retained 70-75% of CVF activity and significant level of antigen-binding capacity. This is the first study to exploit the oligosaccharide chains of CVF for the preparation of active immunoconjugates.     

Syntheses of dendritic linkers containing chlorambucil residues for the preparation of antibody-multidrug immunoconjugates

A novel dendritic molecule with nine chlorambucil (CBL) residues on the surface and a maleimide moiety at the core terminus was synthesized using a convergent synthetic methodology. This molecule is ready for attachment to single-chain Fv antibodies (scFvs) to form antibody-multidrug immunoconjugates in an effort to study the relevance of drug/antibody molar ratio and the potency of these drug-antibody immunoconjugates. A monomer and a trimer with a similar structural motif were also prepared for comparative purposes.     

Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.     

In vitro antitumor activity of 2'-deoxy-5-fluorouridine-monoclonal antibody conjugates

5-Fluorouracil (5-FU) is an anticancer drug used in patients for the treatment of gastric and breast cancer and used either alone or in combination with methotrexate is one of the few drugs with some effect on colon cancer. 2'-Deoxy-5-fluorouridine (5-FUdr) (1) is an analogue based on 5-FU and can be covalently linked to a murine anti-Ly-2.1 monoclonal antibody (mAb) with the active ester derivative of 2'-deoxy-5-fluoro-3'-O-(carboxypropanoyl)uridine (5-FUdr-succ) (4). Such immunoconjugates can contain up to 42 residues of drug, although the most antibody activity was retained when substitution ratios were between 10 and 25 molecules of drug to mAb. In a cytotoxicity assay, 50% inhibition of [3H]deoxyuridine incorporation (IC50) with a murine Ly-2.1+ve thymoma cell line was 6 nM for 5-FUdr-anti-Ly-2.1, which is 12-fold more than that for free 5-FUdr (IC50 = 0.51 nM) but similar to that of 5-FUdr-succ (IC50 = 5.2 nM). The 5-FUdr-monoclonal antibody conjugates (5-FUdr-mAb) were 100-fold more active on the Ly-2.1+ve E3 cell line than on the Ly-2.1-ve BW5147 OU- cell line. The high in vitro activity and specificity of 5-FUdr-MoAb conjugates indicates that potent in vivo activity of these conjugates should be expected.     

High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA–dsDNA triplexes—mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na⁺). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.     

Technical Considerations in the Preparation of Fluorescent-Antibody Conjugates

A comparison was made of (NH₄)₂SO₄, HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.     

Modification of monoclonal antibody carbohydrates by oxidation,conjugation, or deoxymannojirimycin does not interfere with antibody effector functions

Site-specific attachment of metal chelators or cytotoxic agents to the carbohydrate region of monoclonal antibodies results in clinically useful immunoconjugates [Doerr et al. (1991) Ann Surg 214: 118, Wynant et al. (1991) Prostate 18: 229]. Since the capacity of monoclonal antibodies (mAb) to mediate tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may accentuate the therapeutic effectiveness of immunoconjugates, we determined whether site-specific modification of mAb carbohydrates interfered with these functions. The chemical modifications examined consisted of periodate oxidation and subsequent conjugation to either a peptide linker/chelator (GYK-DTPA) or a cytotoxic drug (doxorubicin adipic dihydrazide). mAb-associated carbohydrates were also modified metabolically by incubating hybridoma cells in the presence of a glucosidase inhibitor deoxymannojirimycin to produce high-mannose antibody. All four forms (unaltered, oxidized, conjugated and high-mannose) of murine mAb OVB-3 mediated tumor cell lysis via CDC. Similarly, equivalent ADCC was observed with native and conjugated forms of mAb OVB-3 and EGFR.1. ADCC was achieved with different murine effector cells such as naive (NS), poly (I^*C)- and lipopolysaccharide-stimulated (SS) spleen cells, orCorynebacterium-parvum-elicited peritoneal cells (PEC). All murine effector cell types mediated tumor cell lysis but differed in potency such that PEC>SS>NS. Excellent ADCC activity was also demonstrable by human peripheral blood mononuclear cells with OVB-3-GYK-DTPA and high-mannose OVB-3 mAb. ADCC activity was detectable in vivo: both native and conjugated OVB-3 inhibited growth of OVCAR-3 xenografts in nude mice primed withC. parvum. In conclusion, modification of mAb carbohydrates did not compromise their in vivo or in vitro biological functions. Therefore, combination therapy using immunomodulators to enhance the effector functions of site-specific immunoconjugates could be seriously contemplated.     

单克隆抗体靶向制剂的研究进展

本文综述了制备单克隆抗体免疫偶合物的三种方法,即抗体与药物直接交联的方法,药物通过小分子与抗体连接的戊二醛法、顺乌头酸酐法、活性酯法、Ｎ－琥珀酰胺基－３－（２－吡啶基二硫）丙酸法（ＳＰＤＰ法）、腙衍生物法和肽键连接等方法,以及用大分子做载体的交联方法,并介绍了葡聚糖、聚谷氨酸、聚赖氨酸和聚合多肽作载体的性质和应用情况。     

Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies

Background: Monoclonal antibodies (MAbs) can target therapy to tumours while minimising normal tissue exposure. Efficacy of immunoconjugates containing peptide 101, designed around the first 22 amino acids of bee venom, melittin, to maintain the amphipathic helix, to enhance water solubility, and to increase hemolytic activity, was assessed in nude mice bearing subcutaneous human prostate cancer xenografts. Methods: Mouse MAbs, J591 and BLCA-38, which recognise human prostate cancer cells, were cross-linked to peptide 101 using SPDP. Tumour-bearing mice were used to compare biodistributions of radiolabeled immunoconjugates and MAb, or received multiple sequential injections of immunoconjugates. Therapeutic efficacy was assessed by delay in tumour growth and increased mouse survival. Results: Radiolabeled immunoconjugates and antibodies showed similar xenograft tropism. Systemic or intratumoural injection of immunoconjugates inhibited tumour growth in mice relative to carrier alone, unconjugated antibody and nonspecific antibody-peptide conjugates and improved survival for treated mice. Conclusions: Immunoconjugates deliver beneficial effects; further peptide modifications may increase cytotoxicity.     

World Antibody Drug Conjugate Summit Europe: February 21–23, 2011; Frankfurt,Germany

The World Antibody Drug Conjugate Summit Europe, organized by Biorbis/Hanson Wade was held in Frankfurt, Germany February 21–23, 2011. Antibody drug conjugates (ADCs), also called immunoconjugates, are becoming an increasingly important class of therapeutics as demonstrated by the attendance of nearly 100 delegates at this highly focused meeting. Updates on three ADCs that are in late-stage clinical development, trastuzumab emtansine (T-DM1), brentuximab vedotin (SGN-35) and inotuzumab ozogamicin (CMC-544), were presented by speakers from ImmunoGen, Genentech, Roche, Seattle Genetics and Pfizer. These ADCs have shown encouraging therapeutic effects against solid tumors (T-DM1) and hematological malignancies (SGN-35, CMC-544). The key feature of the new generation of ADCs is the effective combination of the cytotoxicity of natural or synthetic highly potent antineoplastic agents, tumor selective monoclonal antibodies and blood-stable optimized linkers. Early clinical data for ADCs were showcased by Progenics Pharmaceuticals (PSMA ADC), Celldex (CDX-011) and Biotest (BT-062). Takeda, MedImmune and sanofi-aventis outlined their strategies for process development and analytical characterization. In addition, presentations on duocarmycin based-ADCs, α emitting immunoconjugates and antibody-conjugated nanoparticles were given by representatives from Syntarga, Algeta and the University of Stuttgart, respectively.Key words: antibody drug conjugates, immunoconjugates, trastuzumab emtansine, brentuximab vedotin, inotuzumab ozogamicin, oncology, cancer     

Individually specified drug immunoconjugates in cancer treatment

Forty-three patients with disseminated refractory malignancies each received an individually-specified combination of either Adriamycin (24 patients) or mitomycin-C (19 patients) conjugated murine monoclonal antibodies. Tumors were typed using a panel of antibodies with both immunohistochemistry and flow cytometry. Cocktails of up to six antibodies were selected based on binding greater than 80% of the malignant cells in the biopsy specimen. These monoclonal antibody cocktails were drug conjugated and administered intravenously. Seventeen out of twenty-four patients had reactions to the administration of Adriamycin immunoconjugates, but these were tolerable in all but two patients. Fever, chills, pruritus and skin rash were by far the most common transitory reactions. All were well controlled with premedication. In several patients it was demonstrated that there was limited antigenic drift among various biopsies within the same patient over time. Up to 1 gram of Adriamycin and up to 5 grams of monoclonal antibody were administered. The limiting factor appeared to be a variable dissociation of active Adriamycin from the antibody which unpredictably caused hemopoietic depression. Similar findings were noted in 19 patients with mitomycin-C conjugates. Thrombocytopenia at a 60mg dose of mitomycin-C in this schedule was dose limiting. Preliminary serological evidence suggests that the development of an IgM antibody which is specific against the mouse monoclonal antibody has the specificity and sensitivity to predict clinical reactions. These antibodies were quantitatively less in mitomycin-C patients. Selected patients were re-treated. One patient with chronic lymphocytic leukemia had re-treatment on three occasions and demonstrated regression of peripheral lymph nodes. Two patients with breast carcinoma had definite improvement in ulcerating skin lesions and two patients with tongue carcinoma had shrinkage of their lesions. No responses were seen with mitomycin-C conjugates but binding was noted to tumors and colon with likely drug induced colitis seen after colon binding. This study demonstrates the feasibility and illustrates technical considerations in preparing drug immunoconjugate cocktails for patients with refractory malignancies. Cocktail formulation and antibody delivery was accomplished. The major technical hurdle appears to be the selection of effective conjugation methods that can be used to optimally bind drugs to monoclonal antibodies for targeted cancer therapy.     

Protein database searches using compositionally adjusted substitution matrices

Almost all protein database search methods use amino acid substitution matrices for scoring, optimizing, and assessing the statistical significance of sequence alignments. Much care and effort has therefore gone into constructing substitution matrices, and the quality of search results can depend strongly upon the choice of the proper matrix. A long-standing problem has been the comparison of sequences with biased amino acid compositions, for which standard substitution matrices are not optimal. To address this problem, we have recently developed a general procedure for transforming a standard matrix into one appropriate for the comparison of two sequences with arbitrary, and possibly differing compositions. Such adjusted matrices yield, on average, improved alignments and alignment scores when applied to the comparison of proteins with markedly biased compositions. Here we review the application of compositionally adjusted matrices and consider whether they may also be applied fruitfully to general purpose protein sequence database searches, in which related sequence pairs do not necessarily have strong compositional biases. Although it is not advisable to apply compositional adjustment indiscriminately, we describe several simple criteria under which invoking such adjustment is on average beneficial. In a typical database search, at least one of these criteria is satisfied by over half the related sequence pairs. Compositional substitution matrix adjustment is now available in NCBI's protein-protein version of blast.     

DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

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-N′-N′′-N′′′-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 ¹⁷⁷Lu 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 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]₂, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)₃-decalysine]₂ and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)₅-decalysine)]₂. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]₂ (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% 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 (5.1±1.0 (DOTA)₃ versus 11.7±1.4% ID/g (DOTA)₅, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)₃ versus 5.8±0.7 (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).