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[Cancer Research 59, 347-352, January 1, 1999]
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[Cancer Research 59, 347-352, January 15, 1999]
© 1999 American Association for Cancer Research

Biochemistry

Increased Binding Affinity and Valence of Recombinant Antibody Fragments Lead to Improved Targeting of Tumoral Angiogenesis1

Francesca Viti, Lorenzo Tarli, Leonardo Giovannoni, Luciano Zardi and Dario Neri2

Institut für Molekularbiologie und Biophysik, ETH Hönggerberg, CH-8093 Zürich, Switzerland [F. V., L. T., D. N.]; Dipartimento di Biologia Molecolare, Sezíone di Biochimica, Università di Siena, 53100 Siena, Italy [L. G.]; and Istituto Nazionale per la Ricerca sul Cancro/Centro di Biotecnologie Avanzate, 16132 Genova, Italy [L. Z.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note Added in Proof
 REFERENCES
 
The formation of new blood vessels (angiogenesis) is an important step in tumor progression. Molecules capable of selectively targeting markers of angiogenesis may offer opportunities for the in vivo imaging of aggressive tumors and for the delivery of toxic agents to the tumoral vasculature. Using antibody phage display libraries and combinatorial mutagenesis, we isolated single-chain Fv antibody fragments, which recognize with different affinities the same epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Two single-chain Fv fragments, E1 and L19, with dissociation constants of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s.c. in nude mice when injected i.v. in either monomeric or homodimeric form (Mr 27,000 and 54,000, respectively). Biodistribution studies, performed at two time points (4 h and 24 h) with radiolabeled samples, showed that the higher affinity antibody targets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 accumulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h were in the (2.1–8.6):1 and (10.3–29.4):1 range, respectively. This study demonstrates that, although vasculature represents only a small fraction of the total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note Added in Proof
 REFERENCES
 
Most aggressive solid tumors cannot grow beyond a certain mass without the formation of new blood vessels (angiogenesis), and a correlation between microvessel density and tumor invasiveness has been reported (1) . Abs3 or other suitable molecules that are capable of selectively targeting markers of angiogenesis would create clinical opportunities for the diagnosis and therapy of tumors and other diseases characterized by vascular proliferation (2, 3, 4, 5, 6, 7, 8) .

The targeting of angiogenesis is attractive for a number of reasons. Because tumor growth and metastatic spread depend on angiogenesis, the selective delivery of toxic agents to new-forming blood vessels should offer a therapeutic benefit (2, 3, 4, 5, 6, 7, 8) . Unlike tumor cells, endothelial cells do not mutate, and resistance is, therefore, less likely to develop. It should be possible to use a single reagent, specific for angiogenesis, for the selective detection and/or ablation of different types of tumor. Furthermore, markers on new-forming blood vessels should be readily accessible to binding molecules (e.g., Abs) injected i.v. and should overcome the well-recognized problem of slow tumor penetration of targeting agents specific for markers on the membrane of tumor cells (9, 10, 11) .

Research into antiangiogenic therapeutic strategies has mainly focused on the search for inhibitory molecules (1, 2, 3) . Only few reports, with no quantitative biodistribution analyses, have indicated that it may indeed be possible to target angiogenesis in vivo (5 , 12) .

The ED-B domain of fibronectin, a sequence of 91 amino acids identical in mouse, rat, dog, and man, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neovascular structures (13, 14, 15, 16, 17) and could represent a target for molecular intervention. Indeed, we have recently shown with fluorescent techniques that anti-ED-B scFvs3 (12) that are injected in tumor-bearing mice selectively accumulate in tumoral blood vessels.

These experiments, performed using Ab fragments with affinities in the low nanomolar range, demonstrated that it is possible to selectively target tumoral angiogenesis in vivo. They also raised the issue of whether Abs against markers of angiogenesis with further enhancement in binding affinity and/or valence would show an improved localization in tumoral neovasculature—an important parameter for the development of diagnostic and therapeutic applications.

Here we present the results of biodistribution studies in tumor-bearing mice performed with two radiolabeled Abs directed against the same epitope of the ED-B domain of fibronectin and differing in affinity by 760-fold. We quantitatively investigated whether binding affinity influences tumor targeting in vivo when Abs are injected i.v. as purified monomers or homodimers.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Note Added in Proof
 REFERENCES
 
Production of Anti-ED-B Affinity Mutants.
The isolation of the E1 and L19 Abs has been described previously (18) . E1 is a scFv binding to the ED-B domain of fibronectin that is isolated from a synthetic human phage display Ab library. L19, a mutant of E1 with 760-fold improved affinity, differs from the parental Ab by eight mutations introduced in hypervariable loops (European Molecular Biology Laboratory accession no. AJ006113; Ref. 18 ).

The genes coding for scFv(E1) and scFv(L19) were subcloned into NcoI/NotI digested pDN5 (19) according to standard techniques (20) . The corresponding Ab fragments, carrying a myc tag epitope at the COOH terminus (21) , were expressed from TG1 Escherichia coli cells harboring the appropriate plasmids and were affinity-purified from bacterial supernatants using an ED-B-Sepharose column, as described previously (12) .

Native PAGE (19) and gel filtration analysis of purified scFvs showed that E1 and L19 consist of a monomeric and a dimeric fraction, which were quantitatively separated and recovered using a Superdex 75 HR 10/30 column (Pharmacia, Uppsala, Sweden). The stability of the purified monomer and dimer was checked by native gel electrophoresis with Homogenous 12.5% gels (Pharmacia) by using a Phast System (Pharmacia) as described previously (19) .

The stability of E1 and L19 in serum was evaluated by incubating the Ab in FCS at 37°C for different time periods (0–4 h) and analyzing the Ab reactivity in the resulting mixture by ELISA as described previously (14) , applying biotinylated ED-B onto streptavidin-coated ELISA plates (Boehringer, Mannheim, Germany).

Epitope Retention Assay.
The ability of L19 to compete for the binding of E1 to a common epitope on the ED-B domain of fibronectin was investigated by real-time interaction analysis with surface plasmon resonance detection, using a BIAcore 1000 instrument (Pharmacia), according to established protocols (12 , 22) . Biotinylated ED-B (12) was applied to streptavidin-coated microsensor chips until the resulting surface bound ~ 1000 RUs of scFv fragments. Binding studies were performed using 1 µM purified scFv solutions in PBS (50 mM phophate buffer (pH 7.2)-100 mM NaCl), with a 5-µl/min flow. For studies of competitive binding to the same epitope of the ED-B domain, the higher affinity scFv(L19) was injected first until saturation of the microsensor chip was achieved, inasmuch as this Ab shows a flat dissociation profile.

Western blot analysis of Ab specificity was performed as described previously (17) .

Radioiodination.
Immediately after their chromatographic separation (see previous section), dimeric and monomeric forms of scFv(L19) were radiolabeled with 125I using the Iodogen method (Pierce, Rockford, IL). Briefly, 100–200 µg of protein were combined with 100–200 µCi of 125I, followed by separation from unincorporated iodine using a PD-10 disposable gel filtration column (Pharmacia). Ab immunoreactivity after labeling was evaluated by loading an aliquot of radiolabeled sample onto 200 µl of ED-B-Sepharose resin (capacity, >3 mg scFv/ml resin; Ref. 12 ) on a pasteur pipette, followed by radioactive counting of the flow-through and eluate fractions. Immunoreactivity, defined as the ratio between the counts of the eluted protein and the sum of the counts of the eluted and flow-through fractions, was 81% for monomeric L19, 92% for dimeric L19, 90% for monomeric E1, and 91% for dimeric E1.

The purity of radiolabeled samples was analyzed by reducing SDS-PAGE using Homogenous 20 Phast gels and a Phast System (Pharmacia), followed by autoradiography with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Native PAGE of the radiolabeled samples, performed using Homogenous 12.5 Phast gels, native buffer strips, and a Phast System (Pharmacia) followed by autoradiography (19) , was performed to ensure that monomeric and dimeric Ab samples retained their oligomeric state after radioiodination.

Biodistribution Studies.
Biodistribution studies were performed under a license (Tumor Targeting, Bewilligung 53/97) issued to D .N. by the Veterinäramt des Kantons Zürich. Nude mice (10–14 week-old CD1 nude mice, females) were obtained from LTK (Zurich, Switzerland). F9 murine teratocarcinomas were implanted s.c. as described previously (12 , 17) . When tumor size reached 100–300 mg, 1 µg (1 µCi) of scFv in 150 µl of saline solution, radiolabeled on the same day, was injected i.v. Previous biodistribution experiments with anti-ED-B Abs and F9 teratocarcinoma had shown that the amount of scFv injected is well below the dose necessary to saturate the tumor antigen pool. In fact, at least 10 µg of scFv per gram of tumor can selectively localize in F9 teratocarcinoma (12) . Furthermore, biodistribution experiments performed with two radiolabeled Ab fragments directed against irrelevant antigens resulted in tumor:blood ratios of <1 at 1–24 h after injection.

Mice were killed at 4 and 24 h after injection, and organs were weighed and radioactively counted. Three animals were used for each time point. Targeting results of representative organs are expressed as %ID of Ab/g of tissue. The mean error and SE for each group of data were calculated, and T:O ratios were determined. Significance levels were calculated using Student’s t test.

Immunohistochemical studies with cryostat sections of F9 murine teratocarcinoma were performed as described previously (17) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Note Added in Proof
 REFERENCES
 
Ab Fragment.
We have recently described (18) the isolation of two human scFv Ab fragments, E1 and L19, with dissociation constants for the ED-B domain of fibronectin of 41 nM and 0.054 nM, respectively. L19 is an affinity-matured version of E1, obtained by combinatorial mutagenesis of judiciously selected residues in the hypervariable loops of E1 and selected using phage display technology (18 , 23) . The enhanced binding affinity of L19 is mainly due to a lower kinetic dissociation constant (18) . Immunohistochemistry studies with tumor sections have demonstrated that both Abs recognize (ED-B)-containing fibronectin in vascular structures (18) .

To assess how binding affinity influences tumor targeting properties, it is essential to examine Abs that recognize the same antigen and the same epitope with different affinity values (10) . Previous experiments that were aimed at affinity-maturing recombinant Abs using phage display technology have shown that affinity mutants retain the epitope of the parental Ab (12 , 24, 25, 26, 27, 28) . To confirm that E1 and L19 recognize the same epitope, we studied their binding to the ED-B domain of fibronectin covalently immobilized on a BIAcore microsensor chip (12 , 22) by real-time interaction analysis with surface plasmon resonance detection. Fig. 1, a and bCitation , shows the BIAcore sensograms of scFv(L19) and scFv(E1) binding to ED-B. The two Abs did not bind to microsensor chips coated with irrelevant antigens (streptavidin, glutathione S-transferase, BSA, and hen egg lysozyme; data not shown). The different kinetic stability of the two Ab-antigen complexes can be observed in the portion of the sensogram during which the complexes are washed with a flow of buffered saline and are allowed to dissociate. We then saturated the ED-B-coated microsensor chip with four successive injections of L19 (amount of Ab bound, 1100 RUs) and tested whether E1 could still bind to the antigen at a comparable level of RUs (Fig. 1c)Citation . As expected, L19 inhibited the binding of E1 to the ED-B domain of fibronectin, which confirmed that the epitope recognized by the two Abs was retained during affinity maturation.



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  		Fig. 1. Epitope retention assay of anti-ED-B Abs. The binding properties of anti-ED-B Abs L19 and E1 were investigated by real-time interaction analysis with surface plasmon resonance, using a BIAcore instrument and a microsensor chip coated with recombinant ED-B domain of fibronectin. L19 and E1 Abs were injected onto the microsensor chip either individually (a and b) or sequentially (c).

 
To further confirm that E1 and L19 retain the same binding specificity, immunoblot analysis was performed with fibronectins and fibronectin fragments. Both E1 and L19 reacted with ED-B-containing fibronectin and fibronectin fragments but not with plasma fibronectin, with fragments devoid of the ED-B domain, or with tenascin-C, a large multidomain protein containing several fibronectin-like modules (Fig. 2)Citation .



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  		Fig. 2. Specificity of E1 and L19 Abs. Coomassie Blue-stained polyacrylamide gel (a) and immunoblot analysis, performed with L19 and E1 Ab (b), of proteins containing fibronectin or fibronectin-like repeats. Lanes: 1, recombinant fibronectin fragment 7B89 (containing repeats 7, ED-B, 8, and 9); 2, recombinant fibronectin fragment 789 (containing repeats 7, 8, and 9); 3, plasma fibronectin; 4, ED-B-containing fibronectin from WI38VA cells; 5, tenascin-C from SKMEL-28 cells. The preparation of these proteins was described previously (14) . The molecular weight standards (M) are expressed in Mr thousands.

 
Most scFvs are a mixture of monomer and homodimer (Fig. 3aCitation ; Refs. 19 , 29, 30, 31 ). Although the affinity of these molecular species toward a monomeric antigen are identical when measured in solution (32) , the functional affinity (or "avidity") of the dimer toward an immobilized antigen on tumoral vasculature can be significantly increased by rebinding effects and by the simultaneous binding of two antigens (32 , 33) . Furthermore, monomeric and dimeric scFvs have different sizes and pharmacokinetics (11 , 34) . It is, therefore, important that one works with purified Abs of a defined oligomeric state to study the role of binding affinity in the targeting of angiogenesis.



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  		Fig. 3. Monomeric and dimeric fractions of scFvs. a, scFvs exist as a slowly interconverting equilibrium between a monomeric and a dimeric form. b, separation of monomeric and dimeric fractions of scFv(L19) by gel filtration on a Superdex 75 HR10/30 column and analysis of the purified fractions by nondenaturing gel electrophoresis. Lanes: 1, input; 2, dimer; 3, mixture of monomer and dimer; 4, monomer. c, autoradiogram of a SDS-PAGE and a nondenaturing PAGE of radioiodinated scFv(L19) monomer (Lane 1) and dimer (Lane 2). Essentially identical results were obtained for scFv(E1).

 
The monomeric and dimeric fractions of scFv(L19) and scFv(E1) were separated and quantitatively recovered by gel-filtration. A representative chromatogram is presented in Fig. 3bCitation , together with the analysis of the recovered fractions performed by native PAGE. The recovered Ab fractions were labeled with 125I using the Iodogen method (35) . Ab immunoreactivity after radiolabeling ranged between 81% and 92% (see "Materials and Methods"). Purified monomeric and dimeric scFvs (Fig. 3c)Citation incubated at 22°C for 5 days were shown by native PAGE to be stable before and after radiolabeling.

Both E1 and L19 monomeric and dimeric fractions showed comparable reactivity in immunohistochemistry with cryostat sections of F9 teratocarcinoma performed as described previously (Ref. 14 ; Fig. 4Citation ), suggesting that the Ab preparations conserve the same specificity toward new-forming blood vessels. We had previously shown that Ab affinity does not affect performance in immunohistochemistry (14) .



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  		Fig. 4. Immunohistochemistry of F9 murine teratocarcinoma. Immunohistochemical findings obtained with a cryostat section of F9 murine teratocarcinoma stained with scFv(L19) monomer. New-forming blood vessels are strongly stained in red by the monoclonal Ab. Cell nuclei are counterstained with Gill’s hematoxylin. Note that the tumor vasculature represents only a small percentage of the total tumor mass. Scale bar, 10 µm.

 
Biodistribution Studies.
We chose the F9 murine teratocarcinoma grafted s.c. in nude mice (12) as a syngeneic tumor model for evaluating Ab performance in the targeting of angiogenesis. Because the ED-B domain of fibronectin is identical in mouse and man, the results of our tumor targeting studies should be predictive of the Ab performance in humans. Fig. 4Citation shows that new-forming blood vessels of a section of F9 teratocarcinoma are strongly and specifically stained in red by scFv(L19) using immunohistochemical techniques. The neovasculature represents only a low percent of the total F9 tumoral mass.

Biodistribution studies at 4 h and 24 h were performed to examine the role of Ab affinity on the specificity and the degree of tumor retention. One µg of purified radioiodinated scFv (1 µCi) in either monomeric or dimeric form was administered to the mice by i.v. injection. The biodistribution results (Tables 1Citation and 2Citation ) revealed a correlation between the affinity and the %ID of radioiodinated Ab/g delivered to the tumor.


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  		Table 1 Biodistribution of 125I-labeled E1 monomer and 125I-labeled E1 dimer in F9 tumor-bearing nude mice over 4 and 24 h

 

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  		Table 2 Biodistribution of 125I-labeled L19 monomer and 125I-labeled L19 dimer in F9 tumor-bearing nude mice over 4 and 24 h

 
The tumor retention of both monomeric and dimeric L19 was significantly better than that of E1 (confidence level, >95% for each pairwise comparison of L19 and E1 at 4 h and 24 h). The amount of E1 monomer in the tumor decreased by more than 10-fold from 4 h to 24 h, compatibly with its more rapid dissociation from the antigen observed in vitro (Fig. 1bCitation ; Ref. 18 ). The time dependence of Ab retention in the tumor progressively improved for E1 dimer, L19 monomer, and L19 dimer. The latter Ab was retained in relatively large amounts in F9 teratocarcinoma (20.2 %ID/g at 4 h; 11.8 %ID/g at 24 h), the decrease in the %ID/g being due, at least in part, to the fact that the F9 teratocarcinoma doubles in size in 24–48 h. As previously described for different recombinant Abs (11 , 34 , 36) , dimerization resulted in prolonged Ab retention in the blood and in larger %ID/g retained in tumor and in normal tissues, compared with the corresponding monomer.

The higher affinity L19 Ab was significantly superior to E1 also in terms of T:O ratios (confidence levels ranged between 80% and 99.99% for all of the pairwise comparisons of T:O ratios at 4 h and 24 h). Comparisons of the results obtained with monomeric and dimeric preparations revealed that T:O ratios were not significantly influenced by the Ab valence. However, T:O values for L19 dimer at 24 h were consistently better than those of the other Abs analyzed because of the longer Ab retention in the tumor.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Note Added in Proof
 REFERENCES
 
To our knowledge, this work represents the first quantitative examination of the effects of affinity and valence on the targeting of tumoral vasculature by recombinant Abs. We have investigated the tumor targeting properties of two Abs, E1 and L19, in monomeric and homodimeric form, which are specific for the ED-B domain of fibronectin, a marker of angiogenesis (13, 14, 15, 16, 17) . E1 and L19 bind to an identical epitope on the antigen with dissociation constants of 41 nM and 0.054 nM, respectively.

Our tumor model was an aggressive murine F9 teratocarcinoma tumor s.c. grafted in mice. It is generally believed that syngeneic models allow a better evaluation of Ab specificity than do models in which a human tumor marker is present in a human tumor xenografted in mice but is otherwise absent in other murine tissues. The situation with the ED-B domain of fibronectin is particularly favorable; because its amino acid sequence is identical in mouse and man, the results of our biodistribution experiments in mice may to some extent be predictive of the performance of the Abs in humans.

High-affinity L19—both monomer and dimer—target the tumoral neovasculature significantly better than their E1 counterparts (Tables 1Citation and 2Citation ). Tumor:blood ratios were already greater than 2 at 4 h after administration, suggesting the potential usefulness of L19 for the immunoscintigraphic detection of angiogenesis in patients—namely an application in which short-lived radionuclides such as 99mTc or 123I have to be used to minimize exposure to radiation. Moreover, tumor:kidney and tumor:liver ratios, which are often problematic for intact monoclonal Abs and for some Ab fragments (37) , were extremely favorable. Additional advantages are that recombinant human Abs: (a) can be expressed in large amounts in bacteria (38 , 39) and yeast (40) ; (b) are not immunogenic; and (c) are easy to validate for clinical applications (41) .

L19 dimer accumulates in the tumor in significantly larger amounts than does L19 monomer, both at 4 h and 24 h, given its longer half-life in blood. This observation was somewhat surprising considering the extremely high affinity of monomeric L19 and the good accessibility of its antigen, which is localized prevalently around new-forming blood vessels (Fig. 4). We can postulate two different explanations for the fact that targeting of the ED-B domain of fibronectin in vivo is a relatively slow process: (a) microcirculation in new-forming blood vessels could be impaired, preventing the Abs from rapidly reaching their target; or (b) the process of crossing the endothelial layer, which precedes the Abs’ binding to the fibronectin isoform located in the abluminal site of the vessel, could be slow because of interstitial pressure (9) . In either case, it is interesting to notice that bivalence and slower clearance of the Ab appear to drive the targeting process, despite the increased Ab size.

The investigation of the role of Ab affinity on tumor targeting performance has been addressed experimentally in the past with binders directed against tumor markers located on the cell membrane (42) . Very recently, Adams et al.(10) reported the first biodistribution study performed with Ab affinity mutants directed against the same epitope of HER2/neu. This more rigorous experimental approach excludes the possibility that differences in targeting performance may be due to different degrees of accessibility of the epitopes within the tumor. Their findings that increased affinity leads to improved selective tumor delivery by monomeric scFvs are in good agreement with our results obtained here with Abs directed against a marker of angiogenesis.

The amount of L19 dimer selectively delivered to the tumor compares favorably with the tumor retention of other recombinant Ab fragments directed against cellular markers assayed in different tumor models in mice (for a review, see Refs. 43 , 10 , 11 , 34 , 37 , and references therein). Considering that tumoral neovasculature represents only a small percentage of the total tumor mass (Fig. 4)Citation , the %ID of L19 retained per gram of vasculature is extremely high and could have an impact in the diagnosis and therapy of tumors with high angiogenetic index. It will be interesting to evaluate the usefulness of L19 for the detection of angiogenesis in patients by immunoscintigraphic (44) and IR fluorescence (12 , 45) methodologies, as well as its performance as a selective vehicle for delivering toxic agents to tumor neovasculature (2, 3, 4, 5, 6, 7, 8 , 46) . Work in this direction is in progress in our laboratories.


   Note Added in Proof
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note Added in Proof
REFERENCES
 
Recently, Brekken et al. have reported the successful qualitative targeting of tumor angiogenesis using specific monoclonal antibodies against VEGF:receptor complex (47) .


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by an internal Grant of the ETH Zürich (to D. N.), by the Stiftung zur Krebsbekämpfung (to D. N.), and by the Associazione Italiana Ricerca sul Cancro (to L. Z.). Back

2 To whom requests for reprints should be addressed, at Institut für Molekularbiologie und Biophysik, ETH Hönggerberg, CH-8093 Zürich, Switzerland. E-mail: dario{at}mol.biol.ethz.ch Back

3 The abbreviations used are: Ab, antibody; scFv, single-chain Fv Ab fragment; T:O, tumor:organ; %ID, percent injected dose; RU, (BIAcore) resonance unit. Back

Received 7/27/98. Accepted 11/11/98.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Note Added in Proof
 REFERENCES
 

  1. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med., 1: 27-31, 1995.[Medline]
  2. O’Reilly M. S., Holmgren L., Chen C., Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat. Med., 2: 689-692, 1996.[Medline]
  3. O’Reilly M. S., Boehm T., Shing Y., Fukai N., Vasios G., Lane W. S., Flynn E., Birkhead J. R., Olsen B. R., Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumour growth. Cell, 88: 277-285, 1997.[Medline]
  4. Friedler M., Brooks P. C., Shaffer R. W., Kincaid C. M., Varner J. A., Cheresh D. A. Definition of two angiogenic pathways by distinct {alpha}-v integrins. Science (Washington DC), 270: 1500-1502, 1995.[Abstract/Free Full Text]
  5. Pasqualini R., Koivunen E., Ruoslahti E. {alpha}-v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol., 15: 542-546, 1997.[Medline]
  6. Huang H., Molema G., King S., Watkims L., Edgington T. S., Thorpe P. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor neo-vasculature. Science (Washington DC), 275: 547-550, 1997.[Abstract/Free Full Text]
  7. Kim K. J., Li B., Winer J., Armanini M., Gillett N., Phillips H. S., Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature (Lond.), 362: 841-844, 1993.[Medline]
  8. Schmidt-Erfurth U., Diddens H., Birngruber R., Hasan T. Photodynamic targeting of human retinoblastoma cells using covalent low-density lipoprotein conjugates. Br. J. Cancer, 75: 54-61, 1997.[Medline]
  9. Clauss M. A., Jain R. K. Interstitial transport of rabbit and sheep antibodies in normal and neoplastic tissues. Cancer Res., 50: 3487-3492, 1990.[Abstract/Free Full Text]
  10. Adams G. P., Schier R., Marshall K., Wolf E. J., McCall A. M., Marks J. D., Weiner L. M. Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies. Cancer Res., 58: 485-490, 1998.[Abstract/Free Full Text]
  11. King D. J., Turner A., Farnsworth A. P. H., Adair J. R., Owens R. J., Pedley R. B., Baldock D., Proudfoot K. A., Lawson A. D. G., Beeley N. R. A., Millar K., Millican T. A., Boyce B. A., Antoniw P., Mountain A., Begent R. H. J., Schocat D., Yarranton G. T. Improved tumor targeting with chemically cross-linked recombinant antibody fragments. Cancer Res., 54: 6176-6185, 1994.[Abstract/Free Full Text]
  12. Neri D., Carnemolla B., Nissim A., Balza E., Leprini A., Querzè G., Pini A., Tarli L., Halin C., Neri P., Zardi L., Winter G. Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform. Nat. Biotechnol., 15: 1271-1275, 1997.[Medline]
  13. Zardi L., Carnemolla B., Siri A., Petersen T. E., Paolella G., Sebastio G., Baralle F. E. Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. EMBO J., 6: 2337-2342, 1987.[Medline]
  14. Carnemolla B., Balza E., Siri A., Zardi L., Nicotra M. R., Bigotti A., Natali P. G. A tumor-associated fibronectin isoform generated by alternative splicing of messanger RNA precursors. J. Cell Biol., 108: 1139-1148, 1989.[Abstract/Free Full Text]
  15. Castellani P., Viale G., Dorcaratto A., Nicolò G, Kaczmarek J., Querzè G., Zardi L. The fibronectin isoform containing the ED-B oncofetal domain: a marker of angiogenesis. Int. J. Cancer, 59: 612-618, 1994.[Medline]
  16. Kaczmarek J., Castellani P., Nicolò G., Spina B., Alemanni G., Zardi L. Distribution of oncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues. Int. J. Cancer, 58: 11-16, 1994.
  17. Carnemolla B., Neri D., Castellani P., Leprini A., Neri G., Pini A., Winter G., Zardi L. High-affinity human recombinant antibodies to the oncofetal angiogenesis marker fibronectin ED-B domain. Int. J. Cancer, 68: 397-405, 1996.[Medline]
  18. Pini A., Viti F., Santucci A., Carnemolla B., Zardi L., Neri P., Neri D. Design and use of a phage-display library: human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J. Biol. Chem., 273: 21769-21776, 1998.[Abstract/Free Full Text]
  19. Neri D., Petrul H., Light Y., Marais R., Britton K. E., Winter G., Creighton A. M. Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [{gamma}-32P]-ATP. Nat. Biotechnol., 14: 385-390, 1996.
  20. Sambrook J., Fritsch E. F., Maniatis T. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, : Cold Spring Harbor Laboratory Press NY 1990.
  21. Munro S., Pelham H. R. B. An Hsp-like protein in the ER: identity with the 78 kD glucose regulated protein and immunoglobulin heavy chain binding protein. Cell, 46: 291-300, 1986.[Medline]
  22. Jönsson. U., Fägerstam L., Ivarsson B., Johnsson B., Karlsson R., Lundh K., Löfås S., Persson B., Roos H., Rönnberg I., Sjöler S., Stenberg E., Ståhlberg,Urbaniczky C, Östlin H., Malmqvist M. Real-time bispecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques, 11: 620-627, 1991.[Medline]
  23. Winter G., Griffiths A. D., Hawkins R. E., Hoogenboom H. R. Making antibodies by phage display technology. Annu. Rev. Immunol., 12: 433-455, 1994.[Medline]
  24. Figini M., Marks J. D., Winter G., Griffiths A. D. In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation. J. Mol. Biol., 239: 68-78, 1994.[Medline]
  25. Jespers L. S., Roberts A., Mahler S. M., Winter G., Hoogenboom H. R. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Bio/Technology, 12: 899-903, 1994.[Medline]
  26. Low N., Holliger P., Winter G. Mimicking somatic hypermutations: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J. Mol. Biol., 260: 359-368, 1996.[Medline]
  27. Yang W., Green K., Pinz-Sweeney S., Briones A. T., Burton D. R., Barbas C. F., III CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-21 antibody into the picomolar range. J. Mol. Biol., 254: 392-403, 1995.[Medline]
  28. Schier R., Balint R. F., McCall A., Apell G., Larrick J. W., Marks J. D. Identification of functional and structural amino acid residues by parsimonious mutagenesis. Gene (Amst.), 169: 147-155, 1996.[Medline]
  29. Huston J. S., Levinson D., Mudgett H. M., Tai M. S., Novotny J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R., Oppermann H. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA, 85: 5879-5883, 1988.[Abstract/Free Full Text]
  30. Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins. Science (Washington DC), 242: 423-426, 1988.[Abstract/Free Full Text]
  31. Holliger P., Prospero T., Winter G. "Diabodies": small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA, 90: 6444-6448, 1993.[Abstract/Free Full Text]
  32. Neri D., Montigiani S., Kirkham P. M. Biophysical methods for the determination of antibody-antigen affinities. Trends Biotechnol., 14: 465-470, 1996.[Medline]
  33. Crothers D. M., Metzger H. The influence of polyvalency on the binding properties of antibodies. Immunochemistry, 9: 341-357, 1972.[Medline]
  34. Adams G. P., McCartney J. E., Tai M. S., Oppermann H., Huston J. S., Stafford W. F., Bookman M. A., Fand I., Houston L. L., Weiner L. M. Highlly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single chain Fv. Cancer Res., 53: 4026-4034, 1993.[Abstract/Free Full Text]
  35. Salacinski P. R. P., McLean C., Sykes J. E. C., Clement-Jones V. V., Lowry P. J. Iodination of proteins, glycoproteins and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3{alpha},6 {alpha} -diphenyl glycoluril (Iodogen). Anal. Biochem., 117: 136-146, 1981.[Medline]
  36. Wu A. M., Chen W., Raubitschek A., Williams L. E., Neumaier M., Fischer R., Hu S., Odom-Maryon T., Wong J. Y. C., Shively J. E. Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnology (Shannon), 2: 21-36, 1996.[Medline]
  37. Rowlinson-Busza G., Deonarain M. P., Epenetos A. A. Comparison of intact monoclonal antibodies, its F(ab')2 and Fab fragments and recombinant scFv in a human tumour xenograft model. Tumor Targeting, 2: 37-48, 1996.
  38. Carter P., Kelley R. F., Rodrigues M., Snedecor B., Covarrubias M., Velligan M. D., Wong W. L. T., Rowland A. M., Kotts C. E., Carver M. E., Yang M., Bourell J. H., Shephard H. M., Henner D. High level Escherichia coli expression anp production of a bivalent humanized antibody fragment. Bio/Technology, 10: 163-167, 1992.[Medline]
  39. Pack P., Kujau M., Schroeckh V., Knüpfer U., Wenderoth R., Riesenberg D., Plückthun A. Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli. Bio/Technology, 11: 1271-1277, 1993.[Medline]
  40. Ridder R., Schmitz R., Legay F., Gram H. Generation of rabbit monoclonal antibody fragments from a combinatorial phage display lybrary and their production in the yeast Pichia Pastoris. Bio/Technology, 13: 255-260, 1995.[Medline]
  41. Casey J. L., Keep K. A., Chester L., Robson L., Hawkins R. E., Begent R. H. Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography. J. Immunol. Methods, 179: 105-116, 1995.[Medline]
  42. Colcher D., Minelli F. M., Roselli M., Muraro R., Simpson-Milenic D., Schlom J. Radioimmunolocalization of human carcinoma xenografts with B72.3 second generation monoclonal antibodies. Cancer Res., 48: 4597-4603, 1988.[Abstract/Free Full Text]
  43. Kosmas C., Linardou H., Epenetos A. A. Advances in monoclonal antibody tumour targeting. J. Drug Target., 1: 81-91, 1993.[Medline]
  44. Begent R. H. J., Verhaar M. J., Chester K. A., Green A. J., Napier M. P., Hope-Stone L. D., Cushen N., Keep P. A., Johnson C. J., Hawkins R. E., Hilson A. J. W., Robson L. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat. Med., 2: 979-984, 1996.[Medline]
  45. Folli S., Westermann P., Braichotte D., Pelegrin A., Weignieres G., van der Bergh H., Mach J. P. Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice. Cancer Res., 54: 2643-2649, 1994.[Abstract/Free Full Text]
  46. Neri D., Zardi L. Affinity reagents against tumor-associated extracellular molecules and new-forming vessels. Adv. Drug Delivery Rev., 31: 43-52, 1998.[Medline]
  47. Brekken R. A., Huang X., King S. W., Thorpe P. E. Vascular endothelial growth factor as a marker of tumor endothelium. Cancer Res., 58: 1952-1958, 1998.[Abstract/Free Full Text]



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