Pharmacokinetics and clinical evaluation of 125I-radiolabeled humanized CC49 monoclonal antibody (HuCC49deltaC(H)2) in recurrent and metastatic colorectal cancer patients

INTRODUCTION: CC49 is an antitumor monoclonal antibody that is promising for use in radioimmunoguided surgery (RIGS). However, the murine antibody has been limited by human antimouse antibody (HAMA) response and slow clearance. This study examined the pharmacokinetics and tissue localization of a humanized domain-deleted CC49 antibody (HuCC49DeltaC(H)2 MAb) in humans. METHODS: Twenty-one patients with colorectal carcinoma were given 1 mg intravenous (I.V.) bolus of HuCC49DeltaC(H)2 MAb radiolabeled with 2 mCi (125)I after thyroid blockade. The level of circulating HuCC49DeltaC(H)2 MAb was measured daily as precordial counts using a handheld gamma-detecting probe. Each patient underwent an exploratory laparotomy on postinjection days 3-20. Gamma counts were measured at normal organs, aortic bifurcation (AB), and both clinically evident and occult tumors. RESULTS: Precordial and AB gamma counts showed an excellent linear correlation. HuCC49DeltaC(H)2 MAb followed a two-compartment pharmacokinetic model. Normal organs and AB showed similar exposures to HuCC49DeltaC(H)2 MAb, while HuCC49DeltaC(H)2 MAb favorably distributed into tumors from day 3. Intestinal and metastatic liver lesions showed the highest partition coefficients. All patients showed no HAMA response. DISCUSSION: C(H)2 region deletion of HuCC49DeltaC(H)2 MAb did not alter the pharmacokinetics compared to murine CC49. The favorable partition coefficient K of HuCC49DeltaC(H)2 MAb into tumors supports its use in RIGS.     

A humanized single-chain variable fragment antibody against beta3 integrin in Escherichia coli

Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique antibody (Ab) against platelet GPIIIa49-66, which is capable of inducing oxidative platelet fragmentation in the absence of complement activation. By screening a human phage antibody library with the GPIIIa49-66 peptide as bait, we have developed several humanized phage Abs, which act similarly to the parental Ab. However, the presence of a stop codon in the heavy chain of the obtained phage clones limits their expression in soluble recombinant form. To circumvent this problem, we mutated the stop codon inside clone 11 that exhibits the highest binding activity to platelet GPIIIa49-66, resulting in a soluble scFv format (named A11) in Escherichia coli Rosseta. In in vitro binding assay, A11 exhibited similar binding specificity to parental Ab at various concentrations. Moreover, A11 is able to induce oxidative platelet fragmentation by preferentially binding to activated versus resting platelets. These findings provide a proof-of-principle for the development of a novel approach to inhibit arterial thrombosis by generating a selective scFv for the lysis of platelet-rich thrombi.     

A CDR-grafted (humanized) domain-deleted antitumor antibody

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.     

A novel monoclonal antibody design for radioimmunotherapy

The generation of chimeric and complementary-determining region (CDR) grafted monoclonal antibodies (MAb) have reduced the immunogenicity problem in the clinical application of radioimmunotherapy with monoclonal antibodies. However, humanization (Hu) has prolonged the circulation (plasma T1/2) of radiolabeled antibodies, resulting in an increased normal tissue exposure to radioactivity and greater dose-limiting bone marrow suppression. To overcome this problem, a tumor-associated glycoprotein (TAG)-72-specific CDR grafted MAb with C(H)2 domain deletion (DeltaC(H)2) was developed from the MAb CC49. Preclinical studies have demonstrated that HuCC49 DeltaC(H)2 clears more rapidly from the plasma of mice than HuCC49. This preliminary report describes the initial human experience with HuCC49 DeltaC(H)2 radiolabeled with 131I and administered to patients with metastatic colorectal carcinoma. In this pilot study we enrolled four patients who received a single infusion of 20 mg of HuCC49 DeltaC(H)2 (total protein dose) labeled with 10 mCi of 131I. Pharmacokinetics, biodistribution, dosimetry, and immune response were evaluated over 2-6 weeks. No toxicity was observed in this group of patients. A one-compartment bolus model using the non-linear (NLIN) procedures in Statistical Analysis Software (SAS; SAS, Incorporated, Cary, NC) best describes the pharmacokinetics of the 131I-HuCC49 DeltaC(H)2 with a plasma mean T1/2 of 20 +/- 3 hours, a mean residence time (MRT) of 29 +/- 4 hours and a clearance rate (Cl) of 1.5 +/- 0.1 mL/hours/kg. The whole body and marrow radiation dose estimates were 0.55 +/- 0.06 rad/mCi and 1.00 +/- 0.14 rad/mCi, respectively. All patients had positive localization of antibody to metastatic tumor sites. The 131I-HuCC49 DeltaC(H)2 biodistribution was similar to murine CC49. Three patients had no evidence of antibody response to HuCC49 DeltaC(H)2 over 6 weeks of observation, and one patient had a marginal response by week 6. Intravenous administration of HuCC49 DeltaC(H)2 is safe and well tolerated. The deleted C(H)2 construct has a shorter half-life compared with prior studies of murine CC49 but with similar biodistribution and low immunogenicity. These studies support the further clinical investigation of this agent in phase I trials by intravenous and intraperitoneal routes.     

Comparisons between two monoclonal antibodies that bind to the same antigen but have differing affinities: uptake kinetics and 125I-antibody therapy efficacy in multicell spheroids.

It has been predicted that low affinity antibodies (Abs) should penetrate into tumors more readily than high affinity Abs. However, the absolute uptake and residence time of a high affinity Ab may be better. It is, therefore, not clear whether a high affinity Ab would have a therapeutic advantage. This is particularly relevant with 125I radioimmunotherapy, where targeting of every cell is important. This study compared the uptake kinetics and toxicity in multicell spheroids of two murine monoclonal Abs labeled with 125I. 17-1A was produced by immunization with a human colon cancer cell line and has an affinity of 5.15 x 10(7) M-1. 323/A3 was produced by immunization with a human breast cancer cell line and has an affinity of 1.87 x 10(9) M-1. Binding of both Abs to LS174T spheroids was similar at 4 degrees C, but binding of 17-1A was 8-10-fold less than that of 323/A3 at 37 degrees C. Despite this difference, the toxicity of 125I-17-1A in spheroids after 7 days of incubation was similar to that of 125I-323/A3. Autoradiography showed that 17-1A penetrated the spheroids much more deeply and evenly than did 323/A3. It appears that much of the radiation dose to spheroids treated with 125I-323/A3 was wasted because of the uneven Ab distribution. This study demonstrates the potential advantage of using Abs of lower affinity for 125I radioimmunotherapy, because of their more even distribution. It also suggests that a large number of binding sites per cell may be a disadvantage if more 125I is bound than is necessary to kill the cell, because this may slow Ab penetration.     

Pharmacokinetics and tumor localization of (111)in-labeled HuCC49DeltaC(H)2 in BALB/c mice and athymic murine colon carcinoma xenograft

The primary limitation of IgG antibodies for radioimmunotherapy of solid tumors is their prolonged serum half-life, leading to dose-limiting bone marrow toxicity at doses providing inadequate radiation to the tumor. A humanized C(H)2 domain-deleted variant of the anti-TAG-72 antibody CC49 (HuCC49DeltaC(H)2) has faster blood clearance, compared to the IgG, while retaining tumor targeting. We compared the pharmacokinetics and tumor uptake of (111)In-HuCC49DeltaC(H)2 in BALB/c mice and a colon carcinoma (LS-174T) mouse xenograft with that of (111)In-labeled chimeric CC49 (cCC49), an antibody with pharmacokinetics similar to the humanized CC49 parent. Immuno-conjugates of HuCC49DeltaC(H)2 and cCC49 prepared with the (111)In chelator Mx-DTPA (1-isothiocyantobenzyl-3-methyldiethylenetriaminepentaacetic acid) retained low nM affinity and radiolabeling protocols provided greater than 95% radio-incorporation with (111)In while retaining greater than 80% immunoreactivity. Blood clearance of (111)In-HuCC49DeltaC(H)2 in BALB/c mice was monoexponential (t(1/2) 5.4 hours) and faster than (111)In-cCC49 (biexponential clearance; t1/2Delta 1.5 hours; t1/2beta 162 hours). The (111)In-HuCC49DeltaC(H)2 also cleared more rapidly from the blood in the murine xenograft. At 1 hour postinjection, blood concentrations for (111)In-HuCC49DeltaC(H)2 and (111)In-cCC49 were comparable (25.5 injected dose per g [%ID/g] and 21.3 %ID/g, respectively); tumor uptake for (111)In- HuCC49DeltaC(H)2 was 7.9 %ID/g, compared to 7.5 %ID/g for (111)In-cCC49. However, at 24 hours, blood concentration for (111)In-HuCC49DeltaC(H)2 was less than (111)In-cCC49 (0.9 %ID/g versus 5.2 %ID/g, respectively) with comparable tumor retention (14.4 %ID/g versus 19.0 %ID/g, respectively). Faster blood clearance of (111)In-HuCC49DeltaC(H)2 and tumor localization comparable to that of (111)In-cCC49 provided a fourfold improved tumor-to-blood ratio for (111)In-HuCC49DeltaC(H)2 at 24 hours postinjection.     

Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs.

One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.     

Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate

Enzyme-antibody (Ab) conjugates specific for IgG are widely used in indirect immunological assays and have been until recently routinely prepared with polyclonal IgG-specific animal Abs. The use of monoclonal Abs (MAbs) could permit a better standardization of the IgG-specific conjugate reagents but is expected to result in lower reactivity due to the recognition of a single epitope by the MAbs. In this work, we have characterized a monoclonal anti-human IgG-peroxidase (HRP) reagent and compared its reactivity with commercial reagents. The murine C5-1 anti-human IgG MAb was selected for conjugation because of its high affinity (K(a) = 1.9 x 10(10)M), pan-IgG reactivity and absence of cross-reactivity with various structures including animal IgGs. The specific activity and binding kinetics of the C5-1:HRP conjugate were similar to the ones of two polyclonal anti-IgG:HRP conjugates when tested with immobilized human IgG. The C5-1:HRP conjugate could detect low amounts of human IgG much more effectively than two commercial monoclonal conjugates although it was slightly less effective than a polyclonal conjugate. However, the C5-1 conjugate yielded reduced background reactivity compared to the polyclonal conjugate, resulting in similar signal-to-noise ratios. These results indicate that the C5-1:HRP conjugate could be a suitable substitute for anti-human IgG conjugates prepared from animal antisera.     

Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients

Heat shock proteins (HSPs) are components of a physiologic stress response that are also over-expressed in various cancers including non-small cell lung cancer (NSCLC). During NSCLC serum-antibody screening of a NSCLC cDNA T7 phage library for immunogenic proteins we isolated HSP70 and HSP90 proteins. Isolation of these proteins suggested that corresponding antibodies could be elevated in NSCLC patient sera, a novel finding that could pilot their use as markers of NSCLC. We showed histochemically that patient sera were more reactive with each phage-expressed protein than normal sera. Antibody affinity for each phage-expressed protein was confirmed by limiting the dilution of individual sera assayed by Ab enzyme-linked immunosorbent assay (ELISA). Sera from 49 NSCLC patients assayed by Ab ELISA and normalized to 40 controls showed that HSP70 antibodies were significantly greater in patient sera than in normals (P=0.0002), while HSP90 antibodies were not significantly different (P=0.11). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that HSP70 antibodies were modest markers of NSCLC (sensitivity 0.74 and specificity 0.73; area under the curve or AUC=0.731), while HSP90 antibodies appeared to be poor in both criteria with an AUC of 0.602. Further evaluation of HSP70 antibodies as potential markers of disease may be rational.     

Intraperitoneal radioimmunotherapy with a humanized anti-TAG-72 (CC49) antibody with a deleted CH2 region

The application of intraperitoneal (i.p.) radioimmunotherapy to treat i.p. tumor loci has been limited by bone marrow toxicity secondary to circulating radiolabeled antibodies. The generation of novel genetically engineered monoclonal antibodies, which can achieve high tumor uptake and rapid blood clearance, should enhance the therapeutic index of i.p. radioimmunotherapy. In this regard, a novel humanized anti-TAG-72 monoclonal antibody with a deleted CH2 region (HuCC49DeltaCH2) has been described, which localized well to subcutaneous xenograft tumors and had a rapid plasma clearance. The aim of this study was to examine the characteristics of this radiolabeled reagent when administered through the i.p. route in mice bearing i.p. tumor (LS174T). The DeltaCH2 molecule and intact humanized CC49 (HuCC49) monoclonal antibody were conjugated to PA-DOTA and radiolabeled with (177)Lu. Both molecules retained high-affinity binding to TAG-72 positive LS174T tumor cells in vitro. The radiolabeled DeltaCH2 molecule had a modest decrease in tumor localization, as compared to the intact molecule when administered i.p. to tumor-bearing mice and a dramatically shorter plasma disappearance T(1/2) at 2.7 hours compared to 61.2 hours for the intact antibody. The radiolabeled DeltaCH2 molecule thus had very high tumor:blood ratios. Using an (131)I-labeled system, the maximum tolerated dose of DeltaCH2 was >3x that of intact HuCC49. Autoradiography of tumors showed low radiation dose rates at tumor centers early (1 and 4 hours), as compared to higher dose rates at tumor periphery but a more uniform distribution by 24 hours. Dose-rate distributions were similar for both reagents. Animals bearing LS174T i.p. tumors were treated with 300 microCi of (177)Lu-labeled DeltaCH2 or intact HuCC49 by i.p. route daily x 3. The (177)Lu-DeltaCH(2) molecule mediated an increase in median survival compared to controls (67.5 +/- 7.5 days versus controls of 32 +/- 3.3) while the same dose of (177)Lu-HuCC49 produced early toxic deaths. These studies suggest that i.p. radioimmunotherapy using radiolabeled HuCC49DeltaCH2 should allow higher radiation doses to be administered with less marrow toxicity and potentially improved efficacy.     

Vaccination with ant-idiotype antibodies mimicking a renal cell carcinoma-associated antigen induces tumor immunity

We have previously isolated and characterized 6 different internal image mouse monoclonal anti-idiotype antibodies (Ab2) directed against the paratope of mouse monoclonal antibody G250 (MAbG250, Abl), which specifically reacts with human renal cell carcinoma (RCC). These Ab2s (NUH31,44,51, 71,82 and 91) demonstrated specificity for the combining site of AbI, and appeared to recognize 2 partly overlapping idiotopes on AbI. In this study, we further characterize the fine specificity of the Ab2, investigate whether the immunogenicity of Ab2 could be enhanced by conjugation to a carrier and investigate the anti-tumor efficacy of Ab3 sera in mice challenged with RCC. Immunization of animals with Ab2 conjugated to keyhole limpet hemocyanin as carrier protein resulted in a 2-fold increase in antigen-specific anti-anti-idiotype antibodies (Ab3) as compared with immunization using Ab2 alone. Specific reactivity was observed with antigen-positive cell lysates, and all Ab3 sera contained immunoglobulin resembling AbI (Abl), as shown by competitive AbI -antigen binding assays. Fine-specificity studies of Ab3 sera revealed that the Ab2s can be divided into 4 mutually exclusive groups, showing that the 6 Ab2s recognize 4 slightly different idiotopes in the Abl binding pocket. Treatment of RCC-challenged mice with Ab3 sera resulted in significant tumor growth inhibition and lower tumor take rates as compared with control groups. Ab3 sera obtained from NUH-91-immunized animals showed superior characteristics as compared to the other Ab3 sera: no tumors remained after 5 weeks of Ab3-NUH91 treatment. Our findings indicate that the Ab2 elicit powerful anti-tumor effects in immune-competent animals.     

Passively transferred anti‐MUC1 antibodies cause neither autoimmune disorders nor immunity against transplanted tumors in MUC1 transgenic mice

C57BL/6 mice transgenic for human MUC1 (MUC1.Tg) have been developed to investigate the autoimmune consequences of producing MUC1 tumor immunity in an animal that expresses MUC1 as a self‐protein on normal ductal epithelia. Previous work showed that MUC1.Tg mice challenged with MUC1‐bearing syngeneic tumors (B16.MUC1) developed progressively growing MUC1‐expressing tumors and no detectable MUC1‐specific antibody (Ab) response. In contrast, wild‐type C57BL/6 (wt) mice developed MUC1‐negative tumors at a significantly slower rate and produced approximately 50 μg IgG₁ Ab reactive with the MUC1 tandem repeat (TR)/ml of sera. One milliliter of these sera was administered passively to MUC1.Tg or wt mice and the concentration of the MUC1 TR‐reactive IgG₁ Abs was monitored over time. The results indicate that circulating MUC1‐reactive Abs were detectable in MUC1.Tg mice and that significant amounts of these Abs were not absorbed by organs that endogenously express MUC1. No evidence of autoimmune disease, either gross or histological, was observed in the MUC1.Tg recipients of sera suggesting that MUC1, an organ‐specific protein expressed primarily by secretory epithelia, is inaccessible to circulating MUC1‐reactive Abs. Additional studies showed that polyclonal sera containing IgG₁ Abs reactive with MUC1 TR were unable to provide protection against the growth of syngeneic tumors expressing MUC1 in the MUC1.Tg animal model. Int. J. Cancer 80:595–599, 1999. © 1999 Wiley‐Liss, Inc.     

Minimizing the immunogenicity of antibodies for clinical application.

The clinical utility of murine monoclonal antibodies has been greatly limited by the human anti-murine antibody responses they effect in patients. To make them less immunogenic, murine antibodies have been genetically engineered to progressively replace their murine content with that of their human counterparts. This review describes the genetic approaches that have been used to humanize murine antibodies, including the generation of mouse-human chimeric antibodies, veneering of the mouse variable regions, and the grafting of murine complementarity-determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining only those murine framework residues deemed essential for the integrity of the antigen-binding site. To minimize the anti-idiotypic responses that could still be evoked by the murine CDRs in humanized antibodies, two approaches have also been described. These are based on grafting onto the human frameworks the 'abbreviated' CDRs or only the specificity-determining residues (SDRs), the CDR residues that are involved in antigen interaction. The SDRs are identified through the help of the database of three-dimensional structures of antibody:antigen complexes or by mutational analysis of the antibody-combining site. In addition, we also describe the use of in vitro affinity maturation to enhance the binding affinity of humanized antibodies, as well as the manipulation of framework residues to maximize their human content and minimize their immunogenic potential.     

Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates.

The effect of the relative affinity (Ka) on the antitumor efficacy of monoclonal antibodies (MAbs) has been questioned. It has previously been shown in experimental models that the use of MAbs with higher relative Kas manifests itself in a higher percentage of injected dose of MAb bound to tumor. On the other hand, mathematical models have proposed that the use of higher affinity MAbs may be disadvantageous for antitumor effects, since higher Ka MAbs would bind more antigen and prevent penetration of MAb through tumor. To test this hypothesis, three MAbs reacting to the human pancarcinoma antigen TAG-72 were used as radioimmunoconjugates for therapeutic efficacy versus the LS-174T human colon carcinoma xenograft. MAbs B72.3, CC49, and CC83 have all been shown by depletion studies to react to the same molecule and to all react with overlapping epitopes. While the relative Ka of B72.3 is 2.5 x 10(9) M-1, the relative Kas of CC49 and CC83 are 16.2 and 27.7 x 10(9) M-1, respectively. Each MAb was radiolabeled with 131I, and each radioimmunoconjugate was assayed at five dose levels for therapeutic efficacy using the human xenograft model. The results of these studies demonstrate substantial therapeutic advantage of the higher affinity MAbs CC49 and CC83 versus B72.3 at every dose level. While 500 microCi of B72.3 were required to reduce tumor growth in only a minority of tumor-bearing animals, the use of the same amount or less of the radioimmunoconjugates of CC49 or CC83 resulted in strong antitumor effects in 80 to 100% of tumor-bearing animals. Thus, stronger antitumor effects were seen using as little as 2.5- to 3-fold less of the higher Ka immunoconjugates CC49 and CC83 as compared with B72.3. While we acknowledge the potential disadvantages of higher Ka MAbs in some situations, at least the experimental studies and model system described here show that a distinct therapeutic advantage exists with the use of higher affinity immunoconjugates.     

Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas

The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP(+) stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcinomas. Importantly, compared with the CDR-grafted humanized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting.     

Enhanced tumor binding using immunohistochemical analyses by second generation anti-tumor-associated glycoprotein 72 monoclonal antibodies versus monoclonal antibody B72.3 in human tissue

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and nonsmall cell lung cancer as well as weak or nondetectable reactivity to the majority of normal adult tissues, with the exception of secretory endometrium. Radiolabeled B72.3 has demonstrated MAb localization of carcinoma in approximately 70% of several hundred colorectal and ovarian carcinoma patients. The B72.3-reactive antigen, tumor-associated glycoprotein 72, has been purified from a human colon cancer xenograft and used as an immunogen to generate second generation MAbs. Twenty-eight of these MAbs, designated CC (colon cancer), were shown to be reactive with tumor-associated glycoprotein 72; direct-binding radioimmunoassays, Western blotting, live cell surface binding assays, liquid competition radioimmunoassays, and affinity constant measurements distinguished CC MAbs from each other and from B72.3. Two of these MAbs, CC49 and CC112, were selected for further immunohistochemical characterization. These MAbs were tested here against a spectrum of normal, benign, and malignant human adult tissues using the avidin-biotin-peroxidase technique, and their reactivity was compared with B72.3. Both CC MAbs were more reactive than B72.3 against a range of tumors. Extensive testing with MAbs CC49 and B72.3 using serial tissue sections demonstrated that both MAbs reacted similarly to most normal adult tissues with MAb CC49 reacting stronger to inflammatory colonic tissue. In 35 of 48 (72%) carcinoma biopsies of the gastrointestinal tract, ovary, breast, and lung in which one of the MAbs reacted to at least 20% of the cells, CC49 reacted to a greater percentage of carcinoma cells and/or tumor-associated mucin than B72.3. The reciprocal was observed in only 2% of the carcinomas. This study thus provides evidence that these second generation anti-tumor-associated glycoprotein MAbs may be more efficient than B72.3 in the further study of human carcinoma cell populations and in the diagnostic and therapeutic procedures presently being pursued with MAb B72.3.     

鼻咽癌人源抗独特型单链抗体的制备及筛选

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。结论:联