High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules.

Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.

Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies.

Mr 25,000 single-chain Fv (scFv) molecules are rapidly eliminated from the circulation of immunodeficient mice, yielding highly specific retention of small quantities of scFv in human tumor xenografts. We postulated that the specific retention of scFv in tumor could be enhanced by engineering significant increases in the affinity of the scFv for its target antigens. Affinity mutants of the human anti-HER2/neu (c-erbB-2) scFv C6.5 were generated by site-directed mutagenesis, which target the same antigenic epitope with a 320-fold range in affinity (3.2 x 10(-7) to 1.0 x 10(-9) M). In vitro, the Kd of each scFv correlated closely with the duration of its retention on the surface of human ovarian carcinoma SK-OV-3 cells overexpressing HER2/neu. In biodistribution studies performed in scid mice bearing established SK-OV-3 tumors, the degree and specificity of tumor localization increased significantly with increasing affinity. At 24 h after injection, tumor retention of the highest affinity scFv was 7-fold greater than that of a mutant with 320-fold lower affinity for HER2/neu. Because the rapid renal clearance of scFv may blunt the impact of improved affinity on tumor targeting, the distributions were also assayed in the absence of renal clearance (e.g., in mice rendered surgically anephric). In this model, the peak tumor retentions of the two higher affinity scFv approximated that reported previously for IgG targeting the same SK-OV-3 tumors in scid mice with intact kidneys. In contrast, the mutant with the lowest affinity for HER2/neu failed to accumulate in tumor, indicating the presence of an affinity threshold that must be exceeded for active in vivo tumor uptake. These results indicate that affinity can significantly impact the in vivo tumor-specific retention of scFv molecules.

Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis.

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.

Characterization of anti-mouse Fc gamma RII single-chain Fv fragments derived from human phage display libraries.

BACKGROUND: Few antibodies are available to study the function of the Fc gamma RII murine immunoglobulin receptor. Human phage display libraries represent a potential source of single-chain Fv (sFv) to facilitate the study of the Fc gamma RII murine immunoglobulin receptor. OBJECTIVES: To isolate human sFv specific for mouse Fc gamma RII. STUDY DESIGN: Two human phage display libraries were selected for reactivity to mouse Fc gamma RII. Those human anti-mouse Fc gamma RII sFv that were derived from the libraries were characterized with respect to kinetics, cellular binding, epitope specificity and amino acid sequence. RESULTS: Nine anti-mouse Fc gamma RII sFv molecules were isolated from two human phage display libraries (Marks et al., J Mol Biol 1991;222:581-597; Sheets et al., Proc Natl Acad Sci USA, in press). Surface plasmon resonance (SPR) analysis revealed that the human anti-mouse Fc gamma RII sFv had off-rates ranging from 10(-2) to 10(-3) s-1, with KD values calculated to range between 10(-7) and 10(-9) M. The binding of the FITC-labeled human anti-mouse Fc gamma RII sFv to mouse peritoneal neutrophils was not detected by flow cytometry, due to the rapid off-rates of these monomeric proteins. However, when the human anti-mouse Fc gamma RII sFv were coated on yellow-green latex particles, all of the human sFv were found to specifically bind to mouse peritoneal neutrophils. Deglycosylation of mouse Fc gamma RII did not diminish the binding of these sFv, suggesting that the sFv molecules recognize a polypeptide epitope on murine Fc gamma RII. In contrast, denaturation of mouse Fc gamma RII dramatically reduced the binding of the human sFv, suggesting that the epitopes are conformational. Sequence analysis of the human anti-mouse Fc gamma RII sFv revealed a high degree of structural similarity among the nine sFv. The DP73 VH gene segment was utilized by four of the nine sFv, while seven of the nine sFv contained the DPL16 V lambda gene segment. The sequence similarities between these sFv suggested that several of the human sFv may recognize a common epitope on mouse Fc gamma RII. Epitope mapping studies demonstrated that eight of the nine human anti-mouse Fc gamma RII sFv recognized overlapping epitopes. All of these human anti-mouse Fc gamma RII sFv competed with the 2.4G2 rat monoclonal anti-mouse Fc gamma RII/III antibody for binding with mouse Fc gamma RII, suggesting that the targeted epitopes reside in or near the Fc binding pocket of mouse Fc gamma RII. CONCLUSIONS: The availability of novel sFv recognizing mouse Fc gamma RII will facilitate the study of receptor triggering events. Such sFv may prove useful to engage murine Fc gamma RII for targeted cytotoxicity or immunization strategies.

Monoclonal anti-H1 histone autoantibodies from unimmunized Balb/c mice. Specificity and VH and VL domain sequences.

Examination of the nuclear reactivities of monoclonal IgM kappa autoantibodies, secreted by GFM-5 1B12 and NU-6 1F12 hybridomas derived from germ-free and nude mice, respectively, demonstrated homogeneous nuclear immunofluorescence staining patterns consistent with the recognition of histones. Under these conditions, GFM-5 1B12 and NU-6 1F12 mAbs produced species non-specific binding to components within the nuclei of mouse, human and Drosophila melanogaster cells. Immunoblotting confirmed the binding of these two autoantibodies to autologous H1 histones as well as bovine and insect H1 histones. Identification of the epitopes bound by GFM-5 1B12 and NU-6 1F12 mAbs within the D. melanogaster H1 histones was undertaken using 248 overlapping octapeptides encompassing the entire sequence of D. melanogaster H1 histones. GFM-5 1B12 mAbs bound several octapeptides derived from the amino- and carboxyl-terminal regions of D. melanogaster H1 histones with accessible KT, AT or VT amino acids. NU-6 1F12 mAbs, which stained nuclei within sections of D. melanogaster lavae, failed to bind to any of the 248 linear octapeptides, implying recognition of a conformational H1 histone epitope by this autoantibody. ELISA analysis of the polyspecific binding properties of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that both antibodies exhibited unique polyspecificity profiles. GFM-5 1B12 mAbs recognized bovine carbonic anhydrase and mouse IgG1, while NU-6 1F12 bound bovine cardiolipin, rat cytochrome c, Escherichia coli beta-galactosidase, toxoid from Clostridium tetani, mouse IgG1 and the haptens, DNP and FITC from the 24 antigen test panel. Comparison of the VH and VL domain sequences of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that the variations in autoreactivity and polyspecificity profiles resulted from amino acid variations in the CDRs of the VH and VL domains of these autoantibodies. Significantly, major differences in the VH domain sequences of the NU-5 1F12 and GFM-5 1B12 mAbs suggest that the VH domains may preferentially contribute to the unique specificities of the two anti-H1 histone autoantibodies.

Production and characterization of mice transgenic for the A and B isoforms of human FcgammaRIII.

Fcgamma receptor (FcgammaR) engagement is pivotal for many effector functions of macrophages, polymorphonuclear neutrophils (PMN), and natural killer (NK) cells. Mice transgenic for the A and B isoforms of human (h) FcgammaRIII on macrophages, PMN, and NK cells were constructed to permit the study of mechanisms and potential in vivo strategies to utilize the cytotoxic effector and antigen-presenting functions of cells expressing the hFcgammaR. The present report characterizes the phenotypic and functional expression of hFcgammaRIII in transgenic mice derived by crossing hFcgammaRIIIA and hFcgammaRIIIB transgenic mice. Interleukin-2 (IL-2) induces hFcgammaRIII expression by myeloid cells and their precursors, and these transgenic receptors promote in vitro cytotoxicity and anti-hFcgammaRIII antibody internalization. Splenocytes from untreated and IL-2-treated hFcgammaRIIIA, hFcgammaRIIIB, and hFcgammaRIIIA/B mice exhibited enhanced in vitro cytotoxicity toward HER-2/neu-overexpressing SK-OV-3 human ovarian carcinoma cells when incubated with the murine bispecific mAb 2B1, which has specificity for HER-2/neu and hFcgammaRIII. These results indicate that hFcgammaRIII transgenes are expressed on relevant murine cellular subsets, exhibit inducible up-regulation patterns similar to those seen in humans, and code for functional proteins. hFcgammaRIII transgenic mice exhibiting specific cellular subset expression will permit the examination of strategies designed to enhance hFcgammaRIII-dependent immunological effector functions and will provide a model system in which to evaluate preclinically potential candidate molecules that recognize hFcgammaRIII for the immunotherapy of cancer.

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu.

Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue (liver, 2.8 %ID g(-1); lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity.

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.