RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives

E.J. Norton, A.B. Diekman, V.A. Westbrook, C.J. Flickinger and J.C. Herr,1,*

Department of Cell Biology, Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia Health System, Charlottesville, Virginia 22908, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG1{kappa}] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of ~60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.

Key words: agglutination/antibody engineering/contraception/recombinant antibody/single-chain Fv


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
S19, a murine IgG1{kappa} monoclonal antibody (mAb), recognizes a tissue-specific carbohydrate epitope on sperm agglutination antigen-1 (SAGA-1), a highly acidic glycoprotein localized on the entire surface of ejaculated human spermatozoa (Diekman et al., 1997Go). The binding of the S19 mAb to SAGA-1 causes the rapid agglutination of spermatozoa in a head-to-head, head-to-tail, and tail-to-tail pattern, blocks pre-fertilization events including cervical mucus penetration and sperm–oocyte interactions (Anderson et al., 1987Go; Mahoney et al., 1991Go), and has a complement-dependent cytotoxic effect on spermatozoa (Diekman et al., 1999Go).

The peptide core of SAGA-1 is identical to the peptide core of CD52, a lymphocyte surface glycoprotein also identified in the epididymal epithelium (Kirchoff, 1996Go). The CD52 glycoprotein is acquired during epididymal transit and inserted into the sperm membrane via its GPI-anchor. Although CD52 is synthesized in both the spleen and the epididymis, the S19 antibody does not recognize the CD52 antigen in spleen, indicating that spermatozoal SAGA-1 and lymphocytic CD52 have carbohydrate structures that are immunologically distinct (Diekman et al., 1999Go). Additional histochemical studies of 27 human tissues have demonstrated that the S19 epitope is absent from female reproductive tract tissues and is restricted to the human epididymis, vas deferens, and both epididymal and ejaculated spermatozoa (Norton et al., unpublished data). Mass spectrometry has revealed significant structural differences between the N-linked oligosaccharides on CD52 isolated from human seminal plasma and CD52 isolated from human spleen (Schröter et al., 1999Go), further confirming the presence of male reproductive tract-specific carbohydrate epitopes. Thus, SAGA-1 is a CD52 glycoform, a glycoprotein with the same core peptide but with a different carbohydrate structure. An interesting mAb, H6-3C4, also recognizes carbohydrate epitopes on SAGA-1 (Diekman et al., 1999Go). The H6-3C4 mAb is secreted by a human-mouse heterohybridoma generated from the peripheral blood lymphocytes of an infertile woman whose serum exhibited sperm-inhibitory titres (Isojima et al., 1987Go), a finding that implicates SAGA-1 as a factor in human antibody-mediated infertility. Thus, the S19 epitope fulfills several essential criteria to be a candidate for the development of an antibody-based, intravaginal spermistatic agent including tissue-specificity and the ability of the S19 mAb to inhibit sperm function in vitro.

Spermicidal research is experiencing resurgence as safe, effective, sperm-specific products are sought to replace current spermicidal agents. The active ingredients in spermicides currently marketed in the USA are non-specific, non-ionic detergents with harmful side effects that have only recently become known. Nonoxynol-9 (N-9), in particular, has been correlated with an increased incidence of urogenital infections, cervicovaginal inflammation, and epithelial changes in women using this method of birth control (Gupta et al., 1998Go). Repeated application of N-9 caused erosion of vaginal and cervical epithelium in pigtail macaques (Patton et al., 1999Go), and up-regulation of inflammatory cytokines IL-1ß, TNF-{alpha} and IL-6 in the human vagina (D.J.Anderson, personal communication). A large phase 3 study of N-9 use by sex-workers in Africa and Thailand has indicated that N-9 increases the risk of HIV transmission (Stephenson, 2000Go). As an alternative to spermicidal products that contain harsh chemical detergents with potentially harmful side effects, it was proposed that sperm-reactive monoclonal antibodies might provide safer active topical agents (Cone and Whaley, 1994Go). Recent studies in the rabbit have demonstrated the contraceptive potential of anti-sperm antibodies with agglutinating activity (Castle et al., 1997Go).

Monospecific antibodies are widely used for therapeutic and diagnostic applications because large quantities of recombinant antibodies and antibody fragments can be generated economically in bacterial systems. Currently, several recombinant antibodies are undergoing advanced clinical trials for application in various cancer therapies (Hudson, 1999Go) and as treatments for transplant rejection, asthma, rheumatoid arthritis, and proliferative vitreo retinopathy (Hollinger and Hoogenboom, 1998Go; Hudson, 1998Go). The majority of these therapeutic agents are chimeric antibodies or large antibody fragments such as F(ab)2s or Fabs. However, many second-generation formulations (Cochet et al., 1999Go; McCall et al., 1999Go; Pavlinkova et al., 1999Go) utilize single chain Fvs (scFvs), small antibody fragments that retain the parent molecule's binding affinity and in which variable heavy (VH) and light (VL) domains are tethered by a flexible polypeptide linker (Dreher et al., 1991Go). The generation of scFvs serves to minimize problems associated with the loss of antigen specificity in recombinant proteins due to incorrect folding (Jager and Pluckthun, 1999Go) because the reduced size of an scFv drastically decreases the number of potential antibody conformations. Large quantities of bacterially-produced scFvs (up to 400 mg/l) can be produced economically (Kipriyanov et al., 1997Go). Additionally, soluble scFvs are advantageous because they are relatively simple to isolate and do not require complex refolding procedures (Hashimoto et al., 1999Go; Sanchez et al., 1999Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Generation of the RASA
Poly A+mRNA was extracted from 5x108 MHS-8 hybridoma cells (Diekman et al., 1997Go) using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Purified mRNA was reverse-transcribed using the Access RT-PCR kit (Promega, Madison, WI, USA) and PCR-amplified following the manufacturer's instructions using murine IgG-specific primers from the Recombinant Phage Antibody System (RPAS; AmershamPharmacia Biotech, Uppsala, Sweden) with the exception of a novel light chain primer 5'-CGAGCTCACTCAGTCTCCATTCTCCCTGCCTGTCAGTC-3'. The resulting heavy (354 bp) and light chain (348 bp) cDNA fragments were purified by 2% agarose gel electrophoresis. Light chain sequences were digested for 2 h. at 37 °C with the Van91I restriction enzyme [cut site CCA(N)4/NTGG, (Boehringer Mannheim, Indianapolis, IN, USA)] to remove endogenous light chain cDNA sequences (Carroll et al., 1988Go) produced by mRNA from the myeloma fusion partner (Schulman et al., 1978Go). Resultant fragments were electrophoresced on 2% agarose gels to separate digested, endogenous light chain cDNAs from non-digested S19 cDNAs. Non-digested fragments were gel-purified using Ultrafree-MC filter units (Millipore, Bedford, MA, USA) and PCR-amplified. The heavy and light chain fragments were sequenced in the University of Virginia Biomolecular Research Facility using an ABI Prism 377 Automated DNA Sequencer.

To link the heavy and light chain PCR fragments, the cDNA fragments were quantified by ethidium bromide staining on agarose gels and equal amounts were linked by PCR using 100 nucleotide (nt) primers designed in our laboratory (covering nt 337–436, see Figure 1Go). The linked fragment was PCR amplified with primers introducing SfiI and NotI restriction sites to the 5' and 3' ends respectively (Amersham-Pharmacia Biotech). The resulting scFv cDNAs were gel-purified, quantified, and sequentially digested with SfiI and NotI (New England Biolabs, Beverly, MA, USA) to generate cohesive ends for insertion into the pCANTAB 5E expression vector (Amersham-Pharmacia Biotech) previously cut with these enzymes. This vector incorporates the 39 base pair (bp) [13 amino-acid (aa), GAPVPYPDPLEPR] E-Tag at the 3' end of the scFv. Following ligation, the vector and insert were transformed into E. coli HB2151 cells and plated on selective media. Enzyme digestion, insert ligation, transformation, and cell plating were performed using RPAS (Amersham-Pharmacia Biotech) following the manufacturer's instructions.



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Figure 1. cDNA and amino acid sequence of the RASA scFv. The amino acid sequence includes the heavy chain (amino acid 1–120), linker (shaded grey box, amino acid 121–135), light chain (amino acid 136–251), and E Tag (open box) sequences. The Gly residue at position 126 that was converted to Asp in the control inactive scFv is shaded in black. The novel primer engineered to amplify the authentic light chain sequence is underlined. (nt = nucleotide; aa = amino acid)

 
Preparation of bacterial extracts
Individual colonies were selected and cultured in 5 ml Luria-Bertani (LB) media cultures overnight at 37°C. These cultures were added to 500 ml of fresh LB containing 25 mg ampicillin and grown for 3 h at 37°C. Cultures were equilibrated to 30°C for 30 min, isopropyl-1-thio-ß-D-galactopyranoside (IPTG) was added to a final concentration of 1 mmol/l, and the cultures were incubated for an additional 3 h at 30°C. Cell pellets and supernatants were collected by centrifugation. Whole cell fractions were prepared by resuspending cell pellets in 10 mmol/l Tris-HCl, pH 8. To generate periplasmic fractions, the pellets were resuspended in 1/100 culture volume of chloroform, incubated at room temperature for 15 min, diluted 1:5 in 10 mmol/l Tris-HCl, pH 8, and centrifuged to remove cellular debris. The resulting supernatant fraction contained periplasmic proteins.

Preparation of sperm extracts
The University of Virginia's Human Research Committee approved all studies involving human semen donors. Informed consent was obtained from each participant after an explanation of the possible risks and outcomes of the studies. Spermatozoa from liquefied ejaculates were washed by centrifugation at 400 g in Ham's F-10 medium. Sperm samples were extracted with H2O/methanol/chlorofrom (3:8:4) following the procedure to isolate lipid-bound molecules (Svennerholm and Fredman, 1980Go) with modifications as described (Diekman et al., 1999Go). Extracted sperm proteins were resuspended in 1% sodium dodecyl sulphate (SDS) and used for polyacrylamide gel electrophoresis (PAGE) as described below.

Electrophoresis and immunoblot analysis
Sperm or bacterial extracts were mixed with reducing or non-reducing Laemmli buffer and electrophoresced by SDS-PAGE (Laemmli, 1970Go) on a 15% polyacrylamide gel. Proteins were electroblotted onto nitrocellulose to be assayed by Western blot analysis (Towbin et al., 1979Go). For immunostaining, blots were blocked in phosphate-buffered saline (PBS) containing 5% non-fat dry milk and incubated with primary antibody diluted in PBS/0.5% non-fat dry milk. Dilution factors for all antibodies are indicated in the figure legends. Blots were washed three times with PBS and incubated in horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated anti-E Tag antibodies at 1:5000 or 1:1000 respectively (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, and Amersham-Pharmacia Biotech). Blots were washed three times in PBS and developed with 3,3',5,5'-tetramethylbenzidene (TMB) reaction substrate (Kirkegaard & Perry, Gaithersburg, MD, USA).

Immunofluorescence microscopy
Human spermatozoa were harvested from semen by the swim-up method (Bronson and Fusi, 1990Go). Spermatozoa were collected and pelleted by centrifugation for 15 min at 400 g. Sperm pellets were fixed in 4% paraformaldehyde/PBS at 4°C for 20 min, washed, and air-dried onto slides. Non-specific protein binding sites were blocked by incubating slides in 5% normal goat serum (NGS)/PBS, incubated with S19 or RASA mAbs (1:100) in 1% NGS/PBS for 1.5 h, and then washed. Slides incubated with RASA were incubated for an additional 1.5 h with anti-E tag unconjugated antibodies (Amersham-Pharmacia Biotech) and washed in 1% NGS/PBS. All slides were incubated for 1.5 h with FITC-conjugated goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch Laboratories), washed in PBS, and mounted with SlowFade reagent (Molecular Probes, Eugene, OR, USA). Slides incubated with an inactive control scFv (containing a single aa change from Gly to Asp in the linker) followed by anti-E Tag antibody, secondary antibody alone, and anti-E tag antibody followed by secondary antibody were included as negative controls. Results were visualized and recorded with a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY, USA) equipped for epifluorescence and differential interference contrast (DIC) microscopy.

Sperm agglutination assay
The standard slide agglutination assay (Rose, 1976Go) was performed with modification (Diekman et al., 1997Go). Human semen samples, provided by healthy donors, were liquefied at room temperature. One part of semen diluted to 20x106 spermatozoa/ml in Ham's F-10 medium was gently mixed with one part of either S19 mAb purified from MHS-8 hybridoma supernatant (Diekman et al., 1999Go) or RASA in the periplasmic fraction, and diluted 1:5 in Ham's F-10. Periplasmic fractions containing inactive E-tagged recombinant antibodies or no antibodies served as negative controls. 20 µl of each mixture was placed on a plastic slide with a coverslip. After ~2 min, sperm agglutination and motility were observed and recorded with DIC microscopy using a Zeiss Axioplan microscope (Carl Zeiss Inc.).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Generation of the RASA antibody expression cassette
The S19 heavy and light chain variable regions were cloned and connected by PCR with primers encoding a (Gly4Ser)3 linker sequence. The cDNA and encoded amino acid sequences of the scFv expression cassette are shown in Figure 1Go.

Initially, expression of two different light chain mRNA transcripts by the MHS-8 hybridoma complicated isolation of the S19 light chain variable region cDNA. The MHS-8 hybridoma was found to express (i) an aberrant, non-functional light chain mRNA transcript (Carroll et al., 1988Go) that is a product of the SP2/0 fusion partner (Schulman et al., 1978Go) and (ii) the authentic S19 light chain mRNA. Identification of a unique Van91I restriction enzyme site at bp 225–236 (Carroll et al., 1988Go) in the hypervariable region of the endogenous aberrant light chain provided a novel technique to isolate the authentic S19 light chain cDNA. Through sequential restriction enzyme digestion of the light chain PCR products, gel-purification, and amplification of the undigested cDNAs, the S19 light chain cDNA was purified.

Furthermore, as previously suggested (Kramer, 1998Go), the supplied RPAS primers were not adequate to accurately amplify all light chain cDNAs, and initial amplifications of S19 light chain cDNA produced clones with a 2 bp frame shift. The frameshift was identified by comparison of the S19 light chain sequence with known murine light chain sequences in GenBank. To isolate the entire authentic S19 light chain, a novel primer was generated (Figure 1Go, underlined region) and the frameshift was corrected through PCR amplification.

Expression and characterization of RASA
Cell culture supernatants, periplasmic fractions, and whole cell extracts were examined by Western blot analysis of SDS-PAGE gels to determine in which bacterial fraction the recombinant antibodies accumulated (Figure 2Go). Only recombinant proteins in the whole cell and periplasmic fractions demonstrated reactivity with the anti-E Tag antibody at the predicted 29 kDa molecular mass. The E-tagged recombinant proteins were not identified in uninduced cultures (shown), or in constructs without inserts (not shown). Periplasmic fractions containing the soluble E-tagged recombinant antibodies were used for all further experiments without further processing.



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Figure 2. Western analysis of bacterial extracts with anti-E Tag antibody. Cell fractions were analysed by 15% SDS-PAGE and stained with Amido Black or immunoblotted with HRP-anti-E Tag antibody (1:1000). Lanes 1 and 2: E. coli HB2151 whole cell cultures before and after IPTG induction, respectively. Lane 3: Culture supernatant fraction. Lane 4: Periplasmic extract. The 29 kDa scFv is present in the whole cell (lane 2) and periplasmic fractions (lane 4).

 
Periplasmic fractions containing the scFv were subjected to either reducing or non-reducing SDS-PAGE, Western blotted, and detected with either Amido black or the anti-E Tag antibody. Under reducing conditions, the scFv migrated at ~29 kDa, while under non-reducing conditions, immunoreactive bands were observed at ~29, 60, and 90 kDa (Figure 3Go). These higher immunoreactive isoforms of 60 and 90 kDa are likely multimers of the 29 kDa RASA monomer.



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Figure 3. Western analysis demonstrating multimerization of RASA. Periplasmic cell fractions were analysed by 15% SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Amido Black (lane 1) or immunoblotted with the HRP-anti-E Tag antibody (lane 2) at 1:1000. Under reducing conditions, a single immunoreactive band is evident at 29 kDa, while under non-reducing conditions immunoreactive bands are apparent at ~29, 60, and 90 kDa. The monomeric form has a lower apparent molecular weight in the non-reduced sample possibly due to the denser conformation afforded by intra-chain disulphide bonding within the scFv, allowing it to migrate through the gel matrix with greater mobility than its reduced, linearized counterpart.

 
Functional properties of RASA
The E-tagged recombinant proteins from the periplasm were tested for binding to the SAGA-1 antigen. Western blots of human sperm extracts were incubated with recombinant antibodies followed by the anti-E Tag secondary antibody. The recombinant antibody and the S19 positive control antibody both identified an identical set of SAGA-1 bands migrating from 15–25 kDa in a smeared pattern frequently observed with glycoproteins (Figure 4Go) indicating that RASA retained proper conformation to react with the S19 epitope. Anti-E Tag antibody alone, included as a negative control, showed no reactivity with human sperm extracts.



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Figure 4. Western analysis of human sperm extracts demonstrating reactivity of RASA with SAGA-1. Chloroform/methanol-extracted spermatozoa were subjected to preparative SDS-PAGE and immunoblot strips were incubated with the S19 mAb (lane 1, 1:1000) or RASA (lane 2, 1:100) followed by goat anti-mouse (1:5000) or anti-E Tag (1:1000) secondary antibodies, respectively. Both mAbs detected the identical polymorphic pattern (~15–25kDa) characteristic of the SAGA-1 glycoprotein. No reactivity was seen with the anti-E Tag antibody alone (lane 3).

 
Reactivity of RASA with SAGA-1 on fixed human spermatozoa was investigated using indirect immunofluorescence. The native S19 mAb and anti-E Tag antibody alone were included as positive and negative controls respectively. Although RASA (Figure 5AGo) did not stain the entire surface of each spermatazoa as brightly or as uniformly as the S19 mAb (Figure 5BGo), all spermatazoa examined did show RASA staining in multiple domains of the head and/or tail, while the anti-E Tag antibody alone showed only very faint background fluorescence (not shown). This finding confirmed that RASA can bind the S19 epitope on the surface of human spermatazoa and led to the hypothesis that RASA might, like the S19 mAb, agglutinate sperm in a tangled pattern.



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Figure 5. Indirect immunofluorescence demonstrating RASA reactivity with SAGA-1 on the surface of human spermatozoa. The (A) RASA (1:50) and (B) S19 (1:100) mAbs immunolocalized over the entirety of paraformaldehyde-fixed, air-dried spermatozoa. Only faint background fluorescence was observed with the secondary antibodies alone (not shown).

 
Agglutination of human spermatozoa with RASA was investigated using the in-vitro slide agglutination assay. Viable human spermatozoa were mixed with RASA and observed by DIC microscopy. Native S19 mAb and periplasmic fractions containing an inactive control scFv were included as positive and negative controls respectively. This inactive recombinant antibody contains a single bp mutation that converts Gly to Asp at position 126 in the RASA linker (shaded black in Figure 1Go) and it was detected with the anti-E Tag antibody as a component of bacterial periplasmic fractions in expression studies. Further, periplasmic fractions containing this inactive scFv did not immunoreact with SAGA-1 on Western blots nor did the inactive scFv appear to form multimers as evidenced by electrophoretic mobility under reducing and non-reducing conditions (data not shown). The anti-E Tag antibody was added to samples containing the active or inactive recombinant antibodies. The native S19 mAb and RASA, with or without anti-E Tag antibody, agglutinated spermatozoa in a tangled pattern, i.e. head-to-head, tail-to-tail, and head-to-tail (Figure 6Go). Neither the periplasmic fraction containing inactive scFv with added anti-E Tag antibody (shown) nor the anti-E Tag antibody alone showed significant agglutination.



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Figure 6. Agglutination of human spermatozoa with RASA. RASA or the S19 mAb was mixed with human spermatozoa at a concentration of 20x106 spermatozoa/ml and 1:10 dilution of antibody. After 2 min, sperm motility was observed and recorded under DIC microscopy. RASA alone (A), RASA with anti-E Tag antibody (1:100), (B), and the S19 mAb positive control (C). Spermatozoa were agglutinated in a head-to-head, head-to-tail, and tail-to-tail pattern. Agglutination was not seen with the inactive scFv and anti-E Tag antibody (D), or in samples with anti-E Tag antibody alone (not shown).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present report demonstrates the generation of RASA, a recombinant scFv mini-antibody form of the murine anti-sperm mAb S19. Using recombinant DNA techniques, cDNAs encoding the variable regions of the S19 heavy and light chains were isolated from the parental MHS-8 hybridoma, linked to one another, ligated into an expression vector, and expressed in E. coli. Identification of the correct light chain cDNA was initially problematic due to a frame shift in the cDNA generated by the RPAS kit primers and was corrected through the generation of a unique primer. The use of alternative primer sequences that terminate prior to the frameshift region (between bp 429 and 430 in Figure 1Go) are well-established (Orlandi et al., 1989Go; Jones and Bendig, 1991Go), however such primers are less specific. Isolation of the correct S19 light chain cDNA was further complicated by the production of an aberrant light chain (Carroll et al., 1988Go) by the myeloma fusion partner (Schulman et al., 1978Go). Use of the Van91I restriction enzyme to delete cDNAs encoding the aberrant endogenous light chain was a novel technique that expedited cloning of the authentic scFv. Both techniques may be of use to other researchers generating recombinant antibodies from murine hybridomas with homologous sequences to RASA.

Expression of the RASA sequence in E. coli HB2151 cells produced soluble recombinant antibodies in the periplasmic fraction. The presence of RASA in this fraction obviated the complex refolding procedures necessary in the generation of other scFvs (Hashimoto et al., 1999Go; Sanchez et al., 1999Go).

Although the RASA scFv was engineered as a single scFv, Western blot analysis of non-reduced gels showed a ladder of immunoreactive bands. The monomer was present at 29 kDa, while dimers (60 kDa) and trimers (90 kDa) of RASA were visible. Such multimerization has been shown previously with some scFvs (Kortt et al., 1994Go; Arndt et al., 1998Go) and, depending on folding, has the potential to make RASA multivalent. Other scFvs form only intrachain disulphide bridges similar to their parent Fab molecules (Kortt et al., 1994Go), thus the presence of a single reduced band on Western blots was expected. The formation of scFv multimers has been correlated with linker length, demonstrating a transition from monomers towards multimers when the scFv linker is 12 amino acids or less (Desplanq et al., 1994; Atwell et al., 1999Go; Hudson and Kortt, 1999Go). These multimers are not believed to be held together by disulphide bridges. Thus, the presence of multiple RASA bands on a non-reducing gel was novel, as it indicates the presence of an interchain bond in RASA susceptible to reducing agents. The interchain bond in RASA may be made by the free cysteine in the heavy chain at amino acid 105 complexing with one of the cysteines in the light chain or the free cysteine on the neighbouring heavy chain to create multimers. This intrinsic multimerization may obviate the requirement for a delivery vehicle, thereby simplifying later product development. Many scFvs are being preferentially generated as multimers for immunotherapy (such as T-cell recruitment into tumours) and immunodiagnostics (such as red blood cell agglutination reagents) as they are more stable than their monomeric counterparts (Hudson and Kortt, 1999Go). It remains to be seen whether multimer formation will be beneficial in the final formulation of RASA as a spermistatic agent.

Western blot, immunofluorescent, and agglutination analyses confirmed that RASA recognizes the SAGA-1 epitope on the human sperm surface. Recognition of SAGA-1 on Western blots provided the initial confirmation of RASA activity. Immunofluorescent labelling analysis demonstrated the binding of RASA over all domains of the sperm head and tail. The localization of RASA to all sperm surface domains increases the agglutinating potential of the mini-antibody. RASA alone was able to agglutinate human spermatazoa indicating that RASA not only recognizes SAGA-1 on live spermatozoa but also does not require a secondary antibody for agglutination. The agglutination seen with RASA alone is likely to be due to the ability of RASA to form multimers as observed by Western blot analysis. Furthermore, since RASA demonstrates agglutination and localization to the entire sperm surface, as observed with the native S19 molecule, we predict that it will block other aspects of fertilization, such as cervical mucus penetration and zona pellucida binding also observed with the native S19 mAb (Anderson et al., 1987Go).

Over the past decade, the use of monoclonal antibodies has been expanded to a variety of clinical applications (Hollinger and Hoogenboom, 1998Go; Hudson, 1998Go, 1999Go). However, adverse effects, such as binding interference, caused by using whole murine antibodies in humans (Kuus-Reichel et al., 1994Go), and the expense of tissue culture have presented obstacles to their utilization. Recombinant antibody technology has presented a solution to both of these impediments because the generation of recombinant antibody fragments, such as scFvs, not only removes the majority of the immunogenic murine sequence but may also be more economical.

The RASA construct may serve as a prototype for recombinant anti-sperm antibodies and for their use as contraceptive agents. Contraceptive efficacy in the rabbit model has been demonstrated with a variety of individual monoclonal sperm-agglutinating antibodies (Castle et al., 1997Go). RASA represents the first example, to our knowledge, of an active recombinant antibody generated to the entire human sperm surface. RASA itself, or in conjunction with additional sperm-agglutinating antibodies generated against sperm surface proteins such as PH-20, localized to the acrosome and equatorial segment (Lin et al., 1993Go), or LDH-C4, on the midpiece (Diekman and Goldberg, 1995Go), may serve as a topical agglutinin. Bispecific antibodies could also be generated (Coloma and Morrison, 1997Go; Merchant et al., 1998Go) to attach such sperm-reactive antibodies to one another. These antibodies could be combined to create a contraceptive with an additive effect. In addition, generation of an active mini-antibody to the sperm surface opens the path to engineering chimeric fusion proteins (Schwarze et al., 1999Go) or complement-recruiting diabodies (Kontermann et al., 1997Go) with specialized cytotoxic properties using RASA as a targeting domain. SAGA-1 is localized over the entire sperm surface, thereby giving RASA a distinct advantage in its ability to access all sperm surface domains. RASA currently represents a prototype active ingredient for a topical contraceptive product. Adequate large-scale expression, purification, and delivery strategies must be developed and in-vivo feasibility trials performed as a prerequisite to commercialization. Nevertheless, RASA, as the first recombinant mini-antibody generated to the entire human sperm surface, sets the stage for new generations of specific protein-based spermicidal agents that may offer improvement over current non-specific, detergent-based products.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank the staff of the Lymphocyte Culture Core and the Biomolecular Research Facility at the University of Virginia. Supported by NIH HD U54 29099, R01 HD35523, D43 TW/HD 00654 from the Fogarty International Center, R43 HD 35771 to ContraVac, Inc., Virginia's Center for Innovative Technology Award BIO-98–002, Contraceptive Research and Development (CONRAD) Award CIG-97–15, the Andrew W. Mellon Foundation and by 2000-IJ-CX-K013 from the Office of Justice Programs, National Institute of Justice, U.S. Department of Justice (Points of view in this document are those of the authors and do not necessarily represent the official position of the U.S. Department of Justice).


    Notes
 
1 To whom correspondence should be addressed. E-mail: jch7k{at}virginia.edu Back

* Dr Herr serves as an officer and member of the Board of Directors of Contra Vac, Inc. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Castle, P.E., Whaley, K.J., Hoen, T.E. et al. (1997) Contraceptive effect of sperm-agglutinating monoclonal antibodies in rabbits. Biol. Reprod., 56, 153–159.[Abstract]

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Coloma, M.J. and Morrison, S.L. (1997) Design and production of novel tetravalent bispecific antibodies. Nat. Biotechnol., 15, 159–163.[ISI][Medline]

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Submitted on December 29, 2000; accepted on May 31, 2001.