Department of Cell Biology, Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia Health System, Charlottesville, Virginia 22908, USA
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Abstract |
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Key words: agglutination/antibody engineering/contraception/recombinant antibody/single-chain Fv
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Introduction |
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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, 1996). 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., 1999
). 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and is restricted to the human epididymis, vas deferens, and both epididymal and ejaculated spermatozoa (Norton et al., unpublished data).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., 1999
), 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., 1999
). 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., 1987
), 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., 1998). Repeated application of N-9 caused erosion of vaginal and cervical epithelium in pigtail macaques (Patton et al., 1999
), and up-regulation of inflammatory cytokines IL-1ß, TNF-
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, 2000
). 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, 1994
). Recent studies in the rabbit have demonstrated the contraceptive potential of anti-sperm antibodies with agglutinating activity (Castle et al., 1997
).
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, 1999) and as treatments for transplant rejection, asthma, rheumatoid arthritis, and proliferative vitreo retinopathy (Hollinger and Hoogenboom, 1998
; Hudson, 1998
). 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., 1999
; McCall et al., 1999
; Pavlinkova et al., 1999
) 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., 1991
). 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, 1999
) 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., 1997
). Additionally, soluble scFvs are advantageous because they are relatively simple to isolate and do not require complex refolding procedures (Hashimoto et al., 1999
; Sanchez et al., 1999
).
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Materials and methods |
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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 337436, see Figure 1). 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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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, 1980) with modifications as described (Diekman et al., 1999
). 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, 1970) on a 15% polyacrylamide gel. Proteins were electroblotted onto nitrocellulose to be assayed by Western blot analysis (Towbin et al., 1979
). 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, 1990). 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, 1976) was performed with modification (Diekman et al., 1997
). 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., 1999
) 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.).
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Results |
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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., 1988) that is a product of the SP2/0 fusion partner (Schulman et al., 1978
) and (ii) the authentic S19 light chain mRNA. Identification of a unique Van91I restriction enzyme site at bp 225236 (Carroll et al., 1988
) 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, 1998), 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 1
, 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 2). 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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Discussion |
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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., 1999; Sanchez et al., 1999
).
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., 1994; Arndt et al., 1998
) 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., 1994
), 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., 1999
; Hudson and Kortt, 1999
). 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, 1999
). 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., 1987).
Over the past decade, the use of monoclonal antibodies has been expanded to a variety of clinical applications (Hollinger and Hoogenboom, 1998; Hudson, 1998
, 1999
). However, adverse effects, such as binding interference, caused by using whole murine antibodies in humans (Kuus-Reichel et al., 1994
), 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., 1997). 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., 1993
), or LDH-C4, on the midpiece (Diekman and Goldberg, 1995
), may serve as a topical agglutinin. Bispecific antibodies could also be generated (Coloma and Morrison, 1997
; Merchant et al., 1998
) 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., 1999
) or complement-recruiting diabodies (Kontermann et al., 1997
) 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.
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Acknowledgements |
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Notes |
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* Dr Herr serves as an officer and member of the Board of Directors of Contra Vac, Inc.
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References |
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Submitted on December 29, 2000; accepted on May 31, 2001.