WO2017180069A2 - Ebola specific cell penetrable antibodies - Google Patents

Ebola specific cell penetrable antibodies Download PDF

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Publication number
WO2017180069A2
WO2017180069A2 PCT/TH2016/000038 TH2016000038W WO2017180069A2 WO 2017180069 A2 WO2017180069 A2 WO 2017180069A2 TH 2016000038 W TH2016000038 W TH 2016000038W WO 2017180069 A2 WO2017180069 A2 WO 2017180069A2
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antibody
ebola
specific cell
amino acid
binds
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PCT/TH2016/000038
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French (fr)
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Wanpen Chaicumpa
Salma TEIMOORI
Watee SEESUAY
Urai CHAISRI
Surasak JITTAVISUTTHIKUL
Nitat SOOKRUNG
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Mahidol University
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Publication of WO2017180069A2 publication Critical patent/WO2017180069A2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • This invention relates to a group of Ebola virus-specific cell penetrable single chain antibodies wherein the antibodies can readily enter the mammalian cells without causing any cytotoxicity, bind to the important intracellular virion protein 40 (VP40) target of the Ebola virus
  • Ebola is a highly contagious zoonotic disease of humans and other primates caused by Ebola virus (EBOV). Although natural outbreaks of the disease are still limited to the African territory, rapid and convenient ways of people communication, high transmissibility of the virus, and exceptionally high mortality rate that the disease causes (25-90%) have made the Ebola a serious global health threat. Currently, there is no FDA approved vaccine or direct acting anti-EBOV drug.
  • Ebola patients are given only supportive and symptomatic treatment. These include maintenance of blood pressure, body fluid, and electrolytes and management of the abnormal blood clotting and hemorrhage for intervention of hypovolemic shock. Antibiotics may be given also for controlling the co-infection (WHO/EVD/Manual/ECU/15.1, 2015). Although these treatment measures improve the chance of survival, they are difficult to carry out in the disease endemic/ affected areas where the health facilities are inadequate and hygiene is substandard. There is an urgent need of an effective anti- Ebola remedy that can control the viral load for mitigation of the disease severity and allow adequate time for the host immunity that had been suppressed by the virus to restore.
  • EBOV is a negative- sense, single stranded RNA virus. Its genome ( ⁇ 19 kb) contains seven linear genes that encode two non- structural and seven structural proteins. The two non- structural proteins that responsible for the viral pathogenicity are secreted glycoprotein (GP) and small secreted GP (Feldman et al., 1999; De la Vega et al., 2015).
  • the EBOV ribonucleoprotein (vRNP) consists of NP (major neucleoprotein), virion protein 35 (VP35) (a polymerase co- factor and the host innate interferon suppressor), VP30 (minor nucleoprotein that functions as an initiator of the
  • Integrity of the vRNP is maintained by viral matrices formed by two matrix proteins, i.e., VP40 (major matrix protein) and VP24 (minor matrix protein which is also an inhibitor of host interferon signaling) (Elliott et al., 1985; Dessen et al., 2000; Ruigrok e al., 2000;
  • the EBOV envelope displays protruding spikes of trimeric GP (Feldman et al., 1991) which is a type I transmembrane protein synthesized as a monomeric precursor from the forth EBOV gene that also encode the small GP and the small secreted GP through different RNA editing (Mehedi et al., 2011).
  • the precursor monomeric GP is nicked by the host furin in the trans-
  • GP1 receptor binding domain
  • GP2 membrane fusion loop for releasing the vRNP into cytoplasm for further replication.
  • the GP1 and GP2 are still linked together by a disulfide bond (Volchkov et al., 1998, 2000).
  • VP40 is most abundant in the viral particle.
  • the protein has several other pivotal roles in the EBOV life cycle through acquisition of different structural rearrangements in the infected cells (Tirnmins et al, 2004; Bomholdt et al., 2013).
  • the VP40 (326 amino acids, ⁇ 40 kDa) was found to contain two distinctly folded domains, i.e., N- and
  • NTD and CTD C- terminal
  • VP40 forms homodimers through NTD hydrophobic surface interaction involving residues A55, H61, F108, T112, A113, M116, and L117 (Bomholdt et al, 2013).
  • L117 extends into a hydrophobic pocket formed by H61, A55, M116, and F108 in the opposing subunit and locks the VP40 protomers together into a dimer (Bomholdt et al. , 2013) .
  • 7PTAPPEY13 motif in the so-called late (L)-domain of the VP40 NTD interacts with specific cellular proteins that have type I WW- domain, including mammalian ubiquitin ligase (Nedd4/Rsp5), TsglOl, and Vsp4 (Harty et al., 2000; Tirnmins et al., 2003).
  • the VP40 dimers move to the plasma membrane and rearrange into linear hexamers which consequently generate a multilayered, filamentous matrix structure that lies underneath the lipid envelope for viral assembly and budding (Ruigrok et al., 2000; Bomholdt et al, 2013).
  • the VP40 CTD is responsible for plasma membrane insertion, oligomerization into the viral matrix, and budding of nascent virions.
  • the CTD uses its basic amino acids, i.e., K221, K224, K225, K270, K274 and K275, in the cationic patch to latch with the negatively charged lipid bilayers (Ruigrok, et al., 2000; Bomholdt et al., 2013; Adu-Gyamfi et al., 2013).
  • VP40 also forms octameric rings with specific RNA binding properties (Gomis-Ruth et al.,
  • VLPs virus-like particles
  • transbodies engineered cell penetrable human single chain antibodies that target the membrane lipid- binding sites of the VP40 CTD were produced.
  • the transbodies specifically from three clones. R9-HuscFV8, R9-HuscFV23, and R9-HuscFV119, inhibited readily the budding of VLP from mammalian cells.
  • Ebola virus (EBOV) species has multiple pivotal roles in the virus life cycle. Inhibition of the VP40 functions would not only lessen the virion integrity, but also interfere with the viral assembly, budding, and spread.
  • Ebola- specific cell penetrable antibodies according to this invention comprises a group of engineered cell penetrable human single chain antibodies (R9- HuscFvs) that bound to the EBOV VP40 were produced using an antibody phage display technology. Gene sequences coding for VP40- bound HuscFvs were subcloned from the phagemids into protein expression plasmids downstream to the nonaarginine (R9) coding sequence. The R9- HuscFvs produced from the plasmid transformed-E.
  • coli clones readily entered the mammalian cells (being transbodies) and were non- toxic to mammalian cells and mice. Scanning electron microscopy revealed that the transbodies from three clones (R9-HuscFv8, R9-HuscFv23 and R9-HuscFvll9) efficiently inhibited egress of the Ebola virus- like particles from the human hepatic cells transduced with pseudo- typed Lentivirus particles carrying EBOV VP40 and GP genes. Binding
  • HuscFv8 binds to K221, K224 and K274; the HuscFv23 binds to K221, K224, and K274; and the HuscFvll9 binds to K221, K224, K274 and K275).
  • the R9-HuscFvs-8, -23 and -119 bind also to the VP40 N- terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that the antibodies exert the VP40 inhibitory activity through additional mechanism(S).
  • the R9-Huscfv61 and R9-HuscFv89 which bind to VP40 C-terminal domain also inhibit the Ebola virus egress (albeit to the less extent than the former three transbodies).
  • the R9- HuscFv6 and R9-HuscFv69 did not bind to any of the tested portions of the VP40; yet, they reduced also the egress of the Ebola virus- like particles when compared with the non- treated transfected cells or transfected cells treated with control R9-HuscFv.
  • the so- invented Ebola- specific cell penetrable antibodies have high potential for developing further as safe therapeutic agents for halting/ slowing down the EBOV budding and spread which should allow adequate time for the host immunity to cope with the infecting agent.
  • Figure 1 illustrates the recombinant full-length VP40, r ⁇ NTD, and rCTD preparations and phage biopannning according to this invention wherein:
  • Figure 1A illustrates the SDS-PAGE-separated preparations that contained recombinant
  • VP40 stained by Coomassie Brilliant Blue G-250 dye M, Standard protein marker; lane 1, homogenate of VP40 pET21a + transformed BL21 (DE3)E. coli; lane 2, purified E. coli inclusion body; Lane 3, purified and refolded rVP40 ( ⁇ 40 kDa) (arrow). Numbers at the left are protein molecular masses in kDa;
  • Figure IB illustrates (from left to right): M, Standard protein marker; lanes 1 and 2, Purified r ⁇ NTD and rCTD, respectively. Numbers at the left are protein molecular masses in kDa; Figure 1C illustrates the amplicons of genes coding for human single chain antibodies (huscfvs; -1,000 bp) carried by representative phage-transformed-HB2151 E. coli clones derived from phage-panning with recombinant VP40. M, DNA size marker; lanes 1-36, amplicons of huscfvs from 36 representative E. coli clones. Numbers at the left are DNA sizes in base pairs (bp);
  • Figure ID illustrates the indirect ELISA results for testing binding of the HuscFvs in lysates of representative huscfv positive E. coli clones.
  • BSA was used as control antigen and lysate of original HB2151 E. co// (HB) served as background binding control.
  • Bacterial clones that their lysates gave ELISA signal (OD 4 o5nm) to the VP40 at least two times above the BSA signal and higher than the HB were chosen. They were clones 6, 8, 23, 61, 89, 118, 158, and 183. Indirect ELISA results of the other clones are not shown;
  • Figure IE shows the Western blot results for testing the binding of the R9-HuscFvs to the
  • Figure 2 illustrates the amino acid sequences, complementary determining regions (CDRs), and immunoglobulin framework regions (FRs) of the VP40 specific HuscFvs, including (from top to bottom) HuscFvll9, HuscFv8, HuscFv61, HuscFv89, HuscFv69, HuscFv6, and HuscFv23;
  • Figure 3 provides toxicity testings of the R9-HuscFvs wherein:
  • Figure 3 A displays the viability testing result of Vero (African Green monkey kidney continuous cell line) and Huh7 (differentiated human hepatocyte derived carcinoma cell line) cells
  • Figure 3B illustrates the body weights of mice injected intraperitoneally with four doses of R9-HuscFv on every alternate day in comparison with control mice that were given buffer. Both groups of mice did not show any sign of illness and instead they gained some weight during the 14 day-period of observation.
  • Figure 4 illustrates the results of scanning electron microscopy (SEM) for determining the SEM
  • Figure 5 illustrates the results of sandwich ELISA for semi- quantification of VP40 in the culture supernatants of Huh7 cells transduced with pseudo- typed Lentiviral particles carrying RNAs coding for EBOV VP40 and GP after incubating with VP40 specific- R9- HuscFvs and controls .
  • the culture supernatants of the transduced cells treated with R9-HuscFvs of clones 8, 23, and 119 had significant reduction of the VP40 amounts in the culture supernatants compared to the non-treated cells )medium (and cells treated with control R9-HuscFv )control scFv .
  • the R9 HuscFvs of clones 6, 61, and 89 were less effective .
  • N normal cells .
  • the table below the graph shows p values )ANOVA, R 3.0.3 program (for comparison of the mean OD405nm of all treatment groups .
  • the efficacies of the R9- HuscFvs of clones 8, 23 and 119 in reducing the VP40 amounts in the culture supernatants of the transduced cells were not different .
  • Figure 6 is a table showing the binding of the R9- HuscFvs to L domain )N-tail peptide(, recombinant truncated N- terminal )r ⁇ NTD or I (domain, and recombinant C- terminal )rCTD or M (domain of Ebola virus VP40 .
  • Ebola-specific cell penetrable antibodies are engineered human single chain antibodies (HuscFvs) as displayed in Figure 2 wherein each antibody comprises its amino acid sequences, complementarily determining regions ( CDRs) and immunoglobulin framework regions (FRs).
  • HuscFvs human single chain antibodies
  • CDRs complementarily determining regions
  • FRs immunoglobulin framework regions
  • Creating phage clones displaying HuscFvs that bound to VP40 and the phage transformed E. coli clone. The full-length rVP40 were used as antigen in the phage bio-panning for selecting the protein- bound phage clones from a human scFv phage display library (Kulkeaw et al., 2009). The antigen-bound phages were transfected into HB2151 E. coli and the bacteria were grown on a selective agar. Colonies were picked randomly from the plate and screened for the presence of the gene sequences coding for HuscFvs (huscjvs) by PCR using pCANTAB5E phagemid specific primers.
  • Figure 1C shows huscfv amplicons (-1,000 bp) carried by representative phage-transformed E. coli clones derived from the panning.
  • the E. coli colonies that were positive for the huscfv amplicons were grown under EPTG induction and their lysates containing the expressed HuscFvs were tested for binding to rVP40 by indirect ELISA From the assay, lysates of 8 E. coli clones (nos. 6, 8, 23, 61, 89, 119, 158 and 183) gave significant ELISA signals above the controls (lysate of the original HB2151 E. coli or HB and BSA) ( Figure ID).
  • the huscjvs of these clones were sequenced. Only nucleotide sequences huscjvs of 6 clones (nos. 6, 8, 23, 61, 89, and
  • HuscFv6, HuscFv8, HuscFv23, HuscFv61, HuscFv89, and HuscFvll9 were linked molecularly downstream to the nonaarginines (R9) nucleotides in the pLATE52 plasmids by means of the ligase independent cloning (LIC) method.
  • the recombinant plasmids were put in the RosettaTM 2 (DE3)E. coli.
  • HuscFvll9 were expressed, purified and refolded from the inclusion bodies of respective plasmid transformed E. coli clones and were retested for binding to the rVP40 by Western blot analysis.
  • the R9-HuscFv61 and R9-HuscFv89 bound modestly only to the CTD (OD 405nm 0.08 and 0.09, respectively).
  • the R9-HuscFvs6 did not give positive binding to any of the VP40 preparations which conformed to the above Western blot result.
  • Huh7 cells were transduced with pseudo-typed-Lentivirus particles carrying RNAs coding for EBOV VP40 and GP at MOI 0.5 followed by treating the cells with 40 ⁇ g of R9- HuscFvs from the 6 E. coli clones (HuscFv6, HuscFv8, HuscFv23, HuscFv61, HuscFv89, and HuscFvll9) and control R9-HuscFv.
  • the tranduced cells maintained in the medium alone served as negative inhibition control.
  • the transduced cells were kept in a 5% CO 2 incubator at 37°C for 48 h.
  • the culture supernatants of all wells were collected for quantification of VP40 while the cells were subjected to scanning electron microscopy (SEM).
  • the effectiveness of the R9-HuscFvs and controls in reducing the VP40 amounts in the cultured fluids of the transduced Huh7 cells are shown in Figure 5.
  • the most VP40 amount was found in the culture supernatant of the transduced cells cultured in the medium alone (the highest OD405nm).
  • the results of the sandwich ELISA were, more or less, conformed to the SEM results.
  • the culture supernatants of the transduced cells treated with R9-HuscFv8, R9-HuscFv23 and R9-HuscFvll9 had the least VP40 amounts and significantly less than the untreated control and those treated with irrelevant R9-HuscFv.
  • the efficacies of the antibodies in the falling order of magnitude were R9-
  • the inserted Table in Figure 5 shows p values for comparison among treatments.
  • the HuscFv8, HuscFv23 and HuscFvll9 which were highly effective in inhibiting the VLP budding from the Huh7 cells tranduced with pseudo-typed-Lentivirus particles carrying VP40 and GP genes were found to dock on several residues of the CTD cationic patch (i.e. CTD basic patch) that are important for membrane- binding of the dimeric VP40 including K221, K224, and K274 for HuscFv8 and HuscFv23 and K221, K224, K274, and K275 for HuscFvll9.
  • CTD cationic patch i.e. CTD basic patch
  • the set of amino acid residues in the CDRs of HuscFv8 that bind to K221 are Y52, D54, and W102 in the VH- CDR2, VH-CDR2 and VH-CDR3 domains, respectively; those that bind to K224 are W33 and
  • the set of amino acid residues in the CDRs of HuscFv23 that bind to K221 is P106 in the VH-CDR2 domain; those that bind to
  • K224 are S195 and G209 in the VL-CDR2 and VL-FR3 domains, respectively; and those that bind to K274 are Dill and Y175 in the VH-CDR3 and VLCDRl domains, respectively.
  • the set of amino acid residues in the CDRs of HuscFvll9 that binds to K221 is P102 in VH-CDR3 domain; those that bind to K224 are S52 and D57 in the VH-CDR2 domain; those that bind to K274 are
  • the HuscFv61 interacted with K270 and K275 through E55 in the VH-CDR2 domain and F228 in the VL-CDR3 domain of the antibody, respectively; while HuscFv89 interacted with K275 of the CTD cationic patch through S225 in the VL-CDR3 domain of the antibody ( Figure 7). Both HuscFvs also bound to other residues in the CTD. Because, the
  • HuscFv6 did not bind to any region of the VP40 by indirect ELISA and Western blotting, thus, the computerized molecular interaction was not performed.

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Description

citle of the Invention
EBOLA SPECIFIC CELL PENETRABLE ANTIBODIES Field of the invention
This invention relates to a group of Ebola virus-specific cell penetrable single chain antibodies wherein the antibodies can readily enter the mammalian cells without causing any cytotoxicity, bind to the important intracellular virion protein 40 (VP40) target of the Ebola virus
(EBOV) and interfere with the VP40 functions which, in turn, lead to an inhibition of the new virion egress and halt the spread of the virus and reduce the viral load.
Background of the invention
Ebola is a highly contagious zoonotic disease of humans and other primates caused by Ebola virus (EBOV). Although natural outbreaks of the disease are still limited to the African territory, rapid and convenient ways of people communication, high transmissibility of the virus, and exceptionally high mortality rate that the disease causes (25-90%) have made the Ebola a serious global health threat. Currently, there is no FDA approved vaccine or direct acting anti-EBOV drug.
Ebola patients are given only supportive and symptomatic treatment. These include maintenance of blood pressure, body fluid, and electrolytes and management of the abnormal blood clotting and hemorrhage for intervention of hypovolemic shock. Antibiotics may be given also for controlling the co-infection (WHO/EVD/Manual/ECU/15.1, 2015). Although these treatment measures improve the chance of survival, they are difficult to carry out in the disease endemic/ affected areas where the health facilities are inadequate and hygiene is substandard. There is an urgent need of an effective anti- Ebola remedy that can control the viral load for mitigation of the disease severity and allow adequate time for the host immunity that had been suppressed by the virus to restore.
EBOV is a negative- sense, single stranded RNA virus. Its genome (~19 kb) contains seven linear genes that encode two non- structural and seven structural proteins. The two non- structural proteins that responsible for the viral pathogenicity are secreted glycoprotein (GP) and small secreted GP (Feldman et al., 1999; De la Vega et al., 2015). The EBOV ribonucleoprotein (vRNP) consists of NP (major neucleoprotein), virion protein 35 (VP35) (a polymerase co- factor and the host innate interferon suppressor), VP30 (minor nucleoprotein that functions as an initiator of the
EBOV transcription), L protein (RNA-dependent RNA polymerase), and genomic RNA (Feldman and Kiley, 1999). Integrity of the vRNP is maintained by viral matrices formed by two matrix proteins, i.e., VP40 (major matrix protein) and VP24 (minor matrix protein which is also an inhibitor of host interferon signaling) (Elliott et al., 1985; Dessen et al., 2000; Ruigrok e al., 2000;
Jasenosky et al., 2001; Noda et al., 2002; Hartlieb and Weissenhorn, 2006; Watanabe et al., 2007;
Harty et al., 2009; Daugherty and Malik, 2014). These two proteins lie underneath a lipid envelope which the virion acquired from the host plasma membrane during budding process (Klenk and
Feldman, 2004). The EBOV envelope displays protruding spikes of trimeric GP (Feldman et al., 1991) which is a type I transmembrane protein synthesized as a monomeric precursor from the forth EBOV gene that also encode the small GP and the small secreted GP through different RNA editing (Mehedi et al., 2011). The precursor monomeric GP is nicked by the host furin in the trans-
Golgi network into GP1 (receptor binding domain; RBD) and GP2 (membrane anchored protein that contains membrane fusion loop for releasing the vRNP into cytoplasm for further replication). The GP1 and GP2 are still linked together by a disulfide bond (Volchkov et al., 1998, 2000).
Among the seven structural proteins of EBOV, VP40 is most abundant in the viral particle.
Besides maintaining the virion structure and integrity, the protein has several other pivotal roles in the EBOV life cycle through acquisition of different structural rearrangements in the infected cells (Tirnmins et al, 2004; Bomholdt et al., 2013). By means of X-ray crystallographic study, the VP40 (326 amino acids, ~40 kDa) was found to contain two distinctly folded domains, i.e., N- and
C- terminal (NTD and CTD, respectively), which are loosely connected by a flexible linker (Dessen et al., 2000). In cytoplasm, VP40 forms homodimers through NTD hydrophobic surface interaction involving residues A55, H61, F108, T112, A113, M116, and L117 (Bomholdt et al, 2013). The
L117 extends into a hydrophobic pocket formed by H61, A55, M116, and F108 in the opposing subunit and locks the VP40 protomers together into a dimer (Bomholdt et al. , 2013) . The
7PTAPPEY13 motif in the so-called late (L)-domain of the VP40 NTD interacts with specific cellular proteins that have type I WW- domain, including mammalian ubiquitin ligase (Nedd4/Rsp5), TsglOl, and Vsp4 (Harty et al., 2000; Tirnmins et al., 2003). As a result of this viral- host protein interaction, the VP40 dimers move to the plasma membrane and rearrange into linear hexamers which consequently generate a multilayered, filamentous matrix structure that lies underneath the lipid envelope for viral assembly and budding (Ruigrok et al., 2000; Bomholdt et al, 2013). The VP40 CTD is responsible for plasma membrane insertion, oligomerization into the viral matrix, and budding of nascent virions. The CTD uses its basic amino acids, i.e., K221, K224, K225, K270, K274 and K275, in the cationic patch to latch with the negatively charged lipid bilayers (Ruigrok, et al., 2000; Bomholdt et al., 2013; Adu-Gyamfi et al., 2013). In the infected host cells, VP40 also forms octameric rings with specific RNA binding properties (Gomis-Ruth et al.,
2003). Expression of the VP40 alone in mammalian cells generates the virus-like particles (VLPs) that are indistinguishable in morphology from the authentic EBOV (Warfield et al., 2005). The VLP formation is improved by co- expression of the VP40 and GP (Noda et al., 2002). Because of the indispensable roles in the EBOV life cycle, VP40 is an attractive target of direct acting anti- EBOV agents (Harty, 2009; Stahelin, 2014). Interfering with the VP40 activities should lead to inhibition of the EBOV assembly and budding which would ultimately reduce the viral load. In this study, a group of engineered cell penetrable human single chain antibodies (transbodies) that target the membrane lipid- binding sites of the VP40 CTD were produced. The transbodies, specifically from three clones. R9-HuscFV8, R9-HuscFV23, and R9-HuscFV119, inhibited readily the budding of VLP from mammalian cells.
Summary of the invention
Virion protein 40 or major matrix protein (VP40), a highly conserved protein across the
Ebola virus (EBOV) species has multiple pivotal roles in the virus life cycle. Inhibition of the VP40 functions would not only lessen the virion integrity, but also interfere with the viral assembly, budding, and spread. Ebola- specific cell penetrable antibodies according to this invention comprises a group of engineered cell penetrable human single chain antibodies (R9- HuscFvs) that bound to the EBOV VP40 were produced using an antibody phage display technology. Gene sequences coding for VP40- bound HuscFvs were subcloned from the phagemids into protein expression plasmids downstream to the nonaarginine (R9) coding sequence. The R9- HuscFvs produced from the plasmid transformed-E. coli clones readily entered the mammalian cells (being transbodies) and were non- toxic to mammalian cells and mice. Scanning electron microscopy revealed that the transbodies from three clones (R9-HuscFv8, R9-HuscFv23 and R9-HuscFvll9) efficiently inhibited egress of the Ebola virus- like particles from the human hepatic cells transduced with pseudo- typed Lentivirus particles carrying EBOV VP40 and GP genes. Binding
ELISA and computerized simulation indicated that the most effective transbodies (R9-HuscFv8, R9-HuscFv23 and R9-HuscFvl 19) bind to critical residues in the cationic patch of VP40 C-terminal domain important for membrane-binding for viral matrix assembly and nascent virion budding (the
HuscFv8 binds to K221, K224 and K274; the HuscFv23 binds to K221, K224, and K274; and the HuscFvll9 binds to K221, K224, K274 and K275). The R9-HuscFvs-8, -23 and -119 bind also to the VP40 N- terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that the antibodies exert the VP40 inhibitory activity through additional mechanism(S). The R9-Huscfv61 and R9-HuscFv89 which bind to VP40 C-terminal domain also inhibit the Ebola virus egress (albeit to the less extent than the former three transbodies). The R9- HuscFv6 and R9-HuscFv69 did not bind to any of the tested portions of the VP40; yet, they reduced also the egress of the Ebola virus- like particles when compared with the non- treated transfected cells or transfected cells treated with control R9-HuscFv. The so- invented Ebola- specific cell penetrable antibodies have high potential for developing further as safe therapeutic agents for halting/ slowing down the EBOV budding and spread which should allow adequate time for the host immunity to cope with the infecting agent. Brief description of the drawings
Figure 1 illustrates the recombinant full-length VP40, rΔNTD, and rCTD preparations and phage biopannning according to this invention wherein:
Figure 1A illustrates the SDS-PAGE-separated preparations that contained recombinant
VP40 stained by Coomassie Brilliant Blue G-250 dye. M, Standard protein marker; lane 1, homogenate of VP40 pET21a+ transformed BL21 (DE3)E. coli; lane 2, purified E. coli inclusion body; Lane 3, purified and refolded rVP40 (~ 40 kDa) (arrow). Numbers at the left are protein molecular masses in kDa;
Figure IB illustrates (from left to right): M, Standard protein marker; lanes 1 and 2, Purified rΔNTD and rCTD, respectively. Numbers at the left are protein molecular masses in kDa; Figure 1C illustrates the amplicons of genes coding for human single chain antibodies (huscfvs; -1,000 bp) carried by representative phage-transformed-HB2151 E. coli clones derived from phage-panning with recombinant VP40. M, DNA size marker; lanes 1-36, amplicons of huscfvs from 36 representative E. coli clones. Numbers at the left are DNA sizes in base pairs (bp);
Figure ID illustrates the indirect ELISA results for testing binding of the HuscFvs in lysates of representative huscfv positive E. coli clones. BSA was used as control antigen and lysate of original HB2151 E. co// (HB) served as background binding control. Bacterial clones that their lysates gave ELISA signal (OD4o5nm) to the VP40 at least two times above the BSA signal and higher than the HB were chosen. They were clones 6, 8, 23, 61, 89, 118, 158, and 183. Indirect ELISA results of the other clones are not shown;
Figure IE shows the Western blot results for testing the binding of the R9-HuscFvs to the
SDS-PAGE-separated full-length rVP40.M, Protein standard; lane 1, VP40 blot probed with PAb to VP40; lanes 2-7, VP40 blots probed with R9-HuscFvs of clones 8, 23, 61, 89, 119, and control
R9-HuscFv, respectively. R9-HuscFv of clones 6 did not bind to the VP40 in the Western blot analysis (data not shown).
Figure 2 illustrates the amino acid sequences, complementary determining regions (CDRs), and immunoglobulin framework regions (FRs) of the VP40 specific HuscFvs, including (from top to bottom) HuscFvll9, HuscFv8, HuscFv61, HuscFv89, HuscFv69, HuscFv6, and HuscFv23; Figure 3 provides toxicity testings of the R9-HuscFvs wherein:
Figure 3 A displays the viability testing result of Vero (African Green monkey kidney continuous cell line) and Huh7 (differentiated human hepatocyte derived carcinoma cell line) cells
(40,000 cells in individual culture wells) that were incubated with 40 μg of R9- HuscFvs for 48 h and the % cell viability were determined by using the Cell Titer- Glo® Luminescent Cell Viability
Assay kit. It can be seen that the R9-HuscFvs did not cause significant reduction of the cell viability compared with the cells cultured in the medium alone;
Figure 3B illustrates the body weights of mice injected intraperitoneally with four doses of R9-HuscFv on every alternate day in comparison with control mice that were given buffer. Both groups of mice did not show any sign of illness and instead they gained some weight during the 14 day-period of observation. Figure 4 illustrates the results of scanning electron microscopy (SEM) for determining the
Ebola VLP budding from Huh7 cells that were transduced with pseudo- typed Lentiviral particles carrying EBOV VP40 and GP genes after incubating with cell penetrable HuscFvs specific to VP40 (R9-HuscFvs) and controls. From A to J, transduced cells cultured in the medium alone, treated with control R9-HuscFv, R9-HuscFv6, R9-HuscFv8, R9-HuscFv23, R9-HuscFv61, R9-
HuscFv89, and R9-HuscFvll9, and normal cell, respectively. There were numerous Ebola VLP budded out from the transduced cells cultured in the medium alone (A) and treated with control R9-
HuscFv (B) . On contrary, negligible amounts (if there were any) of the VLP egress from the transduced cells treated with R9-HuscFv8 (D), R9-HuscFV23 (E) and R9-HuscFvll9 (H) indicating that these transbodies effectively inhibited the VLP budding. The R9-HuscFv6 (C), R9-HuscFV61
(F), and R9-HuscFv89 (G) were relatively ineffective.
Figure 5 illustrates the results of sandwich ELISA for semi- quantification of VP40 in the culture supernatants of Huh7 cells transduced with pseudo- typed Lentiviral particles carrying RNAs coding for EBOV VP40 and GP after incubating with VP40 specific- R9- HuscFvs and controls .The culture supernatants of the transduced cells treated with R9-HuscFvs of clones 8, 23, and 119 had significant reduction of the VP40 amounts in the culture supernatants compared to the non-treated cells )medium (and cells treated with control R9-HuscFv )control scFv .(The R9 HuscFvs of clones 6, 61, and 89 were less effective . N, normal cells . The table below the graph shows p values )ANOVA, R 3.0.3 program (for comparison of the mean OD405nm of all treatment groups .The efficacies of the R9- HuscFvs of clones 8, 23 and 119 in reducing the VP40 amounts in the culture supernatants of the transduced cells were not different .
Figure 6 is a table showing the binding of the R9- HuscFvs to L domain )N-tail peptide(, recombinant truncated N- terminal )rΔNTD or I (domain, and recombinant C- terminal )rCTD or M (domain of Ebola virus VP40 .
Detailed description of the invention
Ebola-specific cell penetrable antibodies according to this invention are engineered human single chain antibodies (HuscFvs) as displayed in Figure 2 wherein each antibody comprises its amino acid sequences, complementarily determining regions ( CDRs) and immunoglobulin framework regions (FRs). The materials and methods that were used, in one embodiment according to this invention, to derive the Ebola-specific cell penetrable antibodies efficient for inhibiting the biologically activities of EBOV VP40 and, thus potentially, their viral assembly, morphogenesis, egress/budding, and replication are generally provided in the following steps:
- Making of recombinant full-klength VP40, rΔNTD and rCTD. The recombinant full klength rVP40 and rCTD were purified from the inclusion bodies and rΔNTD from soluble fraction of transformed BL21 (DE3) E. coli clones carrying the recombinant pET21a+plasmids with the respective EBOV VP40 gene inserts are shown in Figure 1A and IB. All recombinant proteins were verified by mass spectrometry as the EBOV VP40.
- Creating phage clones displaying HuscFvs that bound to VP40 and the phage transformed E. coli clone.- The full-length rVP40 were used as antigen in the phage bio-panning for selecting the protein- bound phage clones from a human scFv phage display library (Kulkeaw et al., 2009). The antigen-bound phages were transfected into HB2151 E. coli and the bacteria were grown on a selective agar. Colonies were picked randomly from the plate and screened for the presence of the gene sequences coding for HuscFvs (huscjvs) by PCR using pCANTAB5E phagemid specific primers. Figure 1C shows huscfv amplicons (-1,000 bp) carried by representative phage-transformed E. coli clones derived from the panning. The E. coli colonies that were positive for the huscfv amplicons were grown under EPTG induction and their lysates containing the expressed HuscFvs were tested for binding to rVP40 by indirect ELISA From the assay, lysates of 8 E. coli clones (nos. 6, 8, 23, 61, 89, 119, 158 and 183) gave significant ELISA signals above the controls (lysate of the original HB2151 E. coli or HB and BSA) (Figure ID). The huscjvs of these clones were sequenced. Only nucleotide sequences huscjvs of 6 clones (nos. 6, 8, 23, 61, 89, and
119) were complete, i.e., contained sequences coding for FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 of both VH and VL domains with nucleotides of (Gly4Ser)3 linker between the two domains. Therefore these 6 clones were tested further. The amino acid sequences, complementarity determining regions and immunoglobulin framework regions of the huscjvs of clones 6, 8, 23, 61, 89, and 119 are shown in Figure 2.
- Producing cell penetrable HuscFvs specific to VP40-. DNA sequences coding for
HuscFv6, HuscFv8, HuscFv23, HuscFv61, HuscFv89, and HuscFvll9 were linked molecularly downstream to the nonaarginines (R9) nucleotides in the pLATE52 plasmids by means of the ligase independent cloning (LIC) method. The recombinant plasmids were put in the Rosetta™ 2 (DE3)E. coli. The R9- HuscFv6, R9- HuscFv8, R9- HuscFv23, R9- HuscFv61, R9- HuscFv89 and R9-
HuscFvll9 were expressed, purified and refolded from the inclusion bodies of respective plasmid transformed E. coli clones and were retested for binding to the rVP40 by Western blot analysis. R9-
HuscFvs of only 5 clones (8, 23, 61, 89 and 119) bound to the rVP40 (Figure IE). R9-HuscFv6 did not bind to the VP40 on the NC blots (data not shown).
- Binding of R9 HuscFvs to different regions of VP40 by indirect ELISA: The VP40 L domain peptide, rΔNTD and rCTD were used to test the binding specificity of the R9-HuscFvs in the indirect ELISA. As shown in Figure 6, The R9-HuscFv8, R9-HuscFv23, and R9-HuscFvll9 bound to the rΔNTD (interactive/ self- interacting for oligomerization or I domain) and the rCTD (membrane-binding or M domain). Also, they gave significant ELISA OD4o5nm to the VP40 L domain peptide. The R9-HuscFv61 and R9-HuscFv89 bound modestly only to the CTD (OD405nm 0.08 and 0.09, respectively). The R9-HuscFvs6 did not give positive binding to any of the VP40 preparations which conformed to the above Western blot result.
- Creating a set of intracellular localization of the R9-HuscFvs: The Huh7 cells incubated with the R9- HuscFvs were stained for nuclei by DAPI and R9-HuscFvs by mouse anti-6x His tag and goat anti-mouse immunoglobulin before subjecting to a confocal microscopy. It was found that the R9- linked- HuscFvs readily entered the mammalian cells. The intracellular localizations of the R9-HuscFvs are not shown in this document.
- Testing for Toxicity of R9-HuscFvs: For the ex vivo cytotoxic assay, Vero and Huh7 cells (40,000 cells in individual culture wells) incubated with 40 μg of R9-HuscFvs for 48 h did not have significant reduction of the cell viability compared with the cells cultured in the medium alone (Figure 3A). Concurrently, mice injected with four doses of R9-HuscFvs at 25 μg/g body weight on every alternate day did not show any sign of illness. They had normal appetite and gained some weight similar to the control mice (Figure 3B). On day 14, their liver enzymes including SGOT and SGPT were similar to the control mice.
- Inhibiting VLP budding by VP40-specific HuscFvs (VP40 neutralization assay): To determine whether the cell penetrable HuscFvs could inhibit the VLP budding, Huh7 cells were transduced with pseudo-typed-Lentivirus particles carrying RNAs coding for EBOV VP40 and GP at MOI 0.5 followed by treating the cells with 40 μg of R9- HuscFvs from the 6 E. coli clones (HuscFv6, HuscFv8, HuscFv23, HuscFv61, HuscFv89, and HuscFvll9) and control R9-HuscFv.
The tranduced cells maintained in the medium alone served as negative inhibition control. The transduced cells were kept in a 5% CO2 incubator at 37°C for 48 h. The culture supernatants of all wells were collected for quantification of VP40 while the cells were subjected to scanning electron microscopy (SEM). It was revealed by the SEM that there were numerous VLP budded out from the cells transduced with the pseudo-typed-Lentivirus particles carrying the EBO V RNAs that were maintained in the medium alone (Figure 4A) Similar results were obtained for the transduced cells cultured in the medium containing 40 μg of control (irrelevant) R9-HuscFv (Figure 4B) as well as the cells treated with R9-HuscFv6, R9-HuscFv61, and R9- scFv89 (Figures 4C, 4F and 4G, respectively). Nevertheless, there were negligible EBOV-like particles (if there were any) budded out from the transduced cells treated with R9-HuscFv8, R9-HuscFv23, and R9-HuscFvll9 (Figures 4D, 4E and 4H, respectively). Culture supernatants from all wells of the VP40 neutralization assay that had been treated with M-per mammalian protein extraction reagent were subjected to sandwich ELISA using PAb to VP40 as the capture reagent, E-tagged-HuscFvll9 as the protein detection reagent, and anti- E tag- HRP conjugate and ABTS substrate for color development. The effectiveness of the R9-HuscFvs and controls in reducing the VP40 amounts in the cultured fluids of the transduced Huh7 cells are shown in Figure 5. The most VP40 amount was found in the culture supernatant of the transduced cells cultured in the medium alone (the highest OD405nm). The results of the sandwich ELISA were, more or less, conformed to the SEM results. The culture supernatants of the transduced cells treated with R9-HuscFv8, R9-HuscFv23 and R9-HuscFvll9 had the least VP40 amounts and significantly less than the untreated control and those treated with irrelevant R9-HuscFv. The efficacies of the antibodies in the falling order of magnitude were R9-
HuscFv8 = R9-HuscFv23 = R9-HuscFvll9 > R9-HuscFv89 > R9-HuscFv61 > R9-HuscFv6 > control R9-HuscFv > medium. The inserted Table in Figure 5 shows p values for comparison among treatments.
- Identifying the binding residues and domain region sites of individual R9-HuscFvs to the VP40: Tentative residues and domains of the VP40 bound by the individual HuscFvs are shown as below.
Figure imgf000011_0001
Figure imgf000012_0001
The HuscFv8, HuscFv23 and HuscFvll9 which were highly effective in inhibiting the VLP budding from the Huh7 cells tranduced with pseudo-typed-Lentivirus particles carrying VP40 and GP genes were found to dock on several residues of the CTD cationic patch (i.e. CTD basic patch) that are important for membrane- binding of the dimeric VP40 including K221, K224, and K274 for HuscFv8 and HuscFv23 and K221, K224, K274, and K275 for HuscFvll9. The set of amino acid residues in the CDRs of HuscFv8 that bind to K221 are Y52, D54, and W102 in the VH- CDR2, VH-CDR2 and VH-CDR3 domains, respectively; those that bind to K224 are W33 and
D57 in the VH-CDR1 and VH-CDR2 domains, respectively; and those that bind to K274 are D54 and D55 in the VH-CDR2 and VH-CDR3 domains, respectively. The set of amino acid residues in the CDRs of HuscFv23 that bind to K221 is P106 in the VH-CDR2 domain; those that bind to
K224 are S195 and G209 in the VL-CDR2 and VL-FR3 domains, respectively; and those that bind to K274 are Dill and Y175 in the VH-CDR3 and VLCDRl domains, respectively. The set of amino acid residues in the CDRs of HuscFvll9 that binds to K221 is P102 in VH-CDR3 domain; those that bind to K224 are S52 and D57 in the VH-CDR2 domain; those that bind to K274 are
T30 and N31 in the VH-CDR1 domain; and those that bind to K275 is G54 in the VH-CDR2 domain. Further, these antibodies also interacted with some residues in the NTD and L domain peptide, as shown by the positive Indirect ELISA results in Figure 6, to inhibit additional VP40 activity through some mechanisms. The HuscFv61 and HuscFv89 which gave positive binding to rCTD by the indirect ELISA as shown in Figure 6 also interacted with residues of CTD by the computerized molecular docking. The HuscFv61 interacted with K270 and K275 through E55 in the VH-CDR2 domain and F228 in the VL-CDR3 domain of the antibody, respectively; while HuscFv89 interacted with K275 of the CTD cationic patch through S225 in the VL-CDR3 domain of the antibody (Figure 7). Both HuscFvs also bound to other residues in the CTD. Because, the
HuscFv6 did not bind to any region of the VP40 by indirect ELISA and Western blotting, thus, the computerized molecular interaction was not performed.

Claims

Claims
1. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 8 (HuscFv8) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000014_0001
2. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 23 (HuscFv23) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000014_0002
3. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 119 (HuscFvll9) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000015_0001
4. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 61 (HuscFv61) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000015_0002
5. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 89 (HuscFv89) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000016_0002
6. An Ebola-specific cell penetrable antibody wherein said antibody is an engineered human single chain antibody 6 (HuscFv6) having the following amino acid sequences, complementarily determining regions (CDRs) and immunoglobulin framework regions (FRs):
Figure imgf000016_0001
7. The Ebola-specific cell penetrable antibody according to any of claims 1-5 wherein the antibody binds to a virion protein 40 (VP40) of an Ebola virus.
8. The Ebola-specific cell penetrable antibody according to any of claims 1-6 wherein the gene sequences coding for the antibody is subcloned from the phagemid into protein expression plasmid downstream to the non-arginine (R9) coding sequence.
9. The Ebola-specific cell penetrable antibody according to any of claims 1-6 wherein the antibody is a transbody produced from a plasmid transformed-E.coli clone that readily enter mammalian cells.
10. The Ebola-specific cell penetrable antibody according to any of claims 1-6 wherein the antibody inhibits an egress of an Ebola virus-like particle from a human hepatic cell transduced with a lentivirus particle carrying Ebola virus's virion protein 40 (VP40) and secreted glycoprotein (GP) genes.
11. The Ebola-specific cell penetrable antibody according to claim 10 wherein the antibody further inhibits viral assembly, morphogenesis, or replication.
12. The Ebola-specific cell penetrable antibody according to any of claims 1-5 wherein the antibody binds to a virion protein 40 (VP40)'s C-terminal domain of the Ebola virus.
13. The Ebola-specific cell penetrable antibody according to any of claims 1-5 wherein at least one of amino acid residues in the CDRs of the antibody binds to at least one of amino acid residues of a virion protein 40 (VP40) s C-terminal domain of the Ebola virus.
14. The Ebola-specific cell penetrable antibody according to claim 1 wherein the antibody
HuscFv8 binds to K221, K224, and K274 residues in a cationic patch of a virion protein 40 (VP40)
C-terminal domain of the Ebola virus.
15. The Ebola-specific cell penetrable antibody according to claim 14 wherein a set of amino acid residues in the CDRs of the antibody that binds to K221 are Y52 and D54 in the VH-CDR2 domain, and W102 in the VH-CDR3 domain of the antibody.
16. The Ebola-specific cell penetrable antibody according to claim 14 wherein a set of amino acid residues in the CDRs of the antibody that binds to K224 are W33 in the VH-CDR1 domain and D57 in the VH-CDR2 domain of the antibody.
17. The Ebola-specific cell penetrable antibody according to claim 14 wherein a set of amino acid residues in the CDRs of the antibody that bind to K274 are D54 in the VH-CDR2 domain and
D55 in the VH-CDR3 domain of the antibody.
18. The Ebola-specific cell penetrable antibody according to claim 2 wherein the antibody HuscFv23 binds to K221, K224, K274 residues in a cationic patch of a virion protein 40 (VP40) C-terminal domain of the Ebola virus.
19. The Ebola-specific cell penetrable antibody according to claim 18 wherein an amino acid residue in the CDR of the antibody that binds to K221 is P106 in the VH-CDR3 domain of the antibody.
20. The Ebola-specific cell penetrable antibody according to claim 18 wherein a set of amino acid residues in the CDR and FR of the antibody that binds to K224 are S195 in the VL-CDR2 domain and G209 in the \^FR3 domain of the antibody.
21. The Ebola-specific cell penetrable antibody according to claim 18 wherein a set of amino acid residues in the CDRs of the antibody that binds to K274 are Dill in the VH-CDR3 domain and Y175 in the VLCDRl domain of the antibody.
22. The Ebola-specific cell penetrable antibody according to claim 3 wherein the antibody
HuscFvll9 binds to K221, K224, K274, and K275 residues in a cationic patch of a virion protein 40 (VP40) C-terminal domain of the Ebola virus.
23. The Ebola-specific cell penetrable antibody according to claim 22 wherein an amino acid residue in the CDR of the antibody that binds to K221 is P102 in the VH-CDR3 domain of the antibody.
24. The Ebola-specific cell penetrable antibody according to claim 22 wherein a set of amino acid residues in the CDRs of the antibody that binds to K224 are S52 and D57 in the VH-CDR2 domain of the antibody.
25. The Ebola-specific cell penetrable antibody according to claim 22 wherein a set of amino acid residues in the CDRs of the antibody that binds to K274 are T30 and N31 in the VH-CDRl domain of the antibody.
26. The Ebola-specific cell penetrable antibody according to claim 22 wherein an amino acid residue in the CDR of the antibody that binds to K275 is G54 in the VH-CDR2 domain of the antibody.
27. The Ebola-specific cell penetrable antibody according to claim 4 wherein the antibody
HuscFv61 binds to K270 and K275 residues in a cationic patch of a virion protein 40 (VP40) C- terminal domain of the Ebola virus.
28. The Ebola-specific cell penetrable antibody according to claim 27 wherein an amino acid residue in the CDR of the antibody that binds to K720 is E55 in the VH-CDR2 domain of the antibody.
29. The Ebola-specific cell penetrable antibody according to claim 27 wherein an amino acid residue in the CDR of the antibody that binds to K275 is F228 in the VL-CDR3 of the antibody.
30. The Ebola-specific cell penetrable antibody according to claim 5 wherein the antibody HuscFv89 binds to K275 residue in a cationic patch of a virion protein 40 (VP40) C-terminal domain of the Ebola virus.
31. The Ebola-specific cell penetrable antibody according to claim 30 wherein an amino acid residue in the CDR of the antibody that binds to K275 is S225 of the VL-CDR3 domain of the antibody.
32. The Ebola-specific cell penetrable antibody according to any of claims 1-3 wherein the antibody binds to a virion protein 40 (VP40)'S C-terminal domain and N-terminal domain of the Ebola virus.
33. The Ebola-specific cell penetrable antibody according to any of claims 1-3 wherein at least one of amino acid residues in the CDRs of the antibody binds to at least one of amino acid residues in a virion protein 40 (VP40)'S C-terminal domain and N-terminal domain of the Ebola virus.
34. The Ebola-specific cell penetrable antibody according to any of claims 1-3 wherein the antibody binds to a virion protein 40 (VP40)'S C-terminal domain, N-terminal domain, and L domain peptide of the Ebola virus.
35. The Ebola-specific cell penetrable antibody according to any of claims 1-3 wherein at least one of amino acid residues in the CDRs of the antibody binds to at least one of amino acid residues in a virion protein 40 (VP40)'S C-terminal domain, N-terminal domain, and L domain peptide of the Ebola virus.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108250293A (en) * 2018-02-11 2018-07-06 浙江大学 Anti- Ebola virus VP40 protein monoclonal antibodies G7A6 and its application
WO2020093672A1 (en) * 2018-11-06 2020-05-14 中国人民解放军军事科学院军事医学研究院 Monoclonal antibody 2g1 for broad-spectrum neutralization of ebola viruses and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108250293A (en) * 2018-02-11 2018-07-06 浙江大学 Anti- Ebola virus VP40 protein monoclonal antibodies G7A6 and its application
WO2020093672A1 (en) * 2018-11-06 2020-05-14 中国人民解放军军事科学院军事医学研究院 Monoclonal antibody 2g1 for broad-spectrum neutralization of ebola viruses and application thereof

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