Methods of treating filovirus infections utilizing bispecific antibodies and fusion proteins that bind EBOV and NPC1

Abstract

Disclosed are bispecific antibodies and bispecific fusion constructs that bind to Niemann-Pick C1 (NPC1) receptor for treating or preventing filovirus infections, pharmaceutical compositions comprising the bispecific antibodies, and therapeutic methods using the bispecific antibodies.

Claims

1. A method of treating or preventing or reducing or inhibiting a filovirus infection in a subject comprising administering to the subject a monoclonal antibody or fragment thereof that binds to Niemann-Pick C1 (NPC1) receptor in an amount effective to treat or prevent or reduce or inhibit a filovirus infection in a subject, wherein the antibody or fragment thereof comprises: a variable region of a light chain that comprises the amino acid sequence set forth in SEQ ID NO:3 and a variable region of a heavy chain that comprises the amino acid sequence set forth in SEQ ID NO:4; or a variable region of a light chain that comprises the amino acid sequence set forth in SEQ ID NO:5 and a variable region of a heavy chain that comprises the amino acid sequence set forth in SEQ ID NO:6; or a variable region of a light chain that comprises the amino acid sequence set forth in SEQ ID NO:7 and a variable region of a heavy chain that comprises the amino acid sequence set forth in SEQ ID NO:8.

2. The method of claim 1, wherein the monoclonal antibody is provided in a bispecific antibody or bispecific fusion construct comprising (i) the antibody or fragment of claim 1 and (ii) an antibody or fragment that binds to an Ebola virus, wherein the antibody or fragment that binds to the Ebola virus is monoclonal antibody KZ52 or a single-chain variable fragment (scFv) sequence derived from monoclonal antibody KZ52.

3. The method of claim 2, wherein a NPC1-specific sequence is fused to a single-chain variable fragment (scFv) sequence derived from EBOV GP-specific monoclonal antibody KZ52.

4. The method of claim 3, wherein fusion is to a N- or C-terminus of an IgG heavy chain or light chain.

5. The method of claim 1, wherein the monoclonal antibody is provided in a fusion construct comprising the antibody or fragment of claim 1 fused to Niemann-Pick C2 (NPC2).

6. The method of claim 2, wherein the bispecific antibody comprises heavy and light chains of a NPC1-specific sequence fused to variable VH and VL domains of KZ52 to generate a dual-variable domain Ig.

7. The method of claim 2, wherein the bispecific antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs:9-20.

8. The method of claim 5, wherein the bispecific fusion construct comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22.

9. The method of claim 1, wherein the subject is infected with a filovirus.

10. The method of claim 1, wherein the subject is at risk for infection with a filovirus.

11. The method of claim 10, wherein the subject is a family member or healthcare worker in an area of an outbreak of a filovirus infection.

12. The method of claim 10, wherein the subject is a medical personnel, first responder or military personnel potentially exposed or exposed to a filovirus as the result of bioterrorism or biological warfare.

13. The method of claim 10, wherein the subject is a biosafety level 3/4 laboratory personnel or animal worker potentially exposed or exposed to a filovirus.

14. The method of claim 1, wherein the filovirus is an Ebola virus.

15. The method of claim 14, wherein the Ebola virus species is Zaire ebolavirus or Sudan ebolavirus.

16. The method of claim 1, wherein the filovirus is a Marburg virus, a Bundibugyo virus, a Sudan virus, a Ravn virus or a Lloviu virus.

17. The method of claim 1, wherein the subject is a mammal.

18. The method of claim 1, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A. NPC1 is required for Ebola virus infection. Niemann-Pick C1 (NPC1), a ubiquitous multi-pass membrane protein localized to late endosomes is required for cytoplasmic entry and infection by all filoviruses. The filovirus spike glycoprotein, GP, must engage NPC1's second luminal domain (domain C; arrow) to drive viral membrane fusion and cytoplasmic escape.

(2) FIG. 1B. NPC1 is required for Ebola in vivo pathogenesis. NPC1-knockout mice (NPC1.sup.−/−), but not their WT littermates (NPC1.sup.+/+), are completely resistant to infection and killing by EBOV and MARV.

(3) FIG. 2A-2D. A illustrates the X-ray crystal structure of a cleaved EBOV GP (GPCL) trimer bound to three copies of a soluble human NPC1 domain C protein. In B, side bottom views of NPC1 domain C are shown, with residues contacting GPCL (left panel) and mAb 548 (right panel) highlighted. C-D demonstrate the kinetics of binding of 548 to human NPC1 domain C by biolayer interferometry (BLI). D shows BLI-derived kinetic binding constants for the interaction of 548 with human and non-human primate (NHP; Colobus guereza) NPC1 domain C. 548 recognizes these proteins with picomolar affinity, at both neutral and acid pH.

(4) FIG. 3. mAbs specific to human NPC1 domain C block GP-NPC1 binding in vitro. Capacity of three anti-NPC1 mAbs to block GP-NPC1 binding in an ELISA. A soluble, flag-tagged form of NPC1 domain C was pre-incubated with the indicated concentrations of each mAb, and the protein-mAb mixtures were then added to plates coated with vesicular stomatitis virus (VSV) particles bearing EBOV GP. Bound domain C was detected with an anti-flag antibody. Irrelevant isotype-matched antibodies had no effect on GP-NPC1 domain C binding (not shown).

(5) FIG. 4. Anti-NPC1 domain C mAbs neutralize VSV-EBOV GP infection weakly or not at all. VSV-GP particles were exposed to U2OS human osteosarcoma cells in the presence of increasing concentrations of NPC1-specific mAbs or a control GP-specific neutralizing mAb (KZ52). The number of infected cells was determined at 16 h post-infection, and normalized to that obtained in the absence of antibody (set to 100%).

(6) FIG. 5A. Engineering antibodies with dual binding specificities for GP and NPC1. Constructs encoding bsAbs were generated by fusing NPC1-specific or control IgG sequences to a single-chain variable fragment (scFv) sequence derived from the EBOV GP-specific mAb KZ52. Fusions were done in four configurations: to the N- or C-terminus of the IgG heavy chain [HC] or light chain [LC].

(7) FIG. 5B. Engineering antibodies with dual binding specificities for GP and NPC1. bsAbs and IgGs were expressed in 293-Freestyle cells by co-transfection of HC and LC expression vectors, and purified by protein A affinity chromatography.

(8) FIG. 5C. bsAbs, but not NPC1-specific IgGs, can simultaneously bind to GP and NPC1 domain C. bsAbs and IgGs were captured onto GP-coated ELISA plates and then incubated with NPC1 domain C-flag. Bound NPC1 domain C was detected with an anti-flag antibody.

(9) FIG. 6. GP-NPC1 bsAbs neutralize VSV-EBOV GP infection with enhanced potency. Left panel, GP-NPC1 bsAbs show enhanced potency at viral neutralization relative to control GP-only bsAbs. VSV-EBOV GP particles were pre-incubated with the indicated concentrations of each bsAb for 1 h at room temp and then allowed to infect U2OS cells. Infected cells were quantified at 16 h post-infection and normalized to a no-antibody control (100%). Right panel, the neutralization curves were fit to a logistic equation to extract the concentration of half-maximal neutralization (IC.sub.50±95% confidence intervals).

(10) FIG. 7. The enhanced neutralization potency of GP-NPC1 bsAbs can be attributed to their receptor blocking activity. Left panel, VSV-EBOV GP particles were pre-incubated with the indicated concentrations of each bsAb for 1 h at room temp and then allowed to infect matched U2OS cell lines expressing endogenous levels of NPC1 or over-expressing NPC1 (NPC1.sup.hi). Infected cells were quantified at 16 h post-infection and normalized to a no-antibody control (100%). Right panel, the neutralization curves were fit to a logistic equation to extract the concentration of half-maximal neutralization (IC.sub.50±95% confidence intervals). The ratio of IC.sub.50s for each bsAb in NPC1.sup.hi vs. WT U2OS cells is shown.

(11) FIG. 8. Fusion of a receptor-blocking IgG to a late endosome-localizing protein facilitates viral neutralization. bsAbs comprising an NPC1-specific IgG (401 or 548) fused to NPC2, a soluble late endosomal/lysosomal protein, were expressed and purified, and examined for their capacity to neutralize VSV-EBOV OP infection, as described in FIGS. 6-7.

(12) FIG. 9A-9D. A illustrates a bispecific antibody strategy for delivery of an NPC1-binding antibody to NPC1-positive endosomal/lysosomal compartments. In this strategy, the heavy and light chains of an NPC1-binding antibody like 548 are genetically fused to the variable VH and VL domains of a delivery antibody like FVM09, to generate a dual-variable domain Ig (DVD-Ig™). FVM09 broadly recognizes ebolavirus GP in extracellular viral particles or on the cell surface. However, the delivery antibody could also target a cell-surface marker, instead of viral GP. In B, the binding of the FVM09˜548 DVD-Ig to each of its antigens (ebolavirus GP, human NPC1-C) is measured by BLI. In C, two-phase BLI binding curves show that FVM09˜548 can simultaneously bind to both of its antigens. D shows BLI-derived kinetic binding constants for the interaction of the FVM09˜548 DVD-Ig with each of its antigens, and compares these constants to those obtained with the respective parent IgGs.

(13) FIG. 10A-10B. A shows an SDS-polyacrylamide gel of purified FVM09˜548 resolved under non-reducing (NR) and reducing (R) conditions and stained with Commassie Brilliant Blue. In B, FVM09˜548 was subjected to size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). The SEC-MALS trace indicates that the majority species of FVM09˜548 (main peak; retention time=5.10 min) is an IgG-like monomer with an apparent molecular weight of ˜175 kDa. A small (typical) amount of soluble aggregates (retention team=4.46 min) is also present.

(14) FIG. 11A-11C measures the capacity of the FVM09˜548 bispecific antibody to neutralize ebolavirus GP-dependent entry and infection in human cells. In A, vesicular stomatitis viruses bearing EBOV GP (VSV-EBOV GP) were incubated with increasing concentrations of FVM09˜548, its parent IgGs FVM09, 548, or an equimolar mixture of the parent IgGs (FVM09+548), and then exposed to cells. Only FVM09˜548 showed potent, dose-dependent neutralization of VSV-EBOV GP infection. B shows that FVM09˜548 can broadly neutralize infection mediated by multiple ebolavirus GPs. C shows that authentic EBOV, BDBV, and SUM can be potently neutralized by FVM09˜548.

(15) FIG. 12A-12C examines the mechanism of action of the FVM09˜548 bispecific antibody. In A, wild-type (WT) VSV-EBOV GP particles, or mutant particles containing two point mutations (E288D/W292R) in the FVM09 epitope ((FVM09mut) were incubated with increasing concentrations of the FVM09˜549 antibody, and then exposed to cells. Loss of FVM09-GP binding is associated with a 99% reduction in the potency of antiviral neutralization, providing evidence that engagement of GP in extracellular virus particles by FVM09˜548 is necessary for its neutralizing activity. In B, VSV-EBOV GP particles were incubated with FVM09˜548 and then exposed to isogenic cell lines expressing physiological levels of human NPC1 (WT), or expressing high levels of human NPC1 (NPC1-overexp). The neutralization potency of FVM09˜548 was abolished by NPC1 over-expression, likely because of the titration of available antibody by excess NPC1. This experiment provides evidence that engagement of NPC1 domain C by 548 in endosomes is required for the neutralizing activity of FVM09˜548. In C, the delivery of FVM09˜548 to NPC1-containing endosomes was directly examined. VSV-EBOV OP particles labeled with the fluorescent protein mNeongreen (UNG) were incubated with antibodies labeled with the fluorophore pHrodo-Red™, which only fluoresces at acid pH (left panel). The virus-antibody mixture was exposed to cells at 37° C. for 30 min, after which time, cells were examined for both virus- and antibody-associated fluorescence by two-color flow cytometry. The flow cytometric analysis is shown in the right panel. Cells were gated into virus-negative and virus-positive populations based on mNeongreen fluorescence, and each subpopulation was father examined for pHrodo-Red™ fluorescence. Strong pHrodo-Red™ fluorescence was seen with FVM09 in the virus-positive sample only, indicating that binding of this antibody to GP on extracellular virus particles affords its delivery to acidic endosomes. Little or no pHrodo-Red™ fluorescence was observed with 548 in the absence or presence of virus, indicating that this antibody cannot access endosomal compartments on its. By contrast FVM09˜548 gave a strong pHrodo-Red™ signal in virus-positive cells. Therefore, combining FVM09 and 548 into a single bispecific antibody promotes efficient delivery of 548 to endosomes through the action of FVM09,

(16) FIG. 13A-13C. The capacity of the human NPC1 domain C binding antibody 548 to bind to a non-human primate (Colobus guereza) NPC1 domain C protein and block EBOV GP-NPC1 binding is shown. In A, BLI is employed to detect and measure 548 binding to C. guereza NPC domain C at the acidic pH of late endosomes. B shows the capacity of 548 to block GP-C. guereza NPC1 domain C binding in an ELISA. A soluble, flag-tagged form of C. guereza NPC1 domain C was pre-incubated with the indicated concentrations of 548, and the protein-mAb mixtures were then added to plates coated with vesicular stomatitis virus (VSV) particles bearing EBOV GP. Bound domain C was detected with an anti-flag antibody. Because the sequences of C. guereza NPC1 that contact GP are identical to those of NPC1 proteins from rhesus macaques (Macaca mulatto) and cynomolgus macaques (Macaca fascicularis) (FIG. 13C), 548 can be evaluated for antiviral protection in these two established non-human primate (NHP) models of filovirus challenge. In C, sequences corresponding to NPC1 residues 485-514 (human NPC1 numbering) are shown. The 548 epitope is indicated—it is identical in humans and the three indicated NHP species. Homo sapiens—SEQ ID NO:24, Macaca mulatto SEQ ID NO:25, Colobus guereza—SEQ ID NO:26, Macaca fascicularis—SEQ ID NO:27.

DETAILED DESCRIPTION OF THE INVENTION

(17) The present invention provides a method for treating or preventing or reducing or inhibiting or reducing the risk or incidence of a filovirus infection in a subject comprising administering to the subject a bispecific antibody or a bispecific fusion construct that binds to Niemann-Pick C1 (NPC1) receptor in an amount effective to treat or prevent or reduce or inhibit or reduce the risk or incidence of a filovirus infection in a subject.

(18) The invention also provides a bispecific antibody or a bispecific fusion construct that hinds to Niemann-Pick C1 (NPC1) receptor for treating or preventing or reducing or inhibiting or reducing the risk or incidence of a filovirus infection in a subject.

(19) The bispecific antibody or bispecific fusion construct can combine both antiviral and anti-NPC1 specificities in the same molecule. For example, the bispecific antibody or bispecific fusion construct can bind both to NPC1 and to filovirus glycoprotein (GP).

(20) The bispecific antibody can be generated by fusing a NPC1-specific sequence to a single-chain variable fragment (scFv) sequence derived from EBOV GP-specific monoclonal antibody KZ52, where the fusion can be to a N- or C-terminus of an IgG heavy chain or light chain.

(21) The bispecific antibody or bispecific fusion construct can combine an anti-NPC1 specificity with an endosomal targeting functionality. This can include an antibody specific for a cellular molecule that affords endosomal delivery (e.g., NPC2) or a peptide or protein that would do the same (not necessarily just an antibody). Thus, the bispecific antibody or bispecific fusion construct can bind both to NPC1 and to Niemann-Pick C2 (NPC2).

(22) The bispecific antibody or bispecific fusion construct can be generated by fusing heavy and light chains of a NPC1-specific sequence to variable VH and VL domains of a delivery antibody to generate a dual-variable domain Ig. The dual variable domain-Ig (DVD-Ig) bispecific antibody format was developed by AbbVie (Wu et al., 2007). The delivery antibody can target, for example, a filovirus glycoprotein (GP) or a cell-surface marker. The delivery antibody can be, for example, FVM09 (Keck et al. 2015). FVM09 broadly recognizes ebolavirus GP in extracellular viral particles or on the cell surface.

(23) The anti-NPC1 amino acid sequence of a variable region of a light chain of the bispecific antibody or bispecific fusion construct can comprise the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. The anti-NPC1 amino acid sequence of a variable region of a heavy chain of the bispecific, antibody or bispecific fusion constrict can comprise the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. The anti-NPC1 amino acid sequence of a variable region of a light chain can comprise the amino acid sequence set forth in SEQ ID NO:3 and a variable region of a heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:4. The anti-NPC1 amino acid sequence of a variable region of a light chain can comprise the amino acid sequence set forth in SEQ ID NO:5 and a variable region of a heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:6. The anti-NPC1 amino acid sequence of a variable region of a light chain can comprise the amino acid sequence set forth in SEQ ID NO:7 and a variable region of a heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:8.

(24) The bispecific antibody can comprise the amino acid sequence set forth in any one of SEQ ID NOs:9-20. The bispecific fusion construct can comprise the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22.

(25) In different uses, the subject can be infected with a filovirus. Alternatively, the subject can be at risk for infection with a filovirus. Subjects who are at risk for infection with filoviruses include subjects who have been exposed to filovirus or are at risk of exposure to filovirus. In addition to the natural occurrence of filoviruses, there is the potential for exposure to these pathogens if they are used as agents of bioterrorism or biological warfare. For example, the subject can be a family member or healthcare worker in an area of an outbreak of a filovirus infection. The subject can be a medical personnel, first responder or military personnel potentially exposed or exposed to a filovirus as the result of bioterrorism or biological warfare. The subject can be a biosafety level 3/4 laboratory personnel or animal worker potentially exposed or exposed to a filovirus.

(26) The family Filoviridae is a family of viruses including genera Ebolavirus and Marburgvirus. The Ebola virus species can be Zaire ebolavirus or Sudan ebolavirus. Additional examples of filovirus include a Bundibugyo virus, a Sudan virus, a Ravn virus and a Lloviu virus.

(27) The subject can be a mammal, such as, for example, livestock, a bat, a primate or a human.

(28) To treat a subject with a filovirus infection means to reduce or stop the spread of filovirus in the subject, or to eliminate the filovirus from the subject, or to reduce or eliminate a sign or symptom of filovirus infection in the subject. Filovirus infection is characterized by hemorrhagic fever, including abnormalities in blood coagulation. As used herein, “preventing” a filovirus infection means reducing the development of, or reducing the extent of, one or more symptoms of the condition, as compared to the development or extent the condition takes in the absence of preventative treatment. In an embodiment, “preventing” as used herein does not mean an absolute prevention, but a lessened extent of the condition brought about prophylactically, or to reduce the risk or incidence of a filovirus infection.

(29) Human NPC1 receptor protein has the amino acid sequence (SEQ ID NO:1) (NCBI Reference Sequence: NM_000271.4):

(30) TABLE-US-00001 MTARGLALGL LLLLLCPAQV FSQSCVWYGE CGIAYGDKRY NCEYSGPPKP KPKDGYDLVQ  60 ELCPGFFFGN VSLCCDVRQL QTLKDNLQLP LQFLSRCPSC FYNLLNLFCE LTCSPRQSQF 120 LNVTATEDYV DPVTNQTKTN VKELQYYVGQ SFANAMYNAC RDVEAPSSND VDEVTAPCSC 180 ADACNATNWI EYMFNKDNGQ APFTITPVFS DFPVHGMEPM NNATKGCDES VDEVTAPCSC 240 QDCSIVCGPK PQPPPPPAPW TILGLDAMYV IMWITYMAFL LVFFGAFFAV WCYRKRYFVS 300 EYTPIDSNIA FSVNASDKGE ASCCDPVSAA FEGCLRRLFT RWGSFCVRNP GCVIFFSLVF 360 ITACSSGLVF VRVTTNPVDL WSAPSSQARL EKEYFDQHFG PFFRTEQLII RAPLTDKHIY 420 QPYPSGADVP FGPPLDIQIL HQVLDLQIAI ENITASYDNE TVTLQDICLA PLSPYNTNCT 480 ILSVLNYFQN SHSVLDHKKG DDFFVYADYH THFLYCVRAP ASLNDTSLLH DPCLGTFGGP 540 VFPWLVLGGY DDQNYNNATA LVITFPVNNY YNDTEKLQRA QAWEKEFINE VKNYKNPNLT 600 ISFTAERSIE DELNRESDSD VFTVVISYAI MFLYISLALG HMKSCRRLLV DSKVSLGIAG 660 ILIVLSSVAC SLGVFSYIGL PLTLIVIEVI PFLVLAVGVD NIFILVQAYQ RDERLQGETL 720 DQQLGRVLGE VAPSMFLSSF SETVAFFLGA LSVMPAVHTF SLFAGLAVFI DFLLQITCFV 780 SLLGLDIKRQ EKNRLDIFCC VRGAEDGTSV QASESCLFRF FKNSYSPLLL KDWMRPIVIA 840 IFVGVLSFSI AVLNKVDIGL SQSLSMPDDS YMVDYFKSIS QYLHAGPPVY FVLEEGHDYT 900 SSKGQNMVCG GMGCNNDSLV QQIFNAAQLD NYTRIGFAPS SWIDDYFDWV KPQSSCCRVD 960 NITDQFCNAS VVDPACVRCR PLTPEGKQRP QGGDFMRFLP MFLSDNPNPK CGKGGHAAYS 1020 SAVNILLGHG TRVGATYFMT YHTVLQTSAD FIDALKKARL IASNVTETMG INGSAYRVFP 1080 YSVFYVFYEQ YLTIIDDTIF NLGVSLGAIF LVTMVLLGCE LWSAVIMCAT IAMVLVNMFG 1140 NMWLWGISLN AVSLVNLVMS CGISVEFCSH ITRAFTVSMN GSRVERAEEA LAHMGSSVFS 1200 GITLTKFGGI VVLAFAKSQI FQIFYFRMYL AMVLLGATHG LIFLPVLLSY IGPSVNKAKS 1260 CATEERYKGT ERERLLNG 1278

(31) Nucleic acid (mRNA) encoding human NPC1 receptor protein has the nucleotide sequence (SEQ ID NO:2) (NCBI Reference Sequence: NM_000271.4):

(32) TABLE-US-00002 1 gaagggcaac acggggacct tgaagcgggg tcgcggcggc gccccagccc gggccaggga 61 gtcccggcag cggcacctcc cagaaagggc ggagccgacg acgccttctt ccttcctgac 121 cggcgcgcgc agcctgctgc cgcggtcagc gcctgctcct gctcctccgc tcctcctgcg 181 cggggtgctg aaacagcccg gggaagtaga gccgcctccg gggagcccaa ccagccgaac 241 gccgccggcg tcagcagcct tgcgcggcca cagcatgacc gctcgcggcc tggcccttgg 301 cctcctcctg ctgctactgt gtccagcgca ggtgttttca cagtcctgtg tttggtatgg 361 agagtgtgga attgcatatg gggacaagag gtacaattgc gaatattctg gcccaccaaa 421 accattgcca aaggatggat atgacttagt gcaggaactc tgtccaggat tcttctttgg 481 caatgtcagt ctctgttgtg atgttcggca gcttcagaca ctaaaagaca acctgcagct 541 gcctctacag tttctgtcca gatgtccatc ctgtttttat aacctactga acctgttttg 601 tgagctgaca tgtagccctc gacagagtca gtttttgaat gttacagcta ctgaagatta 661 tgttgatcct gttacaaacc agacgaaaac aaatgtgaaa gagttacaat actacgtcgg 721 acagagtttt gccaatgcaa tgtacaatgc ctgccgggat gtggaggccc cctcaagtaa 781 tgacaaggcc ctgggactcc tgtgtgggaa ggacgctgac gcctgtaatg ccaccaactg 841 gattgaatac atgttcaata aggacaatgg acaggcacct tttaccatca ctcctgtgtt 901 ttcagatttt ccagtccatg ggatggagcc catgaacaat gccaccaaag gctgtgacga 961 gtctgtggat gaggtcacag caccatgtag ctgccaagac tgctctattg tctgtggccc 1021 caagccccag cccccacctc ctcctgctcc ctggacgatc cttggcttgg acgccatgta 1081 tgtcatcatg tggatcacct acatggcgtt tttgcttgtg ttttttggag cattttttgc 1141 agtgtggtgc tacagaaaac ggtattttgt ctccgagtac actcccatcg atagcaatat 1201 agctttttct gttaatgcaa gtgacaaagg agaggcgtcc tgctgtgacc ctgtcagcgc 1261 agcatttgag ggctgcttga ggcggctgtt cacacgctgg gggtctttct gcgtccgaaa 1321 ccctggctgt gtcattttct tctcgctggt cttcattact gcgtgttcgt caggcctggt 1381 gtttgtccgg gtcacaacca atccagttga cctctggtca gcccccagca gccaggctcg 1441 cctggaaaaa gagtactttg accagcactt tgggcctttc ttccggacgg agcagctcat 1501 catccgggcc cctctcactg acaaacacat ttaccagcca tacccttcgg gagctgatgt 1561 accctttgga cctccgcttg acatacagat actgcaccag gttcttgact tacaaatagc 1621 catcgaaaac attactgcct cttatgacaa tgagactgtg acacttcaag acatctgctt 1681 ggcccctctt tcaccgtata acacgaactg caccattttg agtgtgttaa attacttcca 1741 gaacagccat tccgtgctgg accacaagaa aggggacgac ttctttgtgt atgccgatta 1801 ccacacgcac tttctgtact gcgtacgggc tcctgcctct ctgaatgata caagtttgct 1861 ccatgaccct tgtctgggta cgtttggtgg accagtgttc ccgtggcttg tgttgggagg 1921 ctatgatgat caaaactaca ataacgccac tgcccttgtg attaccttcc ctgtcaataa 1981 ttactataat gatacagaga agctccagag ggcccaggcc tgggaaaaag agtttattaa 2041 ttttgtgaaa aactacaaga atcccaatct gaccatttcc ttcactgctg aacgaagtat 2101 tgaagatgaa ctaaatcgtg aaagtgacag tgatgtcttc accgttgtaa ttagctatgc 2161 catcatgttt ctatatattt ccctagcctt ggggcacatg aaaagctgtc gcaggcttct 2221 ggtggattcg aaggtctcac taggcatcgc gggcatcttg atcgtgctga gctcggtggc 2281 ttgctccttg ggtgtcttca gctacattgg gttgcccttg accctcattg tgattgaagt 2341 catcccgttc ctggtgctgg ctgttggagt ggacaacatc ttcattctgg tgcaggccta 2401 ccagagagat gaacgtcttc aaggggaaac cctggatcag cagctgggca gggtcctagg 2461 agaagtggct cccagtatgt tcctgtcatc cttttctgag actgtagcat ttttcttagg 2521 agcattgtcc gtgatgccag ccgtgcacac cttctctctc tttgcgggat tggcagtctt 2581 cattgacttt cttctgcaga ttacctgttt cgtgagtctc ttggggttag acattaaacg 2641 tcaagagaaa aatcggctag acatcttttg ctgtgtcaga ggtgctgaag atggaacaag 2701 cgtccaggcc tcagagagct gtttgtttcg cttcttcaaa aactcctatt ctccacttct 2761 gctaaaggac tggatgagac caattgtgat agcaatattt gtgggtgttc tgtcattcag 2821 catcgcagtc ctgaacaaag tagatattgg attggatcag tctctttcga tgccagatga 2881 ctcctacatg gtggattatt tcaaatccat cagtcagtac ctgcatgcgg gtccgcctgt 2941 gtactttgtc ctggaggaag ggcacgacta cacttcttcc aaggggcaga acatggtgtg 3001 cggcggcatg ggctgcaaca atgattccct ggtgcagcag atatttaacg cggcgcagct 3061 ggacaactat acccgaatag gcttcgcccc ctcgtcctgg atcgacgatt atttcgactg 3121 ggtgaagcca cagtcgtctt gctgtcgagt ggacaatatc actgaccagt tctgcaatgc 3181 ttcagtggtt gaccctgcct gcgttcgctg caggcctctg actccggaag gcaaacagag 3241 gcctcagggg ggagacttca tgagattcct gcccatgttc ctttcggata accctaaccc 3301 caagtgtggc aaagggggac atgctgccta tagttctgca gttaacatcc tccttggcca 3361 tggcaccagg gtcggagcca cgtacttcat gacctaccac accgtgctgc agacctctgc 3421 tgactttatt gacgctctga agaaagcccg acttatagcc agtaatgtca ccgaaaccat 3481 gggcattaac ggcagtgcct accgagtatt tccttacagt gtgttttatg tcttctacga 3541 acagtacctg accatcattg acgacactat cttcaacctc ggtgtgtccc tgggcgcgat 3601 atttctggtg accatggtcc tcctgggctg tgagctctgg tctgcagtca tcatgtgtgc 3661 caccatcgcc atggtcttgg tcaacatgtt tggagttatg tggctctggg gcatcagtct 3721 gaacgctgta tccttggtca acctggtgat gagctgtggc atctccgtgg agttctgcag 3781 ccacataacc agagcgttca cggtgagcat gaaaggcagc cgcgtggagc gcgcggaaga 3841 ggcacttgcc cacatgggca gctccgtgtt cagtggaatc acacttacaa aatttggagg 3901 gattgtggtg ttggcttttg ccaaatctca aattttccag atattctact tcaggatgta 3961 tttggccatg gtcttactgg gagccactca cggattaata tttctccctg tcttactcag 4021 ttacataggg ccatcagtaa ataaagccaa aagttgtgcc actgaagagc gatacaaagg 4081 aacagagcgc gaacggcttc taaatttcta gccctctcgc agggcatcct gactgaactg 4141 tgtctaaggg tcggtcggtt taccactgga cgggtgctgc atcggcaagg ccaagttgaa 4201 caccggatgg tgccaaccat cggttgtttg gcagcagctt tgaacgtagc gcctgtgaac 4261 tcaggaatgc acagttgact tgggaagcag tattactaga tctggaggca accacaggac 4321 actaaacttc tcccagcctc ttcaggaaag aaacctcatt ctttggcaag caggaggtga 4381 cactagatgg ctgtgaatgt gatccgctca ctgacactct gtaaaggcca atcaatgcac 4441 tgtctgtctc tccttttagg agtaagccat cccacaagtt ctataccata tttttagtga 4501 cagttgaggt tgtagataca ctttataaca ttttatagtt taaagagctt tattaatgca 4561 ataaattaac tttgtacaca tttttatata aaaaaacagc aagtgatttc agaatgttgt 4621 aggcctcatt agagcttggt ctccaaaaat ctgtttgaaa aaagcaacat gttcttcaca 4681 gtgttcccct agaaaggaag agatttaatt gccagttaga tgtggcatga aatgagggac 4741 aaagaaagca tctcgtaggt gtgtctactg ggttttaact tatttttctt taataaaata 4801 cattgttttc ctaaaaaaaa aaaaaaa

(33) The invention also provides monoclonal antibodies to NPC1.

(34) Examples of anti-NPC1 antibody amino acid sequences of variable regions for light and heavy chains include:

(35) TABLE-US-00003 401-variable light chain (mouse) (SEQ ID NO: 3) DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYN AKTLVEAVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGSPWTFGG GTKLEIK, 401-variable heavy chain (mouse) (SEQ ID NO: 4) EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGR IDPANGNTEYDTKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRGY YWGRGTTLVTVSS, 548-variable light chain (mouse) (SEQ ID NO: 5) DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYN AKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHHYGSPWAFGG GTKLEIK, 548-variable heavy chain (mouse) (SEQ ID NO: 6) EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEEGLEWIGR IDPADGNTEYVPKFQGKATITADTFSNTVYLQLSGLTSEDTAVYYCSRGY YWGQGTTLVTVSS, 952-variable light chain (mouse) (SEQ ID NO: 7) DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVVWYQQKPGQSPKLLIYW ASTRHTGVPDRFTGSGSGTDFTLTISSVQAEDLALYYCQQHYTSPWTFGG GTKLEIK, and 952-variable heavy chain (mouse) (SEQ ID NO: 8) DVQLQESGPDLVKPXQSLSLTCTVTGYSITSGYSWHWIRQFPGNRLEWMD YIHYSGSINYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWG ATGFDYWGQGTTLVTVSS.

(36) Examples of bispecific Ab design include the following. The underlined region designates glycine-rich linker polypeptide. The bold region designates fusion linker polypeptide. The italicized region corresponds to the constant sequences of the pMAZ-encoded heavy or light constant region sequences for human IgG1 (heavy) and kappa constant domain (light).

(37) TABLE-US-00004 401-scFv-KZ52-HCN (heavy chain N-terminal fusion) (in all of the following scFv-KZ52 bispecifics: scFv is human, IgG HC and LC constant domains are human IgG1, IgG VH and VL domains are mouse) (SEQ ID NO: 9) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHW VKQRPEQGLEWIGRIDPANGNTEYDTKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRGYYWGRGTTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSLGYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK, 401-scFv-KZ52-LCN (light chain N-terminal fusion) (SEQ ID NO: 10) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGLGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDIQMTQSPASLSASVGETVTITCRASENIYSYLAWY QQKQGKSPQLLVYNAKTLVEAVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGSPWTFGGGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC, 401-scFv-KZ52-HCC (heavy chain C-terminal fusion) (SEQ ID NO: 11) EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTEYDTKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCSRGYYWGRGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSLGYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSAGSAGSAGSGG SEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNA ENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSAPLTFGGGTKVEIK, 401-scFv-KZ52-LCC (light chain C-terminal fusion) (SEQ ID NO: 12) DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLVEAVPSRFSGSGSGTQFSLKIN SLQPEDFGTYYCQHHYGSPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSAGSAGSAGSGG SEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNA ENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSAPLTFGGGTKVEIK, 548-scFv-KZ52-HCN (heavy chain N-terminal fusion) (SEQ ID NO: 13) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHW VKQRPEEGLEWIGRIDPADGNTYEVPKFQGKATITADTFSNTVYLQLSGLTSEDTAVYYCSRGYYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSLGYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK, 548-scFv-KZ52-LCN (light chain N-terminal fusion) (SEQ ID NO: 14) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDIQMTQSPASLSASVGETVTITCRASENIYSYLAWY QQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHHYGSPWAFGGGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALWSGNSQESVTEQDSKSDTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC, 548-scFv-KZ52-HCC (heavy chain C-terminal fusion) (SEQ ID NO: 15) EVQLQQSAGELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEEGLEWIGRIDPADGNTEYVPKFQGKATITADTFS NTVYLQLSGLTSEDTAVYYCSRGYYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSLGYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKDNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSAGSAGSAGSGG SEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNA ENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSAPLTFGGGTKVEIK, 548-scFv-KZ52-LCC (light chain C-terminal fusion) (SEQ ID NO: 16) DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKIN SLQPEDFGIYYCQHHYGSPWAFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSAGSAGSAGSGG SEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNA ENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSAPLTFGGGTKVEIK, 952-scFv-KZ52-HCN (heavy chain N-terminal fusion) (SEQ ID NO: 17) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDVQLQESGPDLVKPXQSLSLTCTVTGYSITSGYSWH WIRQFPGNRLEWMDYIHYSGSINYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWGATGFDYWGQGTT LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, 952-scFv-KZ52-LCN (light chain N-terminal fusion) (SEQ ID NO: 18) EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAE NSLYLQMNSLRAEDTAVYYCVREGPARTGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLA VSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVVWY QQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSVQAEDLALYYCQQHYTSPWTFGGGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTQEDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC, 952-scFv-KZ52-HCC (heavy chain C-terminal fusion) (SEQ ID NO: 19) DVQLQESGPDLVKPXQSLSLTCTVTGYSITSGYSWHWIRQFPGNRLEWMDYIHYSGSINYNPSLKSRISITRDTSK NQFFLQLNSVTTEDTATYYCARWGATGDFYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSLGYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP HTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSA GSAGSAGSGGSEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVK GRFTISRDNAENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSE LVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSLQAEDVAVYYCQQYYSAPLTFGGGTKVEIK, and 952-scFv-KZ52-LCC (light chain C-terminal fusion) (SEQ ID NO: 20) DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVVWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIS SVQAEDLALYYCQQHYTSPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSAGSAGSAGSGG SEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNA ENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSAPLTFGGGTKVEIK.

(38) Additional examples include the following. The italicized region corresponds to the constant sequences of the pMAZ-encoded heavy or light constant region sequences for human IgG1. The underlined region designates fusion linker polypeptide. The bold region designates the sequence of human NPC2.

(39) TABLE-US-00005 401-NPC2-HCC (NPC2 C-terminal fusion to heavy chain of 401 IgG1) (in all of the following bispecifics: NPC2 is human, IgG HC and LC constant domains are human IgGl, IgG VH and VL domains are mouse) (SEQ ID NO: 21) EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRI DPANGNTEYDTKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCSRGYYW GRGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGRPSVFLLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREFQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSAGSAGSAGSGGSE PVQFKDCGSVDGVIKEVNVSPCPTQPCQLSKGQSYSVNVTFTSNIQSKSSK AVVHGILMGVPVPFPIPEPDGCKSGINCPIQKDKTYSYLNKLPVKSEYPSI KLVVEWQLQDDHNQSLFCWEIPVQIVSHL, and 548-NPC2-HCC (NPC2 C-terminal fusion to heavy chain of 548 IgG1)  (SEQ ID NO: 22) EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEEGLEWIGRI DPADGNTEYVPKFQGKATITADTFSNTVYLQLSGLTSEDTAVYYCSRGYYW GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGKGGSAGSAGSAGSGGSEP VQFKDCGSVDGVIKEVNVSPCPTQPCQLSKGQSYSVNVTFTSNIQSKSSKA VVHGILMGVPVPFPIPEPDGCKSGINCPIQKDKTYSYLNKLPVKSEYPSIK LVVEWQLQDDKNQSLFCWEIPVQIVSHL.

(40) Preferably, 548 has the following epitope, DFFVYADYHT (SEQ ID NO: 23), which corresponds to residues 502-511 of human NPC1.

(41) Preferably, the monoclonal antibody or bispecific antibody is a human antibody or humanized antibody. “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin variable domain are replaced by corresponding non-human residues. These modifications may be made to further refine antibody performance. Furthermore, in a specific embodiment, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In an embodiment, the humanized antibodies do not comprise residues that are not found in the recipient antibody or in the donor antibody. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522-525 (1986): Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409, the contents of each of which references and patents are hereby incorporated by reference in their entirety. In one embodiment where the humanized antibodies do comprise residues that are not found in the recipient antibody or in the donor antibody, the Fc regions of the antibodies are modified as described in WO 99/58572, the content of which is hereby incorporated by reference in its entirety.

(42) Techniques to humanize a monoclonal antibody are described in U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866.692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370, the content of each of which is hereby incorporated by reference in its entirety.

(43) A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains. See, for example, Winter et al. Nature 349: 293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220-4224 (1989), Shaw et al. J. Immunol. 138: 4534-4538 (1987), and Brown et al. Cancer Res. 47: 3577-3583 (1987), the content of each of which is hereby incorporated by reference in its entirety. Other references describe rodent hypervariable regions or CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain. See, for example, Riechmann et al. Nature 332: 323-327 (1988), Verhoeyen et al. Science 239: 1534-1536 (1988), and Jones et al. Nature 321: 522-525 (1986), the content of each of which is hereby incorporated by reference in its entirety. Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions—European Patent Publication No. 0519596 (incorporated by reference in its entirety). These “humanized” molecules are designed to minimize unwanted immunological response toward rodent anti-human antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. The antibody constant region can be engineered such that it is immunologically inert (e.g., does not trigger complement lysis). See, e.g. PCT Publication No. WO99/58572, UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that may also be utilized are disclosed by Daugherty et al., Nucl. Acids Res. 19: 2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01/27160 (each incorporated by reference in their entirety).

(44) Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

(45) The invention also provises pharmaceutical compositions for treating or preventing or reducing or inhibiting a Filovirus infection in a subject comprising any of the bispecific antibodies or bispecific fusion constructs disclosed herein and a pharmaceutically acceptable carrier. Examples of acceptable pharmaceutical carriers include, but are not limited to, additive solution-3 (AS-3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate acid dextrose solution. The bispecific antibody or bispecific fusion construct can be administered to the subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, oral administration, parenteral administration, intravenous administration and administration through an osmotic mini-pump.

(46) The invention further provides an isolated nucleic acid encoding any of the bispecific antibodies or bispecific fusion constructs disclosed herein. The isolated nucleic acid can be, or comprise, a cDNA.

(47) This invention will be better understood from the Experimental Details, which follow. However, one skilled in the an will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

Experimental Details

(48) Introduction and Overview: Ebola virus (EBOV) and related filoviruses are associated with sporadic outbreaks of highly lethal hemorrhagic fever in Middle and West Africa. The ongoing regional EBOV epidemic in West Africa has underscored the urgent need for antiviral treatments and demonstrated the potential of passive immunotherapy to reverse advanced filovirus disease. However, existing monoclonal antibody (mAb) cocktails such as ZMapp are limited by a narrow spectrum of antiviral action, which stems from viral strain-specific neutralization of the highly variable entry glycoprotein, GP, by most mAbs. Accordingly, set forth herein is an immunotherapeutic strategy targeting the broadly required and highly conserved filovirus entry receptor Niemann-Pick C1 (NPC1) instead of GP. Unfortunately, anti-NPC1 mAbs that efficiently blocked GP-NPC1 interaction in vitro failed to neutralize viral infection in cells, presumably because of an unusual feature of filovirus receptor recognition—the GP-NPC1 interaction can occur only in cellular endosomes where both virus and receptor are likely protected from extracellular antibodies. To overcome this limitation, bispecific Abs (bsAbs) were generated that combine both antiviral and anti-receptor specificities in the same molecule. These bsAbs potently neutralized EBOV infection in a manner that required their engagement of both GP and NPC1, indicating a two-step mechanism of action in which they exploit extracellular virus particles to gain access to NPC1-containing endosomes. bsAbs that combine broadly-reactive (hut non-neutralizing) anti-GP Abs with anti-NPC1 Abs are expected to afford broad-spectrum protection against filovirus infection in vivo.

(49) NPC1 is required for Ebola virus infection and in vivo pathogenesis. Niemann-Pick C1 (NPC1), a ubiquitous multi-pass membrane protein localized to late endosomes is required for cytoplasmic entry and infection by all filoviruses. The filovirus spike glycoprotein, GP, must engage NPC1's second luminal domain (FIG. 1A, domain C) to drive viral membrane fusion and cytoplasmic escape (Miller. EMBO J, 2012). As shown in FIG. 1B, NPC1-knockout mice (NPC1.sup.−/−), but not their WT littermates (NPC1.sup.+/+), are completely resistant to infection and killing by EBOV and MARV (Carette, Nature 2011; Herbert, submitted).

(50) Mouse hybridomas were raised that express mAbs specific for human NPC1 domain C (data not shown). Screens identified two mAbs (548 and 952) that hound with high affinity to NPC1 and potently blocked its interaction with the EBOV glycoprotein GP in vitro. A third mAb (401) had much more modest receptor-blocking activity, FIG. 3 shows the capacity of three anti-NPC1 mAbs to block GP-NPC1 binding in an ELISA. A soluble, flag-tagged form of NPC1 domain C was pre-incubated with the indicated concentrations of each mAb, and the protein-mAb mixtures were then added to plates coated with vesicular stomatitis virus (VSV) particles bearing EBOV GP. Bound domain C was detected with an anti-flag antibody. Irrelevant isotype-matched antibodies had no effect on GP-NPC1 domain C binding (not shown).

(51) As exemplified in FIG. 4, anti-NPC1 domain C mAbs neutralize VSV-EBOV GP infection weakly or not at all. VSV-GP particles were exposed to U2OS human osteosarcoma cells in the presence of increasing concentrations of NPC1-specific mAbs or a control GP-specific neutralizing mAb (KZ52). The number of infected cells was determined at 16 h post-infection, and normalized to that obtained in the absence of antibody (set to 100%). The NPC1-specific mAbs 548 and 401 neutralized VSV-EBOV OP infection weakly or not at all, respectively. By contrast, the previously described GP-specific neutralizing mAb KZ52 was highly effective at neutralization.

(52) FIG. 5 illustrates engineering antibodies with dual binding specificities for GP and NPC1. In FIG. 5A, constructs encoding bsAbs were generated by fusing NPC1-specific or control IgG sequences to a single-chain variable fragment (scFv) sequence derived from the EBOV GP-specific mAb KZ52. Fusions were done in four configurations: to the N- or C-terminus of the IgG heavy chain [HC] or light chain [LC] in FIG. 5B, bsAbs and IgGs were expressed in 293-Freestyle cells by co-transfection of HC and LC expression vectors, and purified by protein A affinity chromatography. In FIG. 5C, it is shown that bsAbs, but not NPC1-specific IgGs, can simultaneously bind to GP and NPC1 domain C. bsAbs and IgGs were captured onto GP-coated ELISA plates and then incubated with NPC1 domain C-flag. Bound NPC1 domain C was detected with an anti-flag antibody.

(53) It was postulated that the NPC1-specific mAbs do not neutralize EBOV entry because they cannot efficiently access NPC1-containing late endosomes. To overcome this obstacle, an exploratory panel of bispecific antibodies (bsAbs) containing both NPC1-binding (401 or 548) and GP-binding (KZ52) specificities was generated. It was reasoned that one or more of these bsAbs might be able to ‘ride’ virus particles into late endosomes and then bind to NPC1, thereby inhibiting viral entry by both GP-blocking and receptor-blocking mechanisms. It was found that all four purified bsAbs could bind simultaneously to EBOV GP and NPC1 (e.g., see FIG. 5C), setting the stage for infection neutralization studies.

(54) As shown in FIG. 6, GP-NPC1 bsAbs neutralize VSV-EBOV GP infection with enhanced potency. In the left panel, GP-NPC1 bsAbs show enhanced potency at viral neutralization relative to control GP-only hsAbs. VSV-EBOV GP particles were pre-incubated with the indicated concentrations of each bsAb for 1 hour at room temperature and then allowed to infect U2OS cells. Infected cells were quantified at 16 hours post-infection and normalized to a no-antibody control (100%). In the right panel, the neutralization curves were fit to a logistic equation to extract the concentration of half-maximal neutralization (IC.sub.50±95% confidence intervals). The GP-NPC1 bsAbs were 10-60 fold more potent at neutralizing EBOV entry than control bsAbs in which an irrelevant (non-NPC1 binding) IgG was fused to the same scFv, derived from the OP-specific mAb, KZ52. These results indicate that the bsAbs possess an enhanced capacity to block GP-NPC1 binding within endosomes, relative to the NPC1-specific IgGs.

(55) As shown in FIG. 6, the enhanced neutralization potency of GP-NPC1 bsAbs can be attributed to their receptor blocking activity. In the left panel, VSV-EBOV GP particles were pre-incubated with the indicated concentrations of each bsAb for 1 hour at room temperature and then allowed to infect matched U2OS cell lines expressing endogenous levels of NPC1 or over-expressing NPC1 (NPC1.sup.hi). Infected cells were quantified at 16 hours post-infection and normalized to a no-antibody control (100%). In the right panel, the neutralization curves were fit to a logistic equation to extract the concentration of half-maximal neutralization (IC.sub.50±95% confidence intervals). The ratio of IC.sub.50s for each bsAb in NPC1.sup.hi vs. WT U2OS cells is shown. It was reasoned that the neutralization potency of GP-NPC1 bsAbs should be reduced in cells engineered to overexpress NPC1, because the excess NPC1 should titrate the avail e NPC1-binding sites in the bsAbs and thereby increase the probability of productive GP-NPC1 binding. This is precisely what was observed. The bsAb 548-HCN′ suffered a 24-fold loss in potency in U2OS—NPC1.sup.hi cells, relative to WT U2OS cells. By contrast, the potency of the Ctrl-HCN' bsAb, which cannot bind to NPC1, was reduced much more modestly (˜2-fold). These findings provide strong evidence that the bsAbs neutralize viral infection by deploying both GP- and NPC1-binding specificities within cells.

(56) An additional strategy to deliver NPC1-specific IgGs to NPC1-positive late endosomes was also developed. The IgGs were fused to NPC2, a late endosomalilysosomal host protein that interacts with NPC1 and that can autonomously traffic to late endosomes when added to cells (Naureckiene, Science 2000). As shown in FIG. 8, fusion of a receptor-blocking IgG to a late endosome-localizing protein facilitates viral neutralization. bsAbs comprising an NPC1-specific IgG (401 or 548) fused to NPC2, a soluble late endosomalllysosomal protein, were expressed and purified, and examined for their capacity to neutralize VSV-EBOV GP infection, as described in FIGS. 6-7. Fusion of the potent receptor-blocking IgG 548 to NPC2 afforded neutralization of infection, whereas fusion of the weak receptor-blocking IgG 401 to NPC2 did not. Therefore, direct, cellular protein-mediated delivery of NPC1-specific IgGs to NPC1+ endosomes is also a feasible strategy for targeting NPC1 and preventing NPC1-mediated filovirus entry into cells.

(57) The NPC1-targeting bispecific antibody approaches described herein provide an unprecedented opportunity for anti-filovirus therapeutics with increased antiviral breadth.

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