MULTI-SPECIFIC ANTIBODIES FOR CROSS-NEUTRALIZATION

OF MULTIPLE FILOVIRUS GLYCOPROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 62/069,516 filed October 28, 2014, the contents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers AI009762 and AI090249 and GM007482 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0004] Ebola virus is an NIAID Category A biodefense pathogen that causes severe and rapidly progressing hemorrhagic fever with human case fatalities of 50-90%. There are currently no FDA-approved vaccines or therapies for Ebola virus infection. Among the five species of Ebola virus, the Zaire and Sudan variants (EBOV and SUDV, respectively) are the most pathogenic and both have resulted in recurring outbreaks. Together, EBOV and SUDV account for over 95% of EBOV-related deaths reported to date.

[0005] Herein are disclosed multi-specific antibodies for cross-neutralization of multiple filovirus glycoproteins.

SUMMARY OF THE INVENTION

[0006] This invention provides a composition comprising (1) a first single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a first species of filovirus, which first scFv is covalently joined to (2) a first polypeptide of an immunoglobulin G (IgG) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a second species of filovirus, wherein the first species of filovirus and second species of filovirus are different species.

[0007] A composition is also provided comprising (i) a first single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre- fusion core of a first species of filovirus, which first scFv is covalently joined to (ii) a first polypeptide of an immunoglobulin G (IgG) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a second species of filovirus, the IgG having covalently joined thereto, at a different location from the attachment of (i) thereto, (iii) a second single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a third species of filovirus,

wherein the first species of filovirus and second species of filovirus and third species of filovirus are all different species. In an embodiment, one species-specific scFv is fused to the light chain of an IgG, and different species-specific scFv is fused to the heavy chain of the IgG.

[0008] Also provided is an isolated nucleic acid encoding any of the compositions described herein.

[0009] Also provided is a method of treating a filovirus infection in a subject comprising administering an amount of a bispecific composition described herein to the subject effective to treat a filovirus infection in a subject.

[0010] Also provided is a method of preventing a filovirus infection in a subject comprising administering an amount of a bispecific composition described herein to the subject effective to inhibit a filovirus infection in a subject.

[0011] Also provided is a method of preventing a filovirus infection of a mammalian cell comprising contacting the filovirus with an amount of a bispecific composition described herein effective to inhibit a filovirus infection of a mammalian cell.

[0012] Also provided is a method of treating a filovirus infection in a subject comprising administering an amount of a trispecific composition described herein to the subject effective to treat a filovirus infection in a subject.

[0013] Also provided is a method of preventing a filovirus infection in a subject comprising administering an amount of a trispecific composition described herein to the subject effective to inhibit a filovirus infection in a subject. [0014] Also provided is a method of preventing a filovirus infection of a mammalian cell comprising contacting the filovirus with an amount of a trispecific composition described herein effective to inhibit a filovirus infection of a mammalian cell.

[0015] Herein are disclosed cross-neutralizing, bispecific monoclonal antibody constructs against Ebola variants such as EBOV and SUDV. Recent structural studies indicate that the base of the envelope glycoprotein (GP) is susceptible to neutralization by antibodies in these two species. However, current EBOV and SUDV antibodies targeting this region are narrowly strain specific and therefore have limited therapeutic utility. Herein a fusion approach is disclosed whereby, for example, the IgG of one species-specific antibody (e.g., SUDV) is genetically fused to the scFv of another species-specific antibody (e.g., EBOV) to create a cross-species neutralizing antibody (or vice versa). This strategy can also be extended to generate trispecific antibody constructs. Antibody therapies against Ebolaviruses and other filoviruses have demonstrated post-exposure efficacy in nonhuman primates, but no cross-neutralizing antibodies exist. Therefore, the cross-neutralizing, bispecific antibodies disclosed herein fill a much needed gap in Ebola therapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1A-1B: Structure of the prefusion GP1-GP2 spike and GP conformational changes that lead to viral membrane fusion. (A) Structure of the GP 1-GP2 spike ectodomain (PDB ID: 3CSY, (13)). The GP1 subunits are shown in surface-shaded view and GP2 as rods and loops. One GP1 subunit is colored to show the subdomains: b, base; h, head; gc, glycan cap. fp, fusion peptide; TM, transmembrane domain. C, GP C- terminus. (B) Membrane fusion-associated conformational rearrangements in GP2 inferred from its pre-fusion and putative post-fusion structures (PDB ID: 1EBO, ref. 17)

[0017] Fig. 2. Survival of NHPs administered EBOV-specific serum IgGs upon viral challenge. Animals 7, 8, and 9 survived but the control animal (5) died 8-days postexposure. (These data are from ref. 21, Dye et al).

[0018] Fig. 3. Head-to-head comparison of KZ52 IgG vs. VSV-GP _EB ov and 16F6 IgG vs. VSV-GPSUDV.

[0019] Fig. 4. Exemplary Bispecific (E10 & F4 IgG + KZ52 scFv) designs.

[0020] Fig. 5. Bispecifics (E10 & F4 IgG + KZ52 scFv) SDS PAGE.

[0021] Fig. 6. Bispecifics (F4 IgG + KZ52 scFv) binding data.

[0022] Fig. 7. Bispecifics (E10 IgG + KZ52 scFv) binding data. [0023] Fig. 8. Bispecific antibodies neutralization assay results.

[0024] Fig. 9A-9B. Virus neutralization. (A) Dose-dependent neutralization of VSV- GPEBOV and VSV-GPSUDV by scKZ52-F4 LCN, scKZ52-F4 HCC, scKZ52-E10 LCN, and scKZ52-E10 HCC. Calculated IC50 values are listed next to each curve. (B) Neutralization of authentic viruses by PRNT assay, with IC50 values listed.

[0025] Fig. 10A-10D. Animal challenge experiments. Percent survival and median percent weight change for mouse-adapted EBOV (A) and SUDV (B) challenges with antibody treatment. Mice were treated with a single post-exposure dose (200 μg at 24 hours) for EBOV or two post-exposure doses (500 μg at days +1 and +4) for SUDV. Statistical p-values are listed against PBS and F4 controls for EBOV and against Z.6D8 control for SUDV (****, p < 0.0001; **, p < 0.01; *, p < 0.05; p-values greater that 0.05 are listed numerically; na, not applicable). (C and D) Rechallenge of surviving mice from initial challenge cohorts, with no further antibody treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Herein is described A composition is provided comprising (1) a first single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a first species of filovirus, which first scFv is covalently joined to (2) a first polypeptide of an immunoglobulin G (IgG) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a second species of filovirus, wherein the first species of filovirus and second species of filovirus are different species.

[0027] In an embodiment of the composition, the first scFv comprises three different heavy chain CDRs and three different light chain CDRs, each CDR directed to the membrane glycoprotein pre-fusion core of the first species of filovirus.

[0028] In an embodiment of the composition, the IgG comprises at least three different heavy chain CDRs and at least three different light chain CDRs, each CDR directed to the membrane glycoprotein pre-fusion core of the second species of filovirus.

[0029] In an embodiment of the compositions, the first scFv is covalently joined at its N terminal to a C terminal of a first polypeptide of the IgG. In an embodiment of the compositions, the first polypeptide of the IgG is a heavy chain. In an embodiment of the compositions, the first polypeptide of the IgG is a light chain. [0030] In an embodiment of the compositions, the first scFv is covalently joined at its C terminal to an N terminal of a first polypeptide of the IgG. In an embodiment of the compositions, the first polypeptide of the IgG is a heavy chain. In an embodiment of the compositions, the first polypeptide of the IgG is a light chain.

[0031] In an embodiment of the compositions, the scFv is covalently joined to the fist polypeptide via a polypeptide linker.

[0032] In an embodiment of the compositions, the polypeptide linker comprises the sequence GGSAGSAGSAGSGGS (SEQ ID NO: 17).

[0033] In an embodiment of the compositions, a VH sequence of the first scFv has a sequence identical to VH sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the first species of filovirus.

[0034] In an embodiment of the compositions, a VL sequence of the first scFv has a sequence identical to VL sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the first species of filovirus.

[0035] In an embodiment of the compositions, the VH sequence of the scFv is joined to the VL sequence of the first scFv by a polypeptide linker. In an embodiment of the compositions, the polypeptide linker is majority glycine residues. In an embodiment of the compositions, the polypeptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 18).

[0036] In an embodiment of the compositions, the composition comprises a second scFv, wherein the second scFv is covalently joined to a second polypeptide of the IgG. In an embodiment of such compositions, a VL sequence of the first scFv has a sequence identical to VL sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the first species of filovirus. In an embodiment of the compositions, a VH sequence of the IgG has a sequence identical to VH sequence of a human or a humanized IgG antibody directed to the membrane glycoprotein pre-fusion core of the second species of filovirus. In an embodiment of the compositions, a VL sequence of the IgG has a sequence identical to VL sequence of a human or a humanized IgG antibody directed to the membrane glycoprotein pre-fusion core of the second species of filovirus.

[0037] In an embodiment of the compositions, the IgG is a neutralizing antibody for the second species of filovirus.

[0038] In an embodiment of the compositions, the first species of filovirus and the second species of filovirus are both Ebolavirus species. In an embodiment of the compositions, the Ebola virus species are Zaire ebolavirus and Sudan ebolavirus. [0039] A composition is also provided comprising (i) a first single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre- fusion core of a first species of filovirus, which first scFv is covalently joined to (ii) a first polypeptide of an immunoglobulin G (IgG) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a second species of filovirus, the IgG having covalently joined thereto, at a different location from the attachment of (i) thereto, (iii) a second single chain variable fragment (scFv) comprising at least one CDR directed to a membrane glycoprotein pre-fusion core of a third species of filovirus,

wherein the first species of filovirus and second species of filovirus and third species of filovirus are all different species.

[0040] In an embodiment of the composition, the first scFv comprises three different heavy chain CDRs and three different light chain CDRs, each CDR directed to the membrane glycoprotein pre-fusion core of the first species of filovirus, and the second scFv comprises three different heavy chain CDRs and three different light chain CDRs, each CDR directed to the membrane glycoprotein pre-fusion core of the third species of filovirus. In an embodiment of the composition, the IgG comprises at least three different heavy chain CDRs and at least three different light chain CDRs, each CDR directed to the membrane glycoprotein pre-fusion core of the second species of filovirus.

[0041] In an embodiment of the compositions, the first scFv is covalently joined at its N terminal to a C terminal of a first polypeptide of the IgG, and the second scFv is covalently joined to a different location on the IgG. In an embodiment of the compositions, the first polypeptide of the IgG is a heavy chain. In an embodiment of the compositions, the first polypeptide of the IgG is a light chain.

[0042] In an embodiment of the compositions, the first scFv is covalently joined at its C terminal to an N terminal of a first polypeptide of the IgG, and the second scFv is covalently joined to a different location on the IgG. In an embodiment of the compositions, the first polypeptide of the IgG is a heavy chain. In an embodiment of the compositions, the first polypeptide of the IgG is a light chain.

[0043] In an embodiment of the compositions, the first and second scFvs are each covalently joined to the first polypeptide, each via a separate polypeptide linker. In an embodiment of the compositions, the first and second scFvs are each covalently joined to different polypeptides of the IgG, each via a separate polypeptide linker. In an embodiment of the compositions, the polypeptide linkers each comprise the sequence GGSAGSAGSAGSGGS (SEQ ID NO: 17).

[0044] In an embodiment of the compositions, a V _H sequence of the first scFv has a sequence identical to VH sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the first species of filovirus. In an embodiment of the compositions, a VH sequence of the second scFv has a sequence identical to VH sequence of a human or a humanized antibody directed to the membrane glycoprotein pre- fusion core of the third species of filovirus.

[0045] In an embodiment of the compositions, a VL sequence of the first scFv has a sequence identical to VL sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the first species of filovirus. In an embodiment of the compositions, a VL sequence of the second scFv has a sequence identical to VL sequence of a human or a humanized antibody directed to the membrane glycoprotein pre-fusion core of the third species of filovirus.

[0046] In an embodiment of the compositions, the VH sequence of the first scFv is joined to the VL sequence of the scFv by a polypeptide linker. In an embodiment of the compositions, the VH sequence of the second scFv is joined to the VL sequence of the scFv by a polypeptide linker. In an embodiment of the compositions, the polypeptide linker is majority glycine residues. In an embodiment of the compositions, the polypeptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 18).

[0047] In an embodiment of the compositions, the IgG is a neutralizing antibody for the second species of filovirus.

[0048] In an embodiment of the compositions, the first species of filovirus and the second species of filovirus and third species of filovirus are all Ebolavirus species. In an embodiment of the compositions, the Ebola virus species include Zaire ebolavirus and Sudan ebolavirus. In an embodiment of the compositions, the at least one species of filovirus and is an Ebolavirus species and at least one species of filovirus is a Marburg virus.

[0049] Also provided is an isolated nucleic acid encoding any of the compositions described herein.

[0050] Also provided is a method of treating a filovirus infection in a subject comprising administering an amount of a bispecific composition described herein to the subject effective to treat a filovirus infection in a subject. [0051] Also provided is a method of preventing a filovirus infection in a subject comprising administering an amount of a bispecific composition described herein to the subject effective to inhibit a filovirus infection in a subject.

[0052] Also provided is a method of preventing a filovirus infection of a mammalian cell comprising contacting the filovirus with an amount of a bispecific composition described herein effective to inhibit a filovirus infection of a mammalian cell. In an embodiment of the method, the cell is an antigen presenting cell. In an embodiment of the method, the cell is a dendritic cell or a macrophage. In an embodiment of the method, the cell is human.

[0053] Also provided is a method of treating a filovirus infection in a subject comprising administering an amount of a trispecific composition described herein to the subject effective to treat a filovirus infection in a subject.

[0054] Also provided is a method of preventing a filovirus infection in a subject comprising administering an amount of a trispecific composition described herein to the subject effective to inhibit a filovirus infection in a subject.

[0055] Also provided is a method of preventing a filovirus infection of a mammalian cell comprising contacting the filovirus with an amount of a trispecific composition described herein effective to inhibit a filovirus infection of a mammalian cell. In an embodiment of the method, the cell is an antigen presenting cell. In an embodiment of the method, the cell is a dendritic cell or a macrophage. In an embodiment of the method, the cell is human.

[0056] In an embodiment of all of the methods described herein, the subject is human.

[0057] As used herein, "treating" a specified condition means ameliorating one or more symptoms of an extant condition, for example, a filovirus infection.

[0058] As used herein, "preventing" a specified condition means reducing the development of, or reducing the extent of, one or more symptoms of the condition, for example a filovirus infection, 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.

[0059] Exemplary E10/F4-KZ52 scFv Bispecifics Sequences: the following exemplary amino acid sequences are provided, for the compositions of the invention as relating to the embodiments of antibodies E10 and F4, with non-limiting exemplary nucleotide sequences (in view of the degeneracy of the genetic code).

[0060] E10-KZ52 HC N' Fusion Sequence:

[0061] Amino Acid (SEQ ID NO: 1):

EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSS

YIHYADSVKGRFTISRDNAENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADV

FDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLAVSLGERATrNCKSS

QSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

LQAEDVAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSEVQLVESGG

GLVQPGGSLRLSCAASGFAFNYYDIHWVRQAPGKGLEWVAYINPGGGNTYYADS

VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQLYGNSFMDYWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEP VTVSWNSGALTSG VHTFPA VLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCDKTHTCP PCP

APELLGRPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAK T

KPREEQYNSTYR VVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0062] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide. Italicized region is CH1-CH3 for the HC and the CL region.

[0063] Nucleotide (SEQ ID NO:2):

GAAGTGCAGTTACTGGAAAGCGGCGGCGGCCTGGTTAAACCTGGCGGTAGTCT

GCGTCTGAGTTGCGCCGCCAGCGGTTTCACCCTGATCAACTACCGCATGAACTG

GGTGCGTCAGGCACCGGGTAAAGGCCTGGAGTGGGTGAGCAGCATTAGCAGCA

GCAGCAGCTATATTCACTACGCCGACAGCGTGAAAGGCCGCTTTACCATCAGCC

GCGACAATGCCGAGAACAGTCTGTATCTGCAGATGAACAGCCTAAGGGCGGAA

GATACAGCCGTGTACTACTGTGTGCGCGAAGGCCCTCGCGCAACCGGCTATAGC

ATGGCAGACGTGTTTGATATCTGGGGTCAGGGCACCATGGTTACAGTTAGCAGT

GGTGGTGGTGGTAGTGGTGGCGGTGGTAGCGGTGGTGGTGGCAGTGAACTGGT

GATGACCCAGAGCCCGGATAGCTTAGCCGTGAGTCTGGGCGAAAGGGCGACCA

TTAACTGCAAAAGCAGCCAGAGCGTGCTGTACAGCAGCAACAACAAGAGCTAC

CTGGCATGGTATCAGCAAAAACCGGGTCAGCCTCCGAAACTGCTGATCTATTGG

GCAAGCACCCGCGAAAGTGGTGTTCCGGATCGCTTCAGCGGTAGTGGCAGCGG

TACCGATTTCACCCTGACCATCAGCAGTCTGCAGGCCGAGGACGTTGCAGTGTA TTACTGTCAGCAGTACTACAGCGCCCCGCTGACCTTTGGCGGCGGCACCAAAGT

TGAAATTAAGGGCGGCAGTGCAGGCAGCGCCGGTAGTGCCGGTAGTGGTGGTA

GCGAAGTTCAGCTGGTTGAAAGTGGCGGCGGTCTGGTGCAGCCTGGTGGTAGTC

TGCGTCTGAGTTGTGCCGCCAGCGGCTTTGCCTTCAATTACTATGACATTCATTG

GGTTCGCCAGGCCCCGGGTAAAGGTCTGGAATGGGTTGCATATATCAACCCGG

GTGGCGGTAACACCTACTATGCCGACAGCGTTAAGGGTCGCTTCACCATCAGCG

CAGATACCAGCAAAAACACCGCCTACCTGCAGATGAATAGCCTGCGTGCAGAA

GATACCGCCGTTTACTACTGTGCCCGCCAGCTGTACGGCAATAGCTTCATGGAC

TATTGGGGCCAGGGCACCTTAGTTACCGTGAGCAGC

[0064] Combined (SEQ ID NO: 1 and 2):

gaagtgcagttactggaaagcggcggcggcctggttaaacctggcggtagtctgcgt ctg

E V Q L L E S G G G L V K P G G S L R L

agttgcgccgccagcggtttcaccctgatcaactaccgcatgaactgggtgcgtcag gca

S C A A S G F T L I N Y R M N W V R Q A

ccgggtaaaggcctggagtgggtgagcagcattagcagcagcagcagctatattcac tac

P G K G L E W V S S I S S S S S Y I H Y

gccgacagcgtgaaaggccgctttaccatcagccgcgacaatgccgagaacagtctg tat

A D S V K G R F T I S R D N A E N S L Y

ctgcagatgaacagcctaagggcggaagatacagccgtgtactactgtgtgcgcgaa ggc

L Q M N S L R A E D T A V Y Y C V R E G

cctcgcgcaaccggctatagcatggcagacgtgtttgatatctggggtcagggcacc atg

P R A T G Y S M A D V F D I W G Q G T M

gttacagttagcagtggtggtggtggtagtggtggcggtggtagcggtggtggtggc agt

V T V S S G G G G S G G G G S G G G G S

gaactggtgatgacccagagcccggatagcttagccgtgagtctgggcgaaagggcg acc

E L V M T Q S P D S L A V S L G E R A T

attaactgcaaaagcagccagagcgtgctgtacagcagcaacaacaagagctacctg gca

I N C K S S Q S V L Y S S N N K S Y L A

tggtatcagcaaaaaccgggtcagcctccgaaactgctgatctattgggcaagcacc cgc

W Y Q Q K P G Q P P K L L I Y W A S T R

gaaagtggtgttccggatcgcttcagcggtagtggcagcggtaccgatttcaccctg acc

E S G V P D R F S G S G S G T D F T L T

atcagcagtctgcaggccgaggacgttgcagtgtattactgtcagcagtactacagc gcc I S S L Q A E D V A V Y Y C Q Q Y Y S A

ccgctgacctttggcggcggcaccaaagttgaaattaagggcggcagtgcaggcagc gcc

P L T F G G G T K V E I K G G S A G S A

ggtagtgccggtagtggtggtagcgaagttcagctggttgaaagtggcggcggtctg gtg

G S A G S G G S E V Q L V E S G G G L V

cagcctggtggtagtctgcgtctgagttgtgccgccagcggctttgccttcaattac tat

Q P G G S L R L S C A A S G F A F N Y Y

gacattcattgggttcgccaggccccgggtaaaggtctggaatgggttgcatatatc aac

D I H W V R Q A P G K G L E W V A Y I N

ccgggtggcggtaacacctactatgccgacagcgttaagggtcgcttcaccatcagc gca

P G G G N T Y Y A D S V K G R F T I S A

gataccagcaaaaacaccgcctacctgcagatgaatagcctgcgtgcagaagatacc gcc

D T S K N T A Y L Q M N S L R A E D T A

gtttactactgtgcccgccagctgtacggcaatagcttcatggactattggggccag ggc

V Y Y C A R Q L Y G N S F M D Y W G Q G

accttagttaccgtgagcagc

T L V T V S S

[0065] E10-KZ52 LC N' Fusion Sequence:

[0066] Amino Acid (SEQ ID NO:3):

EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSS

YIHYADSVKGRFTISRDNAENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADV

FDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLAVSLGERATINCKSS

QSVLYSS KSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

LQAEDVAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDIQMTQSPSS

LSASVGDRVTITCRASQDVTTAVAWYQQKPGKAPKLLIYWASRLHNGVPSRFSGS

GSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVEIKRr^ S' / S' ^g

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKA

D YEKHKVYA CE VTHQGLSSP VTKSFNR GECGGSD YKDDDDK

[0067] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide. Italicized region is CH1-CH3 for the HC and the CL region.

[0068] Nucleotide (SEQ ID NO:4):

GAAGTGCAGCTGCTGGAAAGCGGTGGCGGTCTGGTTAAACCTGGCGGTAGTCT GCGCCTGAGTTGCGCCGCCAGCGGTTTTACACTGATCAACTATCGCATGAACTG GGTGCGTCAGGCACCGGGTAAGGGTCTGGAGTGGGTTAGCAGCATTAGTAGCA

GCAGCAGTTACATTCACTACGCCGATAGCGTGAAAGGCCGCTTCACAATTAGCC

GCGATAACGCCGAGAACAGCCTGTATCTGCAGATGAACAGTTTACGCGCCGAA

GATACCGCCGTGTATTATTGCGTTCGCGAAGGTCCGCGTGCAACCGGCTACAGC

ATGGCCGACGTTTTCGATATTTGGGGTCAGGGCACCATGGTGACAGTTAGTAGC

GGTGGTGGTGGTAGTGGTGGTGGCGGCAGCGGTGGTGGTGGTAGTGAACTGGT

GATGACCCAGAGCCCGGATAGCCTGGCAGTGAGCCTGGGTGAGCGTGCCACCA

TCAATTGCAAAAGCAGCCAGAGCGTGCTGTACAGCAGCAACAACAAGAGTTAC

CTGGCCTGGTACCAACAGAAACCGGGCCAGCCGCCGAAACTGCTGATTTATTGG

GCCAGTACCCGCGAAAGCGGCGTGCCTGATCGTTTTAGTGGCAGCGGTAGCGG

CACCGACTTTACCCTGACCATTAGCAGCCTGCAGGCCGAGGATGTGGCAGTGTA

TTACTGCCAGCAGTATTACAGCGCCCCGTTAACCTTTGGCGGCGGTACCAAAGT

GGAGATCAAAGGTGGCAGTGCAGGCAGCGCCGGTAGTGCAGGTAGTGGTGGTA

GCGACATCCAGATGACACAGAGTCCGAGCAGCCTGAGTGCCAGCGTTGGTGAC

CGTGTGACCATTACCTGCCGTGCCAGCCAGGATGTTACCACAGCCGTTGCATGG

TATCAGCAGAAGCCGGGTAAGGCCCCTAAGTTACTGATCTACTGGGCAAGCCG

CCTGCATAACGGTGTGCCGAGCCGCTTTAGCGGCAGTGGTAGCGGTACCGATTT

CACCCTGACCATCAGCAGTCTGCAGCCGGAAGATTTCGCAACCTACTACTGTCA

GCAGCATTACAGCACCCCGCTGACCTTTGGCCAGGGCACCAAAGTGGAAATTA

AA

[0069] Combined (SEQ ID NO: 3 and 4):

gaagtgcagctgctggaaagcggtggcggtctggttaaacctggcggtagtctgcgc ctg

E V Q L L E S G G G L V K P G G S L R L

agttgcgccgccagcggttttacactgatcaactatcgcatgaactgggtgcgtcag gca

S C A A S G F T L I N Y R M N W V R Q A

ccgggtaagggtctggagtgggttagcagcattagtagcagcagcagttacattcac tac

P G K G L E W V S S I S S S S S Y I H Y

gccgatagcgtgaaaggccgcttcacaattagccgcgataacgccgagaacagcctg tat

A D S V K G R F T I S R D N A E N S L Y

ctgcagatgaacagtttacgcgccgaagataccgccgtgtattattgcgttcgcgaa ggt

L Q M N S L R A E D T A V Y Y C V R E G

ccgcgtgcaaccggctacagcatggccgacgttttcgatatttggggtcagggcacc atg P R A T G Y S M A D V F D I W G Q G T M

gtgacagttagtagcggtggtggtggtagtggtggtggcggcagcggtggtggtggt agt

V T V S S G G G G S G G G G S G G G G S

gaactggtgatgacccagagcccggatagcctggcagtgagcctgggtgagcgtgcc acc

E L V M T Q S P D S L A V S L G E R A T

atcaattgcaaaagcagccagagcgtgctgtacagcagcaacaacaagagttacctg gcc

I N C K S S Q S V L Y S S N N K S Y L A

tggtaccaacagaaaccgggccagccgccgaaactgctgatttattgggccagtacc cgc

W Y Q Q K P G Q P P K L L I Y W A S T R

gaaagcggcgtgcctgatcgttttagtggcagcggtagcggcaccgactttaccctg acc

E S G V P D R F S G S G S G T D F T L T

attagcagcctgcaggccgaggatgtggcagtgtattactgccagcagtattacagc gcc

I S S L Q A E D V A V Y Y C Q Q Y Y S A

ccgttaacctttggcggcggtaccaaagtggagatcaaaggtggcagtgcaggcagc gcc

P L T F G G G T K V E I K G G S A G S A

ggtagtgcaggtagtggtggtagcgacatccagatgacacagagtccgagcagcctg agt

G S A G S G G S D I Q M T Q S P S S L S

gccagcgttggtgaccgtgtgaccattacctgccgtgccagccaggatgttaccaca gcc

A S V G D R V T I T C R A S Q D V T T A

gttgcatggtatcagcagaagccgggtaaggcccctaagttactgatctactgggca agc

V A W Y Q Q K P G K A P K L L I Y W A S

cgcctgcataacggtgtgccgagccgctttagcggcagtggtagcggtaccgatttc acc

R L H N G V P S R F S G S G S G T D F T

ctgaccatcagcagtctgcagccggaagatttcgcaacctactactgtcagcagcat tac

L T I S S L Q P E D F A T Y Y C Q Q H Y

agcaccccgctgacctttggccagggcaccaaagtggaaattaaa

S T P L T F G Q G T K V E I K

[0070] E10-KZ52 HC C Fusion Sequence:

[0071] Amino Acid (SEQID O:5):

[0072] EVQLVESGGGLVQPGGSLRLSCAASGFAFNYYDIHWVRQAPGKGLEWV AYINPGGGNTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQLYGN SFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS W SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV HKPSNTKVDK KVEPKSCDKTHTCDKTHTCPPCPAPELLGRPSVFLFPPKPKD7LMISRTPEVTCVVV

DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY STYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPE YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYrQKSLSLSPGKGGSAGSAGSAGSGGSEVQLLESGGGLVKPGGSLRLSCA

ASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAENSLY

LQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGG

GSGGGGSELVMTQSPDSLAVSLGERATINCKSSQSVLYSS KSYLAWYQQKPGQP

PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGG

GTKVEIK

[0073] Underlined region is linker polypeptide. Bold region is fusion linker polypeptide.

[0074] Nucleotide (SEQ ID O:6):

GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACT

CCGTTTGTCCTGTGCAGCTTCTGGCTTCGCGTTTAACTATTATGATATTCATTGG

GTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATATATTAACCCGGG

CGGTGGCAACACCTATTATGCTGATAGCGTCAAGGGCCGTTTCACTATAAGCGC

AGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGG

ACACTGCCGTCTATTATTGTGCTCGCCAGCTGTATGGCAACAGCTTTATGGACT

ACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGTCCGA

GCGTGTTTCCTCTGGCACCTAGCAGTAAAAGCACCAGTGGTGGTACAGCAGCCC

TGGGTTGCCTGGTGAAGGATTACTTTCCGGAGCCGGTGACCGTTAGTTGGAATA

GCGGCGCCCTGACCAGTGGCGTTCATACATTTCCGGCCGTGCTGCAGAGTAGTG

GCCTGTACAGCCTGAGTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCC

AGACCTATATTTGCAATGTTAACCATAAACCGAGCAACACAAAAGTTGATAAA

AAAGTTGAACCGAAGAGCTGTGACAAAACCCATACATGTGACAAAACACACAC

CTGCCCGCCTTGTCCGGCACCTGAGCTGCTGGGTCGCCCGAGCGTTTTTCTGTTT

CCTCCGAAACCGAAAGAC^CC ..7^CCCAGAAGAGTCTGAGCCTGAGTCCTGGC

AAAGGTGGATCCGCCGGTAGCGCAGGTAGTGCAGGTAGTGGCGGCAGCGAAGT

TCAGCTGTTAGAAAGTGGCGGTGGTCTGGTTAAGCCGGGCGGTAGTCTGCGCCT

GAGCTGTGCAGCAAGTGGTTTCACCCTGATCAATTATCGTATGAACTGGGTGCG

CCAAGCCCCGGGTAAAGGTCTGGAGTGGGTTAGTAGTATCAGCAGCAGCAGCA

GTTACATCCACTATGCCGATAGCGTTAAGGGCCGCTTTACAATCAGCCGCGATA

ATGCCGAGAATAGCTTATACCTGCAAATGAACAGTCTAAGGGCGGAAGATACC GCCGTTTACTACTGCGTTCGTGAAGGCCCTCGCGCAACAGGCTATAGCATGGCA

GACGTGTTCGACATTTGGGGTCAGGGCACCATGGTGACCGTTAGTAGCGGCGGT

GGTGGTAGTGGTGGTGGCGGTAGTGGTGGCGGTGGCAGCGAACTGGTGATGAC

CCAGAGTCCGGATAGCCTGGCCGTGAGCTTAGGCGAGCGTGCAACCATTAATTG

TAAAAGCAGTCAGAGTGTTCTGTATAGTAGCAATAACAAGAGCTATCTGGCCTG

GTATCAGCAGAAGCCGGGCCAGCCGCCGAAACTGCTGATTTACTGGGCAAGCA

CCCGCGAAAGTGGCGTGCCTGATCGCTTTAGTGGTAGCGGCAGCGGCACCGATT

TTACCCTGACCATTAGCAGTCTGCAGGCCGAGGACGTTGCCGTTTATTACTGCC

AGCAGTACTATAGCGCACCGCTGACATTTGGCGGTGGCACCAAGGTGGAAATT

AAATAA

[0075] Combined (SEQ ID NO:5 and 6):

gaggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgt ttg

E V Q L V E S G G G L V Q P G G S L R L

tcctgtgcagcttctggcttcgcgtttaactattatgatattcattgggtgcgtcag gcc

S C A A S G F A F N Y Y D I H W V R Q A

ccgggtaagggcctggaatgggttgcatatattaacccgggcggtggcaacacctat tat

P G K G L E W V A Y I N P G G G N T Y Y

gctgatagcgtcaagggccgtttcactataagcgcagacacatccaaaaacacagcc tac

A D S V K G R F T I S A D T S K N T A Y

ctacaaatgaacagcttaagagctgaggacactgccgtctattattgtgctcgccag ctg

L Q M N S L R A E D T A V Y Y C A R Q L

tatggcaacagctttatggactactggggtcaaggaaccctggtcaccgtctcctcg gct

Y G N S F M D Y W G Q G T L V T V S S A

agcaccaagggtccgagcgtgtttcctctggcacctagcagtaaaagcaccagtggt ggt

S T K G P S V F P L A P S S K S T S G G

acagcagccctgggttgcctggtgaaggattactttccggagccggtgaccgttagt tgg

T A A L G C L V K D Y F P E P V T V S W

aatagcggcgccctgaccagtggcgttcatacatttccggccgtgctgcagagtagt ggc

N S G A L T S G V H T F P A V L Q S S G

ctgtacagcctgagtagcgttgttaccgttccgagcagcagcctgggcacccagacc tat

L Y S L S S V V T V P S S S L G T Q T Y

atttgcaatgttaaccataaaccgagcaacacaaaagttgataaaaaagttgaaccg aag

I C N V N H K P S N T K V D K K V E P K agctgtgacaaaacccatacatgtgacaaaacacacacctgcccgccttgtccggcacct

S C D K T H T C D K T H T C P P C P A P

gagctgctgggtcgcccgagcgtttttctgtttcctccgaaaccgaaagacacc [...]

E L L G R P S V F L F P P K P K D T [...] acccagaagagtct gage ct gag tcctggcaaaggtggatccgccggtagcgcaggtagt

T Q K S L S L S P G K G G S A G S A G S

gcaggtagtggcggcagcgaagttcagctgttagaaagtggcggtggtctggttaag ccg

A G S G G S E V Q L L E S G G G L V K P

ggcggtagtctgcgcctgagctgtgcagcaagtggtttcaccctgatcaattatcgt atg

G G S L R L S C A A S G F T L I N Y R M

aactgggtgcgccaagccccgggtaaaggtctggagtgggttagtagtatcagcagc agc

N W V R Q A P G K G L E W V S S I S S S

agcagttacatccactatgccgatagcgttaagggccgctttacaatcagccgcgat aat

S S Y I H Y A D S V K G R F T I S R D N

gccgagaatagcttatacctgcaaatgaacagtctaagggcggaagataccgccgtt tac

A E N S L Y L Q M N S L R A E D T A V Y

tactgcgttcgtgaaggccctcgcgcaacaggctatagcatggcagacgtgttcgac att

Y C V R E G P R A T G Y S M A D V F D I

tggggtcagggcaccatggtgaccgttagtagcggcggtggtggtagtggtggtggc ggt

W G Q G T M V T V S S G G G G S G G G G

agtggtggcggtggcagcgaactggtgatgacccagagtccggatagcctggccgtg agc

S G G G G S E L V M T Q S P D S L A V S

ttaggcgagcgtgcaaccattaattgtaaaagcagtcagagtgttctgtatagtagc aat

L G E R A T I N C K S S Q S V L Y S S N

aacaagagctatctggcctggtatcagcagaagccgggccagccgccgaaactgctg att

N K S Y L A W Y Q Q K P G Q P P K L L I

tactgggcaagcacccgcgaaagtggcgtgcctgatcgctttagtggtagcggcagc ggc

Y W A S T R E S G V P D R F S G S G S G

accgattttaccctgaccattagcagtctgcaggccgaggacgttgccgtttattac tgc

T D F T L T I S S L Q A E D V A V Y Y C

cagcagtactatagcgcaccgctgacatttggcggtggcaccaaggtggaaattaaa taa Q Q Y Y S A P L T F G G G T K V E I K - [0076] E10-KZ52 LC C Fusion Sequence: [0077] Amino Acid (SEQ ID O:7):

[0078] DIQMTQSPSSLSASVGDRVTITCKASQDVTTAVAWYQQKPGKAPKLLIY

WASRLHNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASVVCLL FYPREAKVQWKVDNALQSGNSQES

VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSG

GSAGSAGSAGSGGSEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAP

GKGLE WVS SIS S S S S YIHYAD S VKGRFTISRDNAENSLYLQMNSLRAEDT A VY YC V

REGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL

AVSLGERATINCKSSQSVLYSS KSYLAWYQQKPGQPPKLLIYWASTRESGVPDR

FSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGGGTKVEIK

[0079] Underlined region is linker polypeptide. Bold region is fusion linker polypeptide.

[0080] Nucleotide (SEQ ID NO:8):

GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGG

GTCACCATCACCTGCCGGGCGAGCCAGGATGTGACCACCGCTGTAGCCTGGTAT

CAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTGGGCGAGCCGTCTT

CATAATGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCTCCGGGACGGATTTCACT

CTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAA

CATTATAGCACCCCGCTGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACG

TACGGTGGCAGCACCGAGCGTGTTTATCTTTCCGCCGAGCGACGAACAACTGAA

AAGTGGCACAGCCAGCGTGGTGTGTTTACTGAACAACTTCTATCCTCGCGAGGC

CAAGGTGCAGTGGAAAGTGGACAATGCACTGCAGAGTGGCAATAGCCAGGAGA

GCGTGACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGTAGCACCCTG

ACCCTGAGCAAGGCCGATTATGAGAAGCACAAGGTGTATGCATGCGAGGTTAC

CCATCAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTTAACCGTGGCGAATGCG

GTGGTAGTGGTGGATCCGCCGGTAGCGCAGGTAGTGCAGGTAGTGGCGGCAGC

GAAGTTCAGCTGTTAGAAAGTGGCGGTGGTCTGGTTAAGCCGGGCGGTAGTCTG

CGCCTGAGCTGTGCAGCAAGTGGTTTCACCCTGATCAATTATCGTATGAACTGG

GTGCGCCAAGCCCCGGGTAAAGGTCTGGAGTGGGTTAGTAGTATCAGCAGCAG

CAGCAGTTACATCCACTATGCCGATAGCGTTAAGGGCCGCTTTACAATCAGCCG

CGATAATGCCGAGAATAGCTTATACCTGCAAATGAACAGTCTAAGGGCGGAAG

ATACCGCCGTTTACTACTGCGTTCGTGAAGGCCCTCGCGCAACAGGCTATAGCA

TGGCAGACGTGTTCGACATTTGGGGTCAGGGCACCATGGTGACCGTTAGTAGCG

GCGGTGGTGGTAGTGGTGGTGGCGGTAGTGGTGGCGGTGGCAGCGAACTGGTG ATGACCCAGAGTCCGGATAGCCTGGCCGTGAGCTTAGGCGAGCGTGCAACCAT

TAATTGTAAAAGCAGTCAGAGTGTTCTGTATAGTAGCAATAACAAGAGCTATCT

GGCCTGGTATCAGCAGAAGCCGGGCCAGCCGCCGAAACTGCTGATTTACTGGG

CAAGCACCCGCGAAAGTGGCGTGCCTGATCGCTTTAGTGGTAGCGGCAGCGGC

ACCGATTTTACCCTGACCATTAGCAGTCTGCAGGCCGAGGACGTTGCCGTTTAT

TACTGCCAGCAGTACTATAGCGCACCGCTGACATTTGGCGGTGGCACCAANGTG

GAAATTAAA

[0081] Combined (SEQ ID NO:7 and 8):

gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtc acc

D I Q M T Q S P S S L S A S V G D R V T

atcacctgccgggcgagccaggatgtgaccaccgctgtagcctggtatcaacagaaa cca

I T C R A S Q D V T T A V A W Y Q Q K P

ggaaaagctccgaagcttctgatttactgggcgagccgtcttcataatggcgtgccg agc

G K A P K L L I Y W A S R L H N G V P S

cgctttagcggcagcggctccgggacggatttcactctgaccatcagcagtctgcag ccg

R F S G S G S G T D F T L T I S S L Q P

gaagacttcgcaacttattactgtcagcaacattatagcaccccgctgacgttcgga cag

E D F A T Y Y C Q Q H Y S T P L T F G Q

ggtaccaaggtggagatcaaacgtacggtggcagcaccgagcgtgtttatctttccg ccg

G T K V E I K R T V A A P S V F I F P P

agcgacgaacaactgaaaagtggcacagccagcgtggtgtgtttactgaacaacttc tat

S D E Q L K S G T A S V V C L L N N F Y

cctcgcgaggccaaggtgcagtggaaagtggacaatgcactgcagagtggcaatagc cag

P R E A K V Q W K V D N A L Q S G N S Q

gagagcgtgaccgaacaggatagcaaagatagcacctatagcctgagtagcaccctg acc

E S V T E Q D S K D S T Y S L S S T L T

ctgagcaaggccgattatgagaagcacaaggtgtatgcatgcgaggttacccatcag ggc

L S K A D Y E K H K V Y A C E V T H Q G

ctgagcagcccggtgaccaaaagctttaaccgtggcgaatgcggtggtagtggtgga tcc

L S S P V T K S F N R G E C G G S G G S

gccggtagcgcaggtagtgcaggtagtggcggcagcgaagttcagctgttagaaagt ggc

A G S A G S A G S G G S E V Q L L E S G

ggtggtctggttaagccgggcggtagtctgcgcctgagctgtgcagcaagtggtttc acc G G L V K P G G S L R L S C A A S G F T

ctgatcaattatcgtatgaactgggtgcgccaagccccgggtaaaggtctggagtgg gtt

L I N Y R M N W V R Q A P G K G L E W V

agtagtatcagcagcagcagcagttacatccactatgccgatagcgttaagggccgc ttt

S S I S S S S S Y I H Y A D S V K G R F

acaatcagccgcgataatgccgagaatagcttatacctgcaaatgaacagtctaagg gcg

T I S R D N A E N S L Y L Q M N S L R A

gaagataccgccgtttactactgcgttcgtgaaggccctcgcgcaacaggctatagc atg

E D T A V Y Y C V R E G P R A T G Y S M

gcagacgtgttcgacatttggggtcagggcaccatggtgaccgttagtagcggcggt ggt

A D V F D I W G Q G T M V T V S S G G G

ggtagtggtggtggcggtagtggtggcggtggcagcgaactggtgatgacccagagt ccg

G S G G G G S G G G G S E L V M T Q S P

gatagcctggccgtgagcttaggcgagcgtgcaaccattaattgtaaaagcagtcag agt

D S L A V S L G E R A T I N C K S S Q S

gttctgtatagtagcaataacaagagctatctggcctggtatcagcagaagccgggc cag

V L Y S S N N K S Y L A W Y Q Q K P G Q

ccgccgaaactgctgatttactgggcaagcacccgcgaaagtggcgtgcctgatcgc ttt

P P K L L I Y W A S T R E S G V P D R F

agtggtagcggcagcggcaccgattttaccctgaccattagcagtctgcaggccgag gac

S G S G S G T D F T L T I S S L Q A E D

gttgccgtttattactgccagcagtactatagcgcaccgctgacatttggcggtggc acc

V A V Y Y C Q Q Y Y S A P L T F G G G T

aangtggaaattaaa

X V E I K

[0082] F4-KZ52 HC N' Fusion Sequence:

[0083] Amino Acid (SEQ ID NO : 9) :

EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSS

YIHYADSVKGRFTISRDNAENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADV

FDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLAVSLGERATINCKSS

QSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

LQAEDVAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSEVQLVESGG

GLVQPGGSLRLSCAASGFAFNYYDMFWVRQAPGKGLEWVAYIKPGGGNTYYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQLYGNSFFDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSS GL YSLSS WTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCDKTHTCPPCPA PELLGRPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQ VSL TCL VKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0084] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide. Italicized region is CH1-CH3 for the HC and the CL region.

[0085] Nucleotide (SEQ ID NO: 10):

GAAGTTCAGTTACTGGAAAGTGGCGGCGGCCTGGTTAAACCGGGTGGTAGCCT

GCGTCTGAGTTGCGCAGCAAGCGGCTTCACCCTGATCAACTATCGCATGAACTG

GGTGCGCCAAGCACCGGGTAAGGGTCTGGAGTGGGTGAGCAGCATCAGCAGCA

GCAGCAGCTACATCCACTACGCAGACAGCGTTAAAGGCCGCTTCACCATTAGCC

GCGATAACGCCGAAAACAGCCTGTACCTGCAGATGAACAGTCTAAGGGCGGAG

GATACCGCAGTGTACTACTGCGTTCGTGAAGGCCCGCGTGCAACCGGCTATAGC

ATGGCCGACGTTTTTGATATTTGGGGCCAGGGCACCATGGTGACCGTGAGTAGT

GGTGGTGGTGGCAGCGGTGGTGGCGGTAGTGGTGGTGGTGGCAGTGAACTGGT

TATGACCCAGAGTCCGGACAGTCTGGCAGTGAGCCTGGGCGAGCGTGCAACCA

TCAACTGTAAGAGCAGTCAGAGCGTGCTGTATAGTAGCAACAATAAAAGCTAT

CTGGCCTGGTATCAGCAGAAGCCGGGTCAGCCGCCTAAGCTGCTGATTTATTGG

GCCAGCACCCGCGAAAGCGGTGTTCCGGATCGCTTTAGCGGTAGCGGCAGCGG

TACCGATTTCACCCTGACCATCAGCAGCCTGCAGGCCGAAGATGTGGCCGTGTA

TTATTGCCAGCAGTACTACAGCGCCCCGCTGACCTTTGGTGGCGGTACCAAGGT

GGAAATTAAAGGCGGCAGTGCCGGTAGTGCCGGTAGTGCAGGTAGCGGCGGTA

GCGAGGTTCAGCTGGTGGAAAGCGGCGGTGGTCTGGTTCAGCCTGGTGGTAGC

CTGCGCCTGAGCTGTGCCGCAAGCGGTTTCGCATTTAACTACTATGACATGTTCT

GGGTTCGCCAGGCACCGGGCAAAGGTCTGGAATGGGTGGCCTATATCAAACCG

GGCGGCGGCAACACCTACTACGCCGATAGCGTTAAGGGTCGTTTCACCATCAGC

GCCGATACCAGCAAAAACACCGCCTATCTGCAGATGAATAGCCTAAGGGCGGA

AGACACCGCAGTGTATTACTGCGCACGCCAGCTGTACGGCAACAGCTTTTTCGA

TTACTGGGGCCAGGGTACCCTGGTTACCGTGAGCAGC

[0086] Combined (SEQ ID NO:9 and 10): gaagttcagttactggaaagtggcggcggcctggttaaaccgggtggtagcctgcgtctg

E V Q L L E S G G G L V K P G G S L R L

agttgcgcagcaagcggcttcaccctgatcaactatcgcatgaactgggtgcgccaa gca

S C A A S G F T L I N Y R M N W V R Q A

ccgggtaagggtctggagtgggtgagcagcatcagcagcagcagcagctacatccac tac

P G K G L E W V S S I S S S S S Y I H Y

gcagacagcgttaaaggccgctt caeca ttagccgcgataacgccgaaaacagcctgtac

A D S V K G R F T I S R D N A E N S L Y

ctgcagatgaacagtctaagggcggaggataccgcagtgtactactgcgttcgtgaa ggc

L Q M N S L R A E D T A V Y Y C V R E G

ccgcgtgcaaccggctatagcatggccgacgtttttgatatttggggccagggcacc atg

P R A T G Y S M A D V F D I W G Q G T M

gtgaccgtgagtagtggtggtggtggcagcggtggtggcggtagtggtggtggtggc agt

V T V S S G G G G S G G G G S G G G G S

gaactggttatgacccagagtccggacagtctggcagtgagcctgggcgagcgtgca acc

E L V M T Q S P D S L A V S L G E R A T

atcaactgtaagagcagtcagagcgtgctgtatagtagcaacaataaaagctatctg gcc

I N C K S S Q S V L Y S S N N K S Y L A

tggtatcagcagaagccgggtcagccgcctaagctgctgatttattgggccagcacc cgc

W Y Q Q K P G Q P P K L L I Y W A S T R

gaaagcggtgttccggatcgctttagcggtagcggcagcggtaccgatttcaccctg acc

E S G V P D R F S G S G S G T D F T L T

atcagcagcctgcaggccgaagatgtggccgtgtattattgccagcagtactacagc gcc

I S S L Q A E D V A V Y Y C Q Q Y Y S A

ccgctgacctttggtggcggtaccaaggtggaaattaaaggcggcagtgccggtagt gcc

P L T F G G G T K V E I K G G S A G S A

ggtagtgcaggtagcggcggtagcgaggttcagctggtggaaagcggcggtggtctg gtt

G S A G S G G S E V Q L V E S G G G L V

cagcctggtggtagcctgcgcctgagctgtgccgcaagcggtttcgcatttaactac tat

Q P G G S L R L S C A A S G F A F N Y Y

gacatgttctgggttcgccaggcaccgggcaaaggtctggaatgggtggcctatatc aaa

D M F W V R Q A P G K G L E W V A Y I K

ccgggcggcggcaacacc tact a cgccgatagcgttaagggtcgttt caeca tcagcgcc P G G G N T Y Y A D S V K G R F T I S A

gataccagcaaaaacaccgcctatctgcagatgaatagcctaagggcggaagacacc gca

D T S K N T A Y L Q M N S L R A E D T A

gtgtattactgcgcacgccagctgtacggcaacagctttttcgattactggggccag ggt

V Y Y C A R Q L Y G N S F F D Y W G Q G

accctggttaccgtgagcagc

T L V T V S S

[0087] F4-KZ52 LC N' Fusion Sequence:

[0088] Amino Acid (SEQ ID NO: 11):

EVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAPGKGLEWVSSISSSSS

YIHYADSVKGRFTISRDNAENSLYLQMNSLRAEDTAVYYCVREGPRATGYSMADV

FDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSLAVSLGERATINCKSS

QSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

LQAEDVAVYYCQQYYSAPLTFGGGTKVEIKGGSAGSAGSAGSGGSDIQMTQSPSS

LSASVGDRVTITCKASQDVTTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGS

GSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVEIKRr^ S' / S' ^g

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKA

D YEKHKVYA CE VTHQGLSSP VTKSFNR GECGGSD YKDDDDK

[0089] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide. Italicized region is CH1-CH3 for the HC and the CL region.

[0090] Nucleotide (SEQ ID NO: 12):

GAAGTTCAGTTACTGGAAAGTGGCGGCGGCCTGGTTAAACCGGGTGGTAGCCT

GCGTCTGAGTTGCGCAGCAAGCGGCTTCACCCTGATCAACTATCGCATGAACTG

GGTGCGCCAAGCACCGGGTAAGGGTCTGGAGTGGGTGAGCAGCATCAGCAGCA

GCAGCAGCTACATCCACTACGCAGACAGCGTTAAAGGCCGCTTCACCATTAGCC

GCGATAACGCCGAAAACAGCCTGTACCTGCAGATGAACAGTCTAAGGGCGGAG

GATACCGCAGTGTACTACTGCGTTCGTGAAGGCCCGCGTGCAACCGGCTATAGC

ATGGCCGACGTTTTTGATATTTGGGGCCAGGGCACCATGGTGACCGTGAGTAGT

GGTGGTGGTGGCAGCGGTGGTGGCGGTAGTGGTGGTGGTGGCAGTGAACTGGT

TATGACCCAGAGTCCGGACAGTCTGGCAGTGAGCCTGGGCGAGCGTGCAACCA

TCAACTGTAAGAGCAGTCAGAGCGTGCTGTATAGTAGCAACAATAAAAGCTAT

CTGGCCTGGTATCAGCAGAAGCCGGGTCAGCCGCCTAAGCTGCTGATTTATTGG

GCCAGCACCCGCGAAAGCGGTGTTCCGGATCGCTTTAGCGGTAGCGGCAGCGG TACCGATTTCACCCTGACCATCAGCAGCCTGCAGGCCGAAGATGTGGCCGTGTA

TTATTGCCAGCAGTACTACAGCGCCCCGCTGACCTTTGGTGGCGGTACCAAGGT

GGAAATTAAAGGCGGCAGTGCCGGTAGTGCCGGTAGTGCAGGTAGCGGCGGTA

GCGATATCCAGATGACCCAGAGTCCGAGTAGTCTGAGCGCCAGCGTTGGTGAC

CGCGTTACCATCACCTGCAAGGCCAGCCAGGATGTTACCACCGCCGTGGCCTGG

TATCAACAGAAACCGGGCAAGGCCCCGAAGCTGCTGATTTATTGGGCCAGTAC

ACGCCATACAGGCGTGCCGAGCCGTTTTAGTGGCAGCGGTAGCGGTACCGACTT

CACCCTGACCATCAGTAGCCTGCAACCGGAGGATTTCGCCACCTACTACTGCCA

GCAGCACTACAGCACCCCGCTGACCTTTGGCCAAGGTACCAAGGTGGAGATTA

AG

[0091] Combined (SEQ ID NO: 11 and 12):

gaagttcagttactggaaagtggcggcggcctggttaaaccgggtggtagcctgcgt ctg

E V Q L L E S G G G L V K P G G S L R L

agttgcgcagcaagcggcttcaccctgatcaactatcgcatgaactgggtgcgccaa gca

S C A A S G F T L I N Y R M N W V R Q A

ccgggtaagggtctggagtgggtgagcagcatcagcagcagcagcagctacatccac tac

P G K G L E W V S S I S S S S S Y I H Y

gcagacagcgttaaaggccgctt caeca ttagccgcgataacgccgaaaacagcctgtac

A D S V K G R F T I S R D N A E N S L Y

ctgcagatgaacagtctaagggcggaggataccgcagtgtactactgcgttcgtgaa ggc

L Q M N S L R A E D T A V Y Y C V R E G

ccgcgtgcaaccggctatagcatggccgacgtttttgatatttggggccagggcacc atg

P R A T G Y S M A D V F D I W G Q G T M

gtgaccgtgagtagtggtggtggtggcagcggtggtggcggtagtggtggtggtggc agt

V T V S S G G G G S G G G G S G G G G S

gaactggttatgacccagagtccggacagtctggcagtgagcctgggcgagcgtgca acc

E L V M T Q S P D S L A V S L G E R A T

atcaactgtaagagcagtcagagcgtgctgtatagtagcaacaataaaagctatctg gcc

I N C K S S Q S V L Y S S N N K S Y L A

tggtatcagcagaagccgggtcagccgcctaagctgctgatttattgggccagcacc cgc

W Y Q Q K P G Q P P K L L I Y W A S T R

gaaagcggtgttccggatcgctttagcggtagcggcagcggtaccgatttcaccctg acc E S G V P D R F S G S G S G T D F T L T

atcagcagcctgcaggccgaagatgtggccgtgtattattgccagcagtactacagc gcc

I S S L Q A E D V A V Y Y C Q Q Y Y S A

ccgctgacctttggtggcggtaccaaggtggaaattaaaggcggcagtgccggtagt gcc

P L T F G G G T K V E I K G G S A G S A

ggtagtgcaggtagcggcggtagcgatatccagatgacccagagtccgagtagtctg agc

G S A G S G G S D I Q M T Q S P S S L S

gccagcgttggtgaccgcgttaccatcacctgcaaggccagccaggatgttaccacc gcc

A S V G D R V T I T C K A S Q D V T T A

gtggcctggtatcaacagaaaccgggcaaggccccgaagctgctgatttattgggcc agt

V A W Y Q Q K P G K A P K L L I Y W A S

acacgccatacaggcgtgccgagccgttttagtggcagcggtagcggtaccgacttc acc

T R H T G V P S R F S G S G S G T D F T

ctgaccatcagtagcctgcaaccggaggatttcgccacctactactgccagcagcac tac

L T I S S L Q P E D F A T Y Y C Q Q H Y

agcaccccgctgacctttggccaaggtaccaaggtggagattaag

S T P L T F G Q G T K V E I K

[0092] F4-KZ52 HC C Fusion Sequence:

[0093] Amino Acid (SEQ ID NO: 13):

[0094] EVQLVESGGGLVQPGGSLRLSCAASGFAFNYYDMFWVRQAPGKGLEW

VAYIKPGGGNTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARQLYG

NSFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

KVEPKSCDKTHTCDKTHTCPPCPAPELLGRPSVFLFPPKPKDTLMISRTPEVTCVVV

DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGGSAGSAGSAGSGGSEVQLLESGGGLVKPGGSLRLSCA

ASGFTLINYRMNWVRQAPGKGLEWVSSISSSSSYIHYADSVKGRFTISRDNAENSLY

LQMNSLRAEDTAVYYCVREGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGG

GSGGGGSELVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQP

PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGG

GTKVEIK [0095] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide.

[0096] Nucleotide (SEQ ID NO: 14):

GAAGTTCAACTGGTTGAGAGTGGCGGTGGCTTAGTTCAACCGGGCGGTAGTTTA

CGCCTGAGTTGTGCAGCCAGCGGTTTCGCCTTCAACTATTATGACATGTTTTGGG

TGCGCCAGGCACCGGGTAAAGGCCTGGAGTGGGTGGCCTATATCAAACCGGGC

GGTGGCAACACCTATTACGCCGATAGCGTGAAAGGTCGCTTTACCATCAGCGCA

GATACCAGCAAGAATACCGCCTACCTGCAGATGAATAGCCTGCGTGCCGAAGA

CACCGCCGTTTATTATTGCGCCCGCCAGCTGTACGGCAATAGCTTTTTCGATTAC

TGGGGCCAGGGCACCCTGGTTACCGTTAGCAGCGCTAGCACCAAGGGTCCGAG

CGTGTTTCCTCTGGCACCTAGCAGTAAAAGCACCAGTGGTGGTACAGCAGCCCT

GGGTTGCCTGGTGAAGGATTACTTTCCGGAGCCGGTGACCGTTAGTTGGAATAG

CGGCGCCCTGACCAGTGGCGTTCATACATTTCCGGCCGTGCTGCAGAGTAGTGG

CCTGTACAGCCTGAGTAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCA

GACCTATATTTGCAATGTTAACCATAAACCGAGCAACACAAAAGTTGATAAAA

AAGTTGAACCGAAGAGCTGTGACAAAACCCATACATGTGACAAAACACACACC

TGCCCGCCTTGTCCGGCACCTGAGCTGCTGGGTCGCCCGAGCGTTTTTCTGTTTC

CTCCGAAACCGAAAGACACCCTGATGATCAGCCGCACACCTGAGGTGACCTGT

GTTGTGGTGGATGTGAGCCACGAAGATCCTGAAGTTAAGTTTAACTGGTATGTG

GATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGTGAAGAGCAGTACAA

CAGCACCTATCGTGTTGTTAGTGTGCTGACCGTTCTGCACCAAGATTGGCTGAA

CGGCAAGGAGTATAAATGCAAGGTTAGCAATAAAGCCCTGCCGGCCCCGATCG

AGAAGACCATCAGCAAAGCCAAAGGTCAGCCGCGTGAGCCTCAGGTGTATACA

CTGCCGCCTAGCCGTGAGGAGATGACCAAGAATCAGGTTAGCCTGACCTGTCTG

GTGAAAGGCTTTTACCCGAGCGATATCGCCGTTGAGTGGGAAAGCAATGGTCA

GCCTGAGAACAACTACAAGACCACCCCGCCTGTTTTAGACAGTGATGGTAGCTT

TTTCTTATACAGCAAACTGACCGTTGATAAGAGCCGCTGGCAGCAGGGCAATGT

GTTTAGCTGCAGTGTTATGCATGAGGCCCTGCATAACCACTATACCCAGAAGAG

TCTGAGCCTGAGTCCTGGCAAAGGTGGATCCGCCGGTAGCGCAGGTAGTGCAG

GTAGTGGCGGCAGCGAAGTTCAGCTGTTAGAAAGTGGCGGTGGTCTGGTTAAG

CCGGGCGGTAGTCTGCGCCTGAGCTGTGCAGCAAGTGGTTTCACCCTGATCAAT

TATCGTATGAACTGGGTGCGCCAAGCCCCGGGTAAAGGTCTGGAGTGGGTTAGT

AGTATCAGCAGCAGCAGCAGTTACATCCACTATGCCGATAGCGTTAAGGGCCG CTTTACAATCAGCCGCGATAATGCCGAGAATAGCTTATACCTGCAAATGAACAG

TCTAAGGGCGGAAGATACCGCCGTTTACTACTGCGTTCGTGAAGGCCCTCGCGC

AACAGGCTATAGCATGGCAGACGTGTTCGACATTTGGGGTCAGGGCACCATGG

TGACCGTTAGTAGCGGCGGTGGTGGTAGTGGTGGTGGCGGTAGTGGTGGCGGT

GGCAGCGAACTGGTGATGACCCAGAGTCCGGATAGCCTGGCCGTGAGCTTAGG

CGAGCGTGCAACCATTAATTGTAAAAGCAGTCAGAGTGTTCTGTATAGTAGCAA

TAACAAGAGCTATCTGGCCTGGTATCAGCAGAAGCCGGGCCAGCCGCCGAAAC

TGCTGATTTACTGGGCAAGCACCCGCGAAAGTGGCGTGCCTGATCGCTTTAGTG

GTAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGTCTGCAGGCCGAGG

ACGTTGCCGTTTATTACTGCCAGCAGTACTATAGCGCACCGCTGACATTTGGCG

GTGGCACCAAGGTGGAAATTAAATAA

[0097] Combined (SEQ ID NO: 13 and 14):

gaagttcaactggttgagagtggcggtggcttagttcaaccgggcggtagtttacgc ctg

E V Q L V E S G G G L V Q P G G S L R L

agttgtgcagccagcggtttcgccttcaactattatgacatgttttgggtgcgccag gca

S C A A S G F A F N Y Y D M F W V R Q A

ccgggtaaaggcctggagtgggtggcctatatcaaaccgggcggtggcaacacctat tac

P G K G L E W V A Y I K P G G G N T Y Y

gccgatagcgtgaaaggtcgctttaccatcagcgcagataccagcaagaataccgcc tac

A D S V K G R F T I S A D T S K N T A Y

ctgcagatgaatagcctgcgtgccgaagacaccgccgtttattattgcgcccgccag ctg

L Q M N S L R A E D T A V Y Y C A R Q L

tacggcaatagctttttcgattactggggccagggcaccctggttaccgttagcagc gct

Y G N S F F D Y W G Q G T L V T V S S A

agcaccaagggtccgagcgtgtttcctctggcacctagcagtaaaagcaccagtggt ggt

S T K G P S V F P L A P S S K S T S G G

acagcagccctgggttgcctggtgaaggattactttccggagccggtgaccgttagt tgg

T A A L G C L V K D Y F P E P V T V S W

aatagcggcgccctgaccagtggcgttcatacatttccggccgtgctgcagagtagt ggc

N S G A L T S G V H T F P A V L Q S S G

ctgtacagcctgagtagcgttgttaccgttccgagcagcagcctgggcacccagacc tat

L Y S L S S V V T V P S S S L G T Q T Y

atttgcaatgttaaccataaaccgagcaacacaaaagttgataaaaaagttgaaccg aag I C N V N H K P S N T K V D K K V E P K agctgtgacaaaacccatacatgtgacaaaacacacacctgcccgccttgtccggcacct

S C D K T H T C D K T H T C P P C P A P

gagctgctgggtcgcccgagcgtttttctgtttcctccgaaaccgaaagacaccctg atg

E L L G R P S V F L F P P K P K D T L M

atcagccgcacacctgaggtgacctgtgttgtggtggatgtgagccacgaagatcct gaa

I S R T P E V T C V V V D V S H E D P E

gttaagtttaactggtatgtggatggcgtggaggtgcataatgccaagacaaagccg cgt

V K F N W Y V D G V E V H N A K T K P R

gaagagcagtacaacagcacctatcgtgttgttagtgtgctgaccgttctgcaccaa gat

E E Q Y N S T Y R V V S V L T V L H Q D

tggctgaacggcaaggagtataaatgcaaggttagcaataaagccctgccggccccg atc

W L N G K E Y K C K V S N K A L P A P I

gagaagaccatcagcaaagccaaaggtcagccgcgtgagcctcaggtgtatacactg ccg

E K T I S K A K G Q P R E P Q V Y T L P

cctagccgtgaggagatgaccaagaatcaggttagcctgacctgtctggtgaaaggc ttt

P S R E E M T K N Q V S L T C L V K G F

tacccgagcgatatcgccgttgagtgggaaagcaatggtcagcctgagaacaactac aag

Y P S D I A V E W E S N G Q P E N N Y K

accaccccgcctgttttagacagtgatggtagctttttcttatacagcaaactgacc gtt

T T P P V L D S D G S F F L Y S K L T V

gataagagccgctggcagcagggcaatgtgtttagctgcagtgttatgcatgaggcc ctg

D K S R W Q Q G N V F S C S V M H E A L

cataaccactatacccagaagagtctgagcctgagtcctggcaaaggtggatccgcc ggt

H N H Y T Q K S L S L S P G K G G S A G

agcgcaggtagtgcaggtagtggcggcagcgaagttcagctgttagaaagtggcggt ggt

S A G S A G S G G S E V Q L L E S G G G

ctggttaagccgggcggtagtctgcgcctgagctgtgcagcaagtggtttcaccctg atc

L V K P G G S L R L S C A A S G F T L I

aattatcgtatgaactgggtgcgccaagccccgggtaaaggtctggagtgggttagt agt

N Y R M N W V R Q A P G K G L E W V S S

atcagcagcagcagcagttacatccactatgccgatagcgttaagggccgctttaca atc I S S S S S Y I H Y A D S V K G R F T I

agccgcgataatgccgagaatagcttatacctgcaaatgaacagtctaagggcggaa gat

S R D N A E N S L Y L Q M N S L R A E D

accgccgtttactactgcgttcgtgaaggccctcgcgcaacaggctatagcatggca gac

T A V Y Y C V R E G P R A T G Y S M A D

gtgttcgacatttggggtcagggcaccatggtgaccgttagtagcggcggtggtggt agt

V F D I W G Q G T M V T V S S G G G G S

ggtggtggcggtagtggtggcggtggcagcgaactggtgatgacccagagtccggat agc

G G G G S G G G G S E L V M T Q S P D S

ctggccgtgagcttaggcgagcgtgcaaccattaattgtaaaagcagtcagagtgtt ctg

L A V S L G E R A T I N C K S S Q S V L

tatagtagcaataacaagagctatctggcctggtatcagcagaagccgggccagccg ccg

Y S S N N K S Y L A W Y Q Q K P G Q P P

aaactgctgatttactgggcaagcacccgcgaaagtggcgtgcctgatcgctttagt ggt

K L L I Y W A S T R E S G V P D R F S G

agcggcagcggcaccgattttaccctgaccattagcagtctgcaggccgaggacgtt gcc

S G S G T D F T L T I S S L Q A E D V A

gtttattactgccagcagtactatagcgcaccgctgacatttggcggtggcaccaag gtg

V Y Y C Q Q Y Y S A P L T F G G G T K V

gaaattaaataa

E I K - [0098] F4-KZ52 LC C Fusion Sequence:

[0099] Amino Acid (SEQ ID NO: 15):

[00100] DIQMTQSPSSLSASVGDRVTITCKASQDVTTAVAWYQQKPGKAPKLLIY

WASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES

VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSG

GSAGSAGSAGSGGSEVQLLESGGGLVKPGGSLRLSCAASGFTLINYRMNWVRQAP

GKGLE WVS SIS S S S S YIHYAD S VKGRFTISRDNAENSLYLQMNSLRAEDT A VY YC V

REGPRATGYSMADVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSELVMTQSPDSL

AVSLGERATINCKSSQSVLYSSNNKSYLAWYQQKPGQPPKLLIYWASTRESGVPDR

FSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGGGTKVEIK [00101] Underlined region is glycine-rich linker polypeptide. Bold region is fusion linker polypeptide.

[00102] Nucleotide (SEQ ID NO: 16):

GACATTCAGATGACCCAAAGTCCGAGCAGCCTGAGTGCCAGTGTTGGTGATCGC

GTGACAATCACATGCAAGGCCAGTCAGGACGTGACCACCGCAGTGGCCTGGTA

TCAGCAGAAACCGGGTAAGGCCCCGAAGCTGCTGATCTATTGGGCCAGTACCC

GCCACACCGGTGTTCCTAGTCGCTTCAGTGGCAGTGGCAGCGGCACAGATTTCA

CCCTGACCATCAGCAGCCTGCAACCGGAAGATTTTGCCACCTACTACTGCCAGC

AGCACTATAGCACCCCGCTGACCTTTGGCCAGGGCACCAAGGTTGAGATTAAAC

GTACGGTGGCAGCACCGAGCGTGTTTATCTTTCCGCCGAGCGACGAACAACTGA

AAAGTGGCACAGCCAGCGTGGTGTGTTTACTGAACAACTTCTATCCTCGCGAGG

CCAAGGTGCAGTGGAAAGTGGACAATGCACTGCAGAGTGGCAATAGCCAGGAG

AGCGTGACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGTAGCACCCT

GACCCTGAGCAAGGCCGATTATGAGAAGCACAAGGTGTATGCATGCGAGGTTA

CCCATCAGGGCCTGAGCAGCCCGGTGACCAAAAGCTTTAACCGTGGCGAATGC

GGTGGTAGTGGTGGATCCGCCGGTAGCGCAGGTAGTGCAGGTAGTGGCGGCAG

CGAAGTTCAGCTGTTAGAAAGTGGCGGTGGTCTGGTTAAGCCGGGCGGTAGTCT

GCGCCTGAGCTGTGCAGCAAGTGGTTTCACCCTGATCAATTATCGTATGAACTG

GGTGCGCCAAGCCCCGGGTAAAGGTCTGGAGTGGGTTAGTAGTATCAGCAGCA

GCAGCAGTTACATCCACTATGCCGATAGCGTTAAGGGCCGCTTTACAATCAGCC

GCGATAATGCCGAGAATAGCTTATACCTGCAAATGAACAGTCTAAGGGCGGAA

GATACCGCCGTTTACTACTGCGTTCGTGAAGGCCCTCGCGCAACAGGCTATAGC

ATGGCAGACGTGTTCGACATTTGGGGTCAGGGCACCATGGTGACCGTTAGTAGC

GGCGGTGGTGGTAGTGGTGGTGGCGGTAGTGGTGGCGGTGGCAGCGAACTGGT

GATGACCCAGAGTCCGGATAGCCTGGCCGTGAGCTTAGGCGAGCGTGCAACCA

TTAATTGTAAAAGCAGTCAGAGTGTTCTGTATAGTAGCAATAACAAGAGCTATC

TGGCCTGGTATCAGCAGAAGCCGGGCCAGCCGCCGAAACTGCTGATTTACTGG

GCAAGCACCCGCGAAAGTGGCGTGCCTGATCGCTTTAGTGGTAGCGGCAGCGG

CACCGATTTTACCCTGACCATTAGCAGTCTGCAGGCCGAGGACGTTGCCGTTTA

TTACTGCCAGCAGTACTATAGCGCACCGCTGACATTTGGCGGTGGCACCAAGGT

GGAAATTAAATAA

[00103] Combined (SEQ ID NO: 15 and 16):

gacattcagatgacccaaagtccgagcagcctgagtgccagtgttggtgatcgcgtg aca D I Q M T Q S P S S L S A S V G D R V T atcacatgcaaggccagtcaggacgtgaccaccgcagtggcctggtatcagcagaaaccg

I T C K A S Q D V T T A V A W Y Q Q K P

ggtaaggccccgaagctgctgatctattgggccagtacccgccacaccggtgttcct agt

G K A P K L L I Y W A S T R H T G V P S

cgcttcagtggcagtggcagcggcacagatttcaccctgaccatcagcagcctgcaa ccg

R F S G S G S G T D F T L T I S S L Q P

gaagattttgccacctactactgccagcagcactatagcaccccgctgacctttggc cag

E D F A T Y Y C Q Q H Y S T P L T F G Q

ggcaccaaggttgagattaaacgtacggtggcagcaccgagcgtgtttatctttccg ccg

G T K V E I K R T V A A P S V F I F P P

agcgacgaacaactgaaaagtggcacagccagcgtggtgtgtttactgaacaacttc tat

S D E Q L K S G T A S V V C L L N N F Y

cctcgcgaggccaaggtgcagtggaaagtggacaatgcactgcagagtggcaatagc cag

P R E A K V Q W K V D N A L Q S G N S Q

gagagcgtgaccgaacaggatagcaaagatagcacctatagcctgagtagcaccctg acc

E S V T E Q D S K D S T Y S L S S T L T

ctgagcaaggccgattatgagaagcacaaggtgtatgcatgcgaggttacccatcag ggc

L S K A D Y E K H K V Y A C E V T H Q G

ctgagcagcccggtgaccaaaagctttaaccgtggcgaatgcggtggtagtggtgga tcc

L S S P V T K S F N R G E C G G S G G S

gccggtagcgcaggtagtgcaggtagtggcggcagcgaagttcagctgttagaaagt ggc

A G S A G S A G S G G S E V Q L L E S G

ggtggtctggttaagccgggcggtagtctgcgcctgagctgtgcagcaagtggtttc acc

G G L V K P G G S L R L S C A A S G F T

ctgatcaattatcgtatgaactgggtgcgccaagccccgggtaaaggtctggagtgg gtt

L I N Y R M N W V R Q A P G K G L E W V

agtagtatcagcagcagcagcagttacatccactatgccgatagcgttaagggccgc ttt

S S I S S S S S Y I H Y A D S V K G R F

acaatcagccgcgataatgccgagaatagcttatacctgcaaatgaacagtctaagg gcg

T I S R D N A E N S L Y L Q M N S L R A

gaagataccgccgtttactactgcgttcgtgaaggccctcgcgcaacaggctatagc atg E D T A V Y Y C V R E G P R A T G Y S M gcagacgtgttcgacatttggggtcagggcaccatggtgaccgttagtagcggcggtggt

A D V F D I W G Q G T M V T V S S G G G

ggtagtggtggtggcggtagtggtggcggtggcagcgaactggtgatgacccagagt ccg

G S G G G G S G G G G S E L V M T Q S P

gatagcctggccgtgagcttaggcgagcgtgcaaccattaattgtaaaagcagtcag agt

D S L A V S L G E R A T I N C K S S Q S

gttctgtatagtagcaataacaagagctatctggcctggtatcagcagaagccgggc cag

V L Y S S N N K S Y L A W Y Q Q K P G Q

ccgccgaaactgctgatttactgggcaagcacccgcgaaagtggcgtgcctgatcgc ttt

P P K L L I Y W A S T R E S G V P D R F

agtggtagcggcagcggcaccgattttaccctgaccattagcagtctgcaggccgag gac

S G S G S G T D F T L T I S S L Q A E D

gttgccgtttattactgccagcagtactatagcgcaccgctgacatttggcggtggc acc

V A V Y Y C Q Q Y Y S A P L T F G G G T

aaggtggaaattaaataa

K V E I K - [00104] In an embodiment of any of the antibodies and/or scFv described herein (of the compositions described herein), the antibody and/or scFv is a neutralizing antibody/scFv with respect to the relevant Ebola virus.

[00105] In an embodiment, the antibody or scFv binds the relevant Ebola virus glycoprotein pre-fusion core, which is a heterohexamer of three copies of the GP 1 and 3 copies of the GP2.

[00106] In an embodiment, the antibody comprises an Fc region having a sequence identical to a human Fc region.

[00107] In an embodiment, the Fc region of the antibody is glycosylated.

[00108] A "humanized" antibody as used herein, unless otherwise indicated, is a chimeric antibody that contains minimal sequence (CDRs) derived from non-human immunoglobulin (e.g. such as a mouse immunoglobulin). In one embodiment, a humanized antibody is an antibody having a sequence of a human immunoglobulin (recipient antibody) in which CDR residues of a hypervariable region (HVR) of the recipient are replaced by CDR residues from a non-human species (donor antibody) such as a mouse having the desired specificity. In some instances, FR residues of the human immunoglobulin variable domain are replaced by corresponding non-human residues, for example by a back-mutation. 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 : 105-1 15 (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. Other 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. The framework regions of the antibodies of the invention having a sequence identical to a human framework region may include amino acid residues not encoded by human germline sequences (e.g., mutations introduced by random or site-specific mutagenesis). In an embodiment, the isolated antibody or antigen-binding antibody fragment comprises a variable domain framework sequence having a sequence identical to a human variable domain framework sequence FR1, FR2, FR3 or FR4. In an embodiment, the isolated antibody or antigen-binding antibody fragment comprises a variable domain framework sequence having a sequence identical to at least two of human variable domain framework sequences FR1, FR2, FR3 or FR4. In an embodiment, the isolated antibody or antigen-binding antibody fragment comprises a variable domain framework sequence having a sequence identical to at least three of human variable domain framework sequences FR1, FR2, FR3 or FR4. In an embodiment, the isolated antibody or antigen-binding antibody fragment comprises a variable domain framework sequence having a sequence identical to all four of human variable domain framework sequences FR1, FR2, FR3 and FR4.

[00109] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, an intact antibody as used herein may be an antibody with or without the otherwise C-terminal lysine.

[00110] In an embodiment, the antibodies of the invention described herein comprise a human Fc region or a variant human Fc region. A variant human Fc region comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence human Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably, from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably, at least about 90% sequence identity therewith, more preferably, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[00111] In an embodiment, the scFv is a variable domain light chain (VL) and a variable domain heavy chain (VH) which are linked N-C or C-N, respectively, via a peptide linker. In an embodiment the linker of the scFv is 5-30 amino acids in length. In an embodiment the linker of the scFv is 10-25 amino acids in length. In an embodiment the peptide linker comprises glycine, serine and/or threonine residues. For example, see Bird et al, Science, 242: 423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988), each of which are hereby incorporated by reference in their entirety.

[00112] In an embodiment, the antibody and or scFv(s) of the composition of the invention do not recognize an Ebola GP1 mucin-like domain. In an embodiment, the antibody and or scFv(s) of the composition of the invention do not recognize an Ebola GP 1 variable glycan cap.

[00113] For trispecific constructs, which target three different Filovirus strains and/or species, an IgG directed at the prefusion glycoprotein core for one strain is fused two different scFvs (each directed at a prefusion glycoprotein core for two other strains, respectively). The scFvs are fused to the IgG at two different positions, with the two scFv having specificity for the different viruses. In a non-limiting example, an SUDV-specific IgG with EBOV-specific scFv as light chain N-terminal fusion and a MARV-specific scFv as a heavy chain C-terminal fusion.

[00114] The phrase "and/or" as used herein, with option A and/or option B for example, encompasses the embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[00115] It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.

[00116] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group subjectly and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[00117] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00118] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art 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

Introduction

[00119] Ebola virus pathogenesis and cell entry: The infectious agents Ebola virus and Marburg virus (MARV) are the two major species of the Filoviridae family of enveloped negative-sense RNA viruses (1-4). Based on nucleotide sequence and outbreak location, isolates of Ebola virus are classified into five species: Zaire (EBOV), Tai Forest (TAFV), Sudan (SUDV), Reston (RESTV), and Bundibugyo (BDBV). There are two MARV variants (Marburg and Ravn). Severe human disease and deaths (30-90% case fatality rates in large outbreaks) are associated with EBOV, SUDV, BDBV, and MARV (2). Although the ecology of these agents remains incompletely understood, several species of African fruit bats appear to be reservoirs for Ebola virus and MARV (5). EBOV and SUDV are the most pathogenic among the ebolaviruses, and both have been associated with recurring outbreaks (6). Among the 13 documented EBOV outbreaks and the six SUDV outbreaks from 1976-2012, the average human case fatality rates are 70% and 52%, respectively. Together, EBOV and SUDV account for over 95% of Ebola virus-related deaths (6); these statistics do not include the ongoing EBOV outbreak in West Africa that is of unprecedented scope and geographic distribution. Therefore, a therapeutic agent that is effective against both EBOV and SUDV would greatly reduce the threat of an Ebola virus pandemic.

[00120] All human outbreaks occur as a result of direct contact with infected wildlife, with subsequent person-to-person transmission, mostly through the mucosa or contaminated needles. Uncontrolled viral replication is central to filovirus-induced disease, both because it is cytopathic and because it induces dysregulation of the host immune system (2, 7, 8). Therefore, antiviral therapies that reduce viral load are expected to increase patient survival, in part, by allowing time to mount an effective immune response. While many cell types can be infected with filovirus in vitro and in vivo, antigen-presenting cells (macrophages and dendritic cells) appear to be early and sustained targets of infection in vivo. Infected macrophages are unable to stimulate a robust immune response, and cause a "cytokine storm" that is proposed to be the primary cause of the blood clotting abnormalities and vascular leakage characteristic of filovirus hemorrhagic fever (9). Damage to other tissues (e.g., liver, kidneys, vascular endothelia) is thought to contribute to the above and to late- stage multi-organ failure. Death typically occurs 8-15 days after infection (10). Because of their high mortality rate, rapid proliferation, and potential for aerosolization, Ebola virus and Marburg virus are classified as Category A biodefense pathogens. There are currently no FDA-approved treatments for these infections.

[00121] The filovirus genome is a ~19 kb single-strand negative-sense RNA genome that encodes seven genes arranged in a linear fashion (1-4). In mature viral particles and infected cells, the genome is intimately associated with four viral proteins: the nucleocapsid protein NP, the polymerase L, the polymerase accessory protein VP35, and the transcriptional activator protein VP30. This nucleocapsid structure is in turn encapsidated in a viral matrix, comprising proteins VP40 and VP24. The host-derived viral membrane bilayer surrounds, and is peripherally associated with, the matrix. Embedded in the viral membrane are trimers of the viral glycoprotein, GP, which mediates the first step in infection: delivery of the viral nucleocapsid "payload" into the cytoplasm of the host cell. GP is the target of virus-directed antibodies that neutralize extracellular filovirus particles (4, 1 1-14).

[00122] The mature filovirus GP spike is a trimer of three disulfide-linked GP1-GP2 heterodimers, generated by endoproteolytic cleavage of the GPO precursor polypeptide by furin during virus assembly (4, 13-15). GP1 mediates viral adhesion to host cells and regulates the activity of the transmembrane subunit GP2, which mediates fusion of viral and cellular membranes during cell entry. The prefusion GP1-GP2 spike has a "chalice-and- bowl" morphology - the three GP2 subunits form the chalice within which the bowl, comprised of the three GP1 subunits, rests (Figure 1A) (13-15). This trimeric assembly is stabilized mainly by GP1-GP2 and GP2-GP2 contacts. The GP 1 subunit is organized into three subdomains. The base ('b', light blue) interacts extensively with GP2 and clamps it in its prefusion conformation. The head ('h', green) contains a putative receptor-binding sequence. Together with GP2, the base and head subdomains of GP1 form the conserved structural core of the GP 1 -GP2 spike. In contrast to the GP 1 -GP2 core, the most external subdomains of GP 1 - the glycan cap ('gc', dark blue) and the mucin-like domain (not shown) - are extensively glycosylated and display a high degree of sequence variation among filovirus isolates. Therefore, antibodies with broadly cross-neutralizing activity must recognize the conserved GP1-GP2 core and not the variable glycan cap or mucin-like domain. In response to a fusion trigger within host cell endosomes, GP2 disengages from GP 1 and undergoes a series of large-scale conformational changes that drive coalescence of viral and cellular membrane bilayers (Figure IB) (4, 16-19). The result of viral membrane fusion is cytoplasmic release of the viral nucleocapsid. KZ52 and 16F6 likely function by inhibiting these fusion-associated conformational changes (4, 13, 14).

[00123] Immunotherapy is a tractable approach to filovirus treatment pre- and postexposure. Until recently, it has been unclear if passive immunotherapy would be effective for treatment or prophylaxis of filovirus infection (20). However, recent studies using non- human primate (NHP) models have provided convincing evidence that immunotherapy can and should be pursued (21, 22). Dr. Dye's laboratory reported that rhesus macaques can be protected up to 48 hours post-exposure from EBOV or MARV infection by passive transfer of fractionated EBOV- or MARV-specific IgG isolated from convalescent animals (same species) (21). In this study, two of the three NHPs that were challenged with EBOV, and then administered serum IgG, had no clinical signs of illness; the third developed mild, delayed signs of the disease but fully recovered (Figure 2). The control animal died eight days post exposure, as is typical for untreated infection. Similar results were obtained with MARV-challenged animals, suggesting that filovirus infection in general can be treated with antibodies. This protection required only three total administrations of the serum IgG (48 hours post exposure, then again at four and eight days). Therefore, antibody-based filovirus therapy is feasible, protective, and can be administered post-exposure. In 2012, three groups (Kobinger, Zeitlin/Olinger and Takada/Feldmann) independently reported that cocktails of mAbs could protect NHPs against EBOV challenge (22-24). The Kobinger study involved a cocktail of three GP-specific murine monoclonal antibodies (mAbs) that were administered at intervals of 3 days beginning 24 or 48 hours post-exposure. Initiation of the treatment at 24 hours conferred complete survival (4/4 macaques) and initiation of treatment 48 hours post-exposure conferred partial protection (2/4 macaques fully recovered) (22). The Olinger/Zeitlin study demonstrated that a cocktail of three mouse/human chimeric mAbs (known as 'MB-003') produced in whole plant cells provided full protection when administered 1 hour post-exposure, and partial protection when administered 24 or 48 hours post-exposure (23). The Takada/Feldmann study demonstrated that a combination of two human-mouse chimeric mAbs could partially protect NHPs against EBOV challenge (24). Three NHPs were administered a cocktail of the two mAbs 24 hours preceding challenge with EBOV, then again 24- and 48-hours post-exposure. One of the three animals survived, one had delayed onset of hemorrhagic fever and was ultimately euthanized, and the other was similar to the control. It was concluded that the protection could be improved if serum half-life of the mAbs were optimized, or if the mAbs were used in combination with other mAbs or therapies. Enhanced neutralization potency would also likely improve protection. More recent studies have shown that an optimized cocktail of three monoclonal antibodies (ZMapp) can provide protection in non-human primates up to six days post-exposure.

[00124] There is a gap in treatment of filovirus infection. Only a handful of animal challenge studies have been performed with mAb therapies, in part because few mAbs that target GP (the primary neutralization target) exist. Most antibodies elicited in natural infection react preferentially with a secreted, dimeric version of the glycoprotein known as sGP and do not neutralize the fusion-relevant GP spike (4, 25, 26). Wilson et al. first demonstrated that GP-specific neutralizing antibodies (nAbs) could protect mice from EBOV challenge (27). However, three of five protective antibodies recognize highly variable sequences within the GP1 mucin-like domain, rendering them unlikely candidates for development of cross-neutralizing mAbs. Antibodies KZ52 and 16F6 are among the few well-characterized nAbs and both bind to the GP prefusion core (13, 14). KZ52 was identified by phage-based panning of a B-cell antibody library isolated from a human survivor of EBOV infection (28). Initial experiments in rodent protection studies were promising, but KZ52 failed to protect in macaques when administered on days - 1 and +4 at 50 mg/kg (12, 20). 16F6, a mouse mAb, was identified recently by Dr. Dye's group by vaccination with vector-based vaccine expressing SUDV GP (14).

[00125] mAb 16F6 is much more potent than is KZ52 against the corresponding virus species, but its murine scaffold limits therapeutic utility at this point. Fully humanized 16F6 variants have been developed (U.S. Patent Application Serial No. 14/291,608, filed May 30, 2014, hereby incorporated by reference). Head-to-head comparison in neutralization assays using a vesicular stomatitis virus pseudotyped with GP (VSV-GP) with KZ52 (against EBOV GP, GPEBOV) and 16F6 (against SUDV GP, GPSUDV) indicates that 16F6 can reduce infectivity by ~100-fold more than KZ52 at high antibody concentrations (Figure 3). Both KZ52 and 16F6 are narrowly strain specific (KZ52 for EBOV and 16F6 for SUDV), and therefore have limited potential as immunotherapeutics because they cannot cross-neutralize (13, 14).

[00126] Several candidate therapies and vaccines are under exploration for filovirus infection (27-33). Multiple promising vaccine candidates are able to protect NHPs from lethal challenge, including adenovirus-vectored, VSV-vectored, and virus-like particle- based vaccines (29-32). While any safe and effective EBOV vaccine will be useful for populations or workers that are at high risk for exposure, it is unlikely that vaccination against EBOV will be practical on a general population level. Therefore, there is still a need for an EBOV therapy that can be used to treat acute exposure or infection. Other biologies are under evaluation, including an antisense therapy undergoing clinical trials, and a promising RNAi therapy (33, 34). However, the use of nucleic acids as therapeutic agents in general is in its infancy and therefore there is a high barrier to FDA approval for such biologies. Furthermore, these therapeutic nucleic acids are strain-specific. Some small molecules against EBOV or host targets are also being explored, but studies are largely limited to early proof-of-concept stage (35-37).

[00127] Based on favorable expression and stability profiles, Bis-mAbs scKZ52-F4 LCN, scKZ52-F4 HCC, scKZ52-E10 LCN, and scKZ52-E10 HCC were chosen for additional analysis. These four Bis-mAbs showed dose-dependent neutralization of both VSV-GP pseudotyped viruses, although the IC ₅₀ values were in general higher for VSV- GPSUDV than VSV-GP _EB ov for the "LCN" constructs (Fig. 9A). For both "HCC" constructs the relative IC ₅₀ values for the two pseudotyped viruses were approximately the same. In all cases, the highest levels (> 95%) of neutralization were achieved at 100 nM Bis-mAb concentration against both pseudotyped viruses. The four Bis-mAbs were also tested for their capacity to neutralize authentic EBOV (Kikwit-1995) and SUDV (Boniface-2000) under BioSafety Level 4 (BSL4) conditions using a plaque reduction neutralization test (PRNT) (Fig. 9B). For all Bis-mAbs, potent neutralization of both EBOV and SUDV were observed in both assays, with low- or sub-nanomolar IC ₅₀ values in the PRNT assay for both viruses.

[00128] In Vivo Efficacy. To explore the protective potential of a single cross-binding monoclonal antibody reagent, scKZ52-F4 LCN and scKZ52-F4 HCC were evaluated for their ability to confer protection of mice in two separate mouse models with post-exposure dosing (Fig. 10). For EBOV, a well-established mouse challenge model was employed with mouse-adapted (ma) EBOV (Mayinga) and WT C57BL/6 mice (40). For SUDV, this laboratory has previously reported a model for pathogenicity that utilizes type I interferon α/β receptor knockout mice infected at 4 weeks old with human lethal SUDV (Boniface) (41, 39). For maEBOV, a single Bis-mAb dose (200 μg) was provided 24 hours post- challenge and for SUDV, two doses (500 μg each) were provided at one and four days postexposure. The SUDV model, which involves immunocompromised mice infected at 4 weeks of age, provides a more stringent requirement for protection, and thus higher antibody doses are required than that used for the maEBOV model. Mono-specific mAbs Z.6D8 (EBOV) and F4 (SUDV) were included as controls as they have previously shown excellent protection against their respective viruses (41).

[00129] Both Bis-mAb treatments resulted in high (>70%) protection in both models, with scKZ52-F4 HCC conferring 100% protection against both viruses. As expected, Z.6D8 as a treatment was not protective against SUDV but was 100% protective against EBOV. SUDV monospecific mAb F4 provided 100% protection against SUDV, as we have previously reported (41) and afforded partial (30%) protection against maEBOV. However, the level of protection for F4 against maEBOV is not statistically distinguishable from a PBS control group, in which all mice succumbed to disease by day 7 (p = 0.13). Murine 16F6 afforded no protection against maEBOV in a group size of n = 5 challenge experiment. The 100% protection from maEBOV observed for scKZ52-F4 HCC was statistically distinguishable from both PBS and F4 controls in this experiment, whereas 70% protection of scKZ52-F4 LCN was distinguishable only from the PBS. These results indicate that both scKZ52-F4 HCC and scKZ52-F4 LCN provide statistically significant protection relative to a PBS control group. For SUDV, both Bis-mAbs and F4 were significantly protective relative to the Z.6D8 negative control treatment group.

[00130] Aggregate weight loss was observed in the Bis-mAb- and F4-treated group during the course of the SUDV challenge, a phenomenon we have previously reported for post-exposure dosing of protective SUDV mAbs (41,39). However, the surviving Bis-mAb or mAb-treated population continued to gain weight (on average) after day 8. For maEBOV, mean weight loss was observed during the initial infection period for scKZ52-F4 LCN- and F4-treated mice, as some mice became sick, but this trend was not observed with either Z.6D8 or scKZ52-F4 HCC mice. All mice from the Z.6D8- and scKZ52-F4 HCC- treated groups survived the infection.

[00131] To examine the capacity for memory immunity in Bis-mAb and mAb-treated mice, the surviving cohort of both EBOV and SUDV challenges were subjected to rechallenge (with the same isolate of virus) without mAb treatment 35 days after the initial challenge (Figs. IOC and 10D). For both EBOV and SUDV, Bis-mAb or mAb treated mice were completely protected against viral rechallenge with no observable aggregate weightloss, indicating memory immunity had been established. In both cases, an untreated negative control group was also included to confirm lethality of the virus.

Materials and Methods

[00132] Bispecific construct cloning using pMAZ-IgH and pMAZ-IgL vectors from Mazor et al. (38):

[00133] Constructs: KZ52 scFv was linked via a flexible peptide linker to either full length E10 or F4 IgG at the N- or C-terminus of the heavy chain (HC) and light chain (LC) (total of 8 constructs) were ordered from Genewiz, South Plainfield, NJ. The nucleotide sequence was optimized for E. coli. The constructs were re-suspended in MilliQ™ water to a final concentration of 100 ng/ μΕ.

[00134] Cloning: KZ52-E10 and KZ52-F4 N-terminal HC fusions: pMAZ-IgH and the constructs were digested using BssHII and Nhel in a final volume of 50 for 3 hours. Digests were purified using the Qiagen Gel Extraction Kit. The antibody inserts were ligated into the digested IgH vector using the Quick Ligase kit from NEB. The ligation product was then transformed into Top 10 cells and plated onto LB/Carb plates. Sequences were verified via Sanger Sequencing by Genewiz.

[00135] KZ52-E10 and KZ52-F4 N-terminal LC fusions: pMAZ-IgL and the constructs were digested using BssHII and BsiWI in a final volume of 50 for 3 hours. Subsequent steps were performed as above.

[00136] KZ52-F4 C-terminal HC and LC fusions: pMAZ-IgH, pMAZ-IgL and the constructs were digested with BssHII and Xbal ina final volume of 50 for 3 hours. Subsequent steps were performed as above.

[00137] KZ52-E10 C-terminal HC and LC fusions: A BamHI restriction site was engineered into the flexible peptide linker for the KZ52-F4 C-terminal HC and LC fusions. To produce KZ52-E10 C-terminal HC and LC fusions, the F4-KZ52 C-terminal HC and LC (in pMAZ vectors) were digested with BssHII and BamHI in a final volume of 50 μϊ _^ for 3 hours. Subsequent steps were performed as above.

[00138] After sequences were verified, all 8 constructs were maxiprepped using the Macherey-Nagel maxiprep kit. Additionally, the wildtype (WT) E10 and F4 IgH and IgL DNA was also maxiprepped.

[00139] Trans fections: Transfections were carried out at the AECOM protein production facility using HEK293F cells in 600 mL cultures as follows:

[00140] For each bispecific construct, the WT E10 or F4 IgH or IgL was paired with the corresponding bispecific DNA (example: E10-KZ52 N-terminal HC-IgH is paired with the E10 IgL WT). Both plasmids were co-transfected into 293F cells at a final amount of 201 μg using 1.2 mg of PEI transfection reagent in 50 mL PBS. Cells were incubated for 6 days at 37°C, 5% C0 ₂ , and 110 rpm.

[00141] Antibody purification: Cells were spun down at 4000 rpm, 4°C, for 15 minutes. The supernatant was brought to a pH of 8.0 using diluted NaOH. Protein A agarose beads (ThermoScientific) were washed in 10 mL Gentle Antibody Binding Buffer (ThermoScientific). Washed beads were then incubated with antibody containing supernatants for approximately 2 hours at 4°C. The flow through was collected. Beads were washed 2 times with 10 mL of Gentle Antibody Binding Buffer. Antibodies were eluted 5 times using 2.5 mL of Gentle Antibody Elution Buffer (ThermoScientific). Elutions were subsequently desalted into 150 mM HEPES/200 mM NaCl using PD-10 Desalting Columns (GE Healthcare). For verification of purity, SDS-PAGE (10-15%) were run and stained with Coomassie Blue dye.

Results

[00142] Multi-specific antibody-based structures are disclosed herein, including the exemplified bispecific antibodies (Bis-mAbs) comprising a genetic fusion of a single chain Fv (scFv) and an IgG, each harboring two separate specificities. In one embodiment, EBOV/SUDV Bis-mAbs were created by fusing the scFv of EBOV-specific mAb KZ52 and the IgG of SUDV specific mAbs E10 or F4. Several formats for the fusion are available, with the scFv as an N-terminal fusion to the IgG heavy chain (N' heavy chain or HCN'), the IgG light chain (N' light chain or LCN'); or as a C-terminal fusion to the IgG heavy and light chains (C heavy chain or HCC, and C light chain or LCC) (see Fig. 4). KZ52 (EBOV-specific) is a human antibody, and E10 and F4 (SUDV-specific) are fully humanized versions of the mouse antibody 16F6 recently reported. All together, a total of eight Bis-mAbs were produced, and could be purified from HEK293F cells in varying yield and stability (see Fig. 5). The binding of all eight for the envelope glycoprotein (GP) from EBOV or SUDV was demonstrated (see Figs. 5 and 7).

[00143] Finally, in a pseudotyped virus infection model, where vesicular stomatitis virus displaying the EBOV or SUDV GP was used as the infecting virus, it was found that all eight Bis-mAbs could neutralize GP-mediated cell entry for both EBOV and SUDV GP at 133 nM (see Fig. 8). Testing of the bispecific antibody compositions under BSL4 conditions is performed against authentic pathogens.

REFERENCES

1. Kuhn, J. H., Becker, S., Ebihara, H., Geisbert, T. W., Johnson, K. M., Kawaoka, Y., Lipkin, W. I., Negredo, A. I., Netesov, S. V., Nichol, S. T., Palacios, G., Peters, C. J., Tenorio, A., Volchkov, V. E., and Jahrling, P. B. (2010) Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch. Virol. 155, 2083-2103.

2. Feldmann, H., and Gesibert, T. W. (2011) Ebola haemorrhagic fever. Lancet 9768, 849-862. 3. Miller, E. H., and Chandran, K. (2012) Filovirus entry into cells- new insights. Curr. Opin. Virol. 2, 206-214.

4. Lee, J. E., and Saphire, E. O. (2009) Neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it. Curr. Opin. Struct. Biol. 19, 408-417.

5. Leroy, E. M., Kumulungui, B., Pourrut, X., Rouquet, P., Hassanin, A., Yaba, P., Delicat, A., Paweska, J. T., Gonzalez, J. P., and Swanepoel, R. (2005) Fruit bats as reservoirs of Ebola virus. Nature 438, 575-576.

6. http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola.ht m

7. Bradfute, S. B., Warfield, K. L., and Bavari, S. (2008) Functional CD8+ T cell responses in lethal Ebola virus infection. J. Immunol. 180, 4058-4066.

8. Zampieri, C. A., Sullivan, N. J., and Nabel, G. J. (2007) Immunopathology of highly virulent pathogens: insights from Ebola virus. Nat. Immunol. 8, 1 159-1164.

9. Geisbert, T. W., Hensley, L. E., Larsen, T., Young, H. A., Reed, D. S., Geisbert, J. B., Scott, D. P., Kagan, E., Jahrling, P. B., and Davis, K. J. (2003) Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection. Am. J. Pathol. 163, 2347-2370.

10. Hensley, L. E., Jones, S. M., Feldmann, FL, Jahrling, P. B., and Geisbert, T. W. (2005) Ebola and Marburg viruses: pathogenesis and development of countermeasures. Curr. Mol. Med. 5, 761-772.

11. Wilson, J. A., and Hart, M. K. (2001) Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J. Virol. 75, 2660-2664.

12. Parren, P. W., Geisbert, T. W., Maruyama, T., Jahrling, P. B., and Burton, D. R. (2002) Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody. J. Virol. 76, 6408-6412.

13. Lee, J. E., Fusco, M. L., Hessell, A. J., Oswald, W. B., Burton, D. R., and Saphire, E. O. (2008) Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 454, 177-182.

14. Dias, J. M., Kuehne, A. I., Abelson, D. M., Bale, S., Wong, A. C, Halfmann, P., Muhammad, M. A., Fusco, M. L., Zak, S. E., Kang, E., Kawaoka, Y., Chandran, K., Dye, J. M., and Saphire, E. O. (201 1) A shared structural solution for neutralizing ebolaviruses. Nat. Struct. Mol. Biol. 18, 1424-1427. 15. Lee, J. E., and Saphire, E. O. (2009) Ebolavirus glycoprotein structure and mechanism of entry. Future Virol. 4, 621-635.

16. Weissenhorn, W., Carfi, A., Lee, K.-FL, Skehel, J. J., and Wiley, D. C. (1998) Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol. Cell 2, 605-616.

17. Malashkevich, V. N., Schneider, B. J., McNally, M. L., Milhollen, M. A., Pang, J. X., and Kim, P. S. (1999) Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution. Proc. Natl. Acad. Sci. USA 96, 2662-2667.

18. Koellhoffer, J. F., Malashkevich, V. N., Harrison, J. S., Toro, R., Bhosle, R. C, Chandran, K., Almo, S. C, Lai, J. R. (2012) Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation. Biochemistry 51, 7665-7675.

19. Harrison, J. S., Koellhoffer, J. F., Chandran, K., and Lai, J. R. (2012) Marburg virus glycoprotein GP2: pH-dependent stability of the ectodomain a-helical bundle. Biochemistry 51, 2515-2525.

20. Oswald, W. B., Geisbert, T. W., Davis, K. J., Geisbert, J. B., Sullivan, N. J., Jahrling, P. B., Parren, P. W., and Burton, D. R. (2007) Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathog. 3, e9.

21. Dye, J. M., Herbert, A. S., Kuehne, A. I., Barth, J. F., Muhammad, M. A., Zak, S. E., Ortiz, R. A., Prugar, L. I., and Pratt, W. D. (2012) Postexposure antibody prophylaxis protects nonhuman primates from filovirus disease. Proc. Natl. Acad. Sci. USA 109, 5034- 5039.

22. Wong, G., Richardson, J. S., Pillet, S., Patel, A., Qiu, X., Alimonti, J., Hogan, J., Zhang, Y., Takada, A., Feldmann, H., Kobinger, G. P. (2012) Immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates. Sci. Transl. Med. 4, 158ral46.

23. Olinger, G. G. Jr., Pettitt, J., Kim, D., Working, C, Bohorov, O., Bratcher, B., Hiatt, E., Hume, S. D., Johnson, A. K., Morton, J., Pauly, M., Whaley, K. J., Lear, C. M., Biggins, J. E., Scully, C, Hensley, L., Zeitlin, L. (2012) Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc. Natl. Acad. Sci. USA 109, 18030-18035.

24. Marzi, A., Yoshida, R., Miyamoto, H., Ishijim, M., Suzuki, Y., Higuchi, M., Matsuyama, Y., Igarashi, M., Nakayama, E., Kuroda, M., Saijo, M., Feldmann, F., Brining, D., Feldmann, H., and Takada A. (2012) Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever. PLoS One 7, e36192.

25. Wilson, J. A., Bosio, C. M., and Hart, M. K. (2001) Ebola virus: the search for vaccines and treatments. Cell Mol. Life Sci. 58, 1826-1841.

26. Sullivan, N. J., Martin, J. E., Graham, B. S., and Nabel, G. J. (2009) Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. Nat. Rev. Microbiol. 7, 393-400.

27. Wilson, J. A., Hevey, M., Bakken, R., Guest, S., Bray, M., Schmaljohn, A. L., and Hart, M. K. (2000) Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664-1666.

28. Maruyama, T., Rodriguez, L. L., Jahrling, P. B., Sanchez, A., Khan, A. S., Nichol, S. T., Peters, C. J., Parren, P. W., and Burton, D. R. (1999) Ebola virus can be effectively neutralized by antibody produced in natural human infection. J. Virol. 73, 6024-6030.

29. Shurtleff, A. C, Warren, T. K., and Bavari, S. (201 1) Non-human primates as models for the discovery and development of ebolavirus therapeutics. Expert Opin. Drug Discov. 6, 233-250.

30. Warfield, K. L., and Aman, M. J. (201 1) Advances in virus-like particle vaccines for filoviruses. J. Infect. Dis. 204 Suppl 3, S1053-1059.

31. Fausther-Bovendo, H., Mulangu, S., and Sullivan, N. J. (2012) Ebolavirus vaccines for humans and apes. Curr. Opin. Virol. [Epub ahead of print] (May 3, PMID: 22560007)

32. Hoenen, T., Grosth, A., and Feldmann, H. (2012) Current ebola vaccines. Expert Opin. Biol. Ther. [Epub ahead of print] (May 5, PMID: 22559078)

33. Warren, T. K., Warfield, K. L., Wells, J., Swenson, D. L., Donner, K. S., Van Tongeren, S. A., Garza, N. L., Dong, L., Mourich, D. V., Crumley, S., Nichols, D. K., Iversen, P. L., and Bavari, S. (2010) Advanced antisense therapies for postexposure protection against lethal filovirus infections. Nat. Med. 16, 991-994.

34. Geisbert, T. W., Lee, A. C, Robbins, M., Geisbert, J. B., Honko, A. N., Sood, V., Johnson, J. C, de Jong, S., Tavakoli, I., Judge, A., Hensley, L. E., and Maclachlan, I. (2010) Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet 375, 1896-1905.

35. Panchal, R. G., Reid, S. P., Tran, J. P., Bergeron, A. A., Wells, J., Kota, K. P., Aman, J., and Bavari, S. (2012) Identification of an antioxidant small-molecule with broad- spectrum antiviral activity. Antiviral Res. 93, 23-29. 36. Cote, M., Misasi, J., Ren, T., Bruchez, A., Lee, K., Filone, C. M., Hensley, L., Li, Q., Ory, D., Chandran, K., and Cunningham, J. (2011) Small molecule inhibitors reveal Niemann-Pick CI is essential for Ebola virus infection. Nature 477, 344-348.

37. Basu, A., Li, B., Mills, D. M., Panchal, R. G., Cardinale, S. C, Butler, M. M., Peet, N. P., Majgier-Baranowska, FL, Williams, J. D., Patel, I., Moir, D. T., Bavari, S., Ray, R., Farzan, M. R., Rong, L., and Bowlin, T. L. (201 1) Identification of a small-molecule entry inhibitor for filoviruses. J. Virol. 85, 3106-31 19.

38. Mazor, Y., Barnea, I., Keydar, I,, and Benhar, I. (2007) Antibody internalization studied using a novel IgG binding toxin fusion. Journal of Immunological Methods, 321, (1), 41-59.

39. Brannan, J. M. et al. Interferon alpha/beta Receptor-Deficient Mice as a Model for Ebola Virus Disease. The Journal of infectious diseases 212 Suppl 2, S282-294, doi: 10.1093/infdis/jiv215 (2015).

40. Bray, M., Davis, K., Geisbert, T., Schmaljohn, C. & Huggins, J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. The Journal of infectious diseases 178, 651-661 (1998).

41. Chen, G. et al. Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge. ACS Chemical Biology 9, 2263-2273, doi: 10.1021/cb5006454 (2014).