Abstract
The present disclosure provides gene therapy that targets complement pathways for treating dry age-related macular degeneration.
Claims
1. A single expression construct comprising a first nucleotide sequence encoding an inhibitor for activated complement subcomponent C1s (C1s inhibitor) and a second nucleotide sequence encoding an inhibitor for complement factor Bb (Bb inhibitor); or a pair of expression constructs, one comprising the first nucleotide sequence and the other comprising the second nucleotide sequence.
2. The expression construct(s) of claim 1, wherein the C1s inhibitor and the Bb inhibitor are each an antibody fragment, optionally wherein the antibody fragment is a single-chain Fv (scFv) or a single-chain Fab (scFab).
3. The expression construct(s) of claim 2, wherein (a) the C1s inhibitor is an anti-C1s antibody fragment comprising heavy chain CDR (HCDR) 1-3 in SEQ ID NO:7, optionally comprising SEQ ID NOs: 1-3, respectively, and light chain CDR (LCDR) 1-3 in SEQ ID NO:8, optionally comprising SEQ ID NOs: 4-6, respectively; and/or (b) the Bb inhibitor is an anti-Bb antibody comprising HCDR1-3 in SEQ ID NO: 19, optionally comprising SEQ ID NOs: 13-15, respectively, and LCDR1-3 in SEQ ID NO:20, optionally comprising SEQ ID NOs: 16-18, respectively.
4. The expression construct(s) of claim 3, wherein (a) the C1s inhibitor comprises a heavy chain variable domain (V.sub.H) comprising SEQ ID NO:7 or an amino acid sequence at least 95% identical thereto, and a light chain variable domain (V.sub.L) comprising SEQ ID NO:8 or an amino acid sequence at least 95% identical thereto; and/or (b) the Bb inhibitor comprises a V.sub.H comprising SEQ ID NO:19 or an amino acid sequence at least 95% identical thereto, and a V.sub.L comprising SEQ ID NO:20 or an amino acid sequence at least 95% identical thereto.
5. The expression construct(s) of claim 3, wherein (a) the C1s inhibitor comprises a heavy chain (HC) comprising SEQ ID NO:10 or an amino acid sequence at least 95% identical thereto, and a light chain (LC) comprising SEQ ID NO:11 or an amino acid sequence at least 95% identical thereto; and/or (b) the Bb inhibitor comprises an HC comprising SEQ ID NO:22 or an amino acid sequence at least 95% identical thereto and an LC comprising SEQ ID NO:23 or an amino acid sequence at least 95% identical thereto.
6. The expression construct(s) of claim 2, wherein the C1s inhibitor and the Bb inhibitor each comprise one or more charge mutations for promoting pairing between heavy and light chains of each inhibitor.
7. The expression construct(s) of claim 6, wherein (a) the charge mutations in the C1s inhibitor comprises Q42E and Q292K, wherein the numbering is in accordance with SEQ ID NO: 12; and (b) the charge mutations in the Bb inhibitor comprises Q38K and Q288E, optionally further comprising S114A, N137K, and T434E, wherein the numbering is in accordance with SEQ ID NO:24.
8. The expression construct(s) of claim 2, wherein the C1s inhibitor is an scFv or scFab in which the HC and the LC are linked by a peptide linker, optionally wherein the peptide linker comprises one or more, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10, G.sub.4S (SEQ ID NO:46) repeats.
9. The expression construct(s) of claim 2, wherein the Bb inhibitor is an scFv or scFab in which the HC and the LC are linked by a peptide linker, optionally wherein the peptide linker comprises one or more, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10, G.sub.4S (SEQ ID NO:46) repeats.
10. The single expression construct of claim 1, comprising a transgene encoding a fusion protein comprising the C1s inhibitor and the Bb inhibitor linked by a peptide linker, optionally wherein the peptide linker comprises one or more, optionally 2, 3, 4, 5, 6, 7, 8, 9, or 10, G.sub.4S (SEQ ID NO:46) repeats, further optionally wherein the transgene is linked operably to a minimal chicken -actin (minCBA) promoter.
11. The single expression construct of claim 1, wherein the expression construct comprises a bidirectional promoter that directs expression of the C1s inhibitor and the Bb inhibitor as separate molecules, optionally wherein the bidirectional promoter is a pair of chicken -actin (CBA) promoters placed in opposite direction and separated by a CMV enhancer, further optionally wherein the bidirectional promoter comprises SEQ ID NO:53 or a nucleotide sequence at least 85% identical thereto.
12. The single expression construct of claim 2, wherein the expression construct expresses a heterodimer comprising (i) a fusion protein comprising a single-chain anti-C1s antibody fragment fused to the HC or LC of an anti-Bb antibody fragment; and (ii) the LC or HC polypeptide of the anti-Bb antibody fragment, wherein the coding sequence for the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-Bb antibody fragment are separated in frame by a coding sequence for a cleavable peptide, optionally wherein the cleavable peptide comprises a 2A sequence and/or a furin cleavage site, further optionally the expression construct comprises a minCBA promoter.
13. The single expression construct of claim 2, wherein the expression construct expresses a heterodimer comprising (i) a fusion protein comprising a single-chain anti-Bb antibody fragment fused to the HC or LC of an anti-C1s antibody fragment; and (ii) the LC or HC polypeptide of the anti-C1s antibody fragment, wherein the coding sequence for the fusion protein and the coding sequence of the LC or HC polypeptide of the anti-C1s antibody fragment are separated in frame by a coding sequence for a cleavable peptide, optionally wherein the cleavable peptide comprises a 2A sequence and/or a furin cleavage site, further optionally the expression construct comprises a minCBA promoter.
14. The single expression construct of claim 2, wherein the expression construct encodes a fusion protein comprises, from N-terminus to C-terminus, (i) an anti-C1s scFv, a (G.sub.4S).sub.2 linker, and an anti-Bb scFv, optionally comprising SEQ ID NO: 55 (with or without the signal peptide) or an amino acid sequence at least 95% identical thereto; (ii) an anti-Bb scFv, a (G.sub.4S).sub.2 linker, and an anti-C1s scFv, optionally comprising SEQ ID NO: 57 (with or without the signal peptide) or an amino acid sequence at least 95% identical thereto; (iii) an anti-C1s scFab, a (G.sub.4S).sub.3 linker, and an anti-Bb scFab, optionally comprising SEQ ID NO: 26 or 28 (with or without the signal peptide), or an amino acid sequence at least 95% identical thereto; (iv) an anti-Bb scFab, a (G.sub.4S).sub.3 linker, and an anti-C1s scFab, optionally comprising SEQ ID NO: 30 or 32 (with or without the signal peptide), or an amino acid sequence at least 95% identical thereto; (v) an anti-C1s scFab, a (G.sub.4S).sub.2 linker, and an anti-Bb scFv, optionally comprising SEQ ID NO: 34 or 36 (with or with the signal peptide), or an amino acid sequence at least 95% identical thereto; or (vi) an anti-C1s scFab, a (G.sub.4S).sub.3 linker, and an anti-Bb scFv, optionally comprising SEQ ID NO: 59 or 61 (with or without the signal peptide), or an amino acid sequence at least 95% identical thereto.
15. The expression construct(s) of claim 2, wherein the expression construct(s) encodes an anti-C1s scFab, optionally comprising SEQ ID NO: 12 or an amino acid sequence at least 95% identical thereto, optionally wherein the amino acid sequence comprises Q42E and Q292K mutations relative to SEQ ID NO: 12; and an anti-Bb scFab, optionally comprising SEQ ID NO: 14 or an amino acid sequence at least 95% identical thereto, optionally wherein the amino acid sequence comprises Q38K and Q288E, and optionally S114A, N137K, and T434E, mutations relative to SEQ ID NO:14.
16. The single expression construct of claim 2, wherein the expression construct encodes a heterodimer comprised of (A) (i) an anti-C1s LC and (ii) a fusion protein comprising an anti-C1s HC fused to an Bb scFab, optionally wherein the expression construct comprises a coding sequence for SEQ ID NO: 39, or an amino acid sequence at least 95% identical thereto; (B) (i) an anti-C1s LC and (ii) a fusion protein comprising an anti-C1s HC fused to an anti-Bb scFab, optionally wherein the expression construct comprises a coding sequence for SEQ ID NO: 41, or an amino acid sequence at least 95% identical thereto; (C) (i) a fusion protein comprising an anti-C1s scFab fused to an anti-Bb HC and (ii) an anti-Bb LC, optionally wherein the expression construct comprises a coding sequence for SEQ ID NO: 43, or an amino acid sequence at least 95% identical thereto; or (D) (i) a fusion protein comprising an anti-C1s scFab fused to an anti-Bb HC and (ii) an anti-Bb LC, optionally wherein the expression construct comprises a coding sequence for SEQ ID NO: 45, or an amino acid sequence at least 95% identical thereto.
17. An isolated nucleic acid comprising a nucleotide sequence selected from SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, 38, 40, 42, 54, 56, 58, 60, 62, 79, or 80, or encodes the same amino acid sequence(s) as the selected nucleotide sequence does.
18. One, two or more recombinant adeno-associated viruses (rAAV) comprising the expression construct(s) of claim 1.
19. The rAAV(s) of claim 18, wherein the genome of the rAAV(s) comprises the expression construct flanked by AAV2 inverted terminal repeats (ITRs).
20. The rAAV(s) of claim 19, wherein the genome comprises SEQ ID NO:50, 51, or 52; or encodes the same amino acid sequence(s) as SEQ ID NO:50, 51, or 52 does.
21. The rAAV(s) of claim 18, comprising a capsid of AAV2, optionally wildtype AAV2.
22. A pharmaceutical composition comprising the rAAV(s) of claim 18 and a pharmaceutically acceptable carrier.
23. A protein or proteins encoded by the expression construct(s) of claim 1.
24. A host cell comprising the expression construct(s) of claim 1.
25. A method for treating dry age-related macular degeneration (AMD) in a patient in need thereof, comprising administering an effective amount of the pharmaceutical composition of claim 22.
26. The method of claim 25, wherein the administering is by intravitreal injection.
27. The method of claim 25, wherein the patient has geographic atrophy (GA) secondary to dry AMD.
28. The method of claim 25, wherein the effective amount is 10.sup.7 to 10.sup.15, optionally 10.sup.8 to 10.sup.14, 10.sup.9 to 10.sup.13, further optionally 210.sup.9, 210.sup.10, or 210.sup.11, vector genomes.
29. A mammalian promoter comprising SEQ ID NO:83 or a sequence at least 85% identical thereto.
30. A bidirectional mammalian promoter comprising a pair of chicken -actin promoters placed in opposite orientation, separated by a CMV enhancer, optionally wherein the bidirectional mammalian promoter comprises SEQ ID NO:53 or a sequence at least 85% identical thereto.
31. One, two or more recombinant adeno-associated viruses (rAAV) comprising the isolated nucleic acid of claim 17.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1A is a diagram illustrating an exemplary monocistronic construct for expressing linked (e.g., through a G.sub.4S linker as shown) anti-C1s (C1s) antibody fragment and anti-Bb (Bb) antibody fragment. minCBA: minimal chicken -actin promoter. scFab: single-chain antibody fragment. scFv: single chain antibody variable domain. BGH: bovine growth hormone.
[0033] FIG. 1B is a diagram illustrating an exemplary bicistronic construct using a bidirectional promoter (a modified minCBA) that allows expression of two separate antibody fragments in opposite directions.
[0034] FIG. 1C is a diagram illustrating an exemplary recombinant AAV genome containing an expression cassette of FIG. 1A or FIG. 1B for expressing an anti-Bb antibody fragment and an anti-C1s antibody fragment. ITR: inverted terminal repeat.
[0035] FIG. 2A is a panel of diagrams illustrating linked anti-C1s/anti-Bb antibody fragments produced from four exemplary configurations (#5-#8) of a monocistronic construct. Heavy chain variable domain: V.sub.H. Light chain variable domain: V.sub.L. Heavy chain constant region: C.sub.H. Light chain constant region: C.sub.L.
[0036] FIG. 2B is a pair of diagrams illustrating two exemplary configurations (#9 and #10) of a construct harboring a bidirectional (BiDir) promoter driving expression of two independent antibody fragments.
[0037] FIG. 2C is a pair of diagrams illustrating exemplary linked anti-C1s/anti-Bb scFab antibody fragments (#11 and #12) with charge mutations (CM; ) that are intended to promote cognate heavy chain and light chain pairing. In the figures herein, indicates the presence of a charge mutation and is not meant to illustrate the exact positions or numbers of the charge mutations in the antibody fragment.
[0038] FIG. 2D is a pair of diagrams illustrating exemplary linked anti-C1s/anti-Bb antibody fragments for C1s scFab-(G.sub.4S).sub.2-Bb scFv with (#14) or without (#13) charge mutations.
[0039] FIG. 2E is a pair of diagrams illustrating exemplary linked anti-C1s/anti-Bb antibody fragments for C1s scFab-(G.sub.4S).sub.3-Bb scFv with (#16) or without (#15) charge mutations.
[0040] FIG. 2F is a panel of diagrams illustrating exemplary linked anti-C1s/anti-Bb antibody fragments containing self-cleaving peptides, F2A or GT2A, between the heavy and light chains of the C1s Fab fragment (#17: C1s F2A Fab-(G.sub.4S).sub.3-Bb scFab; and #18: C1s GT2A Fab-(G.sub.4S).sub.3-Bb scFab) or between the heavy and light chains of the Bb Fab fragment (#19: C1s scFab-(G.sub.4S).sub.3-Bb F2A Fab; and #20: C1s scFab-(G.sub.4S).sub.3-Bb GT2A Fab). F2A: a self-cleaving peptide comprising a furin cleavage site linked by a SGSG (SEQ ID NO:81) linker to a foot-and-mouth disease virus 2A peptide (Fuchs et al., PLOS One (2016) doi: 10.1371/journal.pone.0158009). GT2A: a self-cleaving peptide comprising a furin cleavage site linked by a GSG linker to a Thosea asigna virus 2A peptide.
[0041] FIG. 2G is a pair of diagrams illustrating exemplary configurations of a construct harboring a bidirectional promoter driving expression of two independent antibody fragments that differ from constructs #9 and #10 by having charge mutations (#21 and #22).
[0042] FIG. 2H is a diagram showing construct #14 of FIG. 2D (C1s scFab-(G.sub.4S).sub.2-Bb scFv-CM) in the context of an AAV vector plasmid, including AAV2 ITRs. C1s: C1s. Bb: Bb.
[0043] FIG. 2I is a diagram showing construct C1s scFab-BiDir-Bb scFab with (#21; FIG. 2G) or without (#9; FIG. 2B) charge mutations in the context of an AAV vector plasmid, including AAV2 ITRs. aC1s: C1s. aBb: Bb.
[0044] FIG. 2J is a diagram showing construct Bb scFab-(G.sub.4S).sub.3-C1s scFab-CM (construct #12 of FIG. 2C) in the context of an AAV vector plasmid, including AAV2 ITRs. C1s: C1s. Bb: Bb.
[0045] FIG. 3 is a representative biolayer interferometry (BLI) sensorgram showing that the protein expressed from construct #19 of FIG. 2F can bind both C1s and Bb simultaneously.
[0046] FIG. 4A is a plot showing dose-dependent inhibition of complement activation by recombinant anti-C1s Fab and the purified protein expressed by construct #2 of FIG. 2A under conditions where both CP and AP are activated simultaneously in vitro.
[0047] FIG. 4B is a plot showing dose-dependent inhibition of complement activation by recombinant anti-Bb Fab and the purified protein expressed by construct #4 of FIG. 2A under conditions where both CP and AP are activated simultaneously in vitro.
[0048] FIG. 4C is a plot showing dose-dependent inhibition of complement activation by an equimolar mixture of recombinant anti-Bb Fab and anti-C1s Fab, tested alongside an equimolar mixture of purified proteins expressed by constructs #2 and #4 of FIG. 2A under conditions where both CP and AP are activated simultaneously in vitro.
[0049] FIG. 5 is a panel of photographs showing representative vector in situ hybridization of the mouse retina 3 weeks after administration of AAV2 #9. Vector-specific probes targeting the vector genome were used.
[0050] FIG. 6 is a panel of graphs showing combined inhibition of CP and AP on ARPE19 cells in a CRP-mediated complement activation model of dry AMD. The data shown are an average of twelve replicates along with standard deviation for each condition across two independent experiments. NHS: normal human serum. CRP: C-reactive protein. **** p<0.0001.
[0051] FIGS. 7A and 7B are graphs showing cell-ELISA data depicting complement deposition on induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE) in a cell model for AMD. Treatment with anti-Bb and anti-C1s scFabs significantly inhibited deposition of complement products C3d (FIG. 7A) and C5b9 (FIG. 7B) on iPSC-RPE relative to the CRP control. Error bars are standard deviation. **** p<0.0001.
[0052] FIGS. 8A and 8B show immunofluorescent staining of C5b9 on iPSC-RPE. FIG. 8A is a panel of confocal microscopy images showing C5b9 deposition (red) on iPSC-RPE. FIG. 8B is a graph showing the quantification analysis of the images in FIG. 8A.
[0053] FIG. 9 is a heat map showing ocular exam results based on the preclinical ocular toxicology scoring (SPOTS) system. The heat map shows the clinical indicators of ocular inflammation and irritation in controls before and after LPS treatment; it shows median severity scored during ocular exams using the SPOTS system.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The present disclosure is based on the discovery that dual targeting of the complement classical and alternative pathways can be used to treat eye diseases associated with a dysregulated or overactivated complement system in the eye. The present disclosure provides gene therapy that delivers to the eye(s) of a patient in need thereof both an inhibitor of activated complement component 1 subcomponents (aC1s or simply referred to as C1s herein) and an inhibitor of activated factor B (aka. Bb fragment, FBb, or Bb). The gene therapy can use a viral vector, such as recombinant adeno-associated virus (AAV, e.g., AAV2), as a vehicle to deliver transgenes that direct expression of the C1s and Bb inhibitors. In some embodiments, the C1s inhibitor and Bb inhibitor are antibody fragments such as single-chain Fab (scFab) or single-chain Fv (scFv). The C1s inhibitor and the Bb inhibitor can be expressed as a single protein, or as two separate proteins.
[0055] In some embodiments, the eye disease to be treated is dry AMD, including associated geographic atrophy. In some embodiments, the patient has a dysregulated/overactivated complement system in the RPE choroid interface. In some embodiments, the present therapy delivers (e.g., intravitreally or subretinally) the present recombinant expression constructs (e.g., recombinant AAV2) to the retinal ganglion cells (RGCs). Intravitreal delivery of rAAV2 transduces RGCs in the retina and facilitates secretion of the inhibitory proteins for distribution to the broader retina. For example, the rAAV2 may be delivered intravitreally to patients with geographic atrophy (GA) secondary to dry AMD to reduce the growth of retinal GA lesion size over a 12-month period and prevent inevitable vision loss. In addition to its benefit as a potential one-time treatment for GA, the presently disclosed gene therapy may have improved efficacy compared to therapeutic approaches that target downstream components in the complement pathway. This is because the present therapy broadly inhibits both proximal and terminal mediators of inflammation, phagocytosis, and membrane attack complex-mediated cell lysis.
[0056] Therapies that have been approved or currently under development involve repeat dosing (e.g., monthly or every other month) of complement inhibitors. A one-time treatment with an outpatient intravitreal delivery of a recombinant vector will provide a best-in-class approach. Further, in other therapies, the complement inhibitors block all complement pathways. By contrast, the present bifunctional complement inhibitors target upstream activation steps in the complement pathways implicated as drivers of dry AMD pathogenesisthe AP and CPrather than targeting downstream convertases common to all three initiating pathways. This approach leaves C1q and the lectin pathway intact to maintain immune surveillance. Furthermore, this approach has a superior mode of action due to inhibition of not only the membrane attack complex (MAC) but also the complement amplification loop and terminal events that are mediated by upstream activation fragments, such as inflammation and opsonization and phagocytosis. The present approach may also reduce target-mediated drug disposition (TMDD) since the inhibitors target activated enzymes that are often present at much lower levels as compared to the intact pro-enzymes.
I. C1s and Bb Inhibitors
[0057] The present gene therapy introduces both a C1s inhibitor and a Bb inhibitor, either linked or unlinked, to the diseased eye of a patient.
[0058] Prior to processing and activation, a human C1s polypeptide may have the amino acid sequence of SEQ ID NO:65 (UniProt. P09871), in which amino acids 1-15 constitute the signal peptide. Upon activation, the C1s polypeptide is cleaved and becomes a disulfide-linked heterodimer in which the heavy chain corresponds to amino acids 16-437 of SEQ ID NO: 65 and the light chain corresponds to amino acids 438-688 of SEQ ID NO:65. Unless otherwise indicated, the C1s inhibitor herein refers to an inhibitor of this activated form of C1s.
[0059] Prior to processing and activation, a human factor B polypeptide may have the amino acid sequence of SEQ ID NO:66 (UniProt. P00751), in which amino acids 1-25 constitute the signal peptide. Upon activation, the polypeptide is cleaved into two subcomponents, factor Ba, which corresponds to amino acids 26-259 of SEQ ID NO:66, and factor Bb, which corresponds to amino acids 260-764 of SEQ ID NO:66. Factor Bb is also simply referred to as Bb herein.
[0060] The C1s inhibitor and the Bb inhibitor herein may be linked recombinantly (e.g., expressed recombinantly as a fusion protein), with or without a peptide linker. Where these proteins are introduced into the cell through expression vectors, they may also be referred to as vectorized proteins (e.g., vectorized antibody fragments).
[0061] In some embodiments, the C1s inhibitor and the Bb inhibitor are antigen-binding fragments of full antibodies. A full antibody (Ab) or immunoglobulin (Ig) refers to a tetrameric protein comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (V.sub.H) and a heavy chain constant region (C.sub.H). Each light chain is composed of a light chain variable domain (V.sub.L) and a light chain constant region (C.sub.L). The V.sub.H and V.sub.L domains can be subdivided further into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each V.sub.H or V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each region may be in accordance with IMGT definitions (Lefranc et al., Dev Comp Immunol. (2003) 27 (1): 55-77; or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. (1987) 196:901-917; or Chothia et al., Nature (1989) 342:878-83. Additional CDR definition systems include the AbM system and the Martin system (see, e.g., Abhinandan and Martin, Mol Immunol. (2008) 45 (14): 3832-9).
[0062] The term antibody fragment, antigen-binding fragment or a similar term refers to the portion of an intact antibody that comprises the amino acid residues that interact with an antigen and confer on the fragment its specificity and affinity for the antigen. The antibody fragment may be a single-chain variable fragment (scFv), which is a fusion protein of the V.sub.H and the V.sub.L of an antibody, connected with a short peptide linker; a diabody, which is a non-covalent dimer of scFv (Zapata et al., Protein Eng. (1995) 8 (10): 1057-62); or a Fab fragment, including a single-chain Fab (scFab) fragment. Fab fragments contain the constant domain of the light chain and the first constant domain (C.sub.H1) of the heavy chain. Other nonlimiting examples of antigen-binding fragments of antibodies include Fd fragments, Fv fragments, dAb fragments and minimal recognition units consisting of the amino acid residues that mimic the hypervariable domain of the antibody. In particular embodiments, the antibody fragment is an scFv, a Fab, or an scFab.
A. Anti-C1s scFv and scFab
[0063] In some embodiments, the active C1s inhibitor is an antibody fragment such as an sc Fab or an scFv derived from anti-C1s antibody VH3/VK2 from WO 2018/071676. Antibody fragments derived from variants of this antibody as described in WO 2018/071676, or in WO 2016/164358, and U.S. Pat. Nos. 10,729,767 and 11,246,926, may also be used herein. In some embodiments, the anti-C1s (also termed herein C1s) scFv or scFab herein comprises CDRs derived from the aforementioned VH3/VK2 antibody. The CDRs may be defined by any one of the well-known systems, including those described above. In some embodiments, the CDRs are defined by the Kabat system, the IMGT system, or the Chothia system as shown in Table A below (SEQ ID NOs are shown in parenthesis).
TABLE-US-00001 TABLEA CDR Kabat IMGT Chothia HCDR1 DDYIH GFNIKDDY GFNIKDD (1) (67) (71) HCDR2 RIDPADGHTKYAPKFQV IDPADGHT DPADGH (2) (68) (72) HCDR3 YGYGREVFDY ARYGYGREVFDY YGYGREVFDY (3) (69) (3) LCDR1 KASQSVDYDGDSYMN QSVDYDGDSY KASQSVDYDGDSYMN (4) (70) (4) LCDR2 DASNLES DAS DASNLES (5) (5) LCDR3 QQSNEDPWT QQSNEDPWT QQSNEDPWT (6) (6) (6)
[0064] In some embodiments, the anti-C1s scFab or scFv comprises heavy chain CDR (HCDR) 1-3 comprising SEQ ID NOs: 1-3, respectively, and light chain CDR (LCDR) 1-3 comprising SEQ ID NOs: 4-6, respectively.
[0065] In particular embodiments, the anti-C1s scFv or scFab comprises a V.sub.H comprising SEQ ID NO:7 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; and a V.sub.L comprising SEQ ID NO:8 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In certain embodiments, the anti-C1s scFv comprises a peptide linker, such as a flexible linker, e.g., a linker comprising (G.sub.4S).sub.n (SEQ ID NO: 46), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the V.sub.H and the V.sub.L. In some embodiments, the linker comprises SEQ ID NO:48 (i.e., n=3). The V.sub.H may be N-terminal, or C-terminal, to the V.sub.L. In some embodiments, the anti-C1s scFv comprises SEQ ID NO:9 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0066] In certain embodiments, the anti-C1s scFab comprises a heavy chain (HC) comprising SEQ ID NO: 10 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; and a light chain (LC) comprising SEQ ID NO:11 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In further embodiments, the HC and the LC are linked by a peptide linker, such as a flexible linker, e.g., a linker comprising (G.sub.4S).sub.n (SEQ ID NO:46), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the HC and the LC. In some embodiments, the linker comprises SEQ ID NO:49 (i.e., n=7). The HC may be N-terminal, or C-terminal to the LC. In some embodiments, the C1s scFab comprises SEQ ID NO: 12 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0067] In some embodiments, the C1s inhibitor is an antibody fragment such as an scFab or an scFv derived from anti-C1s antibody disclosed in US2022/0380483A1. For example, the C1s inhibitor may comprise the heavy and light chain CDRs, or V.sub.H and V.sub.L, of the parental anti-C1s antibody.
B. Anti-Bb scFv and scFab
[0068] In some embodiments, the Bb inhibitor is an antibody fragment such as an scFab or an scFv derived from anti-Bb antibody V.sub.H6/V7-IgG4v2 from U.S. Pat. No. 11,242,382 and WO 2021/216458. Antibody fragments derived from variants of this antibody as described in WO 2021/216458 may also be used herein. In some embodiments, the anti-Bb (also termed herein Bb) scFv or scFab herein comprises CDRs derived from the aforementioned V.sub.H6/V7-IgG4v2 antibody. The CDRs may be defined by any one of the well-known systems, including those described above. In some embodiments, the CDRs are defined by the Kabat system, the IMGT system, or the Chothia system as shown in Table B below (SEQ ID NOs are shown in parenthesis).
TABLE-US-00002 TABLEB CDR Kabat IMGT Chothia HCDR1 NYAMS GFTFSNYA GFTFSNY (13) (73) (77) HCDR2 TISNRGSYTYYPDSVKG ISNRGSYT SNRGSY (14) (74) (78) HCDR3 ERPMDY ARERPMDY ERPMDY (15) (75) (15) LCDR1 KASQDVGTAVA QDVGTA KASQDVGTAVA (16) (76) (16) LCDR2 WASTRHT WAS WASTRHT (17) (17) LCDR3 HQHSSNPLT HQHSSNPLT HQHSSNPLT (18) (18) (18)
[0069] In some embodiments, the anti-Bb scFab or scFv comprises heavy chain CDR (HCDR) 1-3 comprising SEQ ID NOs: 13-15, respectively, and light chain CDR (LCDR) 1-3 comprising SEQ ID NOs: 16-18, respectively.
[0070] In particular embodiments, the anti-Bb scFv or scFab comprises a V.sub.H comprising SEQ ID NO:19 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; and a V.sub.L comprising SEQ ID NO:20 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In certain embodiments, the anti-Bb scFv comprises a peptide linker, such as a flexible linker, e.g., a linker comprising (G.sub.4S) n (SEQ ID NO:46), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the V.sub.H and the V.sub.L. In some embodiments, the linker comprises SEQ ID NO:48 (i.e., n=3). The V.sub.H may be N-terminal, or C-terminal, to the V.sub.L. In some embodiments, the anti-Bb scFv comprises SEQ ID NO:21 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0071] In certain embodiments, the anti-Bb scFab comprises a heavy chain (HC) comprising SEQ ID NO:22 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; and a light chain (LC) comprising SEQ ID NO:23 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In further embodiments, the HC and the LC are linked by a peptide linker, such as a flexible linker, e.g., a linker comprising (G.sub.4S).sub.n (SEQ ID NO:46), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the HC and the LC. In some embodiments, the linker comprises SEQ ID NO:49 (i.e., n=7). The HC may be N-terminal, or C-terminal to the LC. In some embodiments, the Bb scFab comprises SEQ ID NO:24 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0072] In some embodiments, the Bb inhibitor is an antibody fragment such as an scFab or an scFv derived from anti-Bb antibody disclosed in U.S. Pat. Nos. 10,131,706; 10,604,563; or 7,964,705. For example, the Bb inhibitor may comprise the heavy and light chain CDRs, or V.sub.H and V.sub.L, of the parental anti-Bb antibody.
C. Anti-C1s/Bb Bispecific Fusion Proteins
[0073] In some embodiments, the C1s inhibitor (e.g., anti-C1s scFab or scFv) and the Bb inhibitor (e.g., anti-Bb scFab or scFv) are linked by a peptide linker, such as a flexible linker, e.g., a linker comprising (G.sub.4S).sub.n (SEQ ID NO:46), where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, linking the two inhibitors. In some embodiments, the peptide linker is SEQ ID NO:47 (n=2) or 48 (n=3). The C1s inhibitor may be N-terminal, or C-terminal to the Bb inhibitor. The C1s/Bb fusion protein may have the following exemplary, nonlimiting configurations (from N-terminus to C-terminus): [0074] C1s scFab-Linker-Bb scFab [0075] C1s scFv-Linker-Bb scFab [0076] C1s scFab-Linker-Bb scFv [0077] C1s scFv-Linker-Bb scFv [0078] Bb scFab-Linker-C1 scFab [0079] Bb scFv-Linker-C1 scFab [0080] Bb scFab-Linker-C1 scFv [0081] Bb scFv-Linker-C1 scFv
wherein the Linker may be one of the peptide linkers described herein (e.g., a flexible linker described herein), such as (G.sub.4S).sub.2 (SEQ ID NO:47) and (G.sub.4S).sub.3 (SEQ ID NO:48), and wherein within each configuration, the scFab and/scFv may have the heavy chain and the light chain in the order of N-heavy-light-C, or N-light-heavy-C.
[0082] To facilitate cognate pairing of heavy and light chains within each antigen-binding domain of the fusion protein, each antigen-binding domain may contain charge mutations. Charge mutations refer to substitution of a charge-neutral amino acid (e.g., Q) by a positively charged (e.g., K) or negatively charged (e.g., E) amino acid, and substitution of a charged amino acid to an amino acid of the opposite charge. To increase pairing of two polypeptide chains, the interactive residues on the two chains may be mutated to amino acid residues of opposite charges. Exemplary charge mutations that may contribute to cognate antibody chain pairing are described in, e.g., Tan et al., Biophys J (1998) 75:1473-82; US2014/0242076A1; and WO 2020/136566. In some embodiments, [0083] the charge mutations in the C1s scFv or scFab comprise Q42E (V.sub.L) and Q292K (V.sub.H) mutations (numbering according to SEQ ID NO:12); [0084] the charge mutations in the Bb scFv comprises Q38K (V.sub.L) and Q288E (V.sub.H) (numbering according to SEQ ID NO:24); and [0085] the charge mutations in the Bb scFab comprises Q38K (V.sub.L) and Q288E (V.sub.H), and optionally further comprises S114A (C.sub.L), N137K (C.sub.L), and T434E (C.sub.H1) (numbering according to SEQ ID NO: 24).
[0086] In some embodiments, the fusion protein has a structure shown in construct #5 (FIG. 2A), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFv-(G.sub.4S).sub.2-Bb scFv. In particular embodiments, this fusion protein is encoded by SEQ ID NO:54, or comprises SEQ ID NO:55 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0087] In some embodiments, the fusion protein has a structure shown in construct #6 (FIG. 2A), where components of the fusion protein are in the order of, from N-terminus to C-terminus, Bb scFv-(G.sub.4S).sub.2-C1s scFv. In particular embodiments, this fusion protein is encoded by SEQ ID NO:56, or comprises SEQ ID NO:57 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0088] In some embodiments, the fusion protein has a structure shown in construct #7 (FIG. 2A), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.3-Bb scFab. In particular embodiments, this fusion protein is encoded by SEQ ID NO:25, or comprises SEQ ID NO:26 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0089] In some embodiments, the fusion protein has a structure shown in construct #8 (FIG. 2A), where components of the fusion protein are in the order of, from N-terminus to C-terminus, Bb scFab-(G.sub.4S).sub.3-C1s scFab. In particular embodiments, this fusion protein is encoded by SEQ ID NO:29, or comprises SEQ ID NO:30 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0090] In some embodiments, the fusion protein has a structure shown in construct #11 (FIG. 2C), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.3-Bb scFab (with CMs). In particular embodiments, this fusion protein is encoded by SEQ ID NO:27, or comprises SEQ ID NO:28 (with or with the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0091] In some embodiments, the fusion protein has a structure shown in construct #12 (FIG. 2C), where components of the fusion protein are in the order of, from N-terminus to C-terminus, Bb scFab-(G.sub.4S).sub.3-C1s scFab (with CMs). In particular embodiments, this fusion protein is encoded by SEQ ID NO:31, or comprises SEQ ID NO:32 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0092] In some embodiments, the fusion protein has a structure shown in construct #13 (FIG. 2D), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.2-Bb scFv. In particular embodiments, this fusion protein is encoded by SEQ ID NO:33, or comprises SEQ ID NO:34 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0093] In some embodiments, the fusion protein has a structure shown in construct #14 (FIG. 2D), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.2-Bb scFv-CM (#13 with CMs in both C1s and Bb). In particular embodiments, this fusion protein is encoded by SEQ ID NO:35, or comprises SEQ ID NO: 36 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0094] In some embodiments, the fusion protein has a structure shown in construct #15 (FIG. 2E), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.3-Bb scFv. In particular embodiments, this fusion protein is encoded by SEQ ID NO:58, or comprises SEQ ID NO:59 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0095] In some embodiments, the fusion protein has a structure shown in construct #16 (FIG. 2E), where components of the fusion protein are in the order of, from N-terminus to C-terminus, C1s scFab-(G.sub.4S).sub.3-Bb scFv-CM (with CMs). In particular embodiments, this fusion protein is encoded by SEQ ID NO:60, or comprises SEQ ID NO:61 (with or without the signal peptide) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
D. Bispecific Heterodimers
[0096] In some embodiments, the dual-targeting complement inhibitors are anti-C1s/anti-Bb bispecific heterodimeric proteins. These proteins are encoded by one single open reading frame, but the HC and LC of one of the antibody fragments are cleaved upon translation and post-translational processing within the cell, yielding two separate polypeptides that are folded into two antigen-binding domains. FIG. 2F illustrates such configurations. In these illustrated configurations, the HC and the LC of one of the antibody fragments are linked by a cleavable peptide (e.g., a self-cleaving 2A peptide with or without a protease (e.g., furin) cleavage site). See also discussions in Section II (Recombinant Expression Constructs).
[0097] In some embodiments, the heterodimer has a structure shown in construct #17 (FIG. 2F), where the heterodimer is comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab. In particular embodiments, this heterodimer is encoded by SEQ ID NO:38, or comprise, pre-cleavage, SEQ ID NO:39 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto (including or not including the two signal peptide sequences).
[0098] In some embodiments, the heterodimer has a structure shown in construct #18 (FIG. 2F), where the heterodimer is comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab. In particular embodiments, this heterodimer is encoded by SEQ ID NO:40, or comprise, pre-cleavage, SEQ ID NO:41 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto (including or not including the two signal peptide sequences).
[0099] In some embodiments, the heterodimer has a structure shown in construct #19 (FIG. 2F), where the heterodimer is comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC. In particular embodiments, this heterodimer is encoded by SEQ ID NO:42, or comprise, pre-cleavage, SEQ ID NO:43 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto (including or not including the two signal peptide sequences).
[0100] In some embodiments, the heterodimer has a structure shown in construct #20 (FIG. 2F), where the heterodimer is comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC. In particular embodiments, this heterodimer is encoded by SEQ ID NO:44, or comprise, pre-cleavage, SEQ ID NO:45 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto (including or not including the two signal peptide sequences).
E. Peptide Linkers
[0101] The peptide linkers linker the various domains of the present antibody fragments and fusion proteins may preferably be flexible linkers so as to allow for proper folding, movement, and interaction of the joined domains. In some embodiments, the flexible peptide linker herein largely comprises small amino acids (e.g., Gly, Ser, or Thr). In some embodiments, the peptide linker herein consists primarily (e.g., more than 50% of the residues) of Gly and Ser residues (GS linker). As described above, such a peptide linker may comprise (G.sub.4S).sub.n (SEQ ID NO:46). By adjusting the copy number n, the length of the linker can be adjusted to achieve the desired distance of the joined functional domains. In some embodiments, the peptide linker may contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility. See, e.g., Chen et al., Adv Drug Deliv Rev. (2013) 65 (10): 1357-69.
II. Recombinant Expression Constructs
[0102] The present disclosure provides recombinant expression constructs for expressing the C1s/Bb inhibitors herein. The expression constructs have an expression cassette comprising coding sequences for the C1s/Bb inhibitors, linked operably to a promoter and a poly (A) signal sequence. The coding sequences may be human codon-optimized to improve expression in human cells. The coding sequences may encode a signal peptide (e.g., a signal peptide from IgG Kappa) to support secretion of the proteins. The expression cassette may also include additional transcription regulatory sequences, such as a Kozak sequence and a sequence that enhances gene expression or RNA stability (e.g., a WPRE element).
A. Configurations of Expression Constructs
1. Expression Constructs Encoding a Single Fusion Protein
[0103] In some embodiments, the expression construct herein is monocistronic and comprises a coding sequence for an C1s/Bb fusion protein. See, e.g., FIGS. 1A and 1C. By way of example, the expression construct may be one of the numbered constructs #5 through #8 and constructs #11 through #16, whose gene products are described in the section above.
2. Expression Constructs Encoding Two Separate Proteins
[0104] In some embodiments, the expression construct encodes the C1s inhibitor and the Bb inhibitor as two separate proteins. Independent target engagement may remove the possibility of steric hindrance.
[0105] For example, the expression construct has two separate expression cassettes, one for each of the C1s inhibitor (e.g., scFv or scFab) and the Bb inhibitor (e.g., scFv or scFab). Each expression cassette has its own transcriptional regulatory sequences such as promoters and enhancers.
[0106] In another configuration, the expression construct has a bicistronic expression cassette and a single promoter. The coding sequences for the C1s inhibitor and the Bb inhibitor are transcribed together under the single promoter, into one mRNA, and then the RNA sequence for each isoform is translated separately through the use of an internal ribosome entry site (IRES) in the mRNA. In another approach, the coding sequences of the C1s and Bb inhibitors are separated by the coding sequence for a self-cleaving peptide and/or a protease (e.g., furin) cleavage site, such that translation of the mRNA transcript and subsequent processing yield two separate gene products (C1s inhibitor and Bb inhibitor). Examples of self-cleaving peptides are 2A peptides, which are viral derived peptides with a typical length of 18-22 amino acids. 2A peptides include T2A, P2A, E2A, and F2A. Translation of the transgene can leave a few amino acid residues from the 2A peptide on one or both of the gene product. A furin cleavage site may be included to allow removal of the extra amino acid residues.
[0107] In yet another configuration, the bicistronic expression construct comprises a bidirectional promoter that allows for individual expression of each inhibitor. See, e.g., By way of example, the expression construct may be one of the numbered constructs #9, #10, #21, and #22 illustrated in FIGS. 2B and 2G and listed below (BiDir: bidirectional promoter) [0108] #9: C1s scFab-BiDir-Bb scFab, producing separate C1s scFab and Bb sc Fab [0109] #10: Bb scFab-BiDir-C1s scFab, producing separate C1s scFab and Bb scFab. [0110] #21: C1s scFab-BiDir-Bb scFab-CM, producing separate C1s scFab-CM and Bb scFab-CM [0111] #22: Bb scFab-BiDir-C1s scFab-CM, producing separate C1s scFab-CM and Bb scFab-CM
[0112] In constructs #21 and #22, both the anti-C1s and anti-Bb scFabs contain charge mutations (CMs) to promote cognate pairing of the heavy and light chains within each antibody fragment.
3. Expression Constructs Encoding Heterodimers
[0113] In some embodiments, the expression construct encodes a heterodimer comprised of a first single-chain antibody fragment (e.g., scFab or scFv) fused to one of the two chains of a second antibody fragment (e.g., Fab), where this fusion polypeptide complexes with the other chain of the second antibody fragment. The heterodimer is bispecific and binds both C1s and Bb.
[0114] Exemplary constructs that encode bispecific heterodimers configurations are illustrated in FIG. 2F and listed below: [0115] #17: C1s F2A Fab-(G.sub.4S).sub.3-Bb scFab, producing a heterodimer comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab [0116] #18: C1s GT2A Fab-(G.sub.4S).sub.3-Bb scFab, producing a heterodimer comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab [0117] #19: C1s scFab-(G.sub.4S).sub.3-Bb F2A Fab, producing a heterodimer comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC [0118] #20: C1s scFab-(G.sub.4S).sub.3-Bb GT2A Fab, producing a heterodimer comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC
In the above constructs, inclusion of a coding sequence for a cleavable peptide, such as F2A and GT2A, lead to production of two separate polypeptides, which subsequently complex and fold into one single, bispecific heterodimer protein. The F2A and GT2A coding and amino acid sequences are shown in SEQ ID NOs: 38-45. Coding sequences for other cleavable peptides (e.g., those described above) may also be used.
4. Separate Expression Constructs for C1s Inhibitor and Bb Inhibitor
[0119] In some embodiments, the C1s inhibitor and the Bb inhibitor may be expressed from two separate constructions, e.g., two separate recombinant AAVs, as further described below. The two AAVs may be of the same or different serotypes.
B. Transcriptional Regulatory Sequences
[0120] In the present expression constructs, the coding sequences for the C1s inhibitor and the Bb inhibitor are linked operably to transcription regulatory sequences such as a promoter and an enhancer, to allow expression of the encoded proteins in the intended target cells.
[0121] In some embodiments, the C1s and Bb inhibitors are produced in recombinant host cells. In such cases, the promoter and enhancer are those active in the host cells.
[0122] In some embodiments, the C1s and Bb inhibitors are delivered through gene therapy and are produced in vivo in the eye of a subject (e.g., a human, a nonhuman primate, or a mouse). In such cases, the promoter may be a constitutive promoter or an inducible promoter that functions in ocular or retina cells (e.g., RGCs and RPE cells of the inner and outer nuclear layers, Mueller cells, and photoreceptors).
[0123] In some embodiments, the promoter is a minCBA promoter comprising a CMV enhancer, a chicken -actin promoter, and an intronic sequence. The minCBA promoter may have a sequence that is at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or completely, identical to SED ID NO: 83.
[0124] In some embodiments, the promoter is a bidirectional promoter. The bidirectional promoter may contain, for example, a pair of CBA promoters placed in opposite orientation, separated by a CMV enhancer. In particular embodiments, the bidirectional promoter comprises a sequence that is at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or completely, identical to SED ID NO: 53.
[0125] In some embodiments, the expression cassette has a poly (A) signal sequence derived from bovine growth hormone gene. In particular embodiments, the poly (A) signal sequence comprises a sequence that is at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or completely, identical to the sequence that is italicized and underlined in SED ID NO: 51 shown in the Sequences section below.
[0126] In some embodiments, the expression cassette contains an enhancer, such as a CMV enhancer. In particular embodiments, the CMV enhancer comprises a sequence that is at least 85% (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or completely, identical to the sequence that is boldfaced and italicized in SED ID NO: 53 shown in the Sequence section below.
[0127] In some embodiments, the expression cassette contains an intron sequence such as a chimeric intron. The intron sequences may increase transgene expression levels by promoting transport of mRNA out of the nucleus and enhancing mRNA stability.
C. Recombinant AAV Expression Vectors
[0128] In some embodiments, a viral vector is used to deliver vectorized antibody fragments to the eye of a patient. In some embodiments, the expression/delivery vector is a recombinant adeno-associated viral (rAAV) expression vector. The expression constructs herein may be rAAV genomes. In the case of rAAV genomes, an expression cassette herein may be flanked by a pair of AAV inverted terminal repeats (ITRs), such as AAV2 ITRs. A nonlimiting example of a unidirectional, monocistronic AAV2 recombinant genome is shown in FIG. 2H. A nonlimiting example of a bidirectional, bicistronic AAV2 recombinant genome is shown in FIG. 2I.
[0129] An exemplary rAAV genome harboring construct #9 may have an exemplary nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence encoding the same amino acid sequences as does SEQ ID NO:50 and comprising a sequence that is at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identical to SEQ ID NO:50.
[0130] An exemplary rAAV genome harboring construct #12 may have an exemplary nucleotide sequence of SEQ ID NO:51, or a nucleotide sequence encoding the same amino acid sequences as does SEQ ID NO:51 and comprising a sequence that is at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identical to SEQ ID NO:51.
[0131] An exemplary rAAV genome harboring construct #14 may have an exemplary nucleotide sequence of SEQ ID NO:52, or a nucleotide sequence encoding the same amino acid sequences as does SEQ ID NO:52 and comprising a sequence that is at least 50% (e.g., at least 60, 65, 70, 75, 80, 85, 90, or 95%) identity to SEQ ID NO:52.
[0132] The rAAV genome can be constructed by inserting the expression cassettes herein into an rAAV genome that has had the major rAAV open reading frames excised therefrom. Other portions of the rAAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
[0133] Any suitable AAV serotype may be used. For example, the AAV may be AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, or AAVrh10, or of a pseudotype or a serotype that is a mutant, variant or derivative of one of the AAV serotypes listed herein (i.e., AAV derived from multiple serotypes). The AAV may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans or nonhuman primates.
[0134] In some embodiments, the rAAV herein has an AAV2 capsid. In particular embodiments, the AAV2 capsid is a wildtype AAV2 capsid. In other embodiments, the AAV2 capsid contains mutations that improve the rAAV2's potency and production yield.
[0135] Viral vectors described herein may be produced using methods known in the art. Any suitable permissive or packaging cells may be employed to produce the viral particles. For example, mammalian (e.g., 293 or HeLa) or insect (e.g., Sf9) cells may be used as the packaging cell line. Recombinant AAV vectors can be replicated and packaged into infectious viral particles when introduced into host cells that have been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and capsid proteins). See, e.g., U.S. Pat. No. 11,261,463.
D. Transfection of Host Cells
[0136] Where the C1s and Bb inhibitors are delivered directly to patients, the inhibitors may be produced in recombinant mammalian host cells such as COS, NS0, 293, HeLa, or CHO cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the inhibitors.
III. Pharmaceutical Compositions and Use
[0137] The present disclosure provides pharmaceutical compositions comprising the dual targeting C1s/Bb inhibitors or recombinant viral vectors such as AAV vectors encoding the inhibitors. The pharmaceutical compositions may comprise pharmacologically, especially ophthalmologically, acceptable carriers, diluents, and/or excipients. For example, the composition may comprise a tonicity agent (e.g., sodium chloride, amino acids, sugars, or combinations thereof), a surfactant (e.g., polysorbate 20 or polysorbate 80), and/or a stabilizer (e.g., a methioninc).
[0138] The pharmaceutical compositions may be delivered by intraocular injection, e.g., injection into the anterior chamber via the temporal limbus, suprachoroidal injection, intracameral injection, intrastromal injection, subretinal injection, intravitreal injection (e.g., front, mid or back vitreous injection).
[0139] The present pharmaceutical compositions may be delivered in a therapeutically effective amount to treat dry AMD and geographic atrophy (GA) secondary to dry AMD. An therapeutically effective amount means a dosage sufficient to produce a desired result, e.g., amelioration of one or more symptoms (e.g., growth of GA lesions, retinal lesions, or destruction of retinal layer) of the disease to be treated, and/or slowing progression of the disease. A desired result may also include improvement in one or more functional symptoms; for example, the desired result may be reduction of visual distortions, improved central vision, improved vision in low light settings, and/or reduced blurriness. By treat is meant amelioration of one or more symptoms of the disease and/or slowing of the progress of the disease.
[0140] The present pharmaceutical compositions may be delivered in a prophylactically effective amount to prevent the onset of dry AMD or geographic atrophy (GA) secondary to dry AMD. An prophylactically effective amount means a dosage sufficient to produce a desired result, e.g., prevention or delay of the onset of dry AMD and/or GA, and/or prevention or delay of the onset of one or more symptoms of dry AMD and/or GA. Patients who are at high risk of developing dry AMD, such as those with genetic predisposition, may be administered with the present pharmaceutical compositions prophylactically.
[0141] In some embodiments, the dosage of recombinant AAV (rAAV) injected into the eye is 10.sup.7 to 10.sup.15 vector genomes (vg), for example, 10.sup.8 to 10.sup.14, 10.sup.9 to 10.sup.13, or 10.sup.9 to 10.sup.12, vg. In some embodiments, the dosage of rAAV is 210.sup.9, 210.sup.10, or 210.sup.11 vg.
[0142] In some embodiments, the patient is treated, before, during, and/or after the rAAV injection, with an anti-inflammatory agent (e.g., a steroid) to prevent or ameliorate potential immune response against the rAAV. In some embodiments, the patient may be pre-treated with an IgG-degrading enzyme, such as IdeS, to reduce pre-existing neutralizing antibodies to the AAV capsid. These immune modulators may be administered locally or systematically. In some embodiments, the modulators may be administered intraocularly (e.g., intravitreally), orally, intravenously, intramuscularly, or subcutaneously.
[0143] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words have and comprise, or variations such as has, having, comprises, or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term approximately or about as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
[0144] As used herein, the percent identity of two amino acid sequences (or of two nucleic acid sequences) may be obtained by, e.g., BLAST using default parameters (available at the U.S. National Library of Medicine's National Center for Biotechnology
[0145] Information website). In some embodiments, the length of a query sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%) of the length of the reference sequence.
[0146] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any compound disclosed herein can be used in any of the treatment methods disclosed herein, wherein the individual to be treated is as defined anywhere herein.
[0147] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
Examples
Example 1: Vectorized Antibodies and Expression Constructs Thereof
[0148] This Example describes the design of bifunctional expression constructs that express inhibitors to C1s and Bb, and the characterization of the recombinant proteins produced from these constructs. These constructs have the following features: (i) either a unidirectional or a bidirectional promoter (e.g., minCBA promoter) to drive constitutive transgene expression; (ii) a transgene (e.g., a transgene that contains human codon-optimized sequences); (iii) different combinations of antibody fragments (e.g., scFab-scFab and scFab-scFv) derived from parental anti-Bb IgG4 antibody (e.g., V.sub.H6/V7-IgG4v2 from U.S. Pat. No. 11,242,382 and WO 2021/216458), and parental anti-C1s IgG4 antibody (e.g., V.sub.H3/V2 from WO 2018/071676); (iv) peptide linkers (e.g., between antibody fragments and between heavy and light chains of each antibody fragment, containing G.sub.4S repeats); (v) the presence or absence of rationally designed charge mutations (CM) that promote accurate heavy/light chain pairing; and (vi) a polyadenylation site (e.g., a bovine growth hormone (bGH) gene polyadenylation signal).
A. Generation of Bifunctional Bicistronic or Monocistronic Constructs
[0149] The bifunctional monocistronic or bicistronic constructs generated herein contain DNA fragments expressing scFv or scFab of the constituent antibody fragments to active C1s and Bb, downstream of the ubiquitous minCBA promoter, and a poly (A) signal sequence from the bovine growth hormone gene. The entire expression cassette was cloned between wildtype inverted terminal repeat (ITR) sequences from AAV serotype 2 (FIGS. 1A-C). Glycine/serine-rich linkers (e.g., linkers with G.sub.4S repeats) were inserted between the heavy and light chains of each single-chain C1s and Bb antibody fragment (scFab or scFv) to facilitate proper folding of each antigen-binding domain formed by a pair of V.sub.H and V.sub.L. In the present studies, a linker with seven G.sub.4S repeats was used to link the heavy and light chains of an scFab and a linker with three G.sub.4S repeats was used to link the V.sub.H and V.sub.L of an scFv.
[0150] For monocistronic constructs, exemplary formats were scFab-scFab, scFv-scFab, scFab-scFv, and ScFv-ScFv (see, e.g., FIGS. 2A, 2D, and 2E). Glycine/serine-rich linkers (e.g., linkers with G.sub.4S repeats such as two or three repeats) were inserted between the two single-chain fragments of a bifunctional fusion protein to allow for the flexibility of the bifunctional fusion protein.
[0151] For some monocistronic constructs, an additional feature was the inclusion of a canonical furin cleavage site (RX (R/K) R) (SEQ ID NO:82), e.g., in linkers F2A and GT2A (FIG. 2F). Linking the heavy chain (HC) and light chain (LC) genes on a single cassette using 2A peptides would allow improved control of LC and HC ratio. Insertion of a furin recognition site upstream of 2A would allow removal of 2A residues that would otherwise be attached to the HC and/or LC (see, e.g., FIG. 2F).
[0152] For bidirectional bifunctional constructs, a novel bidirectional promoter was designed based on the ubiquitous minimal chicken -actin (minCBA) promoter. This promoter supports the concurrent expression of individual antibody fragments to factor C1s and factor Bb. MinCBA contains a CBA promoter and an CMV enhancer but with an abbreviated intronic sequence. The bidirectional promoter contains a pair of CBA promoters placed in opposite directions and separated by an CMV enhancer (SEQ ID NO:53). The bidirectional expression construct produces separate anti-C1s and anti-Bb antibody fragments for independent target engagement, which removes the possibility of steric hindrance.
[0153] The monocistronic or bicistronic expression cassette was cloned between AAV2 ITR sequences (see, e.g., FIGS. 1C, 2H, and 2I) for AAV delivery.
[0154] Some experiments used antibody fragments containing charge mutations that promote accurate pairing between heavy and light chains of each constituent antibody fragment. To generate charge mutants (CM), specific amino acids were substituted in the variable and/or constant domains of the C1s and Bb antibody fragments. The following amino acid changes were introduced for the following mutated antibody fragments: [0155] C1s scFab-CM: Q42E and Q292K (numbering in accordance with SEQ ID NO: 12) [0156] Bb scFab-CM: Q38K, S114A, N137K, Q288E, and T434E (numbering in accordance with SEQ ID NO:24)
[0157] Exemplary monodirectional construct configurations are illustrated in FIGS. 2A and 2C-F and listed below: [0158] #5: C1s scFv-(G.sub.4S).sub.2-Bb scFv [0159] #6: Bb scFv-(G.sub.4S).sub.2-C1s scFv [0160] #7: C1s scFab-(G.sub.4S).sub.3-Bb scFab [0161] #8: Bb scFab-(G.sub.4S).sub.3-C1s scFab [0162] #11: C1s scFab-(G.sub.4S).sub.3-Bb scFab-CM (#7 with CMs in both C1s and Bb). [0163] #12: Bb scFab-(G.sub.4S).sub.3-C1s scFab-CM (#8 with CMs in both C1s and Bb). [0164] #13: C1s scFab-(G.sub.4S).sub.2-Bb scFv. [0165] #14: C1s scFab-(G.sub.4S).sub.2-Bb scFv-CM (#13 with CMs in both C1s and Bb). [0166] #15: C1s scFab-(G.sub.4S).sub.3-Bb scFv. [0167] #16: C1s scFab-(G.sub.4S).sub.3-Bb scFv-CM (#15 with CMs in both C1s and Bb) [0168] #17: C1s F2A Fab-(G.sub.4S).sub.3-Bb scFab, producing a heterodimer comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab [0169] #18: C1s GT2A Fab-(G.sub.4S).sub.3-Bb scFab, producing a heterodimer comprised of (i) an C1s LC and (ii) a fusion protein comprising an C1s HC fused to an Bb scFab [0170] #19: C1s scFab-(G.sub.4S).sub.3-Bb F2A Fab, producing a heterodimer comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC [0171] #20: C1s scFab-(G.sub.4S).sub.3-Bb GT2A Fab, producing a heterodimer comprised of (i) a fusion protein comprising an C1s scFab fused to an Bb HC and (ii) an Bb LC
[0172] Exemplary bidirectional construct configurations are illustrated in FIG. 2B and listed below: [0173] #9: C1s scFab-BiDir-Bb scFab, producing separate C1s scFab and Bb scFab [0174] #10: Bb scFab-BiDir-C1s scFab, producing separate C1s scFab and Bb scFab [0175] #21: C1s scFab-BiDir-Bb scFab-CM, producing separate C1s scFab-CM and Bb scFab-CM [0176] #22: Bb scFab-BiDir-C1s scFab-CM, producing separate C1s scFab-CM and Bb scFab-CM
B. Evaluation of Bb- and C1s-Binding
[0177] Each DNA construct was transfected into HEK293 cells. Supernatants containing the secreted recombinant proteins were harvested and purified over Protein L beads. More specifically, the supernatant was incubated with Protein L beads for 1 hour at room temperature. The beads were then washed three times with PBS containing polysorbate 20. The bead column was then eluted with 0.1 M glycine (pH 2.0) for ten minutes at room temperature. The eluate was neutralized with 15% v/v 1 M Tris (pH 8.5) and then desalted through buffer exchange into PBST.
[0178] Purity of the recombinant proteins was evaluated on SDS-PAGE (non-reduced and reduced) and on mass photometer (mass distribution). Concentrations of the proteins were measured on NanoDrop (Thermo Fisher).
[0179] Bio-layer interferometry (BLI) was used to assess the recombinant proteins' target engagement to and binding affinity for complement C1s enzyme (active C1s or C1s herein) and factor Bb (Bb) (Complement Technology, Tyler, TX, USA). C1s and Bb were biotinylated with EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher, Waltham, MA, USA) according to manufacturer instructions. Biotinylated C1s or Bb was loaded on Octet Streptavidin (SA) Biosensors (Sartorius, Gttingen, Germany), followed by a concentration range of purified proteins. To assess dual target engagement, biotinylated active C1s or Bb was loaded onto sensors, followed by purified proteins (first association phase), followed by the non-captured complement target (Bb or active C1s, non-biotinylated; second association phase). The assays were performed at 30 C. using PBS with 0.1% Tween 20 as a diluent (FIG. 3).
[0180] Additionally, inhibition of the classical and alternative complement pathways was evaluated by Wieslab Complement System Classical Pathway and Wieslab Complement System Alternative Pathway kits (Svar, Malm, Sweden). Assays were performed according to manufacturer instructions. Serial dilutions of constructs were performed in the respective assay diluents for each assay.
C. Results
[0181] To confirm vector-derived antibody fragments were expressed and secreted, supernatants were harvested from HEK293 cells transfected with plasmids encoding the transgenes and kappa light chain-containing antibody fragments were enriched from the supernatant by affinity purification using protein L beads. Western blot analyses of the enriched supernatants demonstrated that all transgenes produced antibody fragments.
[0182] Target engagement of antibody fragments from cell supernatants was evaluated using the Octet binding assay. The data demonstrated that proteins produced from all expression constructs all exhibited dual target engagement for C1s and Bb. Across all tested constructs, the binding affinity of the partially purified bifunctional antibody fragments was within 2- to 10-fold of the purified parental anti-C1s and anti-Bb Fabs.
[0183] Exemplary data are shown in FIG. 3, which shows that when partially purified antibody fragments produced by construct #19 (FIG. 2F) were added in first association phase (for binding to Bb), an increase in signal was observed. When the second target C1s was added in second association phase, an additional increase in signal was observed (FIG. 3). All antibody fragments produced by the tested bifunctional constructs, except construct #5 (FIG. 2A), exhibited similar levels of dual target engagement (see Example 2 below).
[0184] The parental monoclonal antibodies used to design the bifunctional complement inhibitors have been previously shown to inhibit either the complement classical (CP; see WO 2016/164358) or alternative (AP; see U.S. Pat. No. 11,242,382) pathway, with neither inhibiting the lectin pathway. The ability of the bifunctional antibody fragments or antibody fragment pairs to inhibit activity of both the CP and AP was assessed in vitro using Wieslab assays. All tested bifunctional antibody constructs inhibited both IgM-stimulated activation of the CP and LPS-stimulated activation of the AP (see Example 2 below). The data show that for all tested constructs, the inhibitory activity was within 4-fold of the parental Fabs.
[0185] The results show that the vector expressed antibody fragments to complement factors Bb and C1s bind to target complement factors and inhibit activated complement with an efficiency that is similar to parental individual Fab proteins. These results were unexpected because parental antibody fragments are Fabs generated using recombinant mAB technology methods, i.e. expressed in a CHO cell and highly purified, in contrast the antibody fragments generated from the AAV pre-viral plasmids are scFab and scFV fragments and were tested as partially purified antibody fragments. Moreover, the plasmid derived antibody fragments, for some constructs, are monocistronic, and are therefore acting like bifunctional antibodies. Despite this difference in design/structure from the parental Fabs, inhibition of each target was largely preserved.
Example 2: Functional Characterization of Anti-C1s and Anti-Bb scFabs
[0186] Constructs #2 and #4 were recombinantly expressed and purified to homogeneity as described above and tested in target binding assays as well as in serum-based and cell-based functional assays. Direct target binding was measured using surface plasmon resonance (SPR).
[0187] The inhibitory activity of the scFabs were tested in serum-based Wieslab enzyme immune assays. In the commercial assay kits, the wells of the microtiter strips are coated with specific activators for each pathway of the complement system. Additionally, the buffers and reagents included in the kits prevent the cross-activation of multiple pathways, maintaining specificity of pathway activation. Test kits for the AP are coated with lipopolysaccharide, while test kits for the CP are coated with human IgM). The final readout is the detection of a neoepitope on the C5b9 complex generated due to the complement pathway activation, measured colorimetrically. The recombinant scFabs were also tested in a modified Wieslab assay, where the microtiter plate was coated with both heat-aggregated (HAGG) IgG) and C3b to allow for simultaneous activation of CP and AP; in this assay, the C5b9 complex generated from the activation of both pathways was also measured colorimetrically.
[0188] Additionally, the recombinant scFabs were tested in an in vitro ARPE19 cell line-based model of dry AMD. In all the functional assays, the recombinant scFabs were tested individually as well as an equimolar mixture to be representative of the vector-derived product.
[0189] Table 1 below shows the characterization of recombinant scFabs and their comparison to parental scFabs (#2 and #4) and mAbs.
TABLE-US-00003 TABLE 1 Wieslab assay IC.sub.50 (nM) K.sub.D (nM) Alternative Classical Lectin Sample Const. Bb C1s Pathway Pathway pathway Const. Purified anti-C1s NA 0.3 NA 6.7 ND #9 scFab Purified anti-Bb 3.7 0.4 NA 190.4 NA ND scFab Equimolar mix of ND ND 461.1 12.9 ND anti-C1s scFab and anti-Bb scFab Parental Purified anti-C1s NA 0.3 NA 5.5 ND scFabs Fab Purified anti-Bb 2.7 0.4 NA 144.1 NA ND Fab Equimolar mix of ND ND 270.4 14.4 ND anti-C1s Fab and anti-Bb Fab Parental anti-C1s mAb NA 1.5 No 22 No mAbs inhibition inhibition anti-Bb mAb 7.3 1.4 NA 189.67 No No inhibition inhibition
[0190] These data show that the recombinant scFabs against both C1s and Bb show similar binding and inhibitory properties as their corresponding parental scFabs.
Example 3: Properties of Exemplary Complement Inhibitors with Charge Mutations
[0191] Three expression constructs were selected for further studies. The first one, construct #14 (FIGS. 2D and 2H), was composed of a unidirectional minCBA promoter driving expression of a single transcript encoding anti-C1s scFab connected to anti-Bb scFv by a flexible (G.sub.4S).sub.2 linker [C1s scFab-(G.sub.4S).sub.2-Bb scFv] and followed by a bGH poly (A) signal. The sequences were human codon-optimized and contain charge mutations to promote accurate chain pairing.
[0192] The second expression construct, construct #12 (FIGS. 2C and 2J), was composed of a unidirectional minCBA promoter driving expression of a single transcript encoding anti-Bb scFab connected to anti-C1s scFab by a flexible (G.sub.4S).sub.3 linker [Bb scFab-(G.sub.4S).sub.3-C1s scFab] and followed by a bGH poly (A) signal. The sequences were human codon-optimized and contain charge mutations to promote accurate chain pairing.
[0193] The third expression construct, construct #9 (FIGS. 2B and 21), is composed of a bidirectional minCBA promoter driving expression of separate transcripts encoding human codon-optimized Bb scFab or C1s scFab [C1s scFab+Bb scFab], each followed by a bGH poly (A) signal. In assays performed as described in Example 1, the complement-binding antibody fragments expressed from constructs #9 and #14 had a binding affinity for both C1s and Bb within 2- to 6-fold of the purified parental Fabs, while complement-binding antibody fragments expressed from construct #12 had a binding affinity for C1s and Bb within about 6- to 7-fold of the purified parental Fabs (Table 2).
TABLE-US-00004 TABLE 2 Inhibitor AP K.sub.D (Bb, nM) CP K.sub.D (C1s, nM) #14 2.5 1.4 #12 6.6 3.6 #9 0.9 1.8 anti-Bb Fab 0.7 N/A anti-C1s Fab N/A 0.3
[0194] In Wieslab assays, the IC.sub.50 values of construct #14-derived complement inhibitors were within about 6-fold of purified anti-C1s Fab (CP inhibition) and purified anti-Bb Fab (AP inhibition). The IC.sub.50 values of #12-derived antibody fragments were within about 7-fold of purified anti-Bb Fab (AP inhibition) and 14-fold of purified anti-C1s (CP inhibition). The IC.sub.50 values of #9-derived antibody fragments were within about 3-fold of purified anti-Bb Fab (AP inhibition) and 25-fold of purified anti-C1s Fab (CP inhibition) (Table 3).
TABLE-US-00005 TABLE 3 Inhibitor AP IC.sub.50 (nM) CP IC.sub.50 (nM) #14 551 38.6 #12 702 84.4 #9 224 157.3 anti-Bb Fab 99 N/A anti-C1s Fab N/A 6
[0195] Additionally, constructs #2, #4, #12, and #14 (FIGS. 2A, 2C, and 2D) were expressed and purified to >98% purity using chromatographic methods (referred to as recombinant constructs) and tested head-to-head with the parental anti-C1s and anti-Bb Fabs in the assays described in Example 1 to characterize the functional properties of these constructs. Constructs #2 and #4 were chosen to represent the two scFabs that would be expressed and secreted by the bidirectional vector construct #9.
[0196] The results of these experiments are summarized in Table 4 below (ND: not determined).
TABLE-US-00006 TABLE 4 IC.sub.50 CP IC.sub.50 AP K.sub.D, C1s K.sub.D, Bb Wieslab Wieslab Construct No. (nM) (nM) EIA (nM) EIA (nM) 2 0.3 NA 7.2 NA 4 NA 0.7 NA 192.9 12 5.4 1.5 10.4 428.1 14 1.3 4.2 5.9 346.6 Equimolar mix of ND ND 12.9 461.1 #2 and #4 Equimolar mix of ND ND 14.4 270.4 anti-C1s Fab and anti-Bb Fab
[0197] Table 5 below summarizes the in vitro binding and functional inhibition results of the proteins expressed by constructs #2, #4, #5, #6, #7, #8, #9, #11, #12, #13, #14, #15, #16, #17, #18, #19, and #20 from FIGS. 2A-2F) in comparison to the recombinant parental anti-anti-C1s Fab and anti-Bb Fab.
TABLE-US-00007 TABLE 5 Dual target binding K.sub.D (nM) k.sub.a (10.sup.5 M.sup.1s.sup.1) k.sub.a (10.sup.4 s.sup.1) IC.sub.50 (nM) Construct No. (Y/N) Bb C1s Bb C1s Bb C1s AP CP 2 NA NA 0.3 NA 2.7 NA 7.6 NA 7.2 4 NA 0.7 NA 6.7 NA 4.9 NA 192.9 NA 5 ND ND ND ND ND ND ND 2826* 216 6 ND ND ND ND ND ND ND 1258* 118 7 Y 9.3 1.8 0.6 2.6 5.4 4.7 570 45.9 8 Y 1.8 2.1 1.2 3 2.1 6.3 420 87.7 9 Y 0.8 1.9 2.6 1.5 2 2.8 224 157 11 Y 8.8 2.7 0.6 2.1 5.1 5.8 1000* 49.7 12 Y 6.6 3.6 0.8 1.8 5.5 6.5 702 84.4 13 Y 3 1 0.7 3 2.2 2.9 201 44.4 14 Y 2.5 1.4 0.7 3.6 1.7 4.9 551 38.6 15 Y 3.9 1.6 1.2 3.7 4.5 5.7 405 34.7 16 Y 2.5 1.4 0.9 3.6 2.3 5.1 586 35.4 17 Y ND ND ND ND ND ND ND ND 18 Y ND ND ND ND ND ND ND ND 19 Y ND ND ND ND ND ND 2093* 34 20 Y ND ND ND ND ND ND ND ND Anti-C1s NA NA 0.3 NA 2.9 NA 8.1 NA 5.4 Fab Anti-Bb NA 0.73 NA 7 NA 5.1 NA 144.1 NA Fab NA: not applicable. ND: not determined. *Curve not saturated; estimated IC.sub.50.
[0198] In addition to direct target binding (BLI) and Wieslab EIA assays, another functional assay was developed to assess the simultaneous inhibition of both CP and AP by these recombinant constructs. In this assay, ELISA plates were coated with both HAGG (heat-aggregated gamma globulin) and C3b and incubated with 12% C1s-depleted serum containing 380 ng/ml proenzyme C1s, to activate both CP and AP simultaneously. The conditions in the assays were optimized to achieve similar levels of CP and AP activation on the plate. Dose responses of constructs #2 and #4 were tested either individually or in an equimolar mix (to represent the expression condition from construct #9). An equimolar mix of the parental anti-C1s Fab and anti-Bb Fab was also tested alongside.
[0199] Under these conditions, constructs #2 and #4 achieved dose-dependent but partial inhibition (70-85%; FIGS. 4A and 4B). However, when these two constructs were mixed together in an equimolar ratio, it resulted in >99% inhibition of complement activation, similar to what was seen for the equimolar mix of the parental Fabs. The IC.sub.50 observed was within 2- to 3-fold of what was observed for the equimolar mix of the parental Fabs (FIG. 4C). See also Table 6, which summarizes the half-maximal inhibitory concentrations of anti-C1s Fab, anti-Bb Fab, the protein expressed from construct #2, the protein expressed from construct #4, or an equimolar mixture of the two, as well as the maximum inhibition achieved, under conditions where both CP and AP were activated simultaneously in vitro.
TABLE-US-00008 TABLE 6 Construct No. IC.sub.50 (nM) Maximal inhibition 2 97.3 75-80% 4 633.4 80-85% anti-C1s Fab 68.2 75-85% anti-Bb Fab 462.6 70-75% Equimolar mix of constructs 119.8 .sup.>99% #2 and #4 Equimolar mix of anti-C1s 51.5 90-92% Fab and anti-Bb Fab
Example 4: In Vivo Retina Studies in Mice
[0200] Based on the above in vitro results, constructs #9, #12, and #14 were selected for in vivo studies, and their ITR plasmid expression cassettes were packaged into AAV2 for delivery to target cells (see, e.g., FIGS. 2H, 2I, and 2J). This Example describes in vivo testing of these vectorized antibody constructs in wildtype mouse retina to confirm transduction of retinal ganglion cells (RGC) and secretion of the antibody fragments into the vitreous. Antibody fragments secreted into the mouse vitreous humor were evaluated in an in vitro assay to demonstrate target engagement with human complement factors C1s and Bb. Tolerability was assessed by optical coherence tomography (OCT).
A. AAV Injection
[0201] More specifically, recombinant AAV2 expressing constructs #9, #12, and #14 flanked by AAV2 ITRs were produced. AAV2 #14, AAV2 #12, and AAV2 #9 were administered to C57BL/6J mice at three doses [10.sup.8, 10.sup.9, or 10.sup.10 vector genomes (vg) per eye] through intravitreal injection, and retinal transduction, transgene expression, antibody secretion, and tolerability were assessed after 3-4 weeks in-life exposure. A recombinant AAV2 encoding a secreted VEGF inhibitor was administered in parallel at 210.sup.9 vg per eye as a positive control. Un-injected, vector-nave mice were used as a negative control.
B. Vector Transduction
[0202] Vector transduction was quantified using a TaqMan assay to detect the vector-derived bGH poly (A) in quantitative PCR analyses of DNA purified from the mouse retinas. The data show that all three vectors successfully transduced the retina, achieving about 10.sup.4-10.sup.5 vg per 500 ng DNA. The levels of transduction from the bifunctional antibody fragment vectors were comparable to what was achieved with the positive control. There was a vector dose-dependent increase in transduction of AAV2 #14 (10.sup.10 vs. 10.sup.8; p=0.01). Similar results are observed for AAV2 #12 (several mice administered 10.sup.9 vg had relatively low levels of transduction; this was likely due to a technical issue with the administration of that dose). AAV2 #9, which has two copies of the bGH poly (A), showed high levels of transduction at all doses.
[0203] Vector transduction and cell targeting in the mouse retina were also assessed using vector-specific probe sets in in situ hybridization (ISH) analyses of sections from fixed, paraffin-embedded eyes. Each probe set included 40 pairs of probes of about 50 bases in length. In eyes administered each of the AAV2 vectors, vector transduction was detected primarily in retinal ganglion cells (RGC) and cells of the inner nuclear layer (INL), to a lesser degree in cells of the outer nuclear layer (ONL), and rarely in the cells of the retinal pigment epithelium (RPE) (FIG. 5).
[0204] Table 7A summarizes the levels of transduction (vector genomes/500 ng genomic DNA) achieved in the mouse retina at 3 weeks after intravitreal administration of AAV2 #9, AAV2 #12, and AAV2 #14 (medianMAD).
TABLE-US-00009 TABLE 7A 3-4 weeks after dosing Dose Construct 1e8 vg 1e9 vg 1e10 vg #14 1.23e4 7.82e3 4.81e4 4.11e4 8.49e4 1.29e4 #12 1.06e4 6.07e3 2.23e3 2.05e3 4.55e4 1.66e4 #9 3.12e4 1.94e4 2.38e4 1.51e4 4.18e4 2.85e4
C. Transgene Expression
[0205] Transgene expression in the retina was measured through quantitative RT-PCR analyses of RNA purified from the mouse retinas, using a TaqMan assay to detect the vector-derived bGH poly (A) sequence. RNA quality was assessed and samples with an RNA integrity number (RIN) lower than 6 were not included in the analyses. The data show that all three AAV vectors produce high levels of transgene expression (10.sup.5 to 10.sup.6 transcripts per 500 ng RNA) in the retina after 3 weeks of in-life exposure. Table 7B summarizes the levels of transgene expression (bGH transcripts/500 ng RNA) achieved in the mouse retina at 3 weeks after intravitreal administration of AAV2 #9, AAV2 #12, and AAV2 #14 (medianMAD).
TABLE-US-00010 TABLE 7B 3-4 weeks after dosing Dose Construct 1e8 vg 1e9 vg 1e10 vg #14 3.16e5 1.32e5 5.76e5 2.37e5 1.39e6 6.92e5 #12 7.90e4 2.96e4 5.34e2 4.99e2 2.92e5 2.11e5 #9 9.42e5 2.55e5 1.25e6 1.91e5 3.02e6 4.28e5
[0206] Across all samples, transcript levels correlate with levels of vector genomes (p=0.59), and expression levels were lower in poorly transduced AAV2 #12 retinas from the 10.sup.9 vg treatment group. AAV2 #9 showed a dose-dependent increase in transgene expression (10.sup.10 vs. 10.sup.8, with p=0.036; 10.sup.10 vs. 10.sup.9, with p=0.0495).
D. Antibody Expression
[0207] Expression and distribution of the vector-derived complement inhibitors in the mouse retina was evaluated through immunohistochemistry (IHC) using an anti-human kappa light chain antibody to detect the vector-derived human antibody fragments. The data show that inhibitors produced by all three vectors were detected in RGCs (retinal ganglion cells) and cells of the INL., (inner nuclear layer.)
E. Antibody Secretion and Target Engagement
[0208] To demonstrate that the viral vectors produced bifunctional complement inhibitors that were secreted, inhibitor levels in the vitreous humor from mice were assessed using an ELISA method. Vector-derived complement inhibitors present in mouse vitreous humor were quantified via target engagement capacity using C1s and Bb ELISA and purified anti-C1s and anti-Bb scFabs as standards. Tables 8A and 8B summarize the ex vivo dual target engagement results of secreted anti-C1s (Table 8A) and anti-Bb (Table 8B) antibody fragments present in mouse vitreous humor at 3 weeks following intravitreal administration of AAV2 #9, AAV2 #12, and AAV2 #14 (meanSD; ng/ml).
TABLE-US-00011 TABLE 8A 3-4 weeks after dosing Dose Construct 1e8 vg 1e9 vg 1e10 vg #14 360 170 159 23 1022 622 #12 72 11 107 19 124 28 #9 145 52 262 194 1011 578
TABLE-US-00012 TABLE 8B 3-4 weeks after dosing Dose Construct 1e8 vg 1e9 vg 1e10 vg #14 384 225 144 29 811 413 #12 84 11 117 17 149 30 #9 144 60 339 215 1159 559
[0209] Overall, the C1s and Bb ELISAs demonstrate that all three rAAVs, when delivered intravitreally, led to expression and secretion from the mouse retinal ganglion cells. Proteins expressed by all three expression vectors could bind to C1s and Bb ex vivo. Overall, the data show that all three expression vectors produced comparable levels of anti-C1s and anti-Bb binding activity in mice. It was unexpected that retinal ganglion cells could support the in vivo production of vectorized antibody fragments that exhibit similar binding properties as parental antibodies generated in vitro using established recombinant antibody production methods.
[0210] AAV2 #14-treated mice have vitreous levels of bifunctional antibodies ranging from about 150 ng/ml to about 900 ng/ml. Vitreous levels of AAV2 #12-derived inhibitors show a slight dose-response across treatment groups, increasing from about 80 ng/mL to about 140 ng/ml. Levels of inhibitors in vitreous from AAV2 #9-treated mice increase in a dose-dependent manner, reaching about 1100 ng/ml at the highest dose. In addition to quantifying inhibitor levels in vitreous, these data demonstrate ex vivo dual target engagement of vector-derived antibody fragments.
[0211] Target engagement and efficacy of vector-derived complement inhibitors cannot be evaluated in vivo in mice because these inhibitors bind only human and nonhuman primate (NHP) C1s and Bb, and do not interact with murine complement factors.
[0212] In mice dosed with the AAV2 positive control (see above), secretion of the VEGF inhibitor into the vitreous was measured by ELISA. Vitreous levels of the VEGF inhibitor average about 57 ng/ml after 2 weeks in-life exposure. Therefore, AAV2 #14, AAV2 #12, and AAV2 #9 all generate higher levels of secreted proteins than the positive control.
F. Tolerability
[0213] Photoreceptor damage can be detected as a thinning of the photoreceptors. Tolerability of the viral vectors was assessed by measuring the thickness of the photoreceptor (PR) layer [outer nuclear layer (ONL)+inner segment/outer segment (IS/OS)] in optical coherence tomography (OCT) images from vector-nave and transduced mouse retinas. Photoreceptor thickness in AAV2 #14-, AAV2 #12-, and AAV2 #9-transduced retinas does not decrease at any dosage (10.sup.8, 10.sup.9, or 10.sup.10 vg) compared to vector-nave retinas, suggesting no impact on photoreceptor tolerability for the doses and time points studied in mice.
Example 5: Inhibition of Complement Activation in a Cell-Based Model of Dry AMD
[0214] C-reactive protein (CRP) is an acute phase reactive protein and an activator of the classical complement pathway (CP). CRP binds to dying cells and activates the CP, labeling those cells for clearance by phagocytes. CRP's levels are elevated under inflammatory conditions. It has been shown that elevated CRP levels is an independent risk factor for the pathogenesis of AMD, and high serum concentrations of CRP are linked to faster AMD progression to advanced disease and higher severity of vision loss in other retinal diseases like retinitis pigmentosa (Chen et al., Trans Vis Sci & Techno. (2021) 10 (7): 7; Molins et al., Front Immunol. (2018) 9:808; and Murakami et al., Acta Ophthalmol. (2018) 96 (2): e174-e179). Additionally, it has been shown that Bruch's membrane, drusen, and choroidal vessel walls stain for elevated levels of CRP in AMD patients' eyes, suggesting that complement activation during the disease is, at least in part, initiated by CRP (Bhutto et al., Br J Ophthalmol. (2011) 95 (9): 1323-30).
[0215] To recapitulate some of these patient characteristics in vitro in a cell-based model, ARPE19 cells (a retinal pigment epithelia (RPE) cell line) were treated with normal human serum (NHS) supplemented with CRP. The extent of complement activation was assessed by monitoring the levels of C3-fragment and C5b9 deposited on the cell surface using an on-cell ELISA protocol. The data show that treatment of ARPE19 cells with NHS supplemented with CRP resulted in elevated levels of both C3-fragments and C5b9 on the cells compared to treatment with NHS alone, indicating a stronger activation of the complement system in the presence of CRP (FIG. 6). When complement inhibitors were included in the treatment, combined inhibition of the CP and the AP (anti-C1s Fab+anti-Bb Fab) resulted in a stronger reduction of both C3-fragment and C5b9 levels as compared to the levels of inhibition achieved by either the anti-C1s Fab (CP) or the anti-Bb Fab (AP) individually (FIG. 6).
Example 6: A New iPSC-Derived Cell Model for AMD
[0216] This Example describes a new cell model developed to demonstrate CRP-initiated complement activation in AMD. This model measures complement deposition on induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE). RPE have many vital roles in the eye and are responsible for the phagocytosis of photoreceptor outer segments and the transfer of nutrients from the choroid to the retina, in addition to many other essential functions. Complement activation on RPE may contribute to inflammation and cell death in AMD. iPSC-RPE were selected for this model because they maintain the morphology of native RPE and share similar cell markers. Measuring complement deposition on the surface of these cells can thus model how certain drug treatments limit complement activation in the retina during AMD disease course.
[0217] A cell-ELISA was used to measure complement deposition on the surface of iPSC-RPE. iPSC-RPE (FujiFilm Cellular Dynamics, Madison, WI) were grown in a fibronectin-coated black/clear bottom 96-well plate. CRP (100 g/mL) (ImmunoPrecise Antibodies, Utrecht, The Netherlands), 10% normal human serum (Complement Technology, Tyler, TX) and complement inhibitors being tested were added to cell culture media and incubated with the iPSC-RPE overnight. The next day, the cells were washed and fixed with 4% paraformaldehyde. After blocking, the cells were incubated with an anti-C3d or anti-C5b9 HRP-conjugated antibody (Novus Biologicals, Centennial CO). QuantaRed Enhanced Chemifluorescent HRP Substrate (Thermo Fisher, Waltham, MA) was used to develop a fluorescent signal that was measured using a plate reader. The data show that individual treatment with either anti-C1s or anti-Bb scFabs led to a significant decrease in C3d and C5b9 deposition on iPSC-RPE; however, the combination of both scFabs decreased deposition of complement products to the greatest extent (FIGS. 7A and 7B).
[0218] A similar method was used for fluorescent imaging of C5b9 deposition on iPSC-RPE. In this method, cells were grown on fibronectin-coated 24-well hanging cell culture inserts. Cells were treated with CRP, 10% normal human serum, and complement inhibitors overnight. Confocal microscopy was used to capture z-stack images at 40 magnification. For image quantification, three regions of interest (ROIs) were randomly imaged from each sample. Total areas of C5b9 were calculated within each ROI and were averaged for each sample. The average of three replicates was measured for each group and error bars were calculated from the average of standard deviations. The fluorescent imaging experiment was repeated three times with three different iPSC-RPE cell lines. The data similarly show that treatment with a combination of anti-C1s and anti-Bb scFabs led to a stark decrease in C5b9 (red) staining (FIGS. 8A and 8B).
[0219] In conclusion, the results from the iPSC-RPE model show that both the classical and alternative pathways likely play a role in AMD pathogenesis. Blocking each pathway separately led to a decrease in complement deposition on RPE cells. However, inhibiting both pathways simultaneously led to the greatest decrease in deposition, suggesting that concurrent classical and alternative pathway inhibition may be beneficial in AMD
Example 7: In Vivo Retina Studies in Non-Human Primates
[0220] This Example describes in vivo testing of exemplary vectorized antibody constructs in non-human primates (NHPs) to confirm transduction and transgene expression in the retina. Activity of the viral vectors following intravitreal administration in NHPs was evaluated in two studies: (1) a 6-week dose-range study of AAV2 #14 and AAV2 #12 and (2) an 8-week single-dose study of AAV2 #14 and AAV2 #9. In each study, NHPs administered ocular formulation buffer were used as controls.
A. Study 1
[0221] In the first study, NHPs were administered through intravitreal injection ocular formulation buffer (N=2 NHPs), or AAV2 #14 or AAV2 #12 at three doses (210.sup.9, 210.sup.10, or 210.sup.11 vg per eye, based on vector titer determined using an assay that detects the BGH poly (A); N=3 NHPs per treatment group). The animals were assessed after six weeks of in-life exposure.
[0222] For evaluation of vector transduction, vector genome levels were quantified by using vector-specific TaqMan assays in quantitative PCR analyses of DNA purified from the NHP retinas. Comparable DNA input across samples was confirmed using TUBB1 as a reference gene. The data show that both AAV2 #12 and AAV2 #14 successfully transduced the NHP retina, resulting in a dose-dependent increase in vector genome levels (dose-response AAV2 #14 p=0.0286, AAV2 #12 p=0.0095). Table 9 below summarizes the level of transduction achieved in the NHP retina at 6 weeks after intravitreal administration (median vector genomes/500 ng genomic DNA).
TABLE-US-00013 TABLE 9 vector genome levels 6 weeks after dosing Dose Construct 2 10.sup.9 vg 2 10.sup.10 vg 2 10.sup.11 vg AAV2#14 4.85 10.sup.3 2.8 10.sup.4 1.2 10.sup.5 AAV2#12 2.3 10.sup.3 4.9 10.sup.4 9.2 10.sup.5
[0223] For evaluation of transgene expression, vector-derived transgene levels were quantified using transcript-specific TaqMan assays in quantitative RT-PCR analyses of RNA purified from the NHP retinas. RNA quality was assessed, and all samples were shown to have an RNA integrity number (RIN) greater than 7.5. One sample was not included in RNA analyses due to low RNA input. Transcript levels were quantified relative to a double-stranded plasmid DNA standard curve. The data show that transduction of both AAV2 #12 and AAV2 #14 leads to dose-dependent levels of transgene expression in the NHP retina (dose-response AAV2 #14 p=0.0286, AAV2 #12 p=0.0286). Table 10 below summarizes transcript abundance in the NHP retina at 6 weeks after intravitreal administration (median transcripts/500 ng RNA).
TABLE-US-00014 TABLE 10 transcript abundance 6 weeks after dosing Dose Construct 2 10.sup.9 vg 2 10.sup.10 vg 2 10.sup.11 vg AAV2#14 1.8 10.sup.4 4.5 10.sup.4 6.3 10.sup.4 AAV2#12 1.1 10.sup.4 2.5 10.sup.4 1.1 10.sup.5
B. Study 2
[0224] In the second study, NHPs were administered through intravitreal injection ocular formulation buffer (N=2 NHPs), or AAV2 #14 or AAV2 #9 at 210.sup.11 vg per eye (N=3 NHPs per vector treatment group). The vector titer was determined based on an assay that detects the BGH poly (A). The animals were assessed over 8 weeks of in-life exposure. Due to the presence of serum AAV2 neutralizing antibodies (Nab), all study 2 NHPs were administered an IgG degrading enzyme (IdeS) by intravitreal administration 2 days prior to vector dosing.
[0225] For evaluation of vector transduction, vector genome levels were quantified using vector-specific TaqMan assays in quantitative PCR analyses of DNA purified from the NHP retinas. For AAV2 #9, vector genome levels were assessed using two different assays that detect the anti-Bb and anti-C1s arms. Comparable DNA input across samples was confirmed using TUBB as a reference gene. Despite potential hindrance by pre-existing AAV2 Nabs, the data show that both AAV2 #14 and AAV2 #9 successfully transduced the NHP retina, with AAV2 #14 achieving about 9.310.sup.3 vg and AAV2 #9 achieving levels between about 7.610.sup.4 and about 2.810.sup.5 vg at 8 weeks after intravitreal administration (median vector genomes/500 ng genomic DNA).
[0226] For evaluation of transgene expression, vector-derived transgene levels were quantified using transcript-specific TaqMan assays in quantitative RT-PCR analyses of RNA purified from the NHP retinas. For AAV2 #9, the anti-Bb and anti-C1s transcripts are expressed independently and were therefore assessed separately. RNA quality was assessed, and all samples were shown to have a RNA integrity number (RIN) greater than 7.5. Transcript levels were quantified relative to a double-stranded plasmid DNA standard curve. After 8 weeks of in-life exposure, AAV2 #14 resulted in abundance levels of about 9.710.sup.4 transcripts and AAV2 #9 resulted in about 1.610.sup.6 anti-Bb transcripts and about 3.310.sup.5 anti-C1s transcripts (median transcripts per 500 ng retina RNA).
C. Persistence Study in NHPs
[0227] The pharmacology and persistence across multiple dose levels of AAV2 #9 were evaluated in a study with a 16-week in-life assessment that included a 6-week interim necropsy.
[0228] NHPs (cynomolgus macaque) were administered through bilateral intravitreal injection the formulation buffer (180 mM NaCl, 5 mM sodium phosphate, 0.01% PS20, pH 7.4), or AAV2 #9 at multiple dose levels (based on vector titer determined by droplet digital PCR (ddPCR) analyses using a vector-specific assay targeting the anti-C1s region of AAV2 #9). All NHPs were given prophylactic steroids (1 mg/kg daily oral prednisolone) beginning two weeks prior to vector dosing and continuing throughout the entire study duration. Vector genome levels in the NHP retina were quantified using vector-specific C1s and Bb Taqman assays in quantitative PCR analyses of DNA purified from the right eye.
[0229] The C1s and Bb assays detected comparable vector genome levels within each sample across the 6- and 16-week timepoints. At 6 weeks, AAV2 #9 transduction resulted in a dose-dependent increase in vector genome levels in the retina. A dose-dependent increase in retina transduction was also observed at 16 weeks.
[0230] Vector biodistribution in the NHP eye was assessed using an AAV2 #9 vector-specific probe set (containing 40 pairs of probes that each span about 50 bases, designed to detect the sense strand of the vector genome) in RNAscope ISH analyses. At 6- and 16-weeks, vector was detected in the retina and iris-ciliary body of eyes administered AAV2 #9. No vector was detected in the optic nerve. In the retina, vector was present in RGCs and in rare cells of the INL, often in the foveal and parafoveal region of the macula.
[0231] Levels of AAV2 #9-derived anti-C1s and anti-Bb transcripts in the NHP retina were quantified using C1s- and Bb-specific Taqman assays in quantitative RT-PCR analyses of RNA purified from the right eye. Transcript levels were quantified relative to a double-stranded plasmid DNA standard curve. Transcript levels in both the 6- and 16-week cohorts were highly correlated with vector genome levels (Spearman r0.97). At 6 weeks, AAV2 #9 transduction resulted in a dose-dependent increase in transcript levels in the retina. A dose-dependent trend of increasing transcript levels was also observed at 16 weeks.
[0232] To assess the kinetics of peak scFab expression and persistence over time, aqueous humor was collected at baseline and during weeks 3, 6, 12, and 16. Vitreous humor was collected at necropsy. In the aqueous humor collected from some NHPs in the 16-week cohort, scFab levels peaked between 3-6 weeks and persisted through the end of the study at 4 months (day 113). Levels of scFabs in the vitreous humor at 4 months were similar to or higher than levels in the aqueous humor.
D. Efficacy Study Evaluating AAV2 #9 Inhibition of LPS-Induced Complement Activation and Ocular Inflammation in NHPs
[0233] The ability of AAV2 #9-derived scFabs to inhibit complement pathway activation in vivo was assessed using an acute model of endotoxin-induced inflammation.
[0234] NHPs (cynomolgus macaque) were administered through bilateral intravitreal injection the formulation buffer (180 mM NaCl, 5 mM sodium phosphate, 0.01% PS20, pH 7.4) or AAV2 #9, followed by bilateral intravitreal lipo-polysaccharide (LPS) administration on day 41 [0.5 endotoxin units (EU) LPS per eye from Escherichia coli 0111: B4; Sigma-Aldrich L4391]. NHPs were given prophylactic steroids (1 mg/kg daily oral prednisolone) beginning two weeks prior to vector dosing and continuing daily for four weeks. NHPs were tapered off prednisolone prior to LPS administration on day 41. Study endpoints were assessed two days after LPS treatment (day 43), which induced high levels of ocular inflammation (FIG. 9).
[0235] Free drug levels of AAV2 #9-derived scFabs in the aqueous and vitreous humors were measured using Bb and C1s target-capture ELISAs. At the end of the study on day 43, aqueous humor and vitreous humor levels of free anti-C1s scFab averaged about 50-100 ng/ml (1-2 nM) and median levels of free anti-Bb scFab reached about 100-200 ng/mL (2-4 nM). The levels of the anti-C1s scFab in both the aqueous humor and vitreous humor were above the equilibrium dissociation constant of the anti-C1s scFab for both human and cynomolgus C1s (human K.sub.D=0.34 nM; cynomolgus K.sub.D=0.016 nM). The anti-Bb scFab had a lower affinity for cynomolgus Bb (K.sub.D=14.8 nM) compared to human Bb (K.sub.D=3.7 nM), and the levels of anti-Bb scFab reached in the aqueous humor and vitreous humor in this study were below the K.sub.D of the anti-Bb scFab for cynomolgus Bb and were not sufficient for inhibition of Bb in NHP eye.
[0236] To assess complement pathway activation, we used a multiplexed ELISA from Quidel to measure activation fragment levels of C4a (classical pathway), Ba (alternative pathway) and sC5b9 (terminal pathway) in the aqueous humor. Compared to non-LPS treated control eyes, LPS-treated eyes had increased levels of Ba, C4a, and sC5b9 in the aqueous humor, demonstrating activation of the alternative, classical, and terminal pathways. LPS-treated eyes dosed with AAV2 #9 had reduced levels of C4a and sC5b9 compared to LPS-treated control eyes, demonstrating inhibition of the classical and terminal pathways. Inhibition of the alternative pathway (Ba) was not detected in AAV2 #9-treated eyes, likely due to the lower affinity of the anti-Bb scFab for the cynomolgus target.
[0237] Ocular exams performed two days after LPS dosing detected ocular inflammation in all treatment groups. However, eyes treated with AAV2 #9 had reduced severity and frequency of clinical indicators of inflammation scored using the SPOTS system.
TABLE-US-00015 SEQUENCES SEQIDNO:1-HCDR1ofanti-C1santibody DDYIH SEQIDNO:2-HCDR2ofanti-C1santibody RIDPADGHTKYAPKFQV SEQIDNO:3-HCDR3ofanti-C1santibody YGYGREVEDY SEQIDNO:4-LCDR1ofanti-C1santibody KASQSVDYDGDSYMN SEQIDNO:5-LCDR2ofanti-C1santibody DASNLES SEQIDNO:6-LCDR3ofanti-C1santibody QQSNEDPWT SEQIDNO:7-V.sub.Hofanti-C1santibody(KabatCDRsunderlined) QVQLVQSGAEVKKPGASVKLSCTASGENIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKY APKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVEDYWGQGTTVTVSS SEQIDNO:8-V.sub.Lofanti-C1santibody(KabatCDRsunderlined) DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGOPPKILIYDASNLES GIPARESGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIK SEQIDNO:9-C1sscFv QVOLVOSGAEVKKPGASVKLSCTASGENIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKY APKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVEDYWGQGTTVTVSSG GGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQ PPKILIYDASNLESGIPARFSGSGSGTDETLTISSLEPEDFAIYYCQQSNEDPWTFGGGT KVEIK SEQIDNO:10-heavychainofanti-C1sFab QVOLVOSGAEVKKPGASVKLSCTASGENIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKY APKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVFDYWGQGTTVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQIDNO:11-lightchainofanti-C1sFab DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGOPPKILIYDASNLES GIPARFSGSGSGTDETLTISSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC SEQIDNO:12-C1sscFab DIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQKPGQPPKILIYDASNLES GIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIKRTVAAPSVE IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKLSCTASGFNIKDDYIHWVKQAPGOGLEW IGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVED YWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQIDNO:13-HCDR1ofanti-Bbantibody NYAMS SEQIDNO:14-HCDR2ofanti-Bbantibody TISNRGSYTYYPDSVKG SEQIDNO:15-HCDR3ofanti-Bbantibody ERPMDY SEQIDNO:16-LCDR1ofanti-Bbantibody KASQDVGTAVA SEQIDNO:17-LCDR2ofanti-Bbantibody WASTRHT SEQIDNO:18-LCDR3ofanti-Bbantibody HQHSSNPLT SEQIDNO:19-V.sub.Hofanti-Bbantibody(KabatCDRsboxed) [00001]![embedded image]()
[00002]![embedded image]()
SEQIDNO:20-V.sub.Lofanti-Bbantibody(KabatCDRsboxed) [00003]![embedded image]()
[00004]![embedded image]()
SEQIDNO:21-BbscFv EVOLVESGGGLVKPGGSLRLSCAASGFTESNYAMSWVRQAPGKRLEWVATISNRGSYTYY PDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSSGGGGS GGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYW ASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK SEQIDNO:22-heavychainofanti-BbFab EVOLVESGGGLVKPGGSLRLSCAASGFTESNYAMSWVRQAPGKRLEWVATISNRGSYTYY PDSVKGRFTISRDNAKNSLYLOMNSLRAEDTALYYCARERPMDYWGQGTLVTVSSASTKG PSVEPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQIDNO:23-lightchainofanti-BbFab DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPD RFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC SEQIDNO:24-BbscFab DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPD RFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVOLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATI SNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLV TVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAV LOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQIDNO:25-[C1sscFab-(G.sub.4S).sub.3-BbscFab]nucleicacid sequence(construct#7,FIG.2A) ATGGAAGCCCCCGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACTGGC GATATCGTGCTGACACAGAGCCCTGATAGCCTGGCTGTTAGCCTGGGCGAACGCGCCACA ATCAGCTGCAAGGCCAGCCAGTCTGTGGATTATGATGGTGACAGCTACATGAACTGGTAC CAGCAGAAGCCCGGACAGCCTCCTAAGATCCTGATCTACGACGCCAGCAACCTGGAATCC GGCATTCCTGCCCGGTTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTGACCATCTCC AGCCTGGAACCCGAGGATTTCGCCATCTACTACTGTCAGCAGAGCAATGAGGACCCATGG ACCTTCGGCGGCGGTACCAAGGTCGAGATCAAGAGAACAGTGGCCGCTCCTAGCGTGTTC ATCTTCCCTCCATCAGACGAGCAGCTGAAGAGCGGAACCGCTTCTGTGGTGTGTCTGCTC AACAATTTCTACCCTAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCTCTCCAGAGC GGCAACAGCCAGGAGAGCGTGACCGAGCAAGATAGCAAGGACAGCACCTACTCTTTAAGC TCTACACTGACGCTGTCCAAGGCTGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTG ACCCACCAGGGCCTGAGCAGCCCTGTGACAAAGAGCTTCAACAGAGGCGAGTGCGGCGGC GGAGGCAGCGGCGGCGGAGGCTCTGGCGGTGGCGGAAGCGGCGGGGGAGGCTCTGGCGGC GGCGGCAGTGGCGGCGGCGGCAGCGGAGGAGGAGGATCGCAAGTGCAACTGGTCCAGTCT GGCGCCGAGGTGAAAAAGCCTGGAGCCAGCGTGAAACTGTCATGCACCGCCTCCGGGTTT AACATCAAAGATGACTACATCCACTGGGTGAAACAGGCTCCAGGACAGGGCCTGGAGTGG ATCGGCAGAATCGACCCTGCGGATGGCCACACCAAGTACGCCCCAAAGTTCCAGGTGAAG GTGACAATCACAGCTGACACCAGCACCAGCACAGCCTACCTGGAACTGAGCAGCCTAAGA AGCGAGGACACCGCCGTGTACTACTGCGCCCGGTACGGCTACGGCCGGGAAGTGTTCGAC TACTGGGGTCAGGGCACCACCGTGACGGTGAGTAGCGCCTCTACAAAAGGCCCTTCCGTG TTCCCCCTGGCCCCTTGCAGCCGGAGCACCAGCGAGAGCACCGCCGCCTTGGGCTGTCTG GTGAAAGACTATTTCCCAGAGCCTGTCACAGTGTCTTGGAACTCCGGAGCCCTCACCTCT GGAGTGCACACATTTCCCGCCGTGCTGCAGAGCAGCGGCTTGTACTCTCTGAGCAGCGTG GTGACAGTGCCCTCTAGCAGCCTGGGCACAAAGACCTACACCTGCAACGTGGACCACAAG CCTTCTAACACCAAGGTGGATAAGAGAGTGGGTGGCGGAGGAAGCGGCGGCGGAGGAAGC GGCGGCGGCGGGTCCGATATTCAGATGACCCAGAGCCCTTCTACCCTTAGTGCCTCTGTT GGAGACCGGGTGACCATCACCTGTAAAGCCTCCCAGGACGTGGGAACAGCAGTTGCTTGG TATCAGCAAAAGCCCGGCAAGGCCCCTAAGTTGCTGATCTACTGGGCCTCCACAAGACAC ACCGGCGTGCCTGATAGATTCAGCGGTAGCGGCAGCGGCACCGATTTTACCCTGACAATC AGCTCTCTGCAGGCCGAGGACTTTGCCGTGTACTTCTGCCACCAGCATTCTAGCAATCCT CTGACTTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGGACAGTAGCCGCTCCTTCTGTA TTTATCTTCCCACCTTCTGACGAGCAGCTGAAGTCTGGTACCGCAAGCGTGGTGTGCCTG CTGAACAACTTCTACCCCAGAGAGGCCAAAGTGCAATGGAAGGTGGACAACGCCCTGCAG AGTGGCAATAGCCAGGAGTCTGTCACTGAGCAGGACTCCAAGGATAGCACCTACAGCCTG TCTTCTACACTCACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAG GTGACACACCAGGGCCTGTCTTCCCCTGTGACCAAAAGCTTCAACCGGGGCGAGTGCGGG GGCGGCGGAAGCGGTGGCGGCGGGTCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGA GGCGGCGGCAGTGGTGGGGGCGGCTCGGGCGGCGGAGGCTCTGAGGTGCAGCTGGTGGAA AGTGGCGGAGGCCTGGTGAAGCCCGGCGGCAGCCTGAGACTGAGTTGCGCCGCGAGCGGA TTCACTTTCTCCAACTACGCCATGTCTTGGGTGAGACAGGCCCCTGGCAAAAGACTGGAA TGGGTCGCTACCATCAGCAACAGAGGTAGCTACACATACTACCCTGATAGCGTGAAAGGC AGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTATCTGCAGATGAACAGCCTG CGGGCCGAAGATACAGCCCTTTATTACTGCGCGAGAGAGAGACCCATGGACTACTGGGGC CAGGGAACACTGGTGACCGTTTCAAGCGCCTCTACCAAGGGCCCCTCTGTGTTTCCTCTG GCCCCTTGTTCTCGGAGCACCTCCGAGAGCACCGCTGCTCTGGGATGCCTCGTGAAGGAC TATTTCCCCGAACCCGTGACCGTGTCCTGGAACAGCGGCGCCCTGACAAGCGGGGTCCAC ACCTTCCCCGCCGTCCTGCAGAGTTCTGGACTGTACAGCCTGAGCAGCGTCGTCACAGTG CCTTCAAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCATAAGCCTTCCAAT ACCAAGGTGGACAAGAGAGTTTGA SEQIDNO:26-[C1sscFab-(G.sub.4S).sub.3-BbscFab]aminoacid(signal peptideboldfaced)(construct#7,FIG.2A) MEAPAQLLFLLLLWLPDTTGDIVLTOSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGF NIKDDYIHWVKQAPGOGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLR SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAW YQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNP LTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVOWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASG FTESNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSL RAEDTALYYCARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRV* SEQIDNO:27-[C1sscFab-(G.sub.4S).sub.3-BbscFab-CM]nucleicacid sequence(construct#11,FIG.2C) ATGGAAGCCCCCGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACTGGC GATATCGTGCTGACACAGAGCCCTGATAGCCTGGCTGTTAGCCTGGGCGAACGCGCCACA ATCAGCTGCAAGGCCAGCCAGTCTGTGGATTATGATGGTGACAGCTACATGAACTGGTAC CAGGAGAAGCCCGGACAGCCTCCTAAGATCCTGATCTACGACGCCAGCAACCTGGAATCC GGCATTCCTGCCCGGTTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTGACCATCTCC AGCCTGGAACCCGAGGATTTCGCCATCTACTACTGTCAGCAGAGCAATGAGGACCCATGG ACCTTCGGCGGCGGTACCAAGGTCGAGATCAAGAGAACAGTGGCCGCTCCTAGCGTGTTC ATCTTCCCTCCATCAGACGAGCAGCTGAAGAGCGGAACCGCTTCTGTGGTGTGTCTGCTC AACAATTTCTACCCTAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCTCTCCAGAGC GGCAACAGCCAGGAGAGCGTGACCGAGCAAGATAGCAAGGACAGCACCTACTCTTTAAGC TCTACACTGACGCTGTCCAAGGCTGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTG ACCCACCAGGGCCTGAGCAGCCCTGTGACAAAGAGCTTCAACAGAGGCGAGTGCGGCGGC GGAGGCAGCGGCGGCGGAGGCTCTGGCGGTGGCGGAAGCGGCGGGGGAGGCTCTGGCGGC GGCGGCAGTGGCGGCGGCGGCAGCGGAGGAGGAGGATCGCAAGTGCAACTGGTCCAGTCT GGCGCCGAGGTGAAAAAGCCTGGAGCCAGCGTGAAACTGTCATGCACCGCCTCCGGGTTT AACATCAAAGATGACTACATCCACTGGGTGAAAAAGGCTCCAGGACAGGGCCTGGAGTGG ATCGGCAGAATCGACCCTGCGGATGGCCACACCAAGTACGCCCCAAAGTTCCAGGTGAAG GTGACAATCACAGCTGACACCAGCACCAGCACAGCCTACCTGGAACTGAGCAGCCTAAGA AGCGAGGACACCGCCGTGTACTACTGCGCCCGGTACGGCTACGGCCGGGAAGTGTTCGAC TACTGGGGTCAGGGCACCACCGTGACGGTGAGTAGCGCCTCTACAAAAGGCCCTTCCGTG TTCCCCCTGGCCCCTTGCAGCCGGAGCACCAGCGAGAGCACCGCCGCCTTGGGCTGTCTG GTGAAAGACTATTTCCCAGAGCCTGTCACAGTGTCTTGGAACTCCGGAGCCCTCACCTCT GGAGTGCACACATTTCCCGCCGTGCTGCAGAGCAGCGGCTTGTACTCTCTGAGCAGCGTG GTGACAGTGCCCTCTAGCAGCCTGGGCACAAAGACCTACACCTGCAACGTGGACCACAAG CCTTCTAACACCAAGGTGGATAAGAGAGTGGGTGGCGGAGGAAGCGGCGGCGGAGGAAGC GGCGGCGGCGGGTCCGATATTCAGATGACCCAGAGCCCTTCTACCCTTAGTGCCTCTGTT GGAGACCGGGTGACCATCACCTGTAAAGCCTCCCAGGACGTGGGAACAGCAGTTGCTTGG TATCAGAAAAAGCCCGGCAAGGCCCCTAAGTTGCTGATCTACTGGGCCTCCACAAGACAC ACCGGCGTGCCTGATAGATTCAGCGGTAGCGGCAGCGGCACCGATTTTACCCTGACAATC AGCTCTCTGCAGGCCGAGGACTTTGCCGTGTACTTCTGCCACCAGCATTCTAGCAATCCT CTGACTTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGGACAGTAGCCGCTCCTGCTGTA TTTATCTTCCCACCTTCTGACGAGCAGCTGAAGTCTGGTACCGCAAGCGTGGTGTGCCTG CTGAAGAACTTCTACCCCAGAGAGGCCAAAGTGCAATGGAAGGTGGACAACGCCCTGCAG AGTGGCAATAGCCAGGAGTCTGTCACTGAGCAGGACTCCAAGGATAGCACCTACAGCCTG TCTTCTACACTCACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAG GTGACACACCAGGGCCTGTCTTCCCCTGTGACCAAAAGCTTCAACCGGGGCGAGTGCGGG GGCGGCGGAAGCGGTGGCGGCGGGTCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGA GGCGGCGGCAGTGGTGGGGGCGGCTCGGGCGGCGGAGGCTCTGAGGTGCAGCTGGTGGAA AGTGGCGGAGGCCTGGTGAAGCCCGGCGGCAGCCTGAGACTGAGTTGCGCCGCGAGCGGA TTCACTTTCTCCAACTACGCCATGTCTTGGGTGAGAGAGGCCCCTGGCAAAAGACTGGAA TGGGTCGCTACCATCAGCAACAGAGGTAGCTACACATACTACCCTGATAGCGTGAAAGGC AGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTATCTGCAGATGAACAGCCTG CGGGCCGAAGATACAGCCCTTTATTACTGCGCGAGAGAGAGACCCATGGACTACTGGGGC CAGGGAACACTGGTGACCGTTTCAAGCGCCTCTACCAAGGGCCCCTCTGTGTTTCCTCTG GCCCCTTGTTCTCGGAGCACCTCCGAGAGCACCGCTGCTCTGGGATGCCTCGTGAAGGAC TATTTCCCCGAACCCGTGACCGTGTCCTGGAACAGCGGCGCCCTGACAAGCGGGGTCCAC ACCTTCCCCGCCGTCCTGCAGAGTTCTGGACTGTACAGCCTGAGCAGCGTCGTCGAAGTG CCTTCAAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCATAAGCCTTCCAAT ACCAAGGTGGACAAGAGAGTTTGA SEQIDNO:28-[ClsscFab-(G.sub.4S).sub.3-BbscFabCM]aminoacid sequence(construct#11,FIG.2C)(signalpeptideboldfaced;charge mutationsboxedanditalicized,numberingexcludingsignalpeptide: Q42EandQ292KinClsscFab,andQ523K,S599A,N622K,Q773Eand T919EinBbscFab) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY [00005]![embedded image]()
TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGF [00006]![embedded image]()
SEDTAVYYCARYGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAW [00007]![embedded image]()
[00008]![embedded image]()
SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASG [00009]![embedded image]()
RAEDTALYYCARERPMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD [00010]![embedded image]()
TKVDKRV* SEQIDNO:29-[BbscFab-(G.sub.4S).sub.3-C1sscFab]nucleicacid sequence(construct#8,FIG.2A) ATGGAAGCCCCCGCCCAGCTGCTTTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGC GATATCCAGATGACCCAGAGCCCTAGCACCTTGAGCGCCTCTGTGGGCGACAGAGTGACC ATCACCTGCAAGGCCAGCCAGGACGTGGGCACAGCCGTGGCTTGGTATCAGCAAAAACCT GGCAAGGCCCCTAAGCTGCTGATTTACTGGGCCAGCACCAGACACACAGGCGTGCCTGAC CGGTTTAGCGGCAGTGGCAGCGGGACAGATTTTACCCTGACCATCAGCTCTCTGCAGGCC GAGGACTTCGCTGTGTACTTCTGCCACCAGCACAGCAGCAACCCCCTGACCTTTGGCCAG GGCACCAAGCTGGAGATCAAGCGGACCGTGGCCGCACCCAGTGTGTTTATCTTCCCCCCC AGCGATGAGCAGCTGAAGAGCGGCACAGCCAGCGTGGTGTGTCTGCTGAACAACTTCTAC CCTAGAGAGGCTAAGGTGCAGTGGAAGGTGGATAATGCTCTGCAGAGCGGAAATAGCCAG GAGTCTGTGACCGAGCAGGACAGCAAGGACTCCACATACAGCCTCTCCTCCACCCTGACA CTGTCCAAGGCCGATTACGAGAAGCACAAAGTGTACGCCTGCGAGGTGACACACCAGGGC CTTAGCAGCCCTGTCACCAAATCTTTCAACAGAGGAGAGTGCGGCGGCGGCGGCTCCGGC GGCGGCGGATCTGGAGGCGGAGGCAGCGGAGGCGGGGGAAGCGGCGGAGGCGGCAGCGGC GGCGGAGGTTCCGGCGGAGGCGGCTCAGAGGTGCAACTCGTGGAAAGCGGTGGCGGCCTG GTTAAGCCCGGCGGCAGCCTGCGCCTGTCATGCGCTGCAAGCGGCTTCACCTTTTCAAAT TACGCCATGAGCTGGGTGCGGCAGGCTCCTGGAAAACGGCTGGAATGGGTGGCTACAATC TCTAACCGGGGCTCTTACACCTACTACCCCGATAGCGTGAAAGGCAGATTCACAATCAGC CGGGACAACGCCAAGAACTCACTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACA GCTCTGTACTACTGTGCCAGAGAAAGACCCATGGACTACTGGGGACAGGGCACACTGGTT ACAGTCTCCTCTGCCTCCACGAAGGGCCCCAGCGTGTTCCCTCTGGCTCCTTGTAGCAGA AGCACTTCTGAATCTACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCT GTGACCGTTAGCTGGAACAGCGGAGCCCTGACAAGCGGAGTGCATACATTCCCTGCCGTG CTGCAGAGCAGCGGCCTCTACAGCCTGTCCTCGGTGGTGACCGTCCCCTCAAGCAGCCTG GGCACCAAGACCTACACTTGCAACGTGGACCATAAGCCTAGCAACACAAAGGTGGACAAG AGAGTCGGAGGCGGAGGTGGCTCCGGCGGCGGTGGCTCTGGCGGAGGCGGCAGCGACATC GTGCTGACCCAAAGCCCTGACAGCCTGGCCGTGTCCCTGGGAGAGCGGGCCACGATCTCC TGCAAGGCCTCCCAATCCGTGGACTATGATGGCGATAGCTACATGAACTGGTACCAGCAG AAGCCTGGCCAGCCTCCAAAGATCCTGATTTACGACGCCTCTAATCTGGAATCCGGCATC CCTGCTAGATTCAGCGGAAGCGGTAGCGGCACCGACTTCACCCTGACAATCAGCAGTCTG GAGCCAGAGGACTTCGCCATCTACTACTGTCAGCAGTCTAACGAGGATCCTTGGACCTTC GGCGGCGGCACCAAGGTGGAAATCAAGAGAACCGTGGCCGCCCCTAGCGTCTTCATCTTC CCTCCTAGTGATGAGCAGCTGAAAAGCGGCACAGCCAGCGTGGTGTGCCTCCTGAACAAC TTCTACCCGCGCGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGTCCGTGACAGAGCAAGATAGCAAGGACAGCACCTACTCCCTGTCGTCTACA CTTACCCTGTCTAAAGCCGACTATGAGAAGCACAAGGTATACGCCTGTGAAGTGACCCAC CAGGGGCTGTCCTCTCCAGTAACCAAGTCCTTCAACAGAGGCGAATGCGGCGGAGGCGGA TCTGGCGGCGGCGGCTCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGGGGCGGAGGC AGCGGCGGCGGAGGAAGCGGAGGCGGAGGCAGCCAGGTGCAGCTGGTGCAGTCAGGCGCT GAGGTGAAAAAGCCTGGCGCCAGCGTCAAGCTGTCTTGCACCGCTTCTGGCTTTAACATC AAGGACGACTACATCCACTGGGTCAAGCAGGCCCCCGGGCAAGGGCTGGAGTGGATCGGC AGAATCGACCCTGCCGACGGCCACACCAAGTACGCCCCTAAGTTCCAGGTGAAGGTGACA ATCACAGCTGATACCAGCACGAGCACCGCCTACCTGGAACTGTCATCCCTCAGATCTGAA GATACAGCCGTTTACTACTGCGCAAGGTACGGGTACGGGCGGGAAGTGTTCGACTATTGG GGCCAGGGCACAACCGTGACCGTGAGCAGCGCCTCTACCAAAGGCCCTAGCGTGTTCCCC CTGGCTCCTTGCAGCAGATCTACAAGCGAGAGCACAGCCGCCCTGGGATGTCTGGTTAAA GATTATTTCCCAGAACCTGTGACAGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTG CACACCTTCCCAGCCGTGCTGCAGTCATCCGGTCTGTATAGCCTGAGCAGCGTGGTTACC GTGCCCAGCTCTAGCCTGGGCACCAAAACCTACACCTGCAATGTGGACCACAAGCCAAGC AATACCAAGGTTGATAAGAGAGTCTGA SEQIDNO:30-[BbscFab-(G.sub.4S).sub.3-QC1sscFab]aminoacidsequence (construct#8,FIG.2A)(signalpeptideboldfaced) MEAPAQLLFLLLLWLPDTTGDIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKP GKAPKLLIYWASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTEGO GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVOWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVOLVESGGGLVKPGGSLRLSCAASGFTESN YAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDT ALYYCARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVGGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWYQQ KPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKLSCTASGENI KDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSE DTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRV* SEQIDNO:31-[BbscFab-(g.sub.4s).sub.3-C1SscfAB-CM]nucleicacid sequence(construct#12,FIG.2C) ATGGAAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTACTGTGGCTGCCTGATACCACCGGC GATATCCAGATGACGCAGAGTCCCAGCACCCTGAGCGCCTCTGTGGGCGACCGGGTGACC ATCACCTGTAAAGCCTCCCAGGACGTGGGCACAGCTGTTGCTTGGTATCAGAAAAAGCCT GGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCAGCACAAGACACACAGGAGTGCCTGAC AGATTCAGCGGCAGCGGCTCTGGGACTGATTTCACCTTGACAATCAGCTCTCTGCAGGCC GAGGACTTTGCCGTGTACTTCTGCCACCAACACAGTTCTAACCCCCTGACCTTCGGCCAA GGAACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCTGCCGTGTTCATCTTCCCTCCA AGCGATGAGCAGCTGAAAAGCGGCACCGCGTCCGTCGTGTGCCTGCTGAAGAACTTCTAC CCGAGAGAAGCGAAGGTGCAGTGGAAAGTCGACAACGCCCTGCAGAGCGGAAATAGCCAG GAGAGCGTGACCGAACAAGACTCTAAGGACAGCACCTACTCGCTGTCCTCCACGCTGACT CTGTCTAAGGCCGACTATGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGC CTGAGCAGCCCCGTTACCAAGAGCTTCAACAGAGGAGAATGCGGCGGAGGTGGCAGCGGC GGCGGCGGGAGCGGCGGCGGCGGCTCAGGCGGAGGGGGAAGTGGCGGCGGCGGCAGCGGC GGCGGAGGCAGCGGCGGTGGCGGCTCTGAGGTGCAACTGGTGGAATCTGGGGGCGGACTG GTGAAGCCTGGCGGCAGTCTGAGACTGAGCTGTGCCGCTTCCGGATTCACCTTTAGCAAT TACGCCATGAGCTGGGTGCGGGAGGCCCCTGGAAAGCGGCTGGAATGGGTTGCTACAATC AGCAATAGAGGCAGCTACACATACTACCCCGACAGTGTCAAAGGCCGGTTTACAATCAGC CGCGACAACGCCAAAAACAGCCTGTACCTGCAGATGAACTCCCTGCGGGCTGAGGATACA GCCCTCTACTACTGTGCCAGAGAACGTCCAATGGACTATTGGGGCCAAGGCACACTGGTG ACCGTGAGCAGCGCGTCTACCAAGGGCCCTTCTGTTTTCCCTCTGGCCCCCTGCAGCAGA AGCACGAGCGAGAGCACCGCTGCCCTGGGCTGTCTGGTGAAGGATTATTTCCCTGAGCCT GTGACCGTGTCTTGGAATAGCGGAGCCCTGACCAGCGGAGTGCATACATTCCCTGCTGTG CTGCAGTCTAGTGGGCTGTACAGCCTGTCTTCCGTTGTGGAAGTCCCTAGCAGCAGCCTG GGCACCAAGACCTACACCTGCAACGTGGATCATAAGCCAAGCAACACCAAGGTGGATAAG AGAGTGGGCGGTGGCGGAGGCTCGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATC GTGCTGACCCAGTCTCCAGATTCTCTGGCCGTGTCACTGGGAGAGAGAGCCACCATTAGC TGCAAGGCCTCTCAGAGCGTAGACTACGACGGCGACTCCTACATGAACTGGTACCAGGAA AAGCCTGGCCAGCCTCCTAAGATCTTGATCTACGATGCCTCCAATCTGGAGAGCGGGATC CCCGCTAGATTCAGCGGGTCTGGAAGTGGAACCGACTTCACACTGACCATCTCTAGCCTG GAGCCCGAGGACTTTGCCATCTACTACTGCCAGCAGAGCAACGAGGACCCCTGGACATTC GGCGGCGGCACAAAGGTTGAGATCAAGAGAACCGTTGCCGCTCCTAGCGTGTTTATCTTC CCTCCCTCTGACGAGCAGCTGAAGAGCGGCACAGCCTCCGTGGTGTGCCTGCTGAACAAC TTCTACCCCAGAGAGGCCAAGGTCCAGTGGAAGGTCGACAATGCCCTTCAGAGCGGCAAC AGCCAGGAGTCCGTGACCGAGCAGGATAGCAAGGACTCTACCTACAGCCTGTCCTCTACG CTGACCCTGAGCAAAGCCGATTACGAAAAGCACAAAGTGTACGCCTGTGAAGTGACACAC CAGGGCCTGTCTAGCCCTGTGACAAAGAGCTTTAACCGGGGCGAGTGCGGCGGCGGTGGA AGCGGAGGTGGAGGTTCAGGAGGCGGCGGAAGCGGAGGCGGAGGCAGTGGGGGCGGCGGC TCCGGCGGCGGCGGCAGCGGAGGCGGCGGTTCCCAAGTGCAGCTCGTGCAGAGCGGCGCC GAGGTGAAAAAGCCCGGAGCCAGCGTGAAGCTGTCTTGCACCGCCTCCGGATTCAACATC AAAGACGACTACATCCACTGGGTCAAGAAAGCCCCAGGGCAGGGGCTGGAGTGGATCGGC AGGATCGACCCTGCTGATGGCCACACCAAATACGCCCCAAAGTTCCAGGTGAAAGTGACA ATTACCGCAGATACCTCCACCAGCACCGCTTATCTGGAACTGAGCTCTCTGCGGAGCGAG GACACAGCCGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAAGTGTTCGACTACTGG GGCCAGGGCACCACAGTGACAGTGAGCTCTGCCAGCACAAAGGGCCCCAGCGTGTTTCCT CTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCACCGCCGCCCTGGGCTGCCTGGTGAAG GACTACTTCCCTGAACCCGTGACCGTCTCCTGGAACAGTGGCGCCTTGACCTCTGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCTCCGGCCTGTACAGCCTGTCTAGCGTGGTGACC GTGCCTAGCTCGAGCCTGGGCACAAAGACATATACCTGTAACGTGGACCACAAGCCCAGC AACACGAAGGTGGACAAGCGAGTGTGA SEQIDNO:32[BbscFab-(G.sub.4S).sub.3-C1sscFab-CM]aminoacidsequence (construct#12,FIG.2C)(signalpeptideboldfaced;chargemutations boxedanditalicized,numberingexcludingsignalpeptide:038K, S114A,N137K,Q288EandT434EinBbscFab,andQ520EandQ770Kin C1sscFab) [00011]![embedded image]()
GKAPKLLIYWASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQ [00012]![embedded image]()
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSN [00013]![embedded image]()
ALYYCARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEP [00014]![embedded image]()
[00015]![embedded image]()
KPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVOWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKLSCTASGENI [00016]![embedded image]()
DTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRV* SEQIDNO:33-[C1SscFAB-(G.sub.4S).sub.2-BbscFV]nucleicacidsequence (construct#13,FIG.2D) ATGGAAGCCCCAGCCCAGCTGCTGTTCCTGCTGCTGTTGTGGCTGCCCGATACAACAGGC GACATCGTGCTGACCCAGAGCCCCGACTCTCTGGCCGTGTCCCTGGGAGAAAGAGCCACA ATCTCCTGTAAAGCCTCTCAGAGCGTGGACTACGACGGCGATTCTTACATGAACTGGTAC CAACAGAAACCTGGACAGCCTCCTAAAATCCTGATCTACGACGCCTCAAACCTGGAAAGC GGCATCCCTGCCAGATTCTCAGGCTCCGGTAGCGGCACCGACTTCACACTGACCATCAGC AGCCTGGAACCTGAGGACTTCGCCATCTACTATTGTCAGCAAAGCAACGAGGACCCTTGG ACCTTCGGAGGCGGCACAAAGGTGGAAATCAAGCGGACCGTGGCAGCACCTTCTGTCTTC ATCTTCCCCCCATCCGATGAGCAGCTGAAGAGCGGCACAGCTAGTGTGGTGTGCCTGCTG AACAACTTCTACCCAAGAGAAGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCT GGTAATAGCCAGGAGAGCGTGACCGAGCAGGATTCTAAGGACAGCACATACAGTCTGTCT AGCACACTCACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTG ACCCACCAGGGCCTGTCTTCTCCGGTGACCAAGTCTTTCAACCGGGGCGAGTGCGGCGGC GGCGGAAGCGGCGGCGGCGGCAGCGGCGGCGGGGGCAGCGGCGGCGGTGGGTCTGGCGGC GGCGGATCAGGCGGAGGCGGCAGCGGCGGAGGCGGATCCCAAGTGCAGTTAGTTCAAAGC GGCGCTGAGGTGAAAAAGCCTGGCGCTTCTGTGAAGCTGAGCTGCACCGCCAGCGGTTTT AACATCAAGGACGACTACATCCACTGGGTGAAGCAGGCCCCTGGCCAGGGACTGGAGTGG ATCGGCAGAATCGACCCCGCTGACGGCCACACCAAATACGCCCCTAAGTTCCAGGTGAAA GTGACCATCACCGCTGATACCTCCACAAGCACCGCCTACCTGGAACTGTCCAGCCTGAGA AGCGAGGATACCGCCGTCTACTACTGTGCCAGATACGGCTACGGCAGAGAGGTGTTCGAC TACTGGGGACAAGGCACCACCGTGACAGTGTCTTCTGCTAGCACGAAAGGCCCTAGCGTG TTTCCTCTGGCTCCATGTAGCAGAAGCACCAGCGAAAGCACCGCCGCCCTGGGCTGCCTG GTGAAAGACTACTTTCCTGAGCCAGTGACCGTGTCCTGGAACTCCGGAGCCCTCACGTCC GGCGTGCACACATTCCCCGCCGTGCTGCAGTCATCCGGCCTGTACAGCCTGAGCTCCGTT GTGACCGTGCCTTCTTCCAGCCTGGGCACAAAGACCTACACATGCAACGTGGACCACAAG CCCAGCAATACCAAGGTGGACAAGAGAGTGGGCGGCGGCGGAAGCGGCGGCGGCGGCAGC GAGGTGCAGCTGGTGGAATCTGGCGGTGGCCTTGTGAAGCCTGGAGGCAGCCTACGGCTG AGCTGCGCCGCTAGCGGCTTCACCTTTAGCAATTACGCCATGAGCTGGGTGCGGCAGGCT CCTGGAAAGCGGCTGGAGTGGGTTGCAACAATCAGCAATAGAGGCAGCTACACCTACTAC CCTGACTCTGTTAAGGGCAGATTTACAATCAGCCGCGACAACGCCAAGAACAGCCTGTAT CTGCAAATGAACAGCCTGAGGGCCGAGGACACCGCCCTGTACTACTGCGCCAGAGAGCGG CCTATGGACTATTGGGGACAGGGCACCCTGGTCACCGTCAGCAGCGGAGGGGGCGGTAGC GGCGGTGGAGGCTCTGGCGGAGGAGGCAGCGACATACAGATGACCCAGAGCCCTAGCACA CTGAGCGCCTCCGTTGGCGACCGGGTGACAATTACCTGCAAGGCCAGCCAGGATGTGGGC ACAGCCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACTGG GCCAGCACCAGACATACAGGCGTCCCCGACAGATTCTCTGGATCAGGCAGCGGCACCGAT TTCACCCTGACTATCAGCAGCCTGCAGGCCGAAGATTTCGCCGTGTACTTCTGCCACCAG CACAGCTCTAACCCCCTGACCTTCGGCCAGGGCACAAAGCTTGAAATCAAGTGA SEQIDNO:34-[C1sscFab-(G.sub.4S).sub.2-BbscFv]aminoacidsequence (construct#13,FIG.2D)(signalpeptideboldfaced) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALOS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGE NIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLR SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSEVOLVESGGGLVKPGGSLRLSCAASGFTESNYAMSWVRQA PGKRLEWVATISNRGSYTYYPDSVKGRETISRDNAKNSLYLOMNSLRAEDTALYYCARER PMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVG TAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQ HSSNPLTFGQGTKLEIK* SEQIDNo:35-[C1sscFab-(G.sub.4S).sub.2-BbscFv]nucleicacidsequence (construct#14,FIG.2D) ATGGAAGCCCCCGCCCAGCTGCTGTTCCTGCTGCTCCTGTGGCTGCCTGATACCACCGGC GATATCGTCCTGACCCAGAGCCCTGATAGCCTGGCCGTTTCACTGGGCGAGCGGGCCACA ATCTCCTGCAAGGCCTCTCAGTCTGTTGACTACGACGGCGACAGCTACATGAACTGGTAC CAGGAGAAACCCGGCCAACCTCCAAAGATCCTGATCTACGACGCCTCTAATCTGGAGAGC GGCATCCCCGCCCGGTTCAGCGGGTCCGGCAGCGGCACCGACTTTACCCTGACCATCTCT AGCCTGGAGCCTGAGGACTTCGCCATCTACTACTGTCAGCAGAGCAACGAGGATCCTTGG ACCTTTGGCGGCGGCACAAAGGTGGAAATCAAGCGGACCGTCGCCGCTCCATCCGTGTTT ATCTTCCCTCCTTCCGACGAGCAGCTCAAGAGCGGTACCGCCAGCGTGGTGTGCCTGCTG AACAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTAGACAACGCCTTGCAGAGC GGCAACTCTCAAGAGAGCGTGACAGAGCAGGACTCTAAGGACAGCACATACAGCCTAAGC TCCACCCTGACCCTCAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTT ACACACCAGGGCCTGAGCAGTCCGGTGACCAAGTCCTTCAACAGAGGCGAATGCGGCGGA GGAGGCTCTGGCGGCGGCGGCAGCGGCGGAGGCGGCAGCGGCGGCGGAGGCTCTGGCGGC GGTGGCAGCGGAGGCGGCGGAAGCGGCGGAGGTGGCAGCCAGGTGCAGCTGGTGCAGAGC GGTGCTGAAGTGAAGAAACCCGGCGCTTCCGTGAAACTGAGCTGCACCGCCAGCGGATTT AACATCAAGGACGACTACATTCACTGGGTGAAAAAGGCCCCTGGCCAGGGCCTGGAATGG ATCGGGAGAATCGACCCCGCCGATGGCCATACCAAGTACGCTCCTAAGTTCCAGGTGAAA GTGACCATCACCGCTGATACAAGCACCTCTACAGCCTACCTGGAGCTGAGCTCCCTGCGG TCTGAGGACACCGCCGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAGGTGTTCGAC TACTGGGGACAGGGCACTACAGTCACCGTGTCTAGTGCTAGCACGAAGGGCCCTAGCGTG TTCCCTCTGGCTCCATGTAGCAGAAGCACCAGCGAAAGCACAGCTGCTCTGGGCTGCCTG GTGAAAGACTACTTCCCCGAGCCTGTGACCGTCAGCTGGAACTCCGGCGCCCTGACCAGC GGAGTGCACACCTTTCCTGCTGTGCTGCAATCCTCTGGCCTGTACTCTCTGAGCTCTGTT GTGACAGTGCCTTCTAGCAGCCTGGGAACCAAGACCTACACCTGCAACGTGGACCACAAG CCCAGCAACACCAAGGTGGATAAGCGCGTGGGCGGCGGCGGATCTGGCGGAGGCGGCAGC GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGAAGCCTGGCGGCTCACTGAGACTG AGCTGTGCCGCCAGCGGCTTCACCTTCTCCAACTACGCCATGAGCTGGGTGCGGGAAGCC CCAGGAAAGCGCCTGGAGTGGGTCGCCACCATCAGCAATAGAGGCTCGTATACATATTAC CCTGATTCCGTCAAAGGCAGATTCACCATCTCTAGAGATAATGCCAAGAACAGCCTGTAC CTGCAGATGAACTCCCTCAGAGCCGAGGATACAGCCCTGTATTACTGCGCCAGAGAACGG CCTATGGACTACTGGGGCCAAGGCACTCTGGTGACAGTGAGCAGCGGCGGCGGTGGTTCC GGCGGCGGAGGCTCTGGAGGAGGCGGCAGCGACATCCAGATGACCCAGAGCCCTAGCACC CTGTCCGCCAGCGTGGGAGATAGAGTGACCATTACCTGTAAAGCGAGCCAGGATGTGGGC ACCGCCGTGGCCTGGTATCAGAAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGG GCCTCTACCCGGCACACAGGCGTGCCCGACAGATTCTCCGGCTCCGGTTCTGGAACAGAC TTCACACTGACCATCAGCTCTCTTCAGGCCGAGGACTTCGCCGTGTACTTCTGCCACCAG CACAGCTCTAATCCTCTGACATTCGGCCAAGGCACAAAGCTGGAAATCAAGTGA SEQIDNo:36-[C1sscFab-(G.sub.4S).sub.2-BbscFv-CM]aminoacid sequence(construct#14,Fig.2D)(signalpeptideboldfaced;charge mutationsboxedanditalicized,numberingexcludingsignalpeptide: Q42EandQ292KinC1sscFab,andQ519EandQ648KinBbscFv) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY [00017]![embedded image]()
TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGF [00018]![embedded image]()
SEDTAVYYCARYGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK [00019]![embedded image]()
PGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARER PMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVG [00020]![embedded image]()
HSSNPLTFGQGTKLEIK* SEQIDNO:37-[C1sscFab-bidirectionalpromoter-BbscFab] nucleicacidsequence(construct#9,FIG.2B) TCACACCCGCTTATCCACCTTGGTGTTGCTGGGCTTGTGGTCCACGTTGCAGGTGTAGGT CTTTGTGCCCAGGCTAGAGCTAGGCACTGTCACGACAGAGGACAGAGAGTACAGGCCGCT GCTCTGCAGCACGGCGGGGAAGGTGTGCACCCCGCTTGTCAGGGCTCCGCTGTTCCAGGA CACGGTCACAGGCTCAGGGAAATAATCCTTGACCAGGCAGCCCAGAGCAGCCGTGCTCTC TGAGGTACTTCTGCTACAAGGAGCCAGTGGGAACACGCTAGGGCCCTTTGTGCTGGCGGA CGACACGGTCACTGTTGTGCCCTGTCCCCAGTAGTCGAACACTTCTCTGCCGTAGCCGTA TCTGGCGCAGTAGTACACAGCGGTGTCCTCGGATCTAAGGCTGCTCAGTTCCAGATAAGC TGTAGAGGTGCTGGTATCGGCGGTGATGGTGACTTTCACCTGGAACTTAGGGGCGTACTT TGTGTGGCCGTCGGCAGGGTCGATTCTGCCGATCCACTCCAGTCCCTGGCCGGGGGCCTG CTTCACCCAGTGGATGTAATCGTCCTTGATATTGAAGCCGCTGGCGGTGCAGCTCAGCTT AACACTAGCGCCAGGCTTTTTCACCTCGGCTCCGCTCTGCACCAGCTGCACCTGGGATCC GCCGCCGCCGCTGCCGCCTCCGCCGCTGCCGCCTCCGCCGCTTCCGCCTCCCCCAGAGCC GCCGCCACCGCTGCCTCCTCCGCCGGAGCCGCCGCCGCCGCACTCGCCCCGGTTGAAGCT TTTGGTCACAGGAGAGGACAGGCCCTGATGTGTCACTTCACAGGCGTACACCTTGTGCTT CTCGTAGTCGGCCTTGCTCAAGGTCAGGGTGCTGGACAGGCTGTATGTTGAGTCCTTGCT GTCCTGCTCGGTCACGCTCTCTTGGCTGTTGCCGCTTTGCAGGGCGTTGTCAACTTTCCA TTGGACCTTTGCCTCTCTGGGGTAGAAGTTATTCAGCAGGCACACCACAGAGGCGGTTCC GCTCTTCAGCTGCTCGTCGCTTGGAGGGAAGATAAAGACAGAAGGGGCGGCCACGGTGCG CTTGATTTCCACCTTGGTGCCGCCTCCAAAGGTCCAGGGGTCCTCGTTGCTCTGCTGGCA GTAGTAGATGGCAAAATCCTCGGGTTCCAGAGAAGAAATTGTCAGGGTGAAATCAGTGCC AGAGCCGCTGCCGCTGAATCTGGCGGGGATGCCGCTTTCCAGATTGCTGGCGTCGTAGAT CAGGATTTTTGGAGGCTGGCCGGGTTTCTGCTGGTACCAGTTCATGTAGCTGTCGCCGTC ATAGTCCACGCTCTGAGAGGCTTTACAGCTGATTGTGGCCCGTTCGCCGAGGCTCACGGC CAGGCTATCAGGGCTCTGCGTCAGCACGATATCGCCGGTGGTGTCAGGCAGCCACAGGAG CAGCAGGAACAGCAGCTGGGCAGGGGCTTCCATGGTGGGCTCTGGCGCCCGCCGCGCGCT TCGCTTTTTATAGGGCCGCCGCCGCCGCCGCCTCGCCATAAAAGGAAACTTTCGGAGCGC GCCGCTCTGATTGGCTGCCGCCGCACCTCTCCGCCTCGCCCCGCCCCGCCCCTCGCCCCG CCCCGCCCCGCCTGGCGCGCGCCCCCCCCCCCCCCCCGCCCCCATCGCTGCACAAAATAA TTAAAAAATAAATAAATACAAAATTGGGGGTGGGGAGGGGGGGGAGATGGGGAGAGTGAA GCAGAACGTGGGGCTCACCTCGCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA CCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGT GCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGGGGGGCGGGGCGAG GGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGG CGGGCGCCAACTAGCCCACCATGGAAGCCCCCGCTCAGCTGCTGTTCCTGCTGCTGCTGT GGCTGCCTGACACCACCGGCGACATCCAGATGACACAGAGCCCTAGCACCCTGAGCGCCT CCGTGGGGGACAGAGTGACAATCACATGTAAAGCCTCCCAGGACGTGGGCACTGCCGTGG CCTGGTACCAGCAAAAACCGGGAAAAGCCCCTAAGCTGCTGATCTACTGGGCCAGCACCA GACACACCGGCGTCCCCGATAGATTCAGCGGCTCTGGCAGCGGAACTGATTTCACCCTGA CCATTTCTTCTCTGCAGGCCGAGGACTTCGCCGTGTACTTTTGCCACCAGCACAGCAGCA ACCCTCTGACCTTCGGACAGGGCACAAAGCTGGAAATCAAGCGGACAGTGGCTGCTCCTT CTGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGAGCGGCACCGCCTCTGTGGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCC TGCAATCTGGCAACAGCCAGGAGAGCGTGACGGAACAAGATAGCAAGGACAGCACCTACT CCCTGAGCAGCACACTGACCTTGTCCAAGGCAGATTACGAGAAGCACAAGGTGTACGCCT GCGAGGTGACCCACCAGGGACTGAGCAGCCCAGTGACCAAGAGCTTCAACAGAGGAGAGT GCGGCGGCGGCGGAAGCGGAGGCGGAGGCAGCGGCGGCGGCGGCAGTGGAGGCGGCGGCT CTGGCGGAGGGGGCAGTGGCGGTGGCGGATCCGGCGGCGGCGGCAGCGAGGTGCAGCTTG TGGAATCCGGCGGCGGCCTGGTGAAGCCCGGCGGTAGCCTGAGACTGTCTTGTGCCGCCT CTGGCTTCACCTTTAGCAATTACGCCATGAGCTGGGTGCGGCAGGCTCCCGGCAAAAGAC TGGAATGGGTCGCCACCATCAGCAACCGGGGATCATATACCTACTACCCTGATAGCGTGA AAGGCAGGTTCACAATCAGCCGGGACAATGCCAAGAACAGCCTGTACCTGCAGATGAACT CACTGCGGGCCGAGGACACCGCCCTGTATTACTGCGCCAGAGAGAGACCTATGGACTACT GGGGCCAGGGCACCCTGGTGACCGTTTCCTCCGCCAGCACCAAGGGCCCTAGCGTGTTCC CTCTGGCCCCATGCAGCAGAAGCACATCTGAGAGCACCGCCGCTCTGGGCTGCCTGGTGA AGGACTACTTCCCCGAGCCTGTGACAGTGAGCTGGAACTCCGGCGCCCTGACCAGCGGCG TGCACACATTTCCAGCTGTGCTGCAGTCTAGCGGCCTGTACAGCCTGAGCAGCGTTGTGA CAGTGCCTTCTAGCAGCCTCGGCACCAAGACCTACACCTGTAACGTGGATCATAAGCCTT CTAATACCAAGGTTGACAAGAGAGTGTGA SEQIDNO:38-[C1sF2AFab-(G.sub.4S).sub.3BbscFab]nucleicacid sequence(construct#17,FIG.2F) ATGGAAGCCCCAGCTCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCCGACACCACCGGC GACATCGTGCTGACCCAGAGCCCTGATAGCCTGGCCGTTTCTCTGGGAGAACGGGCAACC ATTAGCTGCAAGGCCAGCCAGTCTGTGGACTACGACGGCGACAGCTACATGAATTGGTAT CAGCAGAAGCCTGGCCAACCTCCCAAGATCCTGATCTACGATGCCAGCAACCTGGAATCC GGAATCCCCGCCCGCTTCAGCGGCAGCGGCTCAGGCACCGACTTCACCCTGACAATCTCC TCGCTGGAACCCGAGGATTTCGCTATCTACTACTGTCAGCAGTCTAACGAGGATCCTTGG ACCTTCGGCGGCGGCACAAAGGTCGAGATCAAGAGAACAGTTGCCGCCCCTTCTGTGTTT ATCTTCCCTCCCTCTGACGAGCAGCTGAAGAGCGGCACTGCCAGCGTCGTGTGCCTGCTG AACAACTTCTACCCACGTGAGGCCAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGAGC GGCAACTCTCAGGAGTCTGTGACAGAGCAGGACAGCAAAGATAGCACCTACTCTCTGTCT AGCACCCTGACCCTGAGCAAGGCCGATTACGAGAAGCACAAAGTGTACGCCTGCGAGGTG ACCCACCAGGGCCTGAGCAGCCCCGTGACAAAGTCCTTCAACAGGGGCGAGTGTCGGAAG AGACGGAGCGGCAGCGGCGCCCCAGTCAAGCAGACCCTGAACTTCGACCTGCTTAAGCTG GCCGGCGATGTAGAAAGCAATCCTGGCCCCATGGAAGCCCCTGCCCAGCTGCTGTTCCTG CTGCTGCTGTGGCTGCCTGACACCACAGGACAAGTGCAACTAGTGCAGTCAGGCGCCGAG GTAAAAAAGCCTGGCGCCAGCGTGAAACTGTCTTGCACCGCCTCCGGCTTCAATATCAAG GACGACTACATACACTGGGTGAAGCAGGCTCCCGGCCAGGGCCTGGAATGGATCGGCCGC ATCGACCCTGCTGACGGCCACACCAAGTATGCCCCTAAGTTCCAGGTCAAAGTGACCATC ACCGCTGATACCAGCACAAGTACAGCCTACCTGGAACTGAGCAGCCTGCGGAGCGAGGAC ACAGCCGTGTACTACTGCGCCCGGTACGGCTATGGCAGAGAGGTGTTCGACTACTGGGGA CAGGGCACCACCGTGACAGTGTCTAGCGCCTCTACAAAGGGCCCTAGCGTGTTCCCGCTG GCCCCCTGCAGCAGAAGCACATCTGAAAGCACAGCAGCTCTCGGCTGCCTCGTCAAGGAC TACTTTCCTGAGCCGGTGACAGTTAGCTGGAACAGCGGCGCCCTGACTAGCGGCGTGCAT ACATTCCCTGCCGTGCTGCAGTCCTCCGGCCTCTACAGCCTGTCCAGCGTGGTGACAGTC CCTTCTTCCAGTCTGGGTACGAAAACCTACACCTGCAACGTGGACCACAAGCCCTCCAAT ACGAAAGTGGACAAGAGAGTGGGCGGGGGAGGCTCTGGCGGAGGTGGCTCTGGCGGGGGC GGAAGCGACATCCAGATGACACAATCCCCTAGCACCCTGAGCGCCAGCGTGGGAGATAGA GTGACGATCACCTGTAAAGCCTCACAGGACGTGGGCACCGCCGTGGCCTGGTACCAGCAG AAACCTGGAAAGGCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTG CCTGACAGATTCAGCGGCTCTGGCAGCGGCACAGACTTTACCCTGACAATCAGCAGCCTG CAGGCTGAAGATTTCGCCGTGTACTTCTGCCACCAACACAGCAGCAACCCCCTGACATTT GGCCAAGGCACCAAGCTGGAGATCAAGAGAACCGTTGCTGCCCCTAGCGTGTTCATCTTC CCGCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCCTCTGTGGTTTGCCTGCTGAACAAC TTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGTGGAAAC TCTCAAGAGAGCGTGACCGAACAGGATAGCAAAGACAGCACCTATAGCTTGTCTAGCACA CTGACCCTGTCTAAGGCTGACTACGAGAAGCACAAGGTGTACGCATGCGAGGTCACCCAT CAGGGACTGAGCAGCCCCGTGACCAAGTCTTTTAACCGGGGCGAGTGCGGCGGAGGAGGC AGTGGCGGCGGGGGATCCGGCGGCGGCGGCAGCGGCGGAGGCGGATCCGGCGGCGGCGGT AGCGGCGGTGGCGGCAGCGGTGGAGGGGGAAGCGAGGTGCAGCTCGTCGAGTCCGGAGGA GGCCTTGTGAAGCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGATTCACCTTC AGCAATTACGCCATGAGCTGGGTGCGGCAGGCCCCTGGCAAGAGACTGGAATGGGTGGCC ACCATCAGCAACAGAGGCAGCTACACCTACTACCCCGACTCCGTGAAGGGCAGATTTACC ATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAG GACACCGCCCTGTACTACTGTGCCAGGGAAAGACCTATGGACTACTGGGGCCAGGGAACA CTGGTGACCGTATCTTCCGCCTCAACCAAAGGCCCCTCGGTGTTTCCACTGGCTCCTTGC TCCAGATCCACCTCCGAGAGCACCGCCGCCCTGGGCTGTCTGGTGAAGGATTACTTCCCA GAACCTGTGACCGTGAGCTGGAATAGCGGCGCTCTCACCTCTGGAGTGCACACCTTCCCT GCCGTGCTGCAGAGCAGCGGCCTGTATAGCTTGTCCAGTGTGGTGACCGTGCCTAGCTCC AGCCTGGGCACTAAGACATATACATGTAACGTGGACCACAAGCCTAGCAACACCAAGGTG GATAAGAGAGTGTGA SEQIDNO:39-[C1sF2AFab-(G.sub.4S).sub.3-BbscFab]aminoacid sequence(construct#17,FIG.2F)(signalpeptidesboldfaced;furin cleavagesiteunderlined;F2Asequenceitalicized) MEAPAQLLFLLLLWLPDTTGDIVLTOSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGOPPKILIYDASNLESGIPARESGSGSGTDETLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECRK RRSGSGAPVKQTLNEDLLKLAGDVESNPGPMEAPAQLLFLLLLWLPDTTGQVOLVOSGAE VKKPGASVKLSCTASGENIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTI TADTSTSTAYLELSSLRSEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVEPL APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCNVDHKPSNTKVDKRVGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDR VTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSL QAEDFAVYFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVOWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVOLVESGG GLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFT ISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSSASTKGPSVFPLAPC SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAVLOSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRV* SEQIDNO:40-[C1sGT2AFab-(G.sub.4S).sub.3-BbscFab]nucleicacid sequence(construct#18,FIG.2F) ATGGAAGCCCCAGCCCAGCTGCTGTTTCTGCTGCTGTTGTGGCTGCCCGATACTACCGGC GATATCGTGCTGACCCAGAGCCCTGATAGCCTGGCTGTGTCTCTGGGGGAGCGGGCTACC ATCTCTTGTAAAGCCAGCCAAAGCGTGGACTACGACGGCGACTCCTACATGAACTGGTAC CAGCAGAAACCTGGCCAGCCTCCAAAGATCCTGATCTACGACGCCAGCAACCTGGAAAGC GGCATCCCTGCTCGGTTCAGCGGATCAGGCTCGGGCACAGACTTTACACTGACAATTAGC TCTCTGGAACCTGAAGATTTTGCTATCTACTATTGCCAGCAGAGCAACGAGGATCCTTGG ACCTTTGGCGGCGGAACAAAGGTGGAAATCAAGCGGACAGTCGCTGCCCCTAGTGTGTTC ATCTTCCCACCTTCCGATGAGCAGCTCAAGTCTGGAACAGCCTCTGTGGTCTGCCTGCTG AACAACTTCTACCCCCGGGAGGCTAAAGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCT GGCAACTCGCAGGAGAGCGTTACAGAGCAGGACTCTAAGGACAGTACCTACAGCCTGTCA TCAACCCTGACCCTGAGCAAGGCCGACTATGAAAAGCACAAGGTCTACGCCTGCGAGGTG ACACACCAGGGCCTGAGCTCTCCTGTGACTAAGTCCTTCAATAGAGGAGAGTGCAGACGG AAGCGCGGCAGCGGAGAAGGCAGAGGCTCCCTGCTAACCTGTGGAGACGTGGAGGAAAAC CCCGGCCCCATGGAAGCCCCTGCTCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCGGAT ACAACCGGACAAGTGCAGCTGGTGCAATCTGGCGCCGAAGTGAAAAAGCCCGGCGCTTCT GTGAAGCTGTCTTGCACCGCCTCTGGATTCAACATCAAGGACGACTACATCCACTGGGTG AAGCAGGCCCCTGGCCAGGGCCTGGAGTGGATCGGCAGAATCGACCCCGCTGATGGCCAC ACAAAATACGCCCCTAAGTTCCAGGTGAAGGTGACCATCACCGCTGACACCTCGACAAGT ACCGCCTACCTGGAGCTGAGCTCTCTGAGATCCGAGGACACAGCAGTGTACTACTGCGCC AGATACGGCTACGGCAGAGAGGTTTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTG TCCAGCGCCAGCACAAAGGGCCCTTCTGTCTTCCCTCTGGCGCCTTGTAGCCGGAGCACA AGCGAGAGCACTGCCGCTCTTGGCTGCCTGGTGAAGGACTACTTTCCTGAACCTGTTACA GTGAGCTGGAACAGCGGCGCCCTGACATCTGGCGTGCACACCTTTCCAGCCGTGCTGCAG TCCTCCGGCCTGTACAGTCTGAGCAGCGTGGTGACCGTGCCTAGCAGCTCTCTGGGCACC AAGACATATACCTGCAATGTGGACCACAAACCTAGCAACACCAAGGTGGACAAGAGAGTG GGCGGCGGCGGGAGTGGAGGTGGAGGCAGCGGAGGTGGTGGCAGCGACATCCAGATGACA CAGAGCCCTAGCACTCTGAGCGCCAGCGTGGGCGATAGAGTGACCATTACCTGCAAGGCC TCCCAGGACGTGGGAACCGCCGTGGCCTGGTATCAGCAAAAGCCAGGCAAGGCCCCCAAG CTTCTGATCTACTGGGCCAGCACAAGACACACCGGCGTCCCCGACAGGTTCAGCGGCAGT GGCTCAGGCACCGACTTCACCCTAACTATCAGCTCTCTGCAAGCTGAAGACTTCGCCGTG TACTTCTGCCACCAGCACAGCTCCAACCCCTTGACCTTCGGCCAAGGCACAAAGCTGGAA ATCAAACGGACAGTCGCCGCACCTAGCGTGTTCATCTTCCCACCTTCTGACGAGCAGCTG AAGAGCGGCACCGCGTCCGTGGTGTGTCTGCTCAACAACTTCTACCCAAGAGAGGCCAAG GTGCAGTGGAAGGTTGACAATGCCCTGCAGAGCGGGAATAGCCAGGAGAGCGTGACCGAG CAGGACAGCAAGGACTCTACCTACAGCCTCAGTTCTACCCTGACCCTGTCCAAGGCCGAT TACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCATCAGGGCCTGAGCAGTCCTGTG ACTAAAAGCTTCAACAGAGGCGAATGCGGCGGCGGAGGCTCCGGCGGAGGCGGCAGCGGC GGAGGCGGATCTGGCGGCGGTGGCTCCGGAGGCGGCGGCAGCGGCGGCGGCGGCTCTGGC GGCGGCGGCTCTGAGGTGCAACTGGTTGAAAGCGGAGGCGGCCTGGTGAAGCCCGGAGGC TCCCTGCGGCTGAGCTGCGCCGCCAGTGGCTTCACCTTCTCTAATTACGCTATGAGCTGG GTCAGACAGGCCCCTGGAAAGCGGTTGGAGTGGGTGGCCACCATCAGCAACCGGGGAAGC TACACCTACTACCCAGATAGCGTGAAAGGCAGGTTTACCATCAGCAGAGATAACGCCAAG AACTCACTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCCTGTACTACTGC GCCAGAGAGAGACCTATGGACTACTGGGGCCAAGGCACATTAGTCACCGTGTCCTCTGCC AGTACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTTGCTCCAGAAGCACCAGCGAGAGC ACAGCCGCACTTGGATGTCTGGTTAAAGATTATTTCCCCGAGCCCGTGACAGTGTCTTGG AACAGCGGGGCCCTGACCAGCGGTGTTCATACCTTCCCTGCTGTGCTCCAGAGCTCCGGC CTGTATTCCCTGAGTTCAGTAGTGACCGTGCCTAGCAGCAGCCTGGGAACCAAGACCTAC ACATGCAACGTGGACCACAAGCCTAGCAATACCAAGGTGGACAAGCGGGTGTGA SEQIDNO:41[C1sGT2AFab-(G.sub.4S).sub.3-BbscFab]aminoacidsequence construct#18,FIG.2F)(signalpeptidesboldfaced;furincleavage siteunderlined;GT2Asequenceitalicized) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECRR KRGSGEGRGSLLTCGDVEENPGPMEAPAQLLFLLLLWLPDTTGQVQLVQSGAEVKKPGAS VKLSCTASGFNIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTS TAYLELSSLRSEDTAVYYCARYGYGREVFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKA SQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAV YFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGG SLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAK NSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRV* SEQIDNO:42-[C1sscFab-(G.sub.4S).sub.3-BbF2AFab]nucleicacid sequence(construct#19,FIG.2F) ATGGAAGCCCCAGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCCGATACCACCGGC GACATCGTGCTGACACAGAGTCCTGATAGCCTGGCCGTGTCTCTGGGGGAAAGAGCCACA ATCTCTTGCAAGGCCTCCCAGAGTGTAGACTACGACGGCGATAGTTACATGAACTGGTAT CAGCAGAAACCTGGACAACCTCCAAAGATCCTGATCTACGACGCCAGCAACCTGGAGAGC GGCATTCCTGCCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACAATCAGC AGCCTGGAGCCCGAGGACTTTGCCATCTACTACTGTCAGCAAAGCAACGAGGACCCCTGG ACATTTGGCGGCGGCACGAAAGTGGAAATCAAGCGGACCGTCGCCGCCCCCAGCGTGTTC ATCTTCCCTCCTTCTGATGAGCAGCTCAAGAGCGGCACAGCCAGCGTGGTGTGCCTGCTG AACAATTTCTACCCTAGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAAAGC GGCAACTCTCAGGAGTCCGTTACCGAGCAAGATAGCAAGGACTCTACATATTCTCTGTCT AGCACCCTGACCTTGAGCAAGGCCGACTATGAAAAGCACAAGGTCTACGCATGCGAGGTG ACTCATCAGGGCCTCAGCTCCCCAGTGACCAAATCCTTCAACCGGGGCGAGTGCGGCGGA GGCGGCAGCGGGGGCGGAGGCAGCGGAGGAGGCGGCTCAGGCGGAGGAGGCAGCGGCGGC GGCGGCTCGGGCGGAGGCGGAAGCGGCGGCGGCGGCAGCCAAGTGCAGCTGGTGCAGAGC GGCGCTGAAGTGAAAAAGCCTGGCGCCAGCGTGAAGCTGTCCTGCACCGCCAGCGGCTTC AATATCAAGGATGATTACATCCACTGGGTGAAACAGGCCCCTGGCCAGGGCCTTGAGTGG ATCGGAAGGATCGACCCTGCCGATGGCCACACCAAGTACGCTCCCAAGTTCCAGGTGAAG GTGACCATCACCGCCGATACCAGCACGAGCACAGCCTACCTGGAACTGTCTTCCCTGAGA AGCGAAGATACCGCCGTGTACTACTGCGCCAGATACGGATATGGCAGAGAGGTATTCGAC TACTGGGGACAGGGCACCACCGTGACCGTGTCCTCTGCCTCCACCAAGGGCCCCTCTGTG TTTCCTCTGGCCCCCTGCTCTAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGTCTG GTGAAAGACTATTTCCCTGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCCCTGACAAGT GGCGTGCACACCTTTCCTGCTGTTCTGCAGAGTAGCGGCCTGTACAGCCTGTCGAGCGTG GTCACAGTGCCTAGCAGCAGTCTGGGCACAAAGACCTACACTTGTAACGTGGATCACAAG CCCTCTAATACCAAGGTGGACAAGCGGGTGGGAGGCGGCGGAAGCGGAGGCGGCGGCTCT GGGGGAGGTGGCTCAGAAGTGCAGCTGGTGGAAAGCGGCGGCGGGCTGGTGAAGCCTGGC GGCTCTCTCCGGCTGAGCTGTGCCGCCAGCGGTTTTACCTTCTCCAATTACGCCATGAGC TGGGTCAGACAGGCCCCAGGCAAGAGACTTGAGTGGGTTGCTACAATCAGCAACAGAGGC AGCTACACCTACTACCCTGACAGCGTGAAGGGCAGATTCACAATCAGCCGGGACAACGCC AAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGATACAGCCCTTTACTAC TGTGCCAGAGAGAGACCTATGGACTACTGGGGCCAGGGCACTCTGGTGACCGTTTCCAGC GCCAGCACCAAAGGCCCAAGCGTGTTCCCTCTGGCTCCCTGCAGCAGAAGCACCAGCGAA AGCACAGCTGCGCTGGGCTGCCTGGTGAAGGATTACTTCCCCGAGCCTGTGACCGTGTCT TGGAACTCCGGCGCTCTGACATCCGGCGTTCACACATTCCCCGCTGTCCTGCAGTCAAGT GGCCTGTACAGCCTGAGCAGTGTGGTGACCGTTCCAAGCTCTTCTCTGGGAACAAAAACA TACACCTGCAACGTGGACCACAAGCCTAGCAACACCAAAGTGGATAAGCGGGTGCGGAAG CGCCGGAGCGGAAGCGGCGCCCCTGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTG GCTGGCGACGTGGAAAGCAACCCTGGCCCTATGGAAGCCCCCGCACAACTGCTGTTCCTG CTGCTGCTCTGGCTGCCTGACACCACAGGCGACATCCAGATGACCCAAAGCCCTAGCACA CTGAGCGCCAGCGTCGGCGACAGAGTGACCATTACATGCAAGGCCTCCCAGGACGTCGGC ACAGCCGTGGCCTGGTACCAGCAGAAGCCTGGAAAGGCCCCAAAGCTGCTGATCTACTGG GCCTCTACCCGGCATACCGGCGTGCCTGACAGATTCAGCGGCAGCGGCTCTGGTACAGAC TTCACCCTGACCATTAGCAGCTTACAGGCCGAGGACTTCGCCGTGTACTTCTGCCACCAG CACAGCAGCAATCCTCTAACCTTCGGCCAGGGAACCAAGCTGGAAATCAAAAGAACCGTG GCCGCCCCTTCTGTATTCATATTTCCTCCAAGCGACGAGCAGCTCAAGAGCGGCACGGCT TCTGTGGTGTGTCTGCTGAACAACTTTTATCCCAGAGAAGCCAAGGTGCAGTGGAAGGTG GATAACGCCCTGCAATCCGGAAACTCTCAGGAGTCTGTCACCGAGCAGGACTCAAAGGAC TCGACGTACAGCCTGAGCAGCACACTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAA GTTTACGCCTGCGAGGTGACACACCAGGGCCTCTCTAGCCCTGTGACAAAGAGCTTCAAC AGGGGCGAGTGCTGA SEQIDNO:43-[C1sscFab-(G.sub.4S).sub.3-BbF2AFab]aminoacid sequence(construct#19,FIG.2F)(signalpeptidesboldfaced;furin cleavagesiteunderlined;F2Asequenceitalicized) MEAPAQLLFLLLLWLPDTTGDIVLTOSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGQPPKILIYDASNLESGIPARESGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALOS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKLSCTASGE NIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLR SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMS WVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLOMNSLRAEDTALYY CARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVRK RRSGSGAPVKQTLNEDLLKLAGDVESNPGPMEAPAQLLFLLLLWLPDTTGDIQMTOSPST LSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTD FTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVOWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSENRGEC* SEQIDNO:44-[C1sscFab-(G.sub.4S).sub.3-BbGT2A-Fab]nucleicacid sequence(construct#20,FIG.2F) ATGGAAGCCCCTGCCCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCCTGACACCACAGGC GACATCGTTCTGACCCAGAGCCCTGACAGCCTGGCCGTGTCCCTAGGCGAACGGGCCACC ATCAGCTGCAAGGCCAGCCAGAGCGTGGACTATGATGGCGACAGCTACATGAACTGGTAT CAGCAAAAGCCCGGACAGCCTCCTAAGATCCTGATCTACGACGCCTCTAACCTGGAATCT GGCATCCCTGCCAGATTTTCTGGCAGCGGTTCTGGCACCGATTTCACCCTGACCATTAGC TCTCTGGAGCCTGAGGACTTCGCCATCTACTACTGCCAGCAGAGCAACGAGGATCCTTGG ACATTCGGCGGCGGTACCAAGGTCGAGATTAAACGGACCGTGGCTGCTCCCAGCGTGTTC ATCTTCCCACCATCTGATGAGCAGCTGAAATCTGGCACGGCCAGCGTCGTGTGCCTGCTG AACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCC GGCAATAGCCAGGAGAGCGTGACTGAACAGGATAGTAAAGACTCTACCTACAGCCTGTCC AGTACACTGACCCTGTCTAAGGCCGATTACGAGAAGCACAAAGTGTACGCCTGTGAAGTG ACACATCAGGGCCTGAGCTCACCTGTGACTAAGTCCTTCAACCGGGGCGAGTGCGGCGGC GGTGGCAGCGGCGGCGGCGGCAGCGGAGGCGGCGGCAGTGGAGGCGGCGGGTCTGGCGGA GGTGGATCTGGTGGCGGCGGTAGCGGCGGCGGCGGCAGCCAGGTGCAACTGGTGCAGTCT GGAGCTGAGGTGAAGAAACCTGGGGCCAGCGTGAAGCTGTCTTGCACCGCCAGCGGCTTC AACATCAAGGACGACTACATCCACTGGGTCAAACAGGCTCCTGGACAGGGCTTGGAATGG ATCGGCAGAATCGACCCCGCCGACGGCCACACCAAGTACGCCCCAAAATTCCAGGTGAAA GTAACAATCACCGCTGATACATCTACTTCCACAGCTTATCTGGAACTGAGCAGCCTGAGG TCTGAGGATACCGCCGTGTACTACTGCGCCCGGTACGGCTACGGCAGAGAGGTGTTCGAC TACTGGGGACAGGGCACCACCGTGACCGTGTCTTCCGCCAGCACTAAGGGACCTAGCGTG TTCCCCCTGGCCCCATGTTCCCGGAGCACCAGCGAGTCTACTGCCGCCCTGGGATGCCTG GTGAAGGACTACTTTCCTGAGCCCGTGACCGTGTCTTGGAACAGCGGCGCCCTGACCAGC GGCGTGCACACATTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCTCTGTG GTGACAGTGCCCTCTAGCTCTCTCGGCACCAAAACCTACACCTGCAACGTGGACCATAAG CCTAGCAACACCAAGGTCGACAAGCGGGTGGGCGGCGGCGGGAGTGGCGGTGGCGGCTCT GGCGGAGGGGGGAGCGAAGTGCAGCTGGTCGAAAGCGGAGGAGGACTAGTGAAGCCTGGC GGCAGCCTGAGACTGAGCTGTGCTGCCAGCGGCTTTACATTCAGCAACTACGCCATGAGC TGGGTGCGTCAGGCCCCCGGCAAGCGGCTGGAATGGGTCGCAACCATCAGCAATAGAGGC AGCTACACTTACTACCCTGACTCCGTCAAGGGCAGATTCACCATCTCCCGCGACAACGCC AAAAACTCCCTGTACCTGCAAATGAATAGCCTGAGAGCCGAGGACACCGCCCTGTACTAT TGCGCCAGAGAGAGACCTATGGACTACTGGGGCCAGGGTACCCTGGTGACCGTGAGCTCT GCTAGCACAAAGGGCCCTTCCGTGTTCCCTCTGGCTCCTTGCAGCAGAAGCACAAGCGAG AGCACAGCCGCCCTGGGCTGCCTGGTTAAGGACTATTTTCCCGAACCTGTGACAGTCTCC TGGAACAGCGGCGCCCTGACCTCTGGGGTGCACACCTTCCCCGCTGTCCTGCAGAGCAGC GGCCTGTACTCGCTGAGCTCTGTGGTGACCGTGCCTAGCAGCAGCCTGGGCACCAAGACA TACACATGTAATGTGGACCACAAGCCCTCCAACACCAAGGTCGATAAGAGAGTGCGGAGA AAGAGAGGTTCCGGCGAGGGCAGAGGCAGCCTGTTAACATGCGGCGACGTGGAGGAAAAC CCAGGACCTATGGAGGCCCCCGCCCAGCTGCTCTTCCTGCTGCTGCTGTGGCTGCCCGAT ACCACCGGCGATATCCAGATGACACAGTCCCCTTCAACCCTTAGTGCCTCGGTTGGCGAT AGAGTGACAATTACATGTAAAGCTAGCCAGGACGTGGGCACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGCAAAGCCCCAAAGCTGCTCATCTACTGGGCCTCGACAAGACACACCGGC GTGCCAGATAGATTCAGCGGCTCTGGCTCAGGCACAGACTTCACCCTGACTATCAGCTCC CTCCAAGCCGAGGATTTCGCCGTTTACTTCTGCCACCAGCACAGCTCCAATCCCCTGACA TTCGGCCAAGGAACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCTAGTGTCTTCATC TTCCCTCCTTCCGACGAGCAGCTGAAGAGCGGCACAGCCTCCGTGGTGTGTCTGCTCAAC AACTTTTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGA AACAGCCAGGAGTCGGTGACCGAGCAAGACAGCAAGGACTCTACGTACAGCCTGTCAAGC ACCCTGACGCTGAGCAAGGCCGATTACGAGAAGCACAAAGTGTACGCCTGCGAGGTGACC CACCAGGGACTGAGCAGCCCTGTGACCAAGAGCTTTAACCGTGGAGAATGCTGA SEQIDNO:45-[C1sscFab-(G.sub.4S).sub.3-BbGT2A-Fab]aminoacid sequence(construct#20,FIG.2F)(signalpeptidesboldfaced;furin cleavagesiteunderlined;GT2Asequenceitalicized) MEAPAQLLFLLLLWLPDTTGDIVLTOSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGOPPKILIYDASNLESGIPARFSGSGSGTDETLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGF NIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLR SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMS WVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYY CARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVRR KRGSGEGRGSLLTCGDVEENPGPMEAPAQLLFLLLLWLPDTTGDIQMTOSPSTLSASVGD RVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDETLTISS LQAEDFAVYFCHQHSSNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSENRGEC* SEQIDNO:46 GGGGS SEQIDNO:47 GGGGSGGGGS SEQIDNO:48 GGGGSGGGGSGGGGS SEQIDNO:49 GGGGSGGGGSGGGGGGGGSGGGGSGGGGSGGGGS SEQIDNO:50-NucleotidesequenceofAAV2#9(FIGs.2Band2I)(5 ITRboldfaced;bGHpolyAsignalunderlined;reversecomplementof C1sscFabcodingsequenceitalicized;IgGkappasignalcoding sequenceitalicizedandunderlined;Kozaksequenceboxed;CBA promoter(reverse)boldedandunderlined;CBApromoterboxedand underlined;CMVenhancerboldfacedanditalicized;BbscFab boldfaced,italicized,andunderlined;and3ITRboxedand italicized) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG CGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG GCCAACTCCATCACTAGGGGTTCCTTACAATTCTAGTTCCCCAGCATGCCTGCTATTGTC TTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCT ACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACC TTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGG CACAGGTTTAAACCCTGCAGGGAGCTCTCACACCCGCTTATCCACCTTGGTGTTGCTGGG CTTGTGGTCCACGTTGCAGGTGTAGGTCTTTGTGCCCAGGCTAGAGCTAGGCACTGTCAC GACAGAGGACAGAGAGTACAGGCCGCTGCTCTGCAGCACGGCGGGGAAGGTGTGCACCCC GCTTGTCAGGGCTCCGCTGTTCCAGGACACGGTCACAGGCTCAGGGAAATAATCCTTGAC CAGGCAGCCCAGAGCAGCCGTGCTCTCTGAGGTACTTCTGCTACAAGGAGCCAGTGGGAA CACGCTAGGGCCCTTTGTGCTGGCGGACGACACGGTCACTGTTGTGCCCTGTCCCCAGTA GTCGAACACTTCTCTGCCGTAGCCGTATCTGGCGCAGTAGTACACAGCGGTGTCCTCGGA TCTAAGGCTGCTCAGTTCCAGATAAGCTGTAGAGGTGCTGGTATCGGCGGTGATGGTGAC TTTCACCTGGAACTTAGGGGCGTACTTTGTGTGGCCGTCGGCAGGGTCGATTCTGCCGAT CCACTCCAGTCCCTGGCCGGGGGCCTGCTTCACCCAGTGGATGTAATCGTCCTTGATATT GAAGCCGCTGGCGGTGCAGCTCAGCTTAACACTAGCGCCAGGCTTTTTCACCTCGGCTCC GCTCTGCACCAGCTGCACCTGGGATCCGCCGCCGCCGCTGCCGCCTCCGCCGCTGCCGCC TCCGCCGCTTCCGCCTCCCCCAGAGCCGCCGCCACCGCTGCCTCCTCCGCCGGAGCCGCC GCCGCCGCACTCGCCCCGGTTGAAGCTTTTGGTCACAGGAGAGGACAGGCCCTGATGTGT CACTTCACAGGCGTACACCTTGTGCTTCTCGTAGTCGGCCTTGCTCAAGGTCAGGGTGCT GGACAGGCTGTATGTTGAGTCCTTGCTGTCCTGCTCGGTCACGCTCTCTTGGCTGTTGCC GCTTTGCAGGGCGTTGTCAACTTTCCATTGGACCTTTGCCTCTCTGGGGTAGAAGTTATT CAGCAGGCACACCACAGAGGCGGTTCCGCTCTTCAGCTGCTCGTCGCTTGGAGGGAAGAT AAAGACAGAAGGGGCGGCCACGGTGCGCTTGATTTCCACCTTGGTGCCGCCTCCAAAGGT CCAGGGGTCCTCGTTGCTCTGCTGGCAGTAGTAGATGGCAAAATCCTCGGGTTCCAGAGA AGAAATTGTCAGGGTGAAATCAGTGCCAGAGCCGCTGCCGCTGAATCTGGCGGGGATGCC GCTTTCCAGATTGCTGGCGTCGTAGATCAGGATTTTTGGAGGCTGGCCGGGTTTCTGCTG GTACCAGTTCATGTAGCTGTCGCCGTCATAGTCCACGCTCTGAGAGGCTTTACAGCTGAT TGTGGCCCGTTCGCCGAGGCTCACGGCCAGGCTATCAGGGCTCTGCGTCAGCACGATATC [00021]![embedded image]()
[00022]![embedded image]()
CGCCATAAAAGGAAACTTTCGGAGCGCGCCGCTCTGATTGGCTGCCGCCGCACCTCTCCG CCTCGCCCCGCCCCGCCCCTCGCCCCGCCCCGCCCCGCCTGGCGCGCGCCCCCCCCCCCC CCCCGCCCCCATCGCTGCACAAAATAATTAAAAAATAAATAAATACAAAATTGGGGGTGG GGAGGGGGGGGAGATGGGGAGAGTGAAGCAGAACGTGGGGCTCACCTCGCTAGTTATTAA TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA [00023]![embedded image]()
[00024]![embedded image]()
[00025]![embedded image]()
[00026]![embedded image]()
[00027]![embedded image]()
[00028]![embedded image]()
CTCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCGACATCCAGATGA CACAGAGCCCTAGCACCCTGAGCGCCTCCGTGGGGGACAGAGTGACAATCACATGTAAAG CCTCCCAGGACGTGGGCACTGCCGTGGCCTGGTACCAGCAAAAACCGGGAAAAGCCCCTA AGCTGCTGATCTACTGGGCCAGCACCAGACACACCGGCGTCCCCGATAGATTCAGCGGCT CTGGCAGCGGAACTGATTTCACCCTGACCATTTCTTCTCTGCAGGCCGAGGACTTCGCCG TGTACTTTTGCCACCAGCACAGCAGCAACCCTCTGACCTTCGGACAGGGCACAAAGCTGG AAATCAAGCGGACAGTGGCTGCTCCTTCTGTGTTCATCTTTCCACCTAGCGACGAGCAGC TGAAGAGCGGCACCGCCTCTGTGGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCA AAGTGCAGTGGAAGGTGGACAACGCCCTGCAATCTGGCAACAGCCAGGAGAGCGTGACGG AACAAGATAGCAAGGACAGCACCTACTCCCTGAGCAGCACACTGACCTTGTCCAAGGCAG ATTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGAGCAGCCCAG TGACCAAGAGCTTCAACAGAGGAGAGTGCGGCGGCGGCGGAAGCGGAGGCGGAGGCAGCG GCGGCGGCGGCAGTGGAGGCGGCGGCTCTGGCGGAGGGGGCAGTGGCGGTGGCGGATCCG GCGGCGGCGGCAGCGAGGTGCAGCTTGTGGAATCCGGCGGCGGCCTGGTGAAGCCCGGCG GTAGCCTGAGACTGTCTTGTGCCGCCTCTGGCTTCACCTTTAGCAATTACGCCATGAGCT GGGTGCGGCAGGCTCCCGGCAAAAGACTGGAATGGGTCGCCACCATCAGCAACCGGGGAT CATATACCTACTACCCTGATAGCGTGAAAGGCAGGTTCACAATCAGCCGGGACAATGCCA AGAACAGCCTGTACCTGCAGATGAACTCACTGCGGGCCGAGGACACCGCCCTGTATTACT GCGCCAGAGAGAGACCTATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTTTCCTCCG CCAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCATGCAGCAGAAGCACATCTGAGA GCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGAGCT GGAACTCCGGCGCCCTGACCAGCGGCGTGCACACATTTCCAGCTGTGCTGCAGTCTAGCG GCCTGTACAGCCTGAGCAGCGTTGTGACAGTGCCTTCTAGCAGCCTCGGCACCAAGACCT ACACCTGTAACGTGGATCATAAGCCTTCTAATACCAAGGTTGACAAGAGAGTGTGAGAGC TCCCTGCAGGGTTTAAACCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG [00029]![embedded image]()
[00030]![embedded image]()
[00031]![embedded image]()
[00032]![embedded image]()
SEQIDNO:51-NucleotidesequenceofAAV2#12(FIGs.2Cand2J)(5 ITRboldfaced;minCBApromoter(whichcomprisesaCMVenhancer,a CBApromoter,andatruncatedchimericintron)underlined;Kozak sequenceboxed;IgGkappasignalcodingsequenceitalicized;Bb scFabcodingsequenceboldedandunderlined;(G.sub.4S).sub.7,linkercoding sequenceinlowercase,boldfacedanditalicized;(G.sub.4S).sub.3linker codingsequenceboxedanditalicized;C1sscFabcodingsequence boldfacedanditalicized;bGHpolyAitalicizedandunderlined;and 3ITRboxedandboldfaced) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG CGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG GCCAACTCCATCACTAGGGGTTCCTTACCGGTGCGGGCCTCTTCGCTATTACGCCAGCTG GCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCA CGACGTTGTAAAACGACGGCCAGTGAATTCGGACCGAGATCTGAATTCGGTACCTAGTTA TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTAC GCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT CGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAAT TTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGG GCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGC GGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCG GCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCG CCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTT ACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGT TTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCT AGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTG CTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAAGATCCGGTACCCAAT [00033]![embedded image]()
CCACCGGCGATATCCAGATGACGCAGAGTCCCAGCACCCTGAGCGCCTCTGTGGGCGACC GGGTGACCATCACCTGTAAAGCCTCCCAGGACGTGGGCACAGCTGTTGCTTGGTATCAGA AAAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCAGCACAAGACACACAGGAG TGCCTGACAGATTCAGCGGCAGCGGCTCTGGGACTGATTTCACCTTGACAATCAGCTCTC TGCAGGCCGAGGACTTTGCCGTGTACTTCTGCCACCAACACAGTTCTAACCCCCTGACCT TCGGCCAAGGAACCAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCTGCCGTGTTCATCT TCCCTCCAAGCGATGAGCAGCTGAAAAGCGGCACCGCGTCCGTCGTGTGCCTGCTGAAGA ACTTCTACCCGAGAGAAGCGAAGGTGCAGTGGAAAGTCGACAACGCCCTGCAGAGCGGAA ATAGCCAGGAGAGCGTGACCGAACAAGACTCTAAGGACAGCACCTACTCGCTGTCCTCCA CGCTGACTCTGTCTAAGGCCGACTATGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCC ACCAGGGCCTGAGCAGCCCCGTTACCAAGAGCTTCAACAGAGGAGAATGCggcggaggtg gcagcggcggcggcgggagoggcggcggcggctcaggcggagggggaagtggcggcggcg gcagcggcggcggaggcagcggcggtggcggctctGAGGTGCAACTGGTGGAATCTGGGG GCGGACTGGTGAAGCCTGGCGGCAGTCTGAGACTGAGCTGTGCCGCTTCCGGATTCACCT TTAGCAATTACGCCATGAGCTGGGTGCGGGAGGCCCCTGGAAAGCGGCTGGAATGGGTTG CTACAATCAGCAATAGAGGCAGCTACACATACTACCCCGACAGTGTCAAAGGCCGGTTTA CAATCAGCCGCGACAACGCCAAAAACAGCCTGTACCTGCAGATGAACTCCCTGCGGGCTG AGGATACAGCCCTCTACTACTGTGCCAGAGAACGTCCAATGGACTATTGGGGCCAAGGCA CACTGGTGACCGTGAGCAGCGCGTCTACCAAGGGCCCTTCTGTTTTCCCTCTGGCCCCCT GCAGCAGAAGCACGAGCGAGAGCACCGCTGCCCTGGGCTGTCTGGTGAAGGATTATTTCC CTGAGCCTGTGACCGTGTCTTGGAATAGCGGAGCOCTGACCAGCGGAGTGCATACATTCC CTGCTGTGCTGCAGTCTAGTGGGCTGTACAGCCTGTCTTCCGTTGTGGAAGTCCCTAGCA GCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGATCATAAGCCAAGCAACACCAAGG [00034]![embedded image]()
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CCATTAGCTGCAAGGCCTCTCAGAGCGTAGACTACGACGGCGACTCCTACATGAACTGGT ACCAGGAAAAGCCTGGCCAGCCTCCTAAGATCTTGATCTACGATGCCTCCAATCTGGAGA GCGGGATCCCCGCTAGATTCAGCGGGTCTGGAAGTGGAACCGACTTCACACTGACCATCT CTAGCCTGGAGCCCGAGGACTTTGCCATCTACTACTGCCAGCAGAGCAACGAGGACCCCT GGACATTCGGCGGCGGCACAAAGGTTGAGATCAAGAGAACCGTTGCCGCTCCTAGCGTGT TTATCTTCCCTCCCTCTGACGAGCAGCTGAAGAGCGGCACAGCCTCCGTGGTGTGCCTGC TGAACAACTTCTACCCCAGAGAGGCCAAGGTCCAGTGGAAGGTCGACAATGCCCTTCAGA GCGGCAACAGCCAGGAGTCCGTGACCGAGCAGGATAGCAAGGACTCTACCTACAGCCTGT CCTCTACGCTGACCCTGAGCAAAGCCGATTACGAAAAGCACAAAGTGTACGCCTGTGAAG TGACACACCAGGGCCTGTCTAGCCCTGTGACAAAGAGCTTTAACCGGGGCGAGTGCggcg gcggtggaagcggaggtggaggttcaggaggeggeggaagcggaggcggaggcagtgggg geggeggctccggaggcagcggcageggaggeggeggttcccAAGTGCAGCTCGTGCAGA GCGGCGCCGAGGTGAAAAAGCCCGGAGCCAGCGTGAAGCTGTCTTGCACCGCCTCCGGAT TCAACATCAAAGACGACTACATCCACTGGGTCAAGAAAGCCCCAGGGCAGGGGCTGGAGT GGATCGGCAGGATCGACCCTGCTGATGGCCACACCAAATACGCCCCAAAGTTCCAGGTGA AAGTGACAATTACCGCAGATACCTCCACCAGCACCGCTTATCTGGAACTGAGCTCTCTGC GGAGCGAGGACACAGCCGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAAGTGTTCG ACTACTGGGGCCAGGGCACCACAGTGACAGTGAGCTCTGCCAGCACAAAGGGCCCCAGCG TGTTTCCTCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCACCGCCGCCCTGGGCTGCC TGGTGAAGGACTACTTCCCTGAACCCGTGACCGTCTCCTGGAACAGTGGCGCCTTGACCT CTGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCTCCGGCCTGTACAGCCTGTCTAGCG TGGTGACCGTGCCTAGCTCGAGCCTGGGCACAAAGACATATACCTGTAACGTGGACCACA AGCCCAGCAACACGAAGGTGGACAAGCGAGTGTGAGTTTAAACCTGTGCCTTCTAGTTGC CAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG [00036]![embedded image]()
[00037]![embedded image]()
[00038]![embedded image]()
SEQIDNO:52-NucleotidesequenceofAAV2#14(FIGs.2Dand2H)(3 ITRboldfaced;minCBApromoterunderlined;Kozaksequenceboxed; IgGkappasignalsequenceitalicized;C1sscFabcodingsequence boldfacedandunderlined;(G.sub.4S).sub.2linkercodingsequenceboxedand italicized;(G4S);codingsequenceinlowercase,boldfaced,and italicized;BbscFvcodingsequenceboldfacedanditalicized; bGHpolyAsignalitalicizedandunderlined;and5ITRboxedand boldfaced) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG GCCAACTCCATCACTAGGGGTTCCTAATTTGATCTGAATTCGGTACCTAGTTATTAATAG TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG ACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTAT TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTG AGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTAT TTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG CCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCA GCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGG CCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTG CCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCA CAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGA CGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTAGAGCCT CTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTA [00039]![embedded image]()
ATGGAAGCCCCCGCCCAGCTGCTGTTCCTGCTGCTCCTGTGGCTGCCTGATACCACCGGC GATATCGTCCTGACCCAGAGCCCTGATAGCCTGGCCGTTTCACTGGGCGAGCGGGCCACA ATCTCCTGCAAGGCCTCTCAGTCTGTTGACTACGACGGCGACAGCTACATGAACTGGTAC CAGGAGAAACCCGGCCAACCTCCAAAGATCCTGATCTACGACGCCTCTAATCTGGAGAGC GGCATCCCCGCCCGGTTCAGCGGGTOCGGCAGCGGCACCGACTTTACCCTGACCATCTCT AGCCTGGAGCCTGAGGACTTCGCCATCTACTACTGTCAGCAGAGCAACGAGGATCCTTGG ACCTTTGGCGGCGGCACAAAGGTGGAAATCAAGCGGACCGTCGCCGCTCCATCCGTGTTT ATCTTCCCTCCTTCCGACGAGCAGCTCAAGAGCGGTACCGCCAGCGTGGTGTGCCTGCTG AACAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTAGACAACGCCTTGCAGAGC GGCAACTCTCAAGAGAGCGTGACAGAGCAGGACTCTAAGGACAGCACATACAGCCTAAGC TCCACCCTGACCCTCAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTT ACACACCAGGGCCTGAGCAGTCCGGTGACCAAGTCCTTCAACAGAGGCGAATGCggcgga ggaggctctggcggcggcggcagcggcggaggcggcagcggcggcggaggctctggcggc ggtggcagcggaggcggcggaagcggcggaggtggcagcCAGGTGCAGCTGGTGCAGAGC GGTGCTGAAGTGAAGAAACCCGGCGCTTCCGTGAAACTGAGCTGCACCGCCAGCGGATTT AACATCAAGGACGACTACATTCACTGGGTGAAAAAGGCCCCTGGCCAGGGCCTGGAATGG ATCGGGAGAATCGACCCCGCCGATGGCCATACCAAGTACGCTCCTAAGTTCCAGGTGAAA GTGACCATCACCGCTGATACAAGCACCTCTACAGCCTACCTGGAGCTGAGCTCCCTGCGG TCTGAGGACACCGCCGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAGGTGTTCGAC TACTGGGGACAGGGCACTACAGTCACCGTGTCTAGTGCTAGCACGAAGGGCCCTAGCGTG TTCCCTCTGGCTCCATGTAGCAGAAGCACCAGCGAAAGCACAGCTGCTCTGGGCTGCCTG GTGAAAGACTACTTCCCCGAGCCTGTGACCGTCAGCTGGAACTCCGGCGCCCTGACCAGC GGAGTGCACACCTTTCCTGCTGTGCTGCAATCCTCTGGCCTGTACTCTCTGAGCTCTGTT GTGACAGTGCCTTCTAGCAGCCTGGGAACCAAGACCTACACCTGCAACGTGGACCACAAG [00040]![embedded image]()
GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGAAGCCTGGCGGCTCACTGAGACTG AGCTGTGCCGCCAGCGGCTTCACCTTCTCCAACTACGCCATGAGCTGGGTGCGGGAAGCC CCAGGAAAGCGCCTGGAGTGGGTCGCCACCATCAGCAATAGAGGCTCGTATACATATTAC CCTGATTCCGTCAAAGGCAGATTCACCATCTCTAGAGATAATGCCAAGAACAGCCTGTAC CTGCAGATGAACTCCCTCAGAGCCGAGGATACAGCCCTGTATTACTGCGCCAGAGAACGG CCTATGGACTACTGGGGCCAAGGCACTCTGGTGACAGTGAGCAGCGGCGGCGGTGGTTCC GGCGGCGGAGGCTCTGGAGGAGGCGGCAGCGACATCCAGATGACCCAGAGCCCTAGCACC CTGTCCGCCAGCGTGGGAGATAGAGTGACCATTACCTGTAAAGCGAGCCAGGATGTGGGC ACCGCCGTGGCCTGGTATCAGAAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGG GCCTCTACCCGGCACACAGGCGTGCCCGACAGATTCTCCGGCTCCGGTTCTGGAACAGAC TTCACACTGACCATCAGCTCTCTTCAGGCCGAGGACTTCGCCGTGTACTTCTGCCACCAG CACAGCTCTAATCCTCTGACATTCGGCCAAGGCACAAAGCTGGAAATCAAGTGAGTTTAA ACCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGA [00041]![embedded image]()
[00042]![embedded image]()
[00043]![embedded image]()
[00044]![embedded image]()
SEQIDNO:53-BidirectionalpromoterandCMVenhancer(CBA promoter(reverse)boldedandunderlined;CMVenhancerboldfacedand italicized;CBApromoterboxedandunderlined) CGCCCGCCGCGCGCTTCGCTTTTTATAGGGCCGCCGCCGCCGCCGCCTCGCCATAAAAGG AAACTTTCGGAGCGCGCCGCTCTGATTGGCTGCCGCCGCACCTCTCCGCCTCGCCCCGCC CCGCCCCTCGCCCCGCCCCGCCCCGCCTGGCGCGCGCCCCCCCCCCCCCCCCGCCCCCAT CGCTGCACAAAATAATTAAAAAATAAATAAATACAAAATTGGGGGTGGGGAGGGGGGGGA GATGGGGAGAGTGAAGCAGAACGTGGGGCTCACCTCGCTAGTTATTAATAGTAATCAATT ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAA ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTC AATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCT [00045]![embedded image]()
[00046]![embedded image]()
[00047]![embedded image]()
[00048]![embedded image]()
[00049]![embedded image]()
[00050]![embedded image]()
SEQIDNO:54-[C1sscFv-(G.sub.4S).sub.2-BbscFv]nucleicacidsequence (construct#5,FIG.2A) ATGGAAGCCCCAGCTCAGCTGCTGTTCCTCCTGCTGCTGTGGCTGCCTGACACAACCGGC CAAGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAAAAGCCAGGAGCCTCCGTCAAACTG AGCTGTACCGCCAGCGGCTTTAACATCAAGGACGACTACATCCACTGGGTGAAGCAGGCC CCTGGCCAAGGTCTGGAATGGATCGGCAGAATCGACCCCGCTGACGGCCACACCAAGTAC GCCCCTAAGTTCCAGGTGAAGGTGACCATCACCGCCGACACCAGCACAAGCACCGCATAC CTGGAGCTGTCCAGCCTGAGAAGCGAGGATACCGCTGTCTACTACTGCGCCAGATACGGC TACGGCAGAGAGGTGTTCGACTACTGGGGACAAGGTACCACCGTGACGGTGTCTAGCGGC GGTGGCGGCAGCGGAGGAGGCGGCTCTGGAGGCGGCGGATCTGATATCGTGCTGACACAG AGTCCTGACAGCCTGGCCGTGAGCTTGGGGGAGCGGGCTACAATCTCTTGTAAAGCCAGC CAGAGCGTGGACTATGATGGCGATAGCTACATGAACTGGTATCAGCAGAAACCCGGCCAG CCCCCCAAGATCCTGATCTACGACGCCAGCAATCTGGAGAGCGGCATCCCCGCCCGGTTC AGCGGCAGCGGCTCGGGCACAGATTTCACCCTGACCATTAGCTCTCTGGAACCTGAGGAC TTCGCTATCTACTACTGCCAGCAGAGCAACGAGGACCCTTGGACCTTCGGCGGAGGTACA AAGGTGGAAATCAAGGGCGGCGGCGGCAGCGGAGGCGGAGGCTCTGAGGTGCAACTGGTG GAGAGCGGCGGCGGACTGGTAAAGCCCGGCGGCTCACTGAGACTGTCCTGCGCTGCCAGC GGCTTCACCTTTTCTAACTACGCCATGAGCTGGGTGCGGCAGGCTCCTGGAAAGCGCCTG GAATGGGTGGCCACAATCAGCAACCGGGGCTCTTACACCTACTATCCTGATTCTGTGAAG GGTAGGTTCACCATTTCAAGAGATAACGCCAAGAACAGCCTCTACCTGCAGATGAACAGC CTGCGGGCCGAAGACACCGCCCTGTACTACTGCGCCAGAGAAAGACCTATGGACTACTGG GGCCAGGGCACCCTGGTGACAGTTTCCTCCGGAGGCGGAGGCTCCGGCGGCGGCGGCTCC GGAGGCGGCGGAAGCGACATCCAGATGACCCAGAGCCCTAGCACTCTGTCCGCCAGCGTG GGCGACAGAGTGACCATCACATGCAAGGCCTCTCAGGACGTGGGCACCGCCGTGGCCTGG TACCAACAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCAGCACAAGACAT ACAGGCGTGCCCGATAGATTCAGCGGCTCCGGCTCTGGCACAGACTTCACACTGACCATC AGCAGCCTCCAGGCCGAGGATTTTGCCGTGTACTTCTGCCACCAGCACAGCAGCAATCCA CTGACATTTGGCCAGGGCACCAAGCTGGAGATCAAATGA SEQIDNO:55-[C1sscFv-(G.sub.4S).sub.2-BbscFv]aminoacidsequence (construct#5,FIG.2A)(signalpeptideboldfaced) MEAPAQLLFLLLLWLPDTTGQVOLVOSGAEVKKPGASVKLSCTASGENIKDDYIHWVKQA PGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAVYYCARYG YGREVEDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKAS QSVDYDGDSYMNWYQQKPGQPPKILIYDASNLESGIPARFSGSGSGTDFTLTISSLEPED FAIYYCQQSNEDPWTFGGGTKVEIKGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAAS GFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNS LRAEDTALYYCARERPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASV GDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDETLTI SSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK* SEQIDNO:56-[BbscFv-(G.sub.4S).sub.2-C1sscFv]nucleicacidsequence (construct#6,FIG.2A) ATGGAAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTACCTGATACCACCGGC GAGGTGCAGCTGGTCGAGAGCGGCGGGGGCCTGGTGAAACCAGGAGGAAGCCTGAGACTG AGCTGCGCCGCCTCTGGCTTCACCTTCAGCAATTACGCTATGAGCTGGGTCAGACAGGCC CCAGGAAAAAGACTGGAATGGGTGGCCACAATTTCTAACCGGGGCTCCTACACCTACTAT CCTGACAGCGTGAAGGGCAGATTCACAATCAGCCGGGACAACGCCAAGAACAGCCTGTAC CTGCAGATGAACAGCCTCAGAGCCGAGGACACCGCCCTGTACTACTGCGCCAGAGAGCGG CCTATGGACTACTGGGGCCAAGGCACACTGGTCACAGTTTCCAGCGGCGGCGGCGGCAGC GGTGGCGGCGGCAGCGGAGGCGGTGGCTCTGATATCCAGATGACCCAGTCCCCTAGCACC CTGTCTGCCTCTGTGGGCGACAGAGTGACCATTACATGCAAGGCCTCTCAGGACGTGGGC ACCGCTGTGGCCTGGTATCAGCAGAAACCCGGCAAGGCTCCCAAGCTGCTGATCTACTGG GCCAGCACAAGACACACAGGCGTGCCTGATAGATTCAGCGGCAGCGGTAGCGGCACCGAC TTCACCCTGACAATCAGCTCCCTCCAGGCTGAAGATTTTGCCGTGTACTTCTGCCACCAG CATAGCAGCAACCCCCTGACATTCGGCCAGGGCACAAAGCTGGAAATCAAGGGAGGCGGC GGCTCTGGAGGCGGCGGAAGCCAAGTGCAGCTGGTGCAAAGCGGCGCCGAGGTGAAAAAG CCCGGCGCATCTGTGAAGCTGAGTTGTACAGCTTCTGGATTTAACATCAAGGACGACTAC ATCCACTGGGTTAAGCAGGCCCCTGGCCAGGGCCTGGAGTGGATCGGCAGAATCGACCCC GCTGATGGCCACACCAAGTACGCCCCTAAGTTCCAGGTGAAGGTGACCATCACGGCCGAC ACCAGCACAAGCACCGCCTACCTGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTG TACTACTGTGCCAGATACGGCTACGGCCGCGAGGTGTTCGACTACTGGGGACAAGGAACA ACCGTGACCGTGTCCAGCGGCGGCGGCGGCAGCGGCGGAGGAGGCTCTGGCGGCGGCGGC AGCGACATCGTGCTGACCCAGAGCCCCGATTCTCTGGCCGTGAGCCTGGGAGAGAGAGCC ACCATCTCCTGCAAGGCTTCCCAATCTGTGGACTATGATGGAGATAGCTACATGAACTGG TACCAGCAGAAGCCTGGCCAGCCTCCAAAGATCCTGATCTACGACGCCAGCAATCTGGAA TCCGGCATCCCTGCTCGGTTTAGCGGCAGCGGCTCCGGAACCGACTTCACCCTGACCATC AGCTCTCTGGAGCCTGAGGATTTCGCCATCTACTACTGCCAGCAGTCCAACGAAGACCCT TGGACCTTTGGCGGCGGCACCAAGGTCGAAATCAAATGA SEQIDNO:57-[BbscFv-(G.sub.4S).sub.2-C1sscFv]aminoacidsequence (construct#6,FIG.2A)(signalpeptideboldfaced) MEAPAQLLFLLLLWLPDTTGEVOLVESGGGLVKPGGSLRLSCAASGFTESNYAMSWVRQA PGKRLEWVATISNRGSYTYYPDSVKGRETISRDNAKNSLYLQMNSLRAEDTALYYCARER PMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKASQDVG TAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDFTLTISSLQAEDFAVYFCHQ HSSNPLTFGQGTKLEIKGGGGSGGGGSQVQLVOSGAEVKKPGASVKLSCTASGENIKDDY IHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLRSEDTAV YYCARYGYGREVEDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERA TISCKASQSVDYDGDSYMNWYQQKPGQPPKILIYDASNLESGIPARESGSGSGTDETLTI SSLEPEDFAIYYCQQSNEDPWTFGGGTKVEIK* SEQIDNO:58-[C1sscFab-(G.sub.4S).sub.3-BbscFv]nucleicacid sequence(construct#15,FIG.2E) ATGGAAGCTCCAGCCCAGCTGCTGTTCCTGCTGCTCCTTTGGCTGCCTGACACAACAGGC GATATCGTGCTGACCCAGAGCCCTGACAGCCTGGCCGTGTCACTGGGCGAGCGGGCCACG ATCAGCTGCAAGGCCAGCCAGTCCGTGGATTACGACGGCGACAGCTACATGAACTGGTAT CAGCAGAAGCCCGGACAGCCTCCCAAGATCCTGATCTACGACGCCAGCAACCTGGAAAGC GGCATCCCTGCCAGATTCAGCGGGTCCGGCAGCGGAACAGACTTCACCCTGACCATCTCC AGCCTGGAACCTGAGGATTTCGCCATCTACTACTGTCAGCAGAGCAACGAGGATCCTTGG ACCTTCGGCGGCGGCACCAAGGTCGAGATCAAGAGAACCGTGGCCGCTCCTAGCGTGTTC ATCTTCCCTCCTTCCGACGAGCAGCTGAAGAGCGGCACCGCCTCTGTGGTGTGCCTACTG AACAACTTCTACCCTAGAGAGGCTAAAGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGC GGCAACAGCCAGGAGTCTGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGTCT TCCACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTG ACACACCAGGGCCTGAGCTCCCCCGTGACCAAAAGCTTCAACAGAGGAGAATGCGGCGGA GGCGGAAGCGGCGGCGGGGGCTCTGGAGGCGGCGGCTCCGGCGGCGGAGGCAGCGGAGGT GGCGGCTCTGGCGGCGGCGGCTCCGGAGGCGGCGGCTCACAGGTGCAGCTGGTGCAATCT GGTGCTGAGGTGAAGAAGCCAGGCGCCAGCGTGAAGCTAAGCTGCACCGCCTCCGGTTTC AACATCAAAGACGACTACATCCACTGGGTGAAACAGGCCCCAGGCCAGGGCCTGGAGTGG ATCGGCAGAATCGACCCTGCCGATGGCCACACCAAGTACGCTCCTAAGTTCCAGGTCAAG GTGACAATCACCGCAGATACCAGCACAAGCACCGCCTACCTGGAGCTGAGCTCGCTGAGA AGCGAGGACACAGCCGTGTACTACTGCGCCAGATACGGCTACGGAAGAGAGGTGTTTGAT TACTGGGGACAGGGCACTACCGTGACCGTGAGCTCCGCCAGCACCAAGGGCCCTAGCGTG TTCCCCCTGGCCCCATGTTCTAGATCTACATCTGAAAGCACCGCTGCTCTGGGCTGCCTG GTAAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCCCTGACCTCT GGCGTGCATACATTTCCTGCCGTGCTGCAGAGCTCAGGCCTGTACTCCCTGAGCTCTGTC GTTACAGTGCCCAGCAGCTCCCTGGGAACAAAGACCTACACCTGCAACGTGGACCACAAG CCTAGCAATACCAAGGTGGACAAGCGGGTGGGGGGCGGTGGATCCGGCGGAGGCGGGAGC GGCGGCGGAGGATCCGAGGTGCAGCTGGTCGAATCCGGCGGGGGCCTGGTGAAACCCGGC GGCTCTCTGAGGCTGTCCTGCGCCGCTAGCGGCTTTACCTTTAGCAACTACGCTATGAGC TGGGTTAGACAGGCCCCTGGCAAGCGGCTCGAATGGGTCGCAACAATTTCTAATAGAGGC AGTTACACATACTACCCCGACTCTGTGAAGGGCCGGTTCACCATTAGCAGAGATAACGCC AAGAACTCTCTGTACCTGCAGATGAATTCACTGCGGGCCGAGGACACCGCCCTGTATTAT TGTGCTCGGGAACGTCCTATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCT GGCGGCGGCGGCAGCGGCGGTGGCGGCAGCGGCGGCGGCGGTAGCGACATCCAGATGACC CAAAGCCCCAGCACCCTGTCTGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAAAGCC TCCCAGGATGTGGGAACAGCCGTTGCCTGGTACCAGCAAAAACCTGGCAAGGCCCCTAAG CTGCTGATCTACTGGGCCAGCACCCGCCACACTGGCGTGCCTGATCGGTTCAGCGGAAGC GGCAGCGGAACAGATTTTACACTGACTATCAGCTCCCTCCAGGCCGAAGATTTCGCCGTG TACTTCTGCCACCAGCACAGCAGCAACCCTCTGACCTTCGGACAAGGGACAAAACTCGAA ATCAAGTGAG SEQIDNO:59-[C1sscFab-(G.sub.4S).sub.3-BbscFv]aminoacidsequence (construct#15,FIG.2E)(signalPeptideboldfaced) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY QQKPGOPPKILIYDASNLESGIPARESGSGSGTDFTLTISSLEPEDFAIYYCQQSNEDPW TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVOLVQSGAEVKKPGASVKLSCTASGE NIKDDYIHWVKQAPGQGLEWIGRIDPADGHTKYAPKFQVKVTITADTSTSTAYLELSSLR SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMS WVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLOMNSLRAEDTALYY CARERPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKA SQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRESGSGSGTDETLTISSLQAEDFAV YFCHQHSSNPLTFGQGTKLEIK* SEQIDNO:60-[C1sscFab-(G.sub.4S).sub.3-BbscFv-CM]nucleicacid sequence(construct#16,FIG.2E) ATGGAAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACAACCGGC GACATCGTGCTGACACAGAGCCCCGACAGCCTCGCCGTTTCCCTCGGCGAGCGGGCCACA ATCTCATGCAAGGCCTCACAGTCCGTGGACTATGACGGCGATAGCTACATGAACTGGTAC CAGGAGAAGCCTGGCCAACCTCCAAAGATCCTGATCTACGACGCCAGCAATCTGGAATCC GGTATTCCTGCCAGATTCAGCGGCTCTGGATCCGGCACCGACTTTACTCTGACCATCAGC TCTCTGGAACCTGAGGACTTTGCTATCTACTACTGCCAGCAGAGCAACGAGGACCCCTGG ACCTTCGGCGGCGGCACCAAAGTGGAAATCAAGCGGACCGTGGCCGCTCCTTCAGTGTTC ATCTTCCCACCTTCCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTG AACAACTTCTACCCCAGAGAGGCTAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAAAGT GGCAACTCTCAGGAGTCTGTGACAGAGCAGGACTCCAAGGACAGCACCTACAGCCTGTCC TCTACCCTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTG ACACACCAGGGGCTGAGCTCCCCTGTGACAAAATCTTTCAACCGGGGCGAGTGCGGCGGA GGAGGCAGCGGCGGCGGCGGCAGCGGGGGCGGAGGCTCCGGCGGCGGCGGTAGCGGTGGG GGCGGATCTGGAGGCGGGGGATCGGGCGGAGGCGGCAGCCAGGTGCAGCTGGTCCAGAGC GGCGCCGAGGTGAAAAAGCCAGGCGCCTCTGTGAAGCTGTCTTGCACCGCCTCTGGTTTT AATATCAAGGACGACTACATCCACTGGGTGAAGAAGGCTCCAGGTCAAGGACTGGAATGG ATCGGCCGGATCGACCCCGCTGATGGCCACACCAAATACGCTCCTAAGTTCCAGGTGAAA GTTACAATTACAGCCGATACCAGCACAAGCACCGCCTACCTGGAGCTGAGCTCTCTGAGA AGCGAAGATACAGCCGTGTACTACTGCGCAAGATACGGCTACGGCAGAGAGGTGTTCGAC TATTGGGGACAGGGCACCACAGTGACCGTGTCTAGTGCCAGCACCAAGGGCCCCAGCGTG TTCCCTCTGGCCCCTTGTAGCAGATCTACCAGCGAGTCCACCGCTGCTCTGGGCTGCCTG GTCAAGGATTACTTCCCCGAGCCTGTGACCGTTAGCTGGAACAGCGGAGCCCTGACCAGC GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTC GTGACAGTGCCCAGCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAG CCCAGCAACACCAAGGTGGACAAGAGAGTGGGCGGCGGAGGCTCTGGCGGCGGCGGCTCT GGGGGCGGCGGAAGCGAGGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGAAGCCTGGC GGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAACTACGCCATGAGC TGGGTTAGAGAAGCCCCTGGAAAAAGACTGGAATGGGTGGCCACCATCTCTAATAGAGGA TCTTATACATACTACCCTGATTCTGTGAAAGGACGGTTCACAATCTCCCGCGACAACGCC AAGAACTCACTGTACCTGCAGATGAACTCTCTGAGGGCCGAGGATACCGCCCTGTACTAC TGTGCCCGAGAAAGACCTATGGATTACTGGGGCCAGGGCACCCTCGTCACAGTTTCCTCT GGCGGGGGCGGTAGCGGCGGCGGCGGATCCGGCGGAGGTGGCAGCGACATCCAGATGACC CAAAGCCCTTCTACACTGAGCGCCAGCGTCGGCGACCGGGTGACCATCACCTGTAAAGCC AGCCAAGACGTGGGCACGGCTGTGGCTTGGTATCAGAAGAAACCTGGCAAGGCCCCCAAG CTGCTTATCTACTGGGCCAGCACAAGACACACAGGCGTTCCTGATAGATTCAGCGGCAGC GGCTCCGGCACAGATTTCACCCTGACCATCTCGAGTCTGCAGGCCGAGGATTTCGCCGTG TACTTCTGCCACCAGCATTCTTCTAACCCTCTGACCTTTGGCCAGGGAACCAAGCTGGAA ATCAAGTGA SEQIDNO:61-SEQIDNO:60-[C1sscFab-(G.sub.4S).sub.3-BbscFv-CM] aminoacidsequence(construct#16,FIG.2E)(signalpeptide boldfaced;chargemutationsboxedanditalicized,numbering excludingsignalpeptide:Q42EandQ292KinC1sscFab,andQ524E andQ653KinaBbscFv) MEAPAQLLFLLLLWLPDTTGDIVLTOSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY [00051]![embedded image]()
TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALOS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVOLVOSGAEVKKPGASVKLSCTASGF [00052]![embedded image]()
SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTESNYAMS [00053]![embedded image]()
CARERPMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCKA [00054]![embedded image]()
YFCHQHSSNPLTFGQGTKLEIK* SEQIDNO:62-[C1sscFab-BiDir-BbscFab-CM]nucleicacid sequence(construct#21,FIG.2G) TCACACCCGCTTATCCACCTTGGTGTTGCTGGGCTTGTGGTCCACGTTGCAGGTGTAGGT CTTTGTGCCCAGGCTAGAGCTAGGCACTGTCACGACAGAGGACAGAGAGTACAGGCCGCT GCTCTGCAGCACGGCGGGGAAGGTGTGCACCCCGCTTGTCAGGGCTCCGCTGTTCCAGGA CACGGTCACAGGCTCAGGGAAATAATCCTTGACCAGGCAGCCCAGAGCAGCCGTGCTCTC TGAGGTACTTCTGCTACAAGGAGCCAGTGGGAACACGCTAGGGCCCTTTGTGCTGGCGGA CGACACGGTCACTGTTGTGCCCTGTCCCCAGTAGTCGAACACTTCTCTGCCGTAGCCGTA TCTGGCGCAGTAGTACACAGCGGTGTCCTCGGATCTAAGGCTGCTCAGTTCCAGATAAGC TGTAGAGGTGCTGGTATCGGCGGTGATGGTGACTTTCACCTGGAACTTAGGGGCGTACTT TGTGTGGCCGTCGGCAGGGTCGATTCTGCCGATCCACTCCAGTCCCTGGCCGGGGGCCTT CTTCACCCAGTGGATGTAATCGTCCTTGATATTGAAGCCGCTGGCGGTGCAGCTCAGCTT AACACTAGCGCCAGGCTTTTTCACCTCGGCTCCGCTCTGCACCAGCTGCACCTGGGATCC GCCGCCGCCGCTGCCGCCTCCGCCGCTGCCGCCTCCGCCGCTTCCGCCTCCCCCAGAGCC GCCGCCACCGCTGCCTCCTCCGCCGGAGCCGCCGCCGCCGCACTCGCCCCGGTTGAAGCT TTTGGTCACAGGAGAGGACAGGCCCTGATGTGTCACTTCACAGGCGTACACCTTGTGCTT CTCGTAGTCGGCCTTGCTCAAGGTCAGGGTGCTGGACAGGCTGTATGTTGAGTCCTTGCT GTCCTGCTCGGTCACGCTCTCTTGGCTGTTGCCGCTTTGCAGGGCGTTGTCAACTTTCCA TTGGACCTTTGCCTCTCTGGGGTAGAAGTTATTCAGCAGGCACACCACAGAGGCGGTTCC GCTCTTCAGCTGCTCGTCGCTTGGAGGGAAGATAAAGACAGAAGGGGCGGCCACGGTGCG CTTGATTTCCACCTTGGTGCCGCCTCCAAAGGTCCAGGGGTCCTCGTTGCTCTGCTGGCA GTAGTAGATGGCAAAATCCTCGGGTTCCAGAGAAGAAATTGTCAGGGTGAAATCAGTGCC AGAGCCGCTGCCGCTGAATCTGGCGGGGATGCCGCTTTCCAGATTGCTGGCGTCGTAGAT CAGGATTTTTGGAGGCTGGCCGGGTTTCTCCTGGTACCAGTTCATGTAGCTGTCGCCGTC ATAGTCCACGCTCTGAGAGGCTTTACAGCTGATTGTGGCCCGTTCGCCGAGGCTCACGGC CAGGCTATCAGGGCTCTGCGTCAGCACGATATCGCCGGTGGTGTCAGGCAGCCACAGGAG CAGCAGGAACAGCAGCTGGGCAGGGGCTTCCATGGTGGGCTCTGGCGCCCGCCGCGCGCT TCGCTTTTTATAGGGCCGCCGCCGCCGCCGCCTCGCCATAAAAGGAAACTTTCGGAGCGC GCCGCTCTGATTGGCTGCCGCCGCACCTCTCCGCCTCGCCCCGCCCCGCCCCTCGCCCCG CCCCGCCCCGCCTGGCGCGCGCCCCCCCCCCCCCCCCGCCCCCATCGCTGCACAAAATAA TTAAAAAATAAATAAATACAAAATTGGGGGTGGGGAGGGGGGGGAGATGGGGAGAGTGAA GCAGAACGTGGGGCTCACCTCGCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA CCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA ATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGT GCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGGGGGGCGGGGCGAG GGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGG CGGGCGCCAACTAGCCCACCATGGAAGCCCCCGCTCAGCTGCTGTTCCTGCTGCTGCTGT GGCTGCCTGACACCACCGGCGACATCCAGATGACACAGAGCCCTAGCACCCTGAGCGCCT CCGTGGGGGACAGAGTGACAATCACATGTAAAGCCTCCCAGGACGTGGGCACTGCCGTGG CCTGGTACCAGAAAAAACCGGGAAAAGCCCCTAAGCTGCTGATCTACTGGGCCAGCACCA GACACACCGGCGTCCCCGATAGATTCAGCGGCTCTGGCAGCGGAACTGATTTCACCCTGA CCATTTCTTCTCTGCAGGCCGAGGACTTCGCCGTGTACTTTTGCCACCAGCACAGCAGCA ACCCTCTGACCTTCGGACAGGGCACAAAGCTGGAAATCAAGCGGACAGTGGCTGCTCCTT CTGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGAGCGGCACCGCCTCTGTGGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCC TGCAATCTGGCAACAGCCAGGAGAGCGTGACGGAACAAGATAGCAAGGACAGCACCTACT CCCTGAGCAGCACACTGACCTTGTCCAAGGCAGATTACGAGAAGCACAAGGTGTACGCCT GCGAGGTGACCCACCAGGGACTGAGCAGCCCAGTGACCAAGAGCTTCAACAGAGGAGAGT GCGGCGGCGGCGGAAGCGGAGGCGGAGGCAGCGGCGGCGGCGGCAGTGGAGGCGGCGGCT CTGGCGGAGGGGGCAGTGGCGGTGGCGGATCCGGCGGCGGCGGCAGCGAGGTGCAGCTTG TGGAATCCGGCGGCGGCCTGGTGAAGCCCGGCGGTAGCCTGAGACTGTCTTGTGCCGCCT CTGGCTTCACCTTTAGCAATTACGCCATGAGCTGGGTGCGGGAGGCTCCCGGCAAAAGAC TGGAATGGGTCGCCACCATCAGCAACCGGGGATCATATACCTACTACCCTGATAGCGTGA AAGGCAGGTTCACAATCAGCCGGGACAATGCCAAGAACAGCCTGTACCTGCAGATGAACT CACTGCGGGCCGAGGACACCGCCCTGTATTACTGCGCCAGAGAGAGACCTATGGACTACT GGGGCCAGGGCACCCTGGTGACCGTTTCCTCCGCCAGCACCAAGGGCCCTAGCGTGTTCC CTCTGGCCCCATGCAGCAGAAGCACATCTGAGAGCACCGCCGCTCTGGGCTGCCTGGTGA AGGACTACTTCCCCGAGCCTGTGACAGTGAGCTGGAACTCCGGCGCCCTGACCAGCGGCG TGCACACATTTCCAGCTGTGCTGCAGTCTAGCGGCCTGTACAGCCTGAGCAGCGTTGTGA CAGTGCCTTCTAGCAGCCTCGGCACCAAGACCTACACCTGTAACGTGGATCATAAGCCTT CTAATACCAAGGTTGACAAGAGAGTGTGA SEQIDNO:63-C1sscFab-CMarm(construct#22,FIG.2G)(signal sequenceboldfaced;chargemutationsboxedanditalicized,numbering excludingsignalpeptide:Q42EandQ292K) MEAPAQLLFLLLLWLPDTTGDIVLTQSPDSLAVSLGERATISCKASQSVDYDGDSYMNWY [00055]![embedded image]()
TFGGGTKVEIKRTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKLSCTASGF [00056]![embedded image]()
SEDTAVYYCARYGYGREVEDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRV* SEQIDNO:64-BbscFab-CMarm(construct#22,FIG.2G)(signal sequenceboldfaced;chargemutationsboxedanditalicized,numbering excludingsignalpeptide:Q38KandQ288E,andS114A,N137K,and T434E) [00057]![embedded image]()
GKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQ [00058]![embedded image]()
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGECGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVOLVESGGGLVKPGGSLRLSCAASGFTFSN [00059]![embedded image]()
ALYYCARERPMDYWGQGTLVTVSSASTKGPSVEPLAPCSRSTSESTAALGCLVKDYFPEP [00060]![embedded image]()
VTVSWNSGAL SEQIDNO:65-HumancomplementC1saminoacidsequencepriorto processingandactivation(signalsequenceboldfaced) MWCIVLFSLLAWVYAEPTMYGEILSPNYPQAYPSEVEKSWDIEVPEGYGIHLYFTHLDIE LSENCAYDSVQIISGDTEEGRLCGQRSSNNPHSPIVEEFQVPYNKLQVIFKSDESNEERE TGFAAYYVATDINECTDEVDVPCSHFCNNFIGGYFCSCPPEYFLHDDMKNCGVNCSGDVE TALIGEIASPNYPKPYPENSRCEYQIRLEKGFQVVVTLRREDEDVEAADSAGNCLDSLVE VAGDRQFGPYCGHGFPGPLNIETKSNALDIIFQTDLTGOKKGWKLRYHGDPMPCPKEDTP NSVWEPAKAKYVERDVVOITCLDGFEVVEGRVGATSFYSTCQSNGKWSNSKLKCQPVDCG IPESIENGKVEDPESTLEGSVIRYTCEEPYYYMENGGGGEYHCAGNGSWVNEVLGPELPK CVPVCGVPREPFEEKQRIIGGSDADIKNEPWQVFFDNPWAGGALINEYWVLTAAHVVEGN REPTMYVGSTSVQTSRLAKSKMLTPEHVFIHPGWKLLEVPEGRTNEDNDIALVRLKDPVK MGPTVSPICLPGTSSDYNLMDGDLGLISGWGRTEKRDRAVRLKAARLPVAPLRKCKEVKV EKPTADAEAYVFTPNMICAGGEKGMDSCKGDSGGAFAVODPNDKTKFYAAGLVSWGPQCG TYGLYTRVKNYVDWIMKTMQENSTPRED SEQIDNO:66-HumancomplementfactorBpriortoprocessingand activation(signalpeptideboldfaced) MGSNLSPQLCLMPFILGLLSGGVTTTPWSLARPQGSCSLEGVEIKGGSERLLQEGQALEY VCPSGFYPYPVOTRTCRSTGSWSTLKTQDQKTVRKAECRAIHCPRPHDFENGEYWPRSPY YNVSDEISFHCYDGYTLRGSANRTCQVNGRWSGQTAICDNGAGYCSNPGIPIGTRKVGSQ YRLEDSVTYHCSRGLTLRGSQRRTCQEGGSWSGTEPSCQDSFMYDTPQEVAEAFLSSLTE TIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIYLVLDGSDSIGASNETGAKKCLVNLIEKV ASYGVKPRYGLVTYATYPKIWVKVSEADSSNADWVTKQLNEINYEDHKLKSGTNTKKALQ AVYSMMSWPDDVPPEGWNRTRHVIILMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPRE DYLDVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDVFYQMIDESQSLSLCGM VWEHRKGTDYHKQPWQAKISVIRPSKGHESCMGAVVSEYFVLTAAHCFTVDDKEHSIKVS VGGEKRDLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLKYGQTIRPICLPCT EGTTRALRLPPTTTCQQQKEELLPAQDIKALEVSEEEKKLTRKEVYIKNGDKKGSCERDA QYAPGYDKVKDISEVVTPRFLCTGGVSPYADPNTCRGDSGGPLIVHKRSRFIQVGVISWG VVDVCKNQKRQKOVPAHARDFHINLFQVLPWLKEKLQDEDLGEL SEQIDNO:67-HCDR1ofanti-C1santibody(IMGT) GENIKDDY SEQIDNO:68-HCDR2ofanti-C1santibody(IMGT) IDPADGHT SEQIDNO:69-HCDR3ofanti-C1santibody(IMGT) ARYGYGREVEDY SEQIDNO:70-LCDR1ofanti-C1santibody(IMGT) QSVDYDGDSY SEQIDNO:71-HCDR1ofanti-C1santibody(Chothia) GFNIKDD SEQIDNO:72-HCDR2ofanti-C1santibody(Chothia) DPADGH SEQIDNO:73-HCDR1ofanti-Bbantibody(IMGR) GFTFSNYA SEQIDNO:74-HCDR2ofanti-Bbantibody(IMGR) ISNRGSYT SEQIDNO:75-HCDR3ofanti-Bbantibody(IMGR) ARERPMDY SEQIDNO:76-LCDR1ofanti-Bbantibody(IMGR) QDVGTA SEQIDNO:77-HCDR1ofanti-Bbantibody(Chothia) GFTFSNY SEQIDNO:78-HCDR2ofanti-Bbantibody(Chothia) SNRGSY SEQIDNO:79-[BbscFab-BiDir-C1sscFab-CM]nucleicacid sequence(construct#22,FIG.2G) TCACACTCTCTTGTCAACCTTGGTATTAGAAGGCTTATGATCCACGTTACAGGTGTAGGT CTTGGTGCCGAGGCTGCTAGAAGGCACTGTCACAACGCTGCTCAGGCTGTACAGGCCGCT AGACTGCAGCACAGCTGGAAATGTGTGCACGCCGCTGGTCAGGGCGCCGGAGTTCCAGCT CACTGTCACAGGCTCGGGGAAGTAGTCCTTCACCAGGCAGCCCAGAGCGGCGGTGCTCTC AGATGTGCTTCTGCTGCATGGGGCCAGAGGGAACACGCTAGGGCCCTTGGTGCTGGCGGA GGAAACGGTCACCAGGGTGCCCTGGCCCCAGTAGTCCATAGGTCTCTCTCTGGCGCAGTA ATACAGGGCGGTGTCCTCGGCCCGCAGTGAGTTCATCTGCAGGTACAGGCTGTTCTTGGC ATTGTCCCGGCTGATTGTGAACCTGCCTTTCACGCTATCAGGGTAGTAGGTATATGATCC CCGGTTGCTGATGGTGGCGACCCATTCCAGTCTTTTGCCGGGAGCCTCCCGCACCCAGCT CATGGCGTAATTGCTAAAGGTGAAGCCAGAGGCGGCACAAGACAGTCTCAGGCTACCGCC GGGCTTCACCAGGCCGCCGCCGGATTCCACAAGCTGCACCTCGCTGCCGCCGCCGCCGGA TCCGCCACCGCCACTGCCCCCTCCGCCAGAGCCGCCGCCTCCACTGCCGCCGCCGCCGCT GCCTCCGCCTCCGCTTCCGCCGCCGCCGCACTCTCCTCTGTTGAAGCTCTTGGTCACTGG GCTGCTCAGTCCCTGGTGGGTCACCTCGCAGGCGTACACCTTGTGCTTCTCGTAATCTGC CTTGGACAAGGTCAGTGTGCTGCTCAGGGAGTAGGTGCTGTCCTTGCTATCTTGTTCCGT CACGCTCTCCTGGCTGTTGCCAGATTGCAGGGCGTTGTCCACCTTCCACTGCACTTTGGC TTCTCTGGGGTAGAAGTIGTTCAGCAGGCACACCACAGAGGCGGTGCCGCTCTTCAGCTG CTCGTCGCTAGGTGGAAAGATGAACACAGAAGGAGCAGCCACTGTCCGCTTGATTTCCAG CTTTGTGCCCTGTCCGAAGGTCAGAGGGTTGCTGCTGTGCTGGTGGCAAAAGTACACGGC GAAGTCCTCGGCCTGCAGAGAAGAAATGGTCAGGGTGAAATCAGTTCCGCTGCCAGAGCC GCTGAATCTATCGGGGACGCCGGTGTGTCTGGTGCTGGCCCAGTAGATCAGCAGCTTAGG GGCTTTTCCCGGTTTTTTCTGGTACCAGGCCACGGCAGTGCCCACGTCCTGGGAGGCTTT ACATGTGATTGTCACTCTGTCCCCCACGGAGGCGCTCAGGGTGCTAGGGCTCTGTGTCAT CTGGATGTCGCCGGTGGTGTCAGGCAGCCACAGCAGCAGCAGGAACAGCAGCTGAGCGGG GGCTTCCATGGTGGGCTAGTTGGCGCCCGCCGCGCGCTTCGCTTTTTATAGGGCCGCCGC CGCCGCCGCCTCGCCATAAAAGGAAACTTTCGGAGCGCGCCGCTCTGATTGGCTGCCGCC GCACCTCTCCGCCTCGCCCCGCCCCGCCCCTCGCCCCGCCCCGCCCCGCCTGGCGCGCGC CCCCCCCCCCCCCCCGCCCCCATCGCTGCACAAAATAATTAAAAAATAAATAAATACAAA ATTGGGGGTGGGGAGGGGGGGGAGATGGGGAGAGTGAAGCAGAACGTGGGGCTCACCTCG ACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAG GTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGC GTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCC ACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATT GACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTAT GTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAA TAACTAGCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGG GGGGGGGGCGCGCGCCAGGCGGGGGGGGGCGGGGCGAGGGGCGGGGGGGGGCGAGGCGGA GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCCAGAGCCCACCATGG AAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTCCTGTGGCTGCCTGACACCACCGGCGATA TCGTGCTGACGCAGAGCCCTGATAGCCTGGCCGTGAGCCTCGGCGAACGGGCCACAATCA GCTGTAAAGCCTCTCAGAGCGTGGACTATGACGGCGACAGCTACATGAACTGGTACCAGG AGAAACCCGGCCAGCCTCCAAAAATCCTGATCTACGACGCCAGCAATCTGGAAAGCGGCA TCCCCGCCAGATTCAGCGGCAGCGGCTCTGGCACTGATTTCACCCTGACAATTTCTTCTC TGGAACCCGAGGATTTTGCCATCTACTACTGCCAGCAGAGCAACGAGGACCCCTGGACCT TTGGAGGCGGCACCAAGGTGGAAATCAAGCGCACCGTGGCCGCCCCTTCTGTCTTTATCT TCCCTCCAAGCGACGAGCAGCTGAAGAGCGGAACCGCCTCTGTGGTGTGCCTGCTGAATA ACTTCTACCCCAGAGAGGCAAAGGTCCAATGGAAAGTTGACAACGCCCTGCAAAGCGGCA ACAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCAACATACAGCCTGTCCAGCA CCCTGACCTTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACAC ATCAGGGCCTGTCCTCTCCTGTGACCAAAAGCTTCAACCGGGGCGAGTGCGGCGGCGGCG GCTCCGGCGGAGGAGGCAGCGGTGGCGGCGGCTCTGGGGGAGGCGGAAGCGGCGGAGGCG GCAGCGGCGGAGGCGGCAGCGGCGGCGGCGGATCCCAGGTGCAGCTGGTGCAGAGCGGAG CCGAGGTGAAAAAGCCTGGCGCTAGTGTTAAGCTGAGCTGCACCGCCAGCGGCTTCAATA TCAAGGACGATTACATCCACTGGGTGAAGAAGGCCCCCGGCCAGGGACTGGAGTGGATCG GCAGAATCGACCCTGCCGACGGCCACACAAAGTACGCCCCTAAGTTCCAGGTGAAAGTCA CCATCACCGCCGATACCAGCACCTCTACAGCTTATCTGGAACTGAGCAGCCTTAGATCCG AGGACACCGCTGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAAGTGTTCGACTACT GGGGACAGGGCACAACAGTGACCGTGTCGTCCGCCAGCACAAAGGGCCCTAGCGTGTTCC CACTGGCTCCTTGTAGCAGAAGTACCTCAGAGAGCACGGCTGCTCTGGGCTGCCTGGTCA AGGATTATTTCCCTGAGCCTGTGACCGTGTCCTGGAACAGCGGAGCCCTGACAAGCGGGG TGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGTCCTCTGTCGTGA CAGTGCCTAGCTCTAGCCTGGGCACAAAGACCTACACCTGCAACGTGGACCACAAGCCCA GCAACACCAAGGTGGATAAGCGGGTGTGA SEQIDNO:80[BbscFab-BiDir-C1sscFab]nucleicacidsequence (construct#10,FIG.2B) TCACACTCTCTTGTCAACCTTGGTATTAGAAGGCTTATGATCCACGTTACAGGTGTAGGT CTTGGTGCCGAGGCTGCTAGAAGGCACTGTCACAACGCTGCTCAGGCTGTACAGGCCGCT AGACTGCAGCACAGCTGGAAATGTGTGCACGCCGCTGGTCAGGGCGCCGGAGTTCCAGCT CACTGTCACAGGCTCGGGGAAGTAGTCCTTCACCAGGCAGCCCAGAGCGGCGGTGCTCTC AGATGTGCTTCTGCTGCATGGGGCCAGAGGGAACACGCTAGGGCCCTTGGTGCTGGCGGA GGAAACGGTCACCAGGGTGCCCTGGCCCCAGTAGTCCATAGGTCTCTCTCTGGCGCAGTA ATACAGGGCGGTGTCCTCGGCCCGCAGTGAGTTCATCTGCAGGTACAGGCTGTTCTTGGC ATTGTCCCGGCTGATTGTGAACCTGCCTTTCACGCTATCAGGGTAGTAGGTATATGATCC CCGGTTGCTGATGGTGGCGACCCATTCCAGTCTTTTGCCGGGAGCCTGCCGCACCCAGCT CATGGCGTAATTGCTAAAGGTGAAGCCAGAGGCGGCACAAGACAGTCTCAGGCTACCGCC GGGCTTCACCAGGCCGCCGCCGGATTCCACAAGCTGCACCTCGCTGCCGCCGCCGCCGGA TCCGCCACCGCCACTGCCCCCTCCGCCAGAGCCGCCGCCTCCACTGCCGCCGCCGCCGCT GCCTCCGCCTCCGCTTCCGCCGCCGCCGCACTCTCCTCTGTTGAAGCTCTTGGTCACTGG GCTGCTCAGTCCCTGGTGGGTCACCTCGCAGGCGTACACCTTGTGCTTCTCGTAATCTGC CTTGGACAAGGTCAGTGTGCTGCTCAGGGAGTAGGTGCTGTCCTTGCTATCTTGTTCCGT CACGCTCTCCTGGCTGTTGCCAGATTGCAGGGCGTTGTCCACCTTCCACTGCACTTTGGC TTCTCTGGGGTAGAAGTTGTTCAGCAGGCACACCACAGAGGCGGTGCCGCTCTTCAGCTG CTCGTCGCTAGGTGGAAAGATGAACACAGAAGGAGCAGCCACTGTCCGCTTGATTTCCAG CTTTGTGCCCTGTCCGAAGGTCAGAGGGTTGCTGCTGTGCTGGTGGCAAAAGTACACGGC GAAGTCCTCGGCCTGCAGAGAAGAAATGGTCAGGGTGAAATCAGTTCCGCTGCCAGAGCC GCTGAATCTATCGGGGACGCCGGTGTGTCTGGTGCTGGCCCAGTAGATCAGCAGCTTAGG GGCTTTTCCCGGTTTTTGCTGGTACCAGGCCACGGCAGTGCCCACGTCCTGGGAGGCTTT ACATGTGATTGTCACTCTGTCCCCCACGGAGGCGCTCAGGGTGCTAGGGCTCTGTGTCAT CTGGATGTCGCCGGTGGTGTCAGGCAGCCACAGCAGCAGCAGGAACAGCAGCTGAGCGGG GGCTTCCATGGTGGGCTAGTTGGCGCCCGCCGCGCGCTTCGCTTTTTATAGGGCCGCCGC CGCCGCCGCCTCGCCATAAAAGGAAACTTTCGGAGCGCGCCGCTCTGATTGGCTGCCGCC GCACCTCTCCGCCTCGCCCCGCCCCGCCCCTCGCCCCGCCCCGCCCCGCCTGGCGCGCGC CCCCCCCCCCCCCCCGCCCCCATCGCTGCACAAAATAATTAAAAAATAAATAAATACAAA ATTGGGGGTGGGGAGGGGGGGGAGATGGGGAGAGTGAAGCAGAACGTGGGGCTCACCTCG ACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAG GTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGC GTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCC ACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATT GACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTAT GTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAA TAACTAGCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGG GGGGGGGGCGCGCGCCAGGCGGGGGGGGGCGGGGCGAGGGGCGGGGGGGGGCGAGGCGGA GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGCCAGAGCCCACCATGG AAGCCCCTGCCCAGCTGCTGTTCCTGCTGCTCCTGTGGCTGCCTGACACCACCGGCGATA TCGTGCTGACGCAGAGCCCTGATAGCCTGGCCGTGAGCCTCGGCGAACGGGCCACAATCA GCTGTAAAGCCTCTCAGAGCGTGGACTATGACGGCGACAGCTACATGAACTGGTACCAGC AGAAACCCGGCCAGCCTCCAAAAATCCTGATCTACGACGCCAGCAATCTGGAAAGCGGCA TCCCCGCCAGATTCAGCGGCAGCGGCTCTGGCACTGATTTCACCCTGACAATTTCTTCTC TGGAACCCGAGGATTTTGCCATCTACTACTGCCAGCAGAGCAACGAGGACCCCTGGACCT TTGGAGGCGGCACCAAGGTGGAAATCAAGCGCACCGTGGCCGCCCCTTCTGTCTTTATCT TCCCTCCAAGCGACGAGCAGCTGAAGAGCGGAACCGCCTCTGTGGTGTGCCTGCTGAATA ACTTCTACCCCAGAGAGGCAAAGGTCCAATGGAAAGTTGACAACGCCCTGCAAAGCGGCA ACAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCAACATACAGCCTGTCCAGCA CCCTGACCTTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACAC ATCAGGGCCTGTCCTCTCCTGTGACCAAAAGCTTCAACCGGGGCGAGTGCGGCGGCGGCG GCTCCGGCGGAGGAGGCAGCGGTGGCGGCGGCTCTGGGGGAGGCGGAAGCGGCGGAGGCG GCAGCGGCGGAGGCGGCAGCGGCGGCGGCGGATCCCAGGTGCAGCTGGTGCAGAGCGGAG CCGAGGTGAAAAAGCCTGGCGCTAGTGTTAAGCTGAGCTGCACCGCCAGCGGCTTCAATA TCAAGGACGATTACATCCACTGGGTGAAGCAGGCCCCCGGCCAGGGACTGGAGTGGATCG GCAGAATCGACCCTGCCGACGGCCACACAAAGTACGCCCCTAAGTTCCAGGTGAAAGTCA CCATCACCGCCGATACCAGCACCTCTACAGCTTATCTGGAACTGAGCAGCCTTAGATCCG AGGACACCGCTGTGTACTACTGCGCCAGATACGGCTACGGCAGAGAAGTGTTCGACTACT GGGGACAGGGCACAACAGTGACCGTGTCGTCCGCCAGCACAAAGGGCCCTAGCGTGTTCC CACTGGCTCCTTGTAGCAGAAGTACCTCAGAGAGCACGGCTGCTCTGGGCTGCCTGGTCA AGGATTATTTCCCTGAGCCTGTGACCGTGTCCTGGAACAGCGGAGCCCTGACAAGCGGGG TGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGTCCTCTGTCGTGA CAGTGCCTAGCTCTAGCCTGGGCACAAAGACCTACACCTGCAACGTGGACCACAAGCCCA GCAACACCAAGGTGGATAAGCGGGTGTGA SEQIDNO:81-Peptidelinker SGSG SEQIDNO:82-furincleavagesite RX.sub.1X.sub.2R,whereX.sub.1=anynaturallyoccurringaminoacid,andX.sub.2= RorK SEQIDNO:83-minCBApromoter(CMVenhancerunderlined;CBA promoterboldfacedanditalicized;truncatedchimericintron: boldfacedandunderlined) GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGG GGGGGGGGCGCGCGCCAGGCGGGGGGGGGCGGGGCGAGGGGCGGGGGGGGGCGAGGCGGA GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCT GCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGA CCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC GCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCC GGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGG CAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCC
