VEGF-a / complement c5 bispecific antibodies and methods of use thereof

Abstract

Described herein are multispecific antigen binding polypeptides comprising a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety. Also described herein are multispecific antigen binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18.

Description

Attorney Docket No. 63209-724.601VEGF-A / COMPLEMENT C5 BISPECIFIC ANTIBODIES AND METHODS OF USE THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 737,378, filed December 20, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Diseases of the eye including age-related macular degeneration and geographic atrophy afflict thousands of individuals each year and can result in vision loss. The proteins vascular endothelial growth factor A (VEGF-A) and complement C5 (C5) play a role in the development of these diseases thus serve as a potential therapeutic target.SUMMARY

[0003] Described herein are multispecific binding polypeptides comprising a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety.

[0004] In some embodiments, the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof. In some embodiments, the light chain variable region is a surrogate light chain variable region. In some embodiments, the surrogate light chain variable region comprises a VpreB sequence. In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the light chain variable region is coupled to the heavy chain variable region via a linker. In some embodiments, the linker sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL). In some embodiments, the light chain constant domain is a kappa light chain constant domain (CK). In some embodiments, the light chain constant domain is a lambda light chain constant domain. In some embodiments, the scFv and the Fab are covalently coupled by a linker. In some embodiments, the scFv and the Fab are directly covalently coupled without a linker. In some embodiments, the C-terminus of the scFv is covalently coupled to the N- terminus of the heavy chain variable region of the Fab. In some embodiments, the N-terminus ofAttomey Docket No. 63209-724.601 the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab. In some embodiments, the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain constant region of the Fab.

[0005] In some embodiments, the first binding moiety is a VEGF-A or a Complement C5 binding moiety. In some embodiments, the first binding moiety is a VEGF-A moiety. In some embodiments, the second binding moiety is a VEGF-A or a Complement C5 binding moiety. In some embodiments, the second binding moiety is a Complement C5 binding moiety. In some embodiments, the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

[0006] In some embodiments, the Complement C5 binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e)an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 19. In some embodiments, the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19. In some embodiments, the Complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 20. In some embodiments, the Complement C5 binding moiety comprises an immunoglobulin lightAttorney Docket No. 63209-724.601 chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

[0007] Described herein are multispecific antigen binding polypeptides comprising a VEGF- A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18.

[0008] In some embodiments, the VEGF-A binding moiety is a scFv or a Fab. In some embodiments, the VEGF-A binding moiety is a scFv. In some embodiments, the Complement C5 binding moiety is a Fab or a scFv. In some embodiments, the Complement C5 binding moiety is a Fab. In some embodiments, the scFv and the Fab are covalently coupled by a linker. In some embodiments, the scFv and the Fab are directly covalently coupled without a linker.

[0009] In some embodiments, the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL). In some embodiments, the C-terminus of the scFv is covalently coupled to the N- terminus of the heavy chain variable region of the Fab. In some embodiments, the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

[0010] In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101. In some embodiments, the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101. In some embodiments, theAttorney Docket No. 63209-724.601VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2. In some embodiments, the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 19. In some embodiments, the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19. In some embodiments, wherein the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 20. In some embodiments, the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

[0011] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 202. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 203. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:Attorney Docket No. 63209-724.601206. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 206. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207. In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 207.

[0012] In some embodiments, the multispecific antigen binding polypeptide binds to VEGF- A with an EC50 of about 70 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide binds to complement C5 with an EC50 of about 180 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide binds to VEGF-A with an ECso of about 70 picomolar or less, and the multispecific antigen binding polypeptide binds to complement C5 with an EC50 of about 180 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 32 nanomolar or less. In some embodiments, the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 15 nanomolar or less in the presence of complement C5. In some embodiments, the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 29 nanomolar or less in the presence of VEGF-A. In some embodiments, the multispecific antigen binding polypeptide inhibits red blood cell hemolysis with an IC50 of about 28 nanomolar or less. In some embodiments, the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1 :600.

[0013] Described herein are pharmaceutical compositions comprising the multispecific antigen binding polypeptide described herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for intravitreal administration. Also described herein are nucleic acids or plurality of nucleic acids encoding the multispecific antigen binding polypeptide described herein. Also described herein are expression vectors comprising the nucleic acid described herein. In someAttomey Docket No. 63209-724.601 embodiments, the expression vector is RNA. In some embodiments, the expression vector is plasmid or linearized DNA. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adenovirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus virus.

[0014] Described herein are pharmaceutical compositions comprising the nucleic acids or plurality of nucleic acids described herein and a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for intravitreal administration.

[0015] Also described herein are methods of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition described herein, thereby treating the disease. In some embodiments, the disease is an ocular disease. In some embodiments, the ocular disease comprises macular degeneration. In some embodiments, the macular degeneration is age-related macular degeneration. In some embodiments, the ocular disease comprises geographic atrophy. In some embodiments, the ocular disease comprises diabetic macular edema. In some embodiments, the ocular disease comprises retinal vein occlusion.

[0016] Described herein are multispecific antigen binding polypeptides comprising a VEGF- A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab. In certain embodiments, the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acidAttomey Docket No. 63209-724.601 sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 206. In certain embodiments, the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 206. In certain embodiments described herein is a pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide and a pharmaceutically acceptable carrier, excipient, or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. In certain embodiments described herein is a method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition to the individual, thereby treating the disease. In certain embodiments, the disease is an ocular disease. In certain embodiments, the ocular disease comprises macular degeneration. In certain embodiments, the macular degeneration is age-related macular degeneration. In certain embodiments, the ocular disease comprises geographic atrophy. In certain embodiments, the ocular disease comprises diabetic macular edema. In certain embodiments, the ocular disease comprises retinal vein occlusion.

[0017] Described herein are multispecific antigen binding polypeptides comprising a VEGF- A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus ofAttorney Docket No. 63209-724.601 the light chain constant region of the Fab. In some embodiments, the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof. In some embodiments, the light chain variable region is a surrogate light chain variable region. In certain embodiments, the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 207. In certain embodiments, the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 207. In certain embodiments described herein is a pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide and a pharmaceutically acceptable carrier, excipient, or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. In certain embodiments described herein is a method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition to the individual, thereby treating the disease. In certain embodiments, the disease is an ocular disease. In certain embodiments, the ocular disease comprises macular degeneration. In certain embodiments, the macular degeneration is age-related macular degeneration. In certain embodiments, the ocular disease comprises geographic atrophy. In certain embodiments, the ocular disease comprises diabetic macular edema. In certain embodiments, the ocular disease comprises retinal vein occlusion.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

[0019] FIGs. 1A-1D are exemplary bispecific antigen binding polypeptides comprising a first binding moiety and a second binding moiety that bind to two different targets. The first binding moiety comprises a single chain variable region fragment (scFv). The scFv can be a single chain surrobody. The second binding moiety comprises a fragment antigen-binding regionAttomey Docket No. 63209-724.601(Fab). The Fab a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL). The CL can be a kappa light chain constant domain (CK). The first binding moiety can bind to VEGF-A. The second binding moiety can bind to Complement C5. FIG. 1A shows the configuration that the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab. FIG. IB shows the configuration that the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab. FIG. 1C shows the configuration that the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain variable region of the Fab. FIG. ID shows the configuration that the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain variable region of the Fab.

[0020] FIGs. 2A-2H illustrate SDS-PAGE and SE-HPLC analysis of the VEGF-A / C5 bispecific antigen-binding polypeptides. FIG. 2A illustrates SDS-PAGE results for reduced and non-reduced polypeptides comprising the bispecific construct shown in FIG. 1 A. FIG. 2B illustrates HPLC-SEC results of the bispecific construct shown in FIG. 1 A. FIG. 2C illustrates SDS-PAGE results for reduced and non-reduced polypeptides comprising the bispecific construct shown in FIG. IB. FIG. 2D illustrates HPLC-SEC results of the bispecific construct shown in FIG. IB. FIG. 2E illustrates SDS-PAGE results for reduced and non-reduced polypeptides comprising the bispecific construct shown in FIG. 1C. FIG. 2F illustrates HPLC-SEC results of the bispecific construct shown in FIG. 1C. FIG. 2G illustrates SDS-PAGE results for reduced and non-reduced polypeptides comprising the bispecific construct shown in FIG. ID. FIG. 2H illustrates HPLC-SEC results of the bispecific construct shown in FIG. ID.

[0021] FIGs. 3A-3B illustrate the binding affinity of four different constructs of VEGF-A / C5 bispecific antigen binding polypeptides to VEGF-A and C5 in comparison with anti-C5 eculizumab. FIG. 3A illustrates ELISA-binding results for bispecific antigen binding polypeptides and eculizumab binding to VEGF-A. FIG. 3B illustrates ELISA-binding results for bispecific antigen binding polypeptides and eculizumab binding to C5.

[0022] FIGs. 4A-4B illustrate the binding affinity of four different constructs of VEGF-A / C5 bispecific antigen binding polypeptides to the one of the targets after being captured by the biotinylated second target. FIG. 4A illustrates ELISA-binding results for the bispecific antigen binding polypeptides to C5 after being captured by biotinylated VEGF-A. FIG. 4B illustrates ELISA-binding results for the bispecific antigen binding polypeptides to VEGF-A after being captured by biotinylated C5.

[0023] FIG. 5 illustrate the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of the interaction between VEGF and its receptors in comparison with anti-VEGF-A antibody and anti-C5 antibody.Attorney Docket No. 63209-724.601

[0024] FIGs. 6A-6B illustrate the VEGF-A / C5 bispecific antigen binding polypeptide’s inhibition of the interaction between VEGF and its receptors. FIG. 6A illustrate the VEGF-A / C5 bispecific antigen binding polypeptide’s inhibition of the interaction between VEGF and its receptors in the presence of C5. FIG. 6B illustrate the VEGF-A / C5 bispecific antigen binding polypeptide’s inhibition of the interaction between VEGF and its receptors in VEGF bioassay.

[0025] FIG. 7 illustrate the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of antibody sensitized sheep red blood cell (SRBC) hemolysis via the classical pathway.

[0026] FIGs. 8A-8B illustrate the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of antibody sensitized sheep red blood cell (SRBC) hemolysis via the classical pathway. FIG. 8A illustrates the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of antibody sensitized sheep red blood cell (SRBC) hemolysis in human serum. FIG. 8B illustrates the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of antibody sensitized sheep red blood cell (SRBC) hemolysis in cynomolgus monkey serum.

[0027] FIG. 9 illustrate the VEGF-A / C5 bispecific antigen binding polypeptides’ inhibition of rabbit red blood cell hemolysis via the alternative pathway.

[0028] FIG. 10 illustrate the binding affinity of the VEGF-A / C5 bispecific antigen binding polypeptides to the recombinant VEGF-A of different species.

[0029] FIG. 11 illustrate the binding affinity of the VEGF-A / C5 bispecific antigen binding polypeptides to the recombinant C5 of different species.DETAILED DESCRIPTION

[0030] Described herein in one aspect, is multispecific antigen binding polypeptide comprising: a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety.

[0031] Described herein in another aspect is a multispecific antigen binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18.Attorney Docket No. 63209-724.601

[0032] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0033] As used herein the term “about” refers to an amount that is near the stated amount by 10% or less.

[0034] As used herein “vascular endothelial growth factor A” or “VEGF-A” or “VEGF-A” refers to the polypeptide encoded by the VEGF-A gene, which amino acid sequence is available at www.uniprot.org / uniprot / P15692. Such sequence includes post-translational modifications, naturally occurring amino acid variants, and naturally occurring splice variants that do not affect the binding of the antigen binding polypeptides and bispecific antigen binding polypeptides described herein.

[0035] As used herein “complement component 5” or “complement C5” or “C5” refers to the polypeptide encoded by the C5 gene, which amino acid sequence is available https: / / www.uniprot.org / uniprotkb / P01031. Such sequence includes post-translational modifications, naturally occurring amino acid variants, and naturally occurring splice variants that do not affect the binding of the antigen binding polypeptides and bispecific antigen binding polypeptides described herein

[0036] As used herein a “surrogate light chain” refers to a light chain formed by fusion of a VpreB polypeptide and a light chain constant region and can promiscuously associate with two or more structurally distinct heavy chains. The light chain constant region can be a 15 polypeptide. The light chain constant region can be a kappa constant light chain. The light chain constant region can be a lambda constant light chain. Such surrogate light chains are described in U.S.8,114,967; U.S. 10,214,580; and 8,969,082, each of which are incorporated herein in their entirety.

[0037] As used herein a “surrogate light chain variable region” refers to a light chain variable region comprises a VpreB polypeptide. The VpreB polypeptide can be a VpreB 1, VpreB2, or VpreB3 polypeptide. In some embodiments, the VpreB sequence comprises an amino acidAttomey Docket No. 63209-724.601 sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequcence set forth in SEQ ID NO: 101. Such surrogate light chain variable regions are described in U.S. 8,114,967; U.S. 10,214,580; and 8,969,082, each of which are incorporated herein in their entirety.

[0038] As used herein the term “individual,” “patient,” or “subject” refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating. In certain embodiments the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[0039] Among the provided antigen binding polypeptides are monoclonal antibodies, multispecific antibodies (for example, bispecific antibodies, trispecific antibodies, tetraspecific antibodies and polyreactive antibodies), and antibody fragments. The antibodies include antibody-conjugates and molecules comprising the antibodies, such as chimeric molecules. Thus, an antibody includes, but is not limited to, full-length and native antibodies, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and / or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigenbinding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab’)2, Fv, and scFv (single chain or related entity). A monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibody can comprise a human IgGl constant region. The monoclonal antibody can comprise a human IgG4 constant region.

[0040] The term “antibody” herein is used in the broadest sense and includes monoclonal antibodies, bispecific antibodies, multispecific antibodies, and includes intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody”Attorney Docket No. 63209-724.601 should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgGl constant region. The antibody can comprise a human IgG4 constant region.

[0041] The term “antigen binding polypeptide” herein is used in the broadest sense and includes polypeptides that bind to an antigen. The term may include monoclonal antibodies, bispecific antibodies, antigen binding polypeptide complexes, and includes intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, surrobodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antigen binding polypeptide” should be understood to encompass functional antigen binding polypeptide and fragments thereof. The term also encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0042] As used herein the terms “heavy chain” or “light chain” relate to polypeptides derived from or similar to heavy chains or light chains of antibodies or antibody like molecules.

[0043] Herein a molecule, peptide, polypeptide, antibody, or antibody fragment can be referred to as “bispecific” or “dual-specific” including grammatical equivalents. A bispecific molecule possesses the ability to specifically bind to at least two structurally distinct targets. The specific binding may be the result of two distinct binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences; or a single binding moiety that is able to specifically bind to two structurally distinct targets with high affinity (e.g., with a KD less than about IxlO'6). A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as “multi-specific” refers to a molecule that possesses the ability to specifically bind to at least three structurally distinct targets. A “bispecific antibody” including grammatical equivalents refers to a bispecific molecule that preserves at least one fragment of an antibody able to specifically bind a target, for example, a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule.

[0044] Herein a molecule, peptide, polypeptide, antibody, or antibody fragment can be referred to as “trispecific” including grammatical equivalents. A trispecific molecule possessesAttomey Docket No. 63209-724.601 the ability to specifically bind to at least three structurally distinct targets. The specific binding may be the result of three distinct binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences; or a single binding moiety that is able to specifically bind to three structurally distinct targets with high affinity (e.g., with a KD less than about IxlO'6). A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as “multi-specific” refers to a molecule that possesses the ability to specifically bind to at least two structurally distinct targets. A “multi-specific antibody” including grammatical equivalents refers to a multi-specific molecule that preserves at least one fragment of an antibody able to specifically bind with a target, for example, a variable region, heavy or light chain, or complementarity determining region from an antibody molecule.

[0045] The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to noncontiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR- Ll, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non- CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering scheme. In certain embodiments, the CDRs of the antigen binding polypeptides described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0046] The boundaries of a given CDR or FR may vary depending on the scheme used forAttorney Docket No. 63209-724.601 identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0047] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91(2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antigen binding polypeptides that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (See e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0048] Specific binding or binding of antigen binding polypeptide molecules described herein refers to binding mediated by one or more CDR portions of the antigen binding polypeptide. Not all CDRs may be required for specific binding. Specific binding can be demonstrated for example by an ELISA against a specific recited target or antigen that shows significant increase in binding compared to an isotype control antigen binding polypeptide.

[0049] As described herein an “epitope” refers to the binding determinant of an antigen binding polypeptide or fragment described herein minimally necessary for specific binding of the antigen binding polypeptide or fragment thereof to a target antigen. When the target antigen is a polypeptide the epitope will be a continuous or discontinuous epitope. A continuous epitope is formed by one region of the target antigen, while a discontinuous epitope may be formed from two or more separate regions. A discontinuous epitope, for example, may form when a target antigen adopts a tertiary structure that brings two amino acid sequences together and forms a three-dimensional structure bound by the antigen binding polypeptide. When the target antigen is a polypeptide the epitope will generally be a plurality of amino acids linked into a polypeptide chain. A continuous epitope may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids. While an epitope may comprise a contiguous polymer of amino acids, not every amino acid of the polymer may be contacted by an amino acid residue of the antibody. Such non-contacted amino acids will still comprise part of the epitope as they may beAttomey Docket No. 63209-724.601 important for the structure and linkage of the contacted amino acids. The skilled artisan may determine if any given antibody binds an epitope of a reference antibody, for example, by crossblocking experiments with a reference antibody. In certain embodiments, described herein, are antigen binding polypeptides that bind the same epitope of the described antigen binding polypeptides. In certain embodiments, described herein, are antigen binding polypeptides that are competitively blocked by the described antigen binding polypeptides. In certain embodiments, described herein, are antigen binding polypeptides that compete for binding with the described antigen binding polypeptides.

[0050] Among the provided antigen binding polypeptides are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv or sFv); single-chain antibody molecules comprising a surrogate light chain variable region fragment (e.g. scSv);and multispecific antigen binding polypeptides formed from antibody fragments. In particular embodiments, the antigen binding polypeptides are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs.

[0051] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antigen binding polypeptides are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., polypeptide linkers, and / or those that are not produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs. In some aspects, the antibody fragments are scSvs.

[0052] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0053] Among the provided antigen binding polypeptides are human antibodies. A “humanAttomey Docket No. 63209-724.601 antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigenbinding regions, such as those in which all or substantially all CDRs are non-human.

[0054] Human antigen binding polypeptides may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antigen binding polypeptides or intact antigen binding polypeptides with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antigen binding polypeptides also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.

[0055] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antigen binding polypeptides, antigen binding polypeptide complexes and antibody chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides are polypeptide complexes. In some embodiments, the polypeptide complex comprises a heavy chain and a light chain. In some embodiments, the heavy chain and the light chain are covalently linked. In some embodiments, the heavy chain and the light chain are non-covalently linked. In In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. In some embodiments, amino acid sequence variants of the antigen binding polypeptides provided herein are contemplated. A variant typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of a number of known techniques. For example, it may beAttorney Docket No. 63209-724.601 desirable to improve the binding affinity and / or other biological properties of the antibody Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0056] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0057] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acidAttomey Docket No. 63209-724.601 sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0058] Amino acid sequence insertions and deletions include amino- and / or carboxyl- terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C- terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Examples of intrasequence insertion variants of the antibody molecules include an insertion of 3 amino acids in the light chain. Examples of terminal deletions include an antibody with a deletion of 7 or less amino acids at an end of the light chain.

[0059] In some embodiments, an antigen binding polypeptide provided herein has a dissociation constant (KD) of about 1 pM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less (e.g., 10sM or less, e.g., from 10sM to 1013M, e.g., from 109M to 1013M) for the antigen binding polypeptide target. In some embodiments, an antigen binding polypeptide provided herein has a dissociation constant (KD) of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or greater (e.g, 10sM or less, e.g., from 10sM to 1013M, e.g., from 109M to 1013M) for the antigen binding polypeptide target. The antigen binding polypeptide target can be an anti-VEGF or anti-C5 antigen binding polypeptide. KD can be measured by any suitable assay. In certain embodiments, KD can be measured using surface plasmon resonance assays (e.g., using a BIACORE®-2000, a BIACORE®-3000 or Octet).

[0060] In certain embodiments, the antigen binding polypeptides disclosed herein bind to VEGF-A. VEGF-A may have several different splice sites thus may have various isoforms depending on splice sites. Accordingly, in certain embodiments, the antigen binding polypeptides disclosed herein bind to VEGF165. In certain embodiments, the antigen binding polypeptides disclosed herein bind to VEGF121. In certain embodiments, the antigen binding polypeptides’ binding affinity to VEGF121 is predicative of the antigen binding polypeptides’ efficacy in treating a disease of an eye. In certain embodiments, the antigen binding polypeptides disclosed herein bind to a peptide comprising a platelet-derived growth factor (PDGF) domain of VEGF-A. In some embodiments, the PDGF domain of VEGF-A comprises a fragment of VEGF-A ranging from about 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 46th, 47th, 48th, 49th, or 50thto about 130th, 131st, 132nd133rd, 134th, 135th, 136th, 137th, 138th, 139th, or 140thamino acids. In certain embodiments, the antigen binding polypeptides disclosed herein bind to a peptide comprising a VEGF C heparin domain of VEGF-A. In some embodiments, the VEGF C heparin domain of VEGF-AAtorney Docket No. 63209-724.601 comprises a fragment of VEGF-A ranging from about 140th, 141st, 142nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, or 150thto about 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 120thamino acids. In certain embodiments, the antigen binding polypeptides disclosed herein bind to VEGF-A from a species of human or a non-human (e.g., rats, rabbits, canines (dogs), monkeys, or porcine (pigs)).

[0061] In certain embodiments, the antigen binding polypeptides are anti -Human VEGF-A antigen binding polypeptides which bind to human VEGF-A at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which bind to human VEGF at a KD or ECso of 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which bind to human VEGF at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0062] In certain embodiments, the antigen binding polypeptides are anti-non-human VEGF- A antigen binding polypeptides which bind to a non-human VEGF-A at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-non-human VEGF-A antigen binding polypeptides which bind to a non-human VEGF at a KD or ECso of 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti- non-human VEGF-A antigen binding polypeptides which bind to human VEGF at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0063] In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which inhibits an interaction of VEGF-A or a fragment thereof and a VEGF receptor. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which inhibits an interaction of VEGF-A or a fragment thereof and a VEGF receptor at an ICso of 100 nM, 80 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM or less. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which inhibits an interaction of VEGF-A or a fragment thereof and a VEGF receptor at an ICso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more, certain embodiments, the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).

[0064] In certain embodiments, the antigen binding polypeptides are anti-Human C5 antigen binding polypeptides which bind to human C5 at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human C5 antigen binding polypeptides which bind toAttorney Docket No. 63209-724.601 human VEGF at a KD or ECso of 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human C5 antigen binding polypeptides which bind to human VEGF at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0065] In certain embodiments, the antigen binding polypeptides are anti-non-human C5 antigen binding polypeptides which bind to a non-human C5 at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-non-human C5 antigen binding polypeptides which bind to a non-human VEGF at a KD or ECso of 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-non-human C5 antigen binding polypeptides which bind to a non-human C5 at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0066] In certain embodiments, the antigen binding polypeptides are anti-Human C5 antigen binding polypeptides which inhibits C5 downstream activity. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis via the classical pathway. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis via the alternative pathway. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A antigen binding polypeptides which inhibits red blood cell hemolysis at an ICso of 100 nM, 80 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM or less. In certain embodiments, the antigen binding polypeptides are anti- Human VEGF-A antigen binding polypeptides which inhibits red blood cell hemolysis at an ICso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0067] In certain embodiments, the antigen binding polypeptides are anti VEGF-A / C5 bispecific antigen binding polypeptides which bind to VEGF-A at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A / C5 bispecific antigen binding polypeptides which bind to human VEGF-A at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0068] In certain embodiments, the antigen binding polypeptides are anti VEGF-A / C5 bispecific antigen binding polypeptides which bind to complement C5 at a KD or ECso of 2000, 1500, 1000, 800, 600, 500, 400, 300, 200, 100, 75, 60, 50, 40, 30, or 25 picomolar or less. In certain embodiments, the antigen binding polypeptides are anti-Human VEGF-A / C5 bispecific antigen binding polypeptides which bind to C5 at a KD or ECso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more.

[0069] In some embodiments, the antigen binding polypeptides disclosed herein are IgGAttomey Docket No. 63209-724.601 antigen binding polypeptides. In some embodiments, the antigen binding polypeptides disclosed herein are IgE antigen binding polypeptides. In some embodiments, the antigen binding polypeptides disclosed herein are IgM antigen binding polypeptides. In some embodiments, the antigen binding polypeptides disclosed herein are IgA antigen binding polypeptides. In some embodiments, the antigen binding polypeptides disclosed herein are Fab, F(ab)2, single-domain antibody, or single chain variable fragment (scFv).

[0070] In some embodiments, paired with one or more surrogate light chains, the antigen binding polypeptides disclosed herein are surrobodies. In some embodiments, the antigen binding fragment thereof is a surrobody antigen-binding fragment (Sab) or single chain variable fragment of a surrobody (scSv).

[0071] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antigen binding polypeptide provided herein, thereby generating an Fc region variant. An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. An Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0072] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antigen binding polypeptide provided herein, thereby generating an Fc region variant. An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. An Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification e.g., a substitution) at one or more amino acid positions.

[0073] In some instances, the Fc region of an immunoglobulin is important for many important antibody functions (e.g. effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and antibody-dependent cell- mediated phagocytosis (ADCP), result in killing of target cells, albeit by different mechanisms. Accordingly, in some embodiments, the antigen binding polypeptides described herein comprise the variable domains of the invention combined with constant domains comprising different Fc regions, selected based on the biological activities of the antibody for the intended use. In certain instances, Human IgGs, for example, can be classified into four subclasses, IgGl, IgG2, IgG3, and IgG4, and each these of these comprises an Fc region having a unique profile for binding to one or more of Fey receptors (activating receptors FcyRI (CD64), FcyRIIA, FcyRIIC (CD32); FcyRIIIA and FcyRIIIB (CD 16) and inhibiting receptor FcyRIIB), and for the first component ofAttorney Docket No. 63209-724.601 complement (Clq). Human IgGl and IgG3 bind to all Fey receptors; IgG2 binds to FcyRIIAmsi, and with lower affinity to FcyRIIARisi FcyRIII Aviss; IgG4 binds to FcyRI, FcyRIIA, FcyRIIB, FcyRIIC, and FcyRIIIAviss; and the inhibitory receptor FcyRIIB has a lower affinity for IgGl, IgG2 and IgG3 than all other Fey receptors. Studies have shown that FcyRI does not bind to IgG2, and FcyRIIIB does not bind to IgG2 or IgG4. Id. In general, with regard to ADCC activity, human IgGl>IgG3»IgG4>IgG2.

[0074] In some embodiments, the antigen binding polypeptides of this disclosure are variants that possess reduced effector functions, which make it a desirable candidate for applications in which certain effector functions (such as complement fixation and ADCC) are unnecessary or deleterious. Such antibodies can have decreased complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), or antibody dependent cellular phagocytosis (ADCP). In some embodiments, the antigen binding polypeptides of this disclosure are variants that possess increased effector functions for applications in which increased immunogenicity would be beneficial. Such antigen binding polypeptides can have increased CDC, ADCC, or ADCP, or a combination thereof. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 and 5,821,337.Alternatively, non-radioactive assays methods may be employed (e.g., ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and Natural Killer (NK) cells.

[0075] Antigen binding polypeptides can have increased half-lives and improved binding to the neonatal Fc receptor (FcRn) (See e.g, US 2005 / 0014934). Such antigen binding polypeptides can comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn, and include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 according to the EU numbering system See e.g., U.S. Pat. No. 7,371,826). Other examples of Fc region variants are also contemplated (See e.g, Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and5,624,821; and WO94 / 29351).

[0076] In some embodiments, it may be desirable to create cysteine engineered antigen binding polypeptides, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups can be positioned at sites for conjugation to other moieties, such as drug moieties or linker drug moieties, to create an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.Attomey Docket No. 63209-724.601

[0077] In some embodiments, an antigen binding polypeptide provided herein may be further modified to contain additional nonproteinaceous moieties that are known and available. The moieties suitable for derivatization of the antigen binding polypeptide include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3 -di oxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylen oxide / ethylene oxide co-polymers, poly oxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antigen binding polypeptide may vary, and if two or more polymers are attached, they can be the same or different molecules.

[0078] The antigen binding polypeptides described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. Plasmid vectors can be linearized for integration into a genomic region. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus. In certain embodiments, the expression vectorAttorney Docket No. 63209-724.601 described herein is used in a gene therapy. Accordingly, in some embodiments, the expression vector described herein is formulated for intravenous administration. In some embodiments, the expression vector described herein is formulated for intravitreal administration.

[0079] As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873- 5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.

[0080] The nucleic acids encoding the antigen binding polypeptides described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of antigen binding polypeptides for commercial or therapeutic uses. Standard cell lines and methods for the production of antigen binding polypeptides from a large-scale cell culture are known in the art. See e.g., Li et al., “Cell culture processes for monoclonal antibody production ” Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cell is a Eukaryotic cell. In certain embodiments, the Eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antigen binding polypeptides is a Chines Hamster Ovary cell (CHO) cell, an NS0 murine myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antigen binding polypeptide is integrated into a genomic locus of a cell useful for producing antigen binding polypeptides. In certain embodiments, described herein is a method of making an antigen binding polypeptide comprising culturing a cell comprising a nucleic acid encoding an antigen binding polypeptide under conditions in vitro sufficient to allow production and secretion of said antigen binding polypeptide.

[0081] In certain embodiments, described herein, is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antigen binding polypeptide or bispecific antigen binding polypeptide described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antigen binding polypeptide or bispecific antigen binding polypeptide described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container able to withstand freezing by liquid nitrogen.Attomey Docket No. 63209-724.601

[0082] Also described herein are methods of making an antigen binding polypeptide described herein. Such methods comprise incubating a cell or cell-line comprising a nucleic acid encoding the antigen binding polypeptide in a cell culture medium under conditions sufficient to allow for expression and secretion of the antigen binding polypeptide and further harvesting the antigen binding polypeptide from the cell culture medium. The harvesting can further comprise one or more purification steps to remove live cells, cellular debris, non-antigen binding polypeptide proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification step(s) include centrifugation, ultracentrifugation, dialysis, ultrafiltration, diafiltration, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0083] Treat,” “treatment,” or “treating,” as used herein refers to, e.g., a deliberate intervention to a physiological disease state resulting in the reduction in severity of a disease or condition; the reduction in the duration of a condition course; the amelioration or elimination of one or more symptoms associated with a disease or condition; or the provision of beneficial effects to a subject with a disease or condition. Treatment does not require curing the underlying disease or condition.

[0084] A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0085] As used herein, “pharmaceutically acceptable” with reference to a carrier” “excipient” or “diluent” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0086] The pharmaceutical compounds described herein can include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt thatAttomey Docket No. 63209-724.601 retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.Multispecific antigen binding polypeptide

[0087] Described herein are multispecific antigen binding polypeptide comprising: a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety.

[0088] In some embodiments, the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof. In some embodiments, the light chain variable region is a surrogate light chain variable region. In some embodiments, the surrogate light chain variable region comprises a VpreB sequence.

[0089] In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the VpreB sequence comprises an amino acid sequence comprising at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the VpreB sequence comprises an amino acid sequence set forth in SEQ ID NO: 101.

[0090] In some embodiments, the light chain variable region is coupled to the heavy chain variable region via a linker. In some embodiments, the linker comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the linkerAttorney Docket No. 63209-724.601 comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence with at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the linker comprises an amino acid sequence of Gly - Ser.

[0091] In some embodiments, the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL). In some embodiments, the light chain constant domain is a kappa light chain constant domain (CK). In some embodiments, the light chain constant domain is a lambda light chain constant domain. In some embodiments, the heavy chain first constant region comprises an amino acid sequence with at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the heavy chain first constant region comprises an amino acid sequence with at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the amino acid sequence set forth in SEQ ID NO: 102.

[0092] In some embodiments, the ScFv and the Fab are covalently coupled by a linker. In some embodiments, the ScFv and the Fab are directly covalently coupled without a linker.

[0093] Exemplary multispecific antigen binding polypeptide configurations are illustrated in FIGs. 1A-1D. In some embodiments, the C-terminus of the scFv is covalently coupled to the N- terminus of the heavy chain variable region of the Fab. In some embodiments, the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

[0094] In some embodiments, the first binding moiety is a VEGF-A or a Complement C5 binding moiety. In some embodiments, the first binding moiety is a VEGF-A moiety. In some embodiments, the second binding moiety is a VEGF-A or a Complement C5 binding moiety. In some embodiments, the second binding moiety is a Complement C5 binding moiety.VEGF-A / C5 bispecific antigen binding polypeptide

[0095] Described herein are bispecific antigen binding polypeptides targeting VEGF-A andC5. Geographic atrophy (GA) is an advanced stage of dry AMD hallmarked by atrophic lesions and irreversible vision loss. It is associated with chronic inflammation and overactivation of theAttomey Docket No. 63209-724.601 complement system. Current therapies for managing GA inhibit C3 or C5 of the complement cascade but double the rate of conversion from dry AMD to the more rapidly progressive wet AMD (3% to 6%). Furthermore, risk of conversion is cumulative year on year. The addition of VEGF-A to an anti-complement has the potential to reduce the rate of conversion for patients treated with complement inhibitors at large for geographic atrophy and offer a single therapy in the event patients with geographic atrophy do end up converting to exudative macular degeneration. In addition, there is a large subset of patients with exudative macular degeneration that develop geographic atrophy. At present, these patients are being treated with multiple agents to address the atrophic and exudative processes. The Bispecific to C5 x VEGF-A will address both of these issues with a single treatment. Bispecific antigen binding polypeptides described herein possess certain advantages, such as high binding affinities to multiple targets to enhance biological activity, reduced risk of immunogenicity, and improved and scalable cost-efficient manufacturability.

[0096] In some embodiments, the bispecific antigen binding polypeptide comprises a VEGF- A binding moiety and a Complement C5 binding moiety. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region comprising: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4; and / or a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the VEGF-A binding moiety is an scFv or a Fab. In some embodiments, the VEGF-A binding moiety is an scFv.

[0097] In some embodiments, the complement C5 binding moiety comprises an immunoglobulin heavy chain variable region comprising: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15; a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and / or a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the Complement C5 binding moiety is a Fab or an ScFv. In some embodiments, the Complement C5 binding moiety is a Fab.

[0098] In some embodiments, the ScFv and the Fab are covalently coupled by a linker. InAttomey Docket No. 63209-724.601 some embodiments, the ScFv and the Fab are directly covalently coupled without a linker. In some embodiments, the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL). In some embodiments, the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab. In some embodiments, the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab. In some embodiments, the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

[0099] In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 90% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 95% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 96% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 97% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 98% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 99% identity to SEQ ID NO: 1. In some embodiments, the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence identical to SEQ ID NO: 1.

[0100] In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 90% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 95% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 96% identity to SEQAttorney Docket No. 63209-724.601ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 97% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 98% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 99% identity to SEQ ID NO: 2. In some embodiments, the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

[0101] In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 90% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 95% identity to SEQ ID NO: 19. In some embodiments, the binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 96% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 97% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 98% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 99% identity to SEQ ID NO: 19. In some embodiments, the C5 binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence identical to SEQ ID NO: 19.

[0102] In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 85% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 90% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 95% identity to SEQ ID NO: 20. In some embodiments, the binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 96% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence atAttomey Docket No. 63209-724.601 least about 97% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 98% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence at least about 99% identity to SEQ ID NO: 20. In some embodiments, the C5 binding moiety comprises an immunoglobulin light chain variable region amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

[0103] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202.

[0104] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 202.

[0105] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203.

[0106] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 203.

[0107] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206.

[0108] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 206.

[0109] In some embodiments, the multispecific binding polypeptide is formed from a firstAttorney Docket No. 63209-724.601 polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207.

[0110] In some embodiments, the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 207.

[0111] Described herein are multispecific antigen binding polypeptides comprising a VEGF- A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

[0112] In some embodiments, the VEGF-A binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Bevacizumab, Ranibizumab, Aflibercept, Faricimab, and Brolucizumab. In some embodiments, the Complement C5 binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Eculizumab, Ravulizumab, Crovalimab, Pozelimab, and IM-101.

[0113] Described herein are multispecific antigen binding polypeptides comprising a VEGF- A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth inAttomey Docket No. 63209-724.601SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab. In some embodiments, the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof. In some embodiments, the light chain variable region is a surrogate light chain variable region.

[0114] Described herein are means for binding to VEGF-A protein comprising a multispecific binding polypeptide comprising a VEGF-A binding moiety comprising determining regions (CDRs) derived from the group consisting of Bevacizumab, Ranibizumab, Aflibercept, Faricimab, and Brolucizumab.

[0115] Described herein are means for binding to Complement C5 protein comprising a multispecific binding polypeptide comprising a C5 binding moiety comprising determining regions (CDRs) derived from the group consisting of Eculizumab, Ravulizumab, Crovalimab, Pozelimab, and IM-101.

[0116] In some embodiments, the multispecific antigen binding polypeptide binds to VEGF- A. In some embodiments, the multispecific antigen binding polypeptide binds to VEGF-A or a fragment thereof with an EC50 of about 1000, 500, 200, 100, 50, 20, 10, 8, 5, 3, 2, or 1 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide binds to VEGF-A or a fragment thereof with an EC50 of about 70 picomolar or less.

[0117] In some embodiments, the multispecific antigen binding polypeptide binds to Complement C5. In some embodiments, the multispecific antigen binding polypeptide binds to Complement C5 or a fragment thereof with an EC50 of about 1000, 500, 200, 100, 50, 20, 10, 8, 5, 3, 2, or 1 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide binds to Complement C5 or a fragment thereof with an EC50 of about 180 picomolar or less. In some embodiments, the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 32 nanomolar or less.

[0118] In certain embodiments, the multispecific antigen binding polypeptide inhibits an interaction of VEGF-A or a fragment thereof and a VEGF receptor. In certain embodiments, the multispecific antigen binding polypeptide inhibits an interaction of VEGF-A or a fragmentAttorney Docket No. 63209-724.601 thereof and a VEGF receptor at an ICso of 100 nM, 80 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM or less. In certain embodiments, the multispecific antigen binding polypeptide inhibits an interaction of VEGF-A or a fragment thereof and a VEGF receptor at an ICso of 32 nM or less. In certain embodiments, the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 15 nanomolar or less in the presence of complement C5. In certain embodiments, the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).

[0119] In certain embodiments, the antigen binding polypeptides are anti-Human C5 antigen binding polypeptides which inhibits C5 downstream activity. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis via the classical pathway. In certain embodiments, the antigen binding polypeptides inhibits the red blood cell hemolysis via the alternative pathway. In certain embodiments, the antigen binding polypeptides inhibits red blood cell hemolysis at an ICso of 100 nM, 80 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM or less. In certain embodiments, the antigen binding polypeptides inhibits red blood cell hemolysis at an ICso of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more. In certain embodiments, the antigen binding polypeptides inhibits red blood cell hemolysis at an ICso of 28 nM or less.

[0120] Elevated levels of pre-existing anti-drug antibodies (AD As) in patients may predict adverse outcomes in response to intravitreal injections with anti -vascular endothelial growth factor (anti-VEGF) agents. Higher levels of pre-existing AD As detected may be correlated with a greater incidence of adverse events such as severe intraocular inflammation (IOI) including occlusive retinal vasculitis upon treatment. In some embodiments, the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1 : 100, 1 :200, 1 :300, 1 :400, 1 :500, 1 :600, 1 :800, 1 : 1000, 1 : 1500, 1 : 1800, 1 :2000, 1 : 1500, 1 :3000, 1 :5000, or 1 : 10,000. In some embodiments, the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1 :2700. In some embodiments, the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1 :600. In some embodiments, the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is between 1 :300 and 1 :600.Vectors

[0121] Some embodiments disclosed herein relate to nucleic acids encoding theAttomey Docket No. 63209-724.601 multispecific antigen binding polypeptide as disclosed herein. Some embodiments disclosed herein relate to expression vectors comprising the nucleic acids as disclosed herein. In some instances, the expression vector is an RNA vector. In some instances, the expression vector is plasmid or linearized DNA vector. In some instances, the expression vector is a viral vector.

[0122] The term “viral vector” is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. In some embodiments, the viral vector is an adenovirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the viral vector is a lentivirus virus.

[0123] The nucleic acids described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector.Suitable vectors for use in eukaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989).

[0124] Accordingly, in some instances, the multispecific antigen binding polypeptides of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.

[0125] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0126] Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s). Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus).Attomey Docket No. 63209-724.601

[0127] In some instances, the nucleic acid molecules are delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for stable or transient expression. Accordingly, in some instances, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some instances, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can also be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA-directed CRISPR / Cas9, DNA-guided endonuclease genome editing NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some instances, the nucleic acid molecule is present in the recombinant host cell as a mini circle expression vector for stable or transient expression.

[0128] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle. Alternatively, endonuclease polypeptide(s) can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. For example, introduction of nucleic acids into cells may be achieved using viral transduction methods. In a non-limiting example, adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0129] Lentiviral systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host cell genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy -target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile (e.g., by targeting a site for integration that has little or no oncogenic potential); and (vii) a relatively easy system for vector manipulation and production.Therapeutic methods

[0130] In certain embodiments, the antigen binding polypeptides can be administered to a subject in need thereof by any route suitable for the administration of antigen binding polypeptide-containing pharmaceutical compositions, such as, for example, topically, subcutaneously, intravenously, or intravitreally. In certain embodiments, the antigen bindingAttomey Docket No. 63209-724.601 polypeptide or pharmaceutical composition comprising the antigen binding polypeptide is administered intravenously. In certain embodiments, the antigen binding polypeptide or pharmaceutical composition comprising the antigen binding polypeptide is administered intravitreally. In certain embodiments, the antigen binding polypeptide or pharmaceutical composition comprising the antigen binding polypeptide is administered topically. In certain embodiments, the antigen binding polypeptide or pharmaceutical composition comprising the antigen binding polypeptide is administered subcutaneously.

[0131] In certain embodiments, the antigen binding polypeptides are administered on a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month, once every two months, once every three months, once every four months, once every five months, or once every six months, etc. The antigen binding polypeptides can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. Intravitreal dosage concentrations can range from 50 mg / ml to 150 mg / ml for intravitreal use. Therapeutically effective amounts include amounts sufficient to ameliorate one or more symptoms associated with the disease or affliction to be treated.

[0132] In certain embodiments, disclosed herein, are antigen binding polypeptides useful for the treatment of a disease of the eye. In certain embodiments, the disease of the eye is associated with age or another comorbidity such as diabetes mellitus. In certain embodiments, the disease of the eye is macular degeneration. In certain embodiments, the macular degeneration is age related. In certain embodiments, the disease of the eye is age related macular degeneration. In certain embodiments, the disease of the eye is geographic atrophy. In certain embodiments, the macular degeneration is diabetes related. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease of the eye is branch retinal vein occlusion. In certain embodiments, the disease of the eye is central retinal vein occlusion.

[0133] In certain embodiments, disclosed herein, are multispecific antigen binding polypeptides useful for the treatment of a cancer.Pharmaceutically acceptable excipients., carriers, and diluents

[0134] In certain embodiments, the multispecific antigen binding polypeptides of the currentAttomey Docket No. 63209-724.601 disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the pharmaceutical composition comprises a nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptides described herein and one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers and diluents can be included to increase shelf-life, stability, or the admini strability of the antigen binding polypeptide. Such compounds include salts, pH buffers, detergents, anti-coagulants, and preservatives. In certain embodiments, the antigen binding polypeptides of the current disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyol / disaccharide / polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA.

[0135] In certain embodiments, the antigen binding polypeptides of the current disclosure can be shipped / stored lyophilized and reconstituted before administration. In certain embodiments, lyophilized antigen binding polypeptide formulations comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulation can be contained in a vial comprised of glass or other suitable non-reactive material. The antigen binding polypeptides when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0136] Also described herein are kits comprising one or more of the antigen binding polypeptides described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration.

[0137] In certain embodiments, described herein is a method of preparing a treatment for a disease of the eye comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and an antigen binding polypeptide of the current disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or shipping comprising lyophilizing one or more antigen binding polypeptides of the current disclosure.Attorney Docket No. 63209-724.601List of Embodiments:

[0138] Embodiment 1. A multispecific binding polypeptide comprising a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety.

[0139] Embodiment 2. The multispecific binding polypeptide of embodiment 1, wherein the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof.

[0140] Embodiment 3. The multispecific binding polypeptide of embodiment 2, wherein the light chain variable region is a surrogate light chain variable region.

[0141] Embodiment 4. The multispecific binding polypeptide of embodiment 1, wherein the surrogate light chain variable region comprises a VpreB sequence.

[0142] Embodiment 5. The multispecific binding polypeptide of embodiment 2, wherein the VpreB sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101.

[0143] Embodiment 6. The multispecific binding polypeptide of any one of embodiments 1- 3, wherein the light chain variable region is coupled to the heavy chain variable region via a linker.

[0144] Embodiment 7. The multispecific binding polypeptide of embodiment 4, wherein the linker sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0145] Embodiment 8. The multispecific binding polypeptide of any one of embodiments 1- 5, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL).

[0146] Embodiment 9. The multispecific binding polypeptide of embodiment 6, wherein the light chain constant domain is a kappa light chain constant domain (CK).

[0147] Embodiment 10. The multispecific binding polypeptide of any one of embodiments 1-7, wherein the scFv and the Fab are covalently coupled by a linker.

[0148] Embodiment 11. The multispecific binding polypeptide of any one of embodiments 1-7, wherein the scFv and the Fab are directly covalently coupled without a linker.

[0149] Embodiment 12. The multispecific binding polypeptide of any one of embodiments 6-9, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab.Atorney Docket No. 63209-724.601

[0150] Embodiment 13. The multispecific binding polypeptide of any one of embodiments 6-9, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab.

[0151] Embodiment 14. The multispecific binding polypeptide of any one of embodiments 6-9, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

[0152] Embodiment 15. The multispecific binding polypeptide of any one of embodiments 6-9, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

[0153] Embodiment 16. The multispecific binding polypeptide of any one of embodiments 1-13, wherein the first binding moiety is a VEGF-A or a Complement C5 binding moiety.

[0154] Embodiment 17. The multispecific binding polypeptide of any one of embodiments 1-13, wherein the first binding moiety is a VEGF-A moiety.

[0155] Embodiment 18. The multispecific binding polypeptide of any one of embodiments 1-15, wherein the second binding moiety is a VEGF-A or a Complement C5 binding moiety.

[0156] Embodiment 19. The multispecific binding polypeptide of any one of embodiments 1-15, wherein the second binding moiety is a Complement C5 binding moiety.

[0157] Embodiment 20. The multispecific binding polypeptide of any one of embodiments 14 to 17, wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106

[0158] Embodiment 21. The multispecific binding polypeptide of any one of embodiments 14 to 18, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1.

[0159] Embodiment 22. The multispecific binding polypeptide of any one of embodiments 14 to 18, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain aminoAttomey Docket No. 63209-724.601 acid sequence comprising a sequence identical to SEQ ID NO: 1.

[0160] Embodiment 23. The multispecific binding polypeptide of any one of embodiments 14 to 20, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2.

[0161] Embodiment 24. The multispecific binding polypeptide of any one of embodiments 14 to 21, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

[0162] Embodiment 25. The multispecific binding polypeptide of any one of embodiments 14 to 22, wherein the Complement C5 binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 18

[0163] Embodiment 26. The multispecific binding polypeptide of any one of embodiments 14 to 23, wherein the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 19.

[0164] Embodiment 27. The multispecific binding polypeptide of any one of embodiments 14 to 23, wherein the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19.

[0165] Embodiment 28. The multispecific binding polypeptide of any one of embodiments 14 to 25, wherein the Complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 20.

[0166] Embodiment 29. The multispecific binding polypeptide of any one of embodiments 14 to 25, wherein the Complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

[0167] Embodiment 30. A multispecific binding polypeptide comprising a VEGF-A bindingAttorney Docket No. 63209-724.601 moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18

[0168] Embodiment 31. The multispecific binding polypeptide of embodiment 28, wherein the VEGF-A binding moiety is a scFv or a Fab.

[0169] Embodiment 32. The multispecific binding polypeptide of embodiment 28, wherein the VEGF-A binding moiety is a scFv.

[0170] Embodiment 33. The multispecific binding polypeptide of any one of embodiments28-30, wherein the Complement C5 binding moiety is a Fab or a scFv.

[0171] Embodiment 34. The multispecific binding polypeptide of any one of embodiments28-30, wherein the Complement C5 binding moiety is a Fab.

[0172] Embodiment 35. The multispecific binding polypeptide of embodiment 31 or 32, wherein the scFv and the Fab are covalently coupled by a linker.

[0173] Embodiment 36. The multispecific binding polypeptide of embodiment 31 or 32, wherein the ScFv and the Fab are directly covalently coupled without a linker.

[0174] Embodiment 37. The multispecific binding polypeptide of any one of embodimentsAttomey Docket No. 63209-724.60129-34, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL).

[0175] Embodiment 38. The multispecific binding polypeptide of embodiment 35, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab.

[0176] Embodiment 39. The multispecific binding polypeptide of embodiment 35, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab.

[0177] Embodiment 40. The multispecific binding polypeptide of embodiment 35, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

[0178] Embodiment 41. The multispecific binding polypeptide of embodiment 35, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

[0179] Embodiment 42. The multispecific binding polypeptide of any one of embodiments 28 to 39, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1.

[0180] Embodiment 43. The multispecific binding polypeptide of any one of embodiments 28 to 39, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 1.

[0181] Embodiment 44. The multispecific binding polypeptide of any one of embodiments 28 to 41, wherein the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101.

[0182] Embodiment 45. The multispecific binding polypeptide of any one of embodiments 28 to 41, wherein the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101.

[0183] Embodiment 46. The multispecific binding polypeptide of any one of embodiments 28 to 43, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2.

[0184] Embodiment 47. The multispecific binding polypeptide of any one of embodiments 28 to 43, wherein the VEGF-A binding moiety comprises a single chain variable region fragmentAttorney Docket No. 63209-724.601 amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

[0185] Embodiment 48. The multispecific binding polypeptide of any one of embodiments 28 to 45, wherein the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 19.

[0186] Embodiment 49. The multispecific binding polypeptide of any one of embodiments 28 to 45, wherein the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19.

[0187] Embodiment 50. The multispecific binding polypeptide of any one of embodiments 28 to 47, wherein the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 20.

[0188] Embodiment 51. The multispecific binding polypeptide of any one of embodiments 28 to 47, wherein the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

[0189] Embodiment 52. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202.

[0190] Embodiment 53. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 202.

[0191] Embodiment 54. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203.

[0192] Embodiment 55. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acidAttomey Docket No. 63209-724.601 sequence identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 203.

[0193] Embodiment 56. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206.

[0194] Embodiment 57. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 206.

[0195] Embodiment 58. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207.

[0196] Embodiment 59. The multispecific binding polypeptide of any one of embodiments 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 207.

[0197] Embodiment 60. The multispecific binding polypeptide of any one of embodiments 1 to 57, wherein the multispecific antigen binding polypeptide binds to VEGF-A with an ECso of about 70 picomolar or less.

[0198] Embodiment 61. The multispecific binding polypeptide of any one of embodiments 1 to 58, wherein the multispecific antigen binding polypeptide binds to complement C5 with an ECso of about 180 picomolar or less.

[0199] Embodiment 62. The multispecific binding polypeptide of any one of embodiments 1 to 59, wherein the multispecific antigen binding polypeptide binds to VEGF-A with an EC50 of about 70 picomolar or less, and wherein the multispecific antigen binding polypeptide binds to complement C5 with an EC50 of about 180 picomolar or less.

[0200] Embodiment 63. The multispecific binding polypeptide of any one of embodiments 1Attomey Docket No. 63209-724.601 to 60, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 32 nanomolar or less.

[0201] Embodiment 64. The multispecific binding polypeptide of any one of embodiments 1 to 61, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 15 nanomolar or less in the presence of complement C5.

[0202] Embodiment 65. The multispecific binding polypeptide of any one of embodiments 1 to 62, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF-A receptor with an IC50 of about 29 nanomolar or less in the presence of VEGF-A.

[0203] Embodiment 66. The multispecific binding polypeptide of any one of embodiments 1 to 63, wherein the multispecific antigen binding polypeptide inhibits red blood cell hemolysis with an IC50 of about 28 nanomolar or less.

[0204] Embodiment 67. The multispecific binding polypeptide of any one of embodiments 1 to 63, wherein the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1:600.

[0205] Embodiment 68. The multispecific binding polypeptide of any one of embodiments 1 to 65, wherein the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide ranges from 1 :300 to 1 :600.

[0206] Embodiment 69. A pharmaceutical composition comprising the multispecific antigen binding polypeptide of any one of embodiments 1 to 66 and a pharmaceutically acceptable carrier, excipient, or diluent.

[0207] Embodiment 70. The pharmaceutical composition of embodiment 67 formulated for intravenous administration.

[0208] Embodiment 71. The pharmaceutical composition of embodiment 67 formulated for intravitreal administration.

[0209] Embodiment 72. A nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of any one of embodiments 1 to 66.

[0210] Embodiment 73. An expression vector comprising the nucleic acid of embodiment 70.

[0211] Embodiment 74. The expression vector of embodiment 71, wherein the expression vector is RNA.

[0212] Embodiment 75. The expression vector of embodiment 71, wherein the expression vector is plasmid or linearized DNA.

[0213] Embodiment 76. The expression vector of embodiment 71, wherein the expression vector is a viral vector.Atorney Docket No. 63209-724.601

[0214] Embodiment 77. The viral vector of embodiment 74, wherein the viral vector is an adenovirus.

[0215] Embodiment 78. The viral vector of embodiment 74, wherein the viral vector is an adeno-associated virus.

[0216] Embodiment 79. The viral vector of embodiment 74, wherein the viral vector is a lentivirus virus.

[0217] Embodiment 80. A pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of embodiment 70 and a pharmaceutically acceptable carrier, excipient, or diluent.

[0218] Embodiment 81. The pharmaceutical composition of embodiment 78 formulated for intravenous administration.

[0219] Embodiment 82. The pharmaceutical composition of embodiment 78 formulated for intravitreal administration.

[0220] Embodiment 83. A method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition of any one of the preceding embodiments, thereby treating the disease.

[0221] Embodiment 84. The method of embodiment 81, wherein the disease is an ocular disease.

[0222] Embodiment 85. The method of embodiment 82, wherein the ocular disease comprises macular degeneration.

[0223] Embodiment 86. The method of embodiment 83, wherein the macular degeneration is age-related macular degeneration.

[0224] Embodiment 87. The method of embodiment 82, wherein the ocular disease comprises geographic atrophy.

[0225] Embodiment 88. The method of embodiment 82, wherein the ocular disease comprises diabetic macular edema.

[0226] Embodiment 89. The method of embodiment 82, wherein the ocular disease comprises retinal vein occlusion.

[0227] Embodiment 90. A multispecific binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5;Attomey Docket No. 63209-724.601 d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

[0228] Embodiment 91. A multispecific binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: g) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; h) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; i) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; j) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; k) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or l) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; g. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; h. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; i. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; j. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; k. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or l. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.Attomey Docket No. 63209-724.601

[0229] Embodiment 92. The multispecific binding polypeptide of embodiment 88 or 91, wherein the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof, and wherein the light chain variable region is a surrogate light chain variable region

[0230] Embodiment 93. The multispecific binding polypeptide of any one of embodiments 90 to 92 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 206.

[0231] Embodiment 94. The multispecific binding polypeptide of any one of embodiments 90 to 92, wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 206.

[0232] Embodiment 95. The multispecific binding polypeptide of any one of embodiments 90 to 92, wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 207.

[0233] Embodiment 96. The multispecific binding polypeptide of any one of embodiments 90 to 92 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 207.

[0234] Embodiment 97. A pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of any one of embodiments 90 to 96 and a pharmaceutically acceptable carrier, excipient, or diluent.

[0235] Embodiment 98. The pharmaceutical composition of embodiment 97 formulated for intravenous administration.

[0236] Embodiment 99. The pharmaceutical composition of embodiment 97 formulated for intravitreal administration.

[0237] Embodiment 100. A method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid orAtorney Docket No. 63209-724.601 plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition of any one of the preceding embodiments, thereby treating the disease.

[0238] Embodiment 101. The method of embodiment 100, wherein the disease is an ocular disease.

[0239] Embodiment 102. The method of embodiment 101, wherein the ocular disease comprises macular degeneration.

[0240] Embodiment 103. The method of embodiment 102, wherein the macular degeneration is age-related macular degeneration.

[0241] Embodiment 104. The method of embodiment 101, wherein the ocular disease comprises geographic atrophy.

[0242] Embodiment 105. The method of embodiment 101, wherein the ocular disease comprises diabetic macular edema.

[0243] Embodiment 106. The method of embodiment 101, wherein the ocular disease comprises retinal vein occlusion.

[0244] Embodiment 107. The multispecific binding polypeptide of any one of embodiments 16-19 and 25-29, wherein the VEGF-A binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Bevacizumab, Ranibizumab, Aflibercept, Faricimab, and Brolucizumab.

[0245] Embodiment 108. The multispecific binding polypeptide of any one of embodiments 16-24, wherein the Complement C5 binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Eculizumab, Ravulizumab, Crovalimab, Pozelimab, and IM-101.EXAMPLES

[0246] The following illustrative examples are representative of embodiments of compositions and methods described herein and are not meant to be limiting in any way.Example 1 -Production of VEGF-A / C5 bispecific antigen-binding polypeptides

[0247] Nucleic acid sequences encoding sequences corresponding to SEQ ID NOs: 201-208 were cloned into plasmid vectors to direct eukaryotic cell expression. An expression plasmid was constructed. Protein expression vectors were co-transfected into HEK293 cells. Transfectants were isolated from supernatants by CaptureSelect IgG-CHl resin. Purified proteins were respectively analyzed for uniformity, purity, and quantity by SEC-HPLC, SDS-PAGE and UV spectrophotometric analysis.Attomey Docket No. 63209-724.601

[0248] For SDS-PAGE analysis, reduced and non-reduced samples of the bispecific antigen binding polypeptides were electrophoresed. FIGs. 2A-2B confirms the presence of bispecific antigen binding polypeptide with the construct shown in FIG. 1 A and good levels of purity were observed. Under non-reducing conditions, A protein band around 64.8 kDa was seen. Under reducing conditions, bands corresponding to the molecular weight of light chain (-21 kDa) and heavy chains (-38.4 kDa) are observed. The purified bispecific polypeptide was also analyzed in HPLC-SEC. The polypeptide showed 95.78% single peak, indicating 95.78% purity (see FIG.2B)

[0249] FIGs. 2C-2D confirm the presence of bispecific antigen binding polypeptide with the construct shown in FIG. IB and good levels of purity were observed. Under non-reducing conditions, a major protein band around 69.3 kDa was seen. Under reducing conditions, bands corresponding to the molecular weight of light chain (-19.7 kDa) and heavy chains (-39.8 kDa) are observed (see FIG. 2C). The purified bispecific polypeptide was also analyzed in HPLC- SEC. The polypeptide showed 95.51% single peak, indicating 95.51% purify (see FIG. 2D).

[0250] FIGs. 2E-2F confirm the presence of bispecific antigen binding polypeptide with the construct shown in FIG. 1C and good levels of purity were observed. Under non-reducing conditions, a protein band around 77.8 kDa was seen. Under reducing conditions, bands corresponding to the molecular weight of light chain (-20.7 kDa) and heavy chains (-40.2 kDa) are observed (see FIG. 2E). The purified bispecific polypeptide was also analyzed in HPLC- SEC. The polypeptide showed 95.90% single peak, indicating 95.9% purity (see FIG. 2F).

[0251] FIGs. 2G-2H confirm the presence of bispecific antigen binding polypeptide with the construct shown in FIG. ID and good levels of purity were observed. Under non-reducing conditions, a protein band around 76.3 kDa was seen. Under reducing conditions, bands corresponding to the molecular weight of light chain (-20.7 kDa) and heavy chains (-42.0 kDa) are observed (see FIG. 2G). The purified bispecific polypeptide was also analyzed in HPLC- SEC. The polypeptide showed 95.40% single peak, indicating 95.4% purity (see FIG. 2H).Example 2-Binding of VEGF-A / C5 bispecific antigen binding polypeptide

[0252] VEGF-A / C5 bispecific antigen-binding polypeptides prepared according to example 1 were tested for their binding capacity to target antigen.

[0253] VEGF-A ELISA- Recombinant human VEGF-A (Peprotech, Cat #100-20) was used to coat plates at 1 pg / mL. The products were diluted in blocking buffer at a starting concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by 100 ng / mL biotinylated anti-IgG-CHl (ThermoFisher Cat #7103202500) followed by 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmuoResearch Lab, Cat #016-030-084) EculizumabAttorney Docket No. 63209-724.601(Selleckchem, Cat #A2538) was run alongside bispecific polypeptides as reference material. OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader. Table 1 shows the EC50 value of the binding affinity to VEGF-A. As illustrated in FIG. 3A and Table 1, all four bispecific polypeptides have high binding affinities with EC50 value around 52 pM. On the contrary, the anti-C5 antibody eculizumab does not bind to VEGF-A.

[0254] COMPLEMENT C5 ELISA Recombinant human C5 (Aero Biosystems, Cat # CO5-H52Ha) was used to coat plates at 1 pg / mL. The products were diluted in blocking buffer at a starting concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by 100 ng / mL biotinylated anti-IgG-CHl (ThermoFisher Cat #7103202500) followed by 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmuoResearch Lab, Cat #016-030-084) Eculizumab (Selleckchem, Cat #A2538) was run alongside bispecific polypeptides as reference material. OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader.Table 1 shows the EC50 value of the binding affinity to Complement C5. As illustrated in FIGs. 3B and Table 1, the results showed high binding affinity for all four bispecific polypeptides with EC50 value around 158.3 pM, which was comparable to the binding affinity of eculizumab.Table 1: VEGF-A / C5 bispecific antigen binding polypeptides EC50 binding affinity to VEGF-A and Complement C5Example 3-Binding of VEGF-A / C5 bispecific antigen binding polypeptides to both VEGF-A and Complement C5 at the same time

[0255] Four different constructs of VEGF-A / C5 bispecific antigen binding polypeptides prepared according to methods in example 1 were tested for their dual binding capacity to target antigens.

[0256] C5-binding ELISA- Recombinant human C5 (Aero Biosystems, Cat # CO5-H52Ha) was used to coat plates at 1 pg / mL. The products were diluted in blocking buffer at a startingAtorney Docket No. 63209-724.601 concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by biotinylated rhVEGF-A (Aero Biosystems, Cat #VE5-H8210) followed by 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmuoResearch Lab, Cat #016-030-084). OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader. Table 2 shows the EC50 value of the binding affinity to Complement C5. As illustrated in FIG. 4A and Table 2, the results showed high binding affinities for the four bispecific antigen polypeptides with EC50 value at 466.2 pM, 304.1 pM, 93.3 pM and 104.8 pM, respectively. On the contrary, the anti- VEGF-A and anti-C5 antibodies did not show binding affinity to VEGF-A or C5.

[0257] VEGF-A-binding ELISA- Recombinant human VEGF-A (Peprotech, Cat #100-20) was used to coat plates at 1 pg / mL. The products were diluted in blocking buffer at a starting concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by biotinylated rhC5 (Aero Biosystems, Cat # CO5-H82E9 ) followed by 1 :5,000 dilution of HRP- conjugated streptavidin (Jackson ImmuoResearch Lab, Cat #016-030-084). OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader. Table 2 shows the EC50 value of the binding affinity to VEGF-A. As illustrated in FIG. 4B and Table 2, the results showed high binding affinities for the four bispecific antigen binding polypeptides with EC50 value at 88.5 pM, 123.6 pM, 30.9 pM, and 37.8 pM, respectively. On the contrary, the anti-VEGF-A and anti- C5 antibodies did not show binding affinity to VEGF-A or C5.Table 2. VEGF-A / C5 bispecific antigen binding polypeptides dual binding affinity to both VEGF-A and Complement C5Attorney Docket No. 63209-724.601

[0258] Overall, the binding affinity results suggested that all four bispecific antigen binding polypeptides bind to both targets with high affinity at the same time.Example 4- The half maximal inhibitory concentration (IC50) of VEGF-A / C5 bispecific antigen binding polypeptides

[0259] Four different constructs of VEGF-A / C5 bispecific antigen binding polypeptides prepared according to methods in example 1 were tested for their IC50 of VEGF-A and VEGF R2 / KDR binding,

[0260] VEGF-A inhibition ELISA- The VEGF-A / C5 bi specific antigen binding polypeptides were diluted in blocking buffer at a starting concentration of 400 nM and then 1 :3 serial dilutions were tested. This 2x bispecific dilutions were mixed with 2x biotinylated VEGF R2 / KDR receptor (Aero Biosystems, Cat # KDR-H82E5). Then lx mixtures of bispecific polypeptides and biotinylated VEGF R2 / KDR were added to plate wells. The binding was detected through a biotinylated receptor VEGF R2 / KDR and 1 : 5,000 dilution of HRP-conjugated streptavidin (Jackson ImmunoResearch Lab, Cat #016-030-084). The anti-VEGF-A antibody and the anti-C5 antibody were run as controls using the same protocol. OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader. Table 3 shows the IC50 value to VEGF- A / VEGF-A receptor interactions. As illustrated in FIG. 5 and Table 3, IC50 of VEGF-A / C5 bispecific antigen binding polypeptides was 29.5 nM, 31.6 nM, 22.7 nM, and 22.06 nM, which was comparable to the IC50 value of the anti-VEGF-A antibody (31. InM).Table 3: The IC50 value of VEGF-A / C5 bispecific antigen binding polypeptides to VEGF-A / VEGF R2 interactionExample 5- The half maximal inhibitory concentration (IC50) of VEGF-A / C5 bispecificAtorney Docket No. 63209-724.601 antigen binding polypeptides in the presence of C5

[0261] The VEGF-A / C5 bispecific antigen binding polypeptide with the construct illustrated in FIG. 1C was prepared according to methods in example 1. The construct was tested for its ICso of VEGF-A / VEGF R2 / KDR binding in the presence of C5.

[0262] VEGF-A inhibition ELISA- The VEGF-A / C5 bi specific antigen binding polypeptide was diluted in blocking buffer at a starting concentration of 400nM and then 1 :3 serial dilutions were tested. This 2x bispecific dilutions were mixed with 2x rhC5 with a concentration of 2nM, 20nM and 200nM, respectively, and lx of the mixtures were added to wells. After 1 hour incubation and washes with PBS-Tween, lOnM biotinylated VEGF R2 / KDR (Aero Biosystems, Cat # KDR-H82E5) was added to wells to compete the binding. The binding was detected through 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmunoResearch Lab, Cat #016-030-084). OD450 nm readings were measured by BioTek Synergy HTX Microplate Reader. Table 4 shows the IC50 value to VEGF-A / VEGF R2 / KDR interaction. As illustrated in FIG. 6A and Table 4, IC50 of VEGF-A / C5 bispecific antigen binding polypeptide decreased gradually in the presence of the increased concentration of C5.Table 4: The IC50 value of VEGF-A / C5 bispecific antigen binding polypeptide to VEGF- A / VEGF R2 interaction in the presence of C5Example 6- The half maximal inhibitory concentration (IC50) of VEGF-A / C5 bispecific antigen binding polypeptides in inhibiting VEGF-A stimulated bioassay

[0263] The VEGF-A / C5 bispecific antigen binding polypeptide with the construct illustrated in FIG. 1C was prepared according to methods in example 1. The construct was tested for its IC50 of VEGF-A / VEGF R2 interaction in VEGF-A stimulated bioassay.

[0264] The inhibition of VEGF bioassay stimulated by rhVEGF-A was carried out by using Promega’s ready -to-use VEGF R2 / KDR-expressing HEK293 bioassay (Promega, Cat#GA2001) and following Promega’s bioassay protocol. The rhVEGF-A (Peprotech, Cat#100-20) was titrated in VEGF stimulation bioassay. To perform RP-060 VEGF blockade bioassay, 25 L / wellAtorney Docket No. 63209-724.601 of KDR / NFAT-RE HEK293 cells were seeded to plate and 25pL / well of 1 :3 serial diluted 3x RP-060 starting at 600nM was added. Then 25pL of 3x rhVEGF-A stimulator with EC80 - EC90 concentration was added to each well. The final volume of each well is 75 L. After incubating for 6 hours at 37°C incubator with 5% CO2, added 75pL / well Bio-Gio Reagent and incubated at room temperature for 10 minutes. Read plate on GloMax (Promega) luminometer and analyzed the results by GraphPad Prism 10.6.1. As shown in Fig. 6B and Table 5, the ICso of VEGF-A / C5 bispecific antigen binding polypeptide RP-060 was 28.9 nM.Table 5: The IC50 value of VEGF-A / C5 bispecific antigen binding polypeptide to VEGF- A / VEGF R2 interaction in the presence of VEGF-AExample 7 — Inhibition of antibody-sensitized sheep red blood cells (SRBCs) hemolysis via the classical pathway by the VEGF-A / C5 bispecific antigen binding polypeptides

[0265] Four different constructs of VEGF-A / C5 bispecific antigen binding polypeptides prepared according to methods in example 1 were tested for their IC50 of inhibiting Ab-sensitized sheep RBC hemolysis via classical complement pathway.

[0266] Hemolysis inhibition assay for classical complement pathway-Hemolysis inhibition assay was carried out with Ab-sensitized SRBCs [Complement Technology, Inc.(CTI), Cat#B202] at the presence of human serum (Fisher Scientific, Cat #P2657-100) following CTI’s protocol, except that the assay was performed in 2mL 96-well block by scaled down the materials used. The total assay volume of each well was 0.2 mL. The VEGF-A / C5 bispecific antigen binding polypeptide or control antibody was diluted in GVB++ buffer (with Ca and Mg, CTI, Cat #B100) at a starting concentration of lOOOnM and then 1 :3 serial dilutions were tested. This 2x bispecific dilutions were mixed with 2x human serum solution (1 :4 dilution), and added to wells containing 0.1 mL SRBCs prepared in GVB++ buffer. The block was incubated for 1 hour at 37°C, 5% CO2 with gently mixed every 15 minutes. The block was centrifuged at 2,000 rpm to pellet cells and the supernatants were transferred to an ELISA plate. OD415 nm readings were measured by BioTek Synergy HTX Microplate. Table 6 shows the IC50 value of inhibiting SRBC hemolysis. As illustrated in FIG. 7 and Table 6, IC50 of VEGF-A / C5 bispecific antigen binding polypeptides were 27.67 nM, 22.49 nM, 24.77 nM, and 24.24 nM, which were comparable to the IC50 value of eculizumab (12.33 nM) and the anti-C5 antibody (25.30 nM).Attorney Docket No. 63209-724.601Table 6: IC50 of VEGF-A / C5 bispecific polypeptides in inhibiting RBC hemolysis via classical pathway

[0267] Comparison to Izevey and Syfoyre in hemolysis inhibition assay for classical complement pathway. Hemolysis inhibition assay was carried out with Ab-sensitized SRBCs (CTI, Cat #B202) using human serum obtained from CTI (Cat #NHS) or cynomolgus monkey serum (CTI, Cat #NCYS). The total assay volume of each well was 0.2 mL. The VEGF-A / C5 bispecific antigen binding polypeptide or control inhibitor / antibody was diluted in GVB++ buffer (with Ca and Mg, CTI, Cat #B100) at a starting concentration of lOOOnM and then 1 :3 serial dilutions were tested. This 2x bispecific dilutions were mixed with 2x human serum solution (1 : 10 dilution) or 2x cynomolgus monkey serum (1 :20 dilution) and added to wells containing 0.1 mL SRBCs prepared in GVB++ buffer. Water (H2O) and GVB++ buffer only served as 100% cell lysis and no lysis control, respectively. The block was incubated for 1 hour at 37°C, 5% CO2 with gently mixed every 15 minutes. The block was centrifuged at 2,000 rpm to pellet cells and the supernatants were transferred to an ELISA plate. OD415 nm readings were measured by BioTek Synergy HTX Microplate. Table 7 shows the ICso value of inhibiting SRBC hemolysis. As illustrated in FIG. 8A and Table 7, ICso of VEGF-A / C5 bispecific antigen binding polypeptides with human serum were 18.29 nM (the test sample was prepared from the protein lot of high bispecific polypeptide concentration) and 20.2 nM (the test sample was prepared from the protein lot of lower bispecific polypeptide concentration), which were comparable to the ICso value of bivalent eculizumab (12.51 nM), the parental anti-C5 antibody RP-047 (18.32 nM), and the C5 inhibitor Izervay (16.11 nM). On the contrary, the VEGF-A / C5 bispecific polypeptide did not inhibit SRBC hemolysis in the cynomolgus monkey serum (see FIG. 8B and Table 7)Attorney Docket No. 63209-724.601Table 7: IC50 of VEGF-A / C5 bispecific polypeptides in inhibiting RBC hemolysis via classical pathwayExample 8 — Inhibition of rabbit red blood cells (RBCs) hemolysis via the alternative pathway by the VEGF-A / C5 bispecific antigen binding polypeptides

[0268] The VEGF-A / C5 bispecific antigen binding polypeptide construct shown in FIG. 1C was prepared according to methods in example 1. The bispecific polypeptide was tested for their ICso of inhibiting rabbit RBC hemolysis via the alternative complement pathway.

[0269] Hemolysis inhibition assay for alternative complement pathway. Hemolysis inhibition assay was carried out with rabbit RBCs (CTI), Cat#B302) at the presence of human serum (CTI, Cat #NHS) following CTI’s protocol, except performing the assay in 2mL 96-well block by scaled down the materials used. The total assay volume of each well was 0.2 mL. The VEGF-A / C5 bispecific antigen binding polypeptide or control antibody was diluted in GVBo (without Ca and Mg) buffer (CTI, Cat #B 100) at a starting concentration of lOOOnM and then 1 :3 serial dilutions were tested. This 2x bispecific dilutions were mixed with 2x human serum solution (1 :2 dilution), and added to wells containing 0.1 mL rabbit RBCs prepared in GVBo buffer. Water / no test sample and buffer only served as 100% cell lysis and no cell lysis control, respectively. The block was incubated for 1 hour at 37°C, 5% CO2 with gently mixed every 15 minutes. The block was centrifuged at 2,000 rpm to pellet cells and the supernatants were transferred to an ELISA plate. OD415 nm readings were measured by BioTek Synergy HTX Microplate. As illustrated in FIG. 9, Izervay, Eculizumab and RP-060 showed low level inhibition between 55.5 nM to 500 nM. Syfovre showed little inhibition at 500 nM.Atorney Docket No. 63209-724.601Example 9-Binding of VEGF-A / C5 bispecific antigen binding polypeptide to VEGF-A from different species

[0270] VEGF-A / C5 bispecific antigen-binding polypeptides prepared according to example 1 were tested for their binding capacity to VEGF-A from different species.

[0271] VEGF-A ELISA- Recombinant human (P eprotech, Cat #100-20), rabbit (Kingfisher, Cat #RP1025U), rat (Sino Biological, Cat #80006-RNAB), pig (AssayGenie, Cat #KPRT0149), and dog (R&D, Cat #1603-CV-010 / CF) VEGF-A were used to coat plates at 1 pg / mL. Human and monkey have an identical VEGF sequence. The products were diluted in blocking buffer at a starting concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by 100 ng / mL biotinylated anti-VpreB Ab followed by 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmuoResearch Lab, Cat #016-030-084). OD450 nm readings were measured by BioTek Synergy HTX Microplate. Table 8 shows the ECso value of the binding affinity to VEGF-A from different species. As illustrated in FIG. 10 and Table 8, the results showed that the VEGF-A / C5 bispecific antigen-binding polypeptide binds to VEGF-A from different species with comparable EC50 between 32.8 pM and 80.4 pM.Table 8: EC50 value of VEGF-A / C5 bispecific polypeptide to VEGF-A from different speciesExample 10-Binding of VEGF-A / C5 bispecific antigen binding polypeptide to C5 from different species

[0272] VEGF-A / C5 bispecific antigen-binding polypeptides prepared according to example 1 were tested for their binding capacity to C5 from different species.

[0273] COMPLEMENT C5 ELISA Recombinant human C5 (Aero Biosystems, at # CO5- H52Ha), cynomolgus monkey (Aero Biosystems, Cat # CO5-C52Hx), rabbit (Aero Biosystems, Cat # CO5-R52H4), and rat C5 (Aero Biosystems, Cat #CO5-R52H5) were used to coat plates at 1 pg / mL. The products were diluted in blocking buffer at a starting concentration of 100 nM and then 1 :3 dilutions were tested. The binding was detected by 100 ng / mL biotinylated anti-VpreB Ab followed by 1 :5,000 dilution of HRP-conjugated streptavidin (Jackson ImmuoResearch Lab,Atorney Docket No. 63209-724.601Cat #016-030-084). Eculizumab was run alongside bispecific polypeptides as reference material. OD450 nm readings were measured by BioTek Synergy HTX Microplate. Table 9 shows the EC50 value of the binding affinity to Complement C5. As illustrated in FIG. 11 and Table 9, the bispecific polypeptide binds to human C5 with much higher affinity of an EC50 at 38.2 pM. The bispecific polypeptide binds to cynomolgus monkey C5 with an EC50 of 43.95 nM, while no binding was observed to rabbit or rat C5.Table 9: EC50 value of VEGF-A / C5 bispecific polypeptide to C5 from different speciesExample 11 -Assessment of pre-existing anti-drug antibodies (Al) As) against VEGF-A / C5 bispecific antigen binding polypeptide

[0274] Elevated levels of pre-existing anti-drug antibodies (AD As) in patients may predict adverse outcomes in response to intravitreal injections with anti -vascular endothelial growth factor (anti-VEGF) agents. Higher levels of pre-existing AD As detected against brolucizumab than ranibizumab may be correlated with a greater incidence of adverse events such as severe intraocular inflammation (IOI) including occlusive retinal vasculitis upon treatment with brolucizumab. Four constructs of the bispecific antigen binding polypeptides prepared according to methods in example 1 were tested against pre-existing AD As to determine which version would be least likely to induce IOI in patients.

[0275] To detect AD As, pooled human serum from Fisher Scientific (Cat #BP2657) was analyzed in an enzyme-linked immunosorbent assay (ELISA). Plate wells were coated with 100 pL of 1 pg / mL of the known most immunogenic agent (brolucizumab), the known least immunogenic agent (ranibizumab), an anti-C5 antibody (RP-047) and four of the bispecific antigen binding polypeptides (RP-058, RP-059, RP-060, RP-061) made in lx coating buffer (BioFx / Surmodics, Cat# COAT-1000-01). All plates were incubated overnight at room temperature. Wells were washed with PBS-Tween, blocked with 300 pL 1% BSA-PBST blocking buffer, washed three times again, and incubated with 100 pL of 1 :3 serially diluted pooled human serum for 1 hour at room temperature. Samples were tested in duplicates. Wells were washed and then incubated with HRP-conjugated anti-human IgG-Fcy secondary antibody (Jackson ImmunoResearch. Labs, Cat#709-035-098) for 1 hour at room temperature. After sixAtorney Docket No. 63209-724.601 washes, the TMB substrate and stop solution were added. ELISA results were visualized at OD450 nm by BioTek Synergy HTX Microplate reader. The ADA titer was determined as the dilution presenting threefold OD450 reading over background control.

[0276] Table 10 shows the ADA titer in pooled human serum against the bispecific antigen polypeptides as well as anti-C5 antibody (RP-047), brolucizumab and ranibizumab. As shown in Table 10, the ADA titer in pooled human serum against RP-060 ranged from 1 : 300-1 :600, which was even lower than ranibizumab (known clinically being the least immunogenic). The ADA titers against RP-058 and RP-059 ranged from 1 : 1800 to 1 :2700, and the ADA titers against RP- 061 ranged from 1 :900 to 1 : 1800. The result suggested that of the four constructs, RP-060 has the lowest amounts of pre-existing ADA in pooled human serum, and are therefore least likely to induce adverse immunogenic responses in patients.Table 10: The ADA titer in pooled human serum against the bispecific antigen binding polypeptidesExample 12-exemplary VEGF-A / C5 bispecific antigen binding polypeptide sequences

[0277] Exemplary sequences of the bispecific antigen binding polypeptides and its components are listed in Table 11.Table 11: Exemplary sequences of the bispecific antigen binding polypeptides and its componentsAtorney Docket No. 63209-724.601Atorney Docket No. 63209-724.601Atorney Docket No. 63209-724.601

[0278] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0279] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

Attorney Docket No. 63209-724.601CLAIMSWHAT IS CLAIMED IS:

1. A multispecific binding polypeptide comprising a single chain variable region fragment (scFv) and a Fragment antigen-binding region (Fab), wherein the scFv comprises a first binding moiety and the Fab comprises a second binding moiety, wherein the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof, and wherein the light chain variable region is a surrogate light chain variable region.

2. The multispecific binding polypeptide of claim 1, wherein the surrogate light chain variable region comprises a VpreB sequence.

3. The multispecific binding polypeptide of claim 2, wherein the VpreB sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101.

4. The multispecific binding polypeptide of any one of claims 1-3, wherein the light chain variable region is coupled to the heavy chain variable region via a linker.

5. The multispecific binding polypeptide of claim 4, wherein the linker sequence comprises an amino acid sequence comprising at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

6. The multispecific binding polypeptide of any one of claims 1-5, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL).

7. The multispecific binding polypeptide of claim 6, wherein the light chain constant domain is a kappa light chain constant domain (CK).

8. The multispecific binding polypeptide of any one of claims 1-7, wherein the scFv and the Fab are covalently coupled by a linker.

9. The multispecific binding polypeptide of any one of claims 1-7, wherein the scFv and the Fab are directly covalently coupled without a linker.

10. The multispecific binding polypeptide of any one of claims 1-9, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab.Attomey Docket No. 63209-724.60111. The multispecific binding polypeptide of any one of claims 1-9, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab.

12. The multispecific binding polypeptide of any one of claims 6-9, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

13. The multispecific binding polypeptide of any one of claims 6-9, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

14. The multispecific binding polypeptide of any one of claims 1-13, wherein the first binding moiety is a VEGF-A or a Complement C5 binding moiety.

15. The multispecific binding polypeptide of any one of claims 1-13, wherein the first binding moiety is a VEGF-A moiety.

16. The multispecific binding polypeptide of any one of claims 1-15, wherein the second binding moiety is a VEGF-A or a Complement C5 binding moiety.

17. The multispecific binding polypeptide of any one of claims 1-15, wherein the second binding moiety is a Complement C5 binding moiety.

18. The multispecific binding polypeptide of any one of claims 14 to 17, wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106.

19. The multispecific binding polypeptide of any one of claims 14 to 18, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1.

20. The multispecific binding polypeptide of any one of claims 14 to 18, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 1.

21. The multispecific binding polypeptide of any one of claims 14 to 20, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequenceAttomey Docket No. 63209-724.601 comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2.

22. The multispecific binding polypeptide of any one of claims 14 to 21, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

23. The multispecific binding polypeptide of any one of claims 14 to 22, wherein the Complement C5 binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 18.

24. The multispecific binding polypeptide of any one of claims 14 to 23, wherein the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 19.

25. The multispecific binding polypeptide of any one of claims 14 to 23, wherein the Complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19.

26. The multispecific binding polypeptide of any one of claims 14 to 25, wherein the Complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 978%, 98%, 99% identity to or is identical to SEQ ID NO: 20.

27. The multispecific binding polypeptide of any one of claims 14 to 25, wherein the Complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

28. A multispecific binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; andAttomey Docket No. 63209-724.601 wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18.

29. The multispecific binding polypeptide of claim 28, wherein the VEGF-A binding moiety is a scFv or a Fab.

30. The multispecific binding polypeptide of claim 28, wherein the VEGF-A binding moiety is a scFv.

31. The multispecific binding polypeptide of any one of claims 28-30, wherein the Complement C5 binding moiety is a Fab or a scFv.

32. The multispecific binding polypeptide of any one of claims 28-30, wherein the Complement C5 binding moiety is a Fab.

33. The multispecific binding polypeptide of claim 31 or 32, wherein the scFv and the Fab are covalently coupled by a linker.

34. The multispecific binding polypeptide of claim 31 or 32, wherein the ScFv and the Fab are directly covalently coupled without a linker.

35. The multispecific binding polypeptide of any one of claims 29-34, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL).

36. The multispecific binding polypeptide of claim 35, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the heavy chain variable region of the Fab.

37. The multispecific binding polypeptide of claim 35, wherein the C-terminus of the scFv is covalently coupled to the N-terminus of the light chain variable region of the Fab.

38. The multispecific binding polypeptide of claim 35, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

39. The multispecific binding polypeptide of claim 35, wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.

40. The multispecific binding polypeptide of any one of claims 28 to 39, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 1.Attomey Docket No. 63209-724.60141. The multispecific binding polypeptide of any one of claims 28 to 39, wherein the VEGF-A binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 1.

42. The multispecific binding polypeptide of any one of claims 28 to 41, wherein the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101.

43. The multispecific binding polypeptide of any one of claims 28 to 41, wherein the VEGF-A binding moiety comprises a light chain variable region amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 101.

44. The multispecific binding polypeptide of any one of claims 28 to 43, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 2.

45. The multispecific binding polypeptide of any one of claims 28 to 43, wherein the VEGF-A binding moiety comprises a single chain variable region fragment amino acid sequence comprising a sequence identical to SEQ ID NO: 2.

46. The multispecific binding polypeptide of any one of claims 28 to 45, wherein the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 19.

47. The multispecific binding polypeptide of any one of claims 28 to 45, wherein the complement C5 binding moiety comprises an immunoglobulin heavy chain amino acid sequence comprising a sequence identical to SEQ ID NO: 19.

48. The multispecific binding polypeptide of any one of claims 28 to 47, wherein the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or is identical to SEQ ID NO: 20.

49. The multispecific binding polypeptide of any one of claims 28 to 47, wherein the complement C5 binding moiety comprises an immunoglobulin light chain amino acid sequence comprising a sequence identical to SEQ ID NO: 20.

50. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence atAtorney Docket No. 63209-724.601 least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202.

51. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 201 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 202.

52. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203.

53. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 204 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 203.

54. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206.

55. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 205 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 206.

56. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 and theAttomey Docket No. 63209-724.601 second polypeptide comprises an amino acid sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207.

57. The multispecific binding polypeptide of any one of claims 1 to 49, wherein the multispecific binding polypeptide is formed from a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an amino acid sequence identical to SEQ ID NO: 208 and the second polypeptide comprises an amino acid sequence identical to SEQ ID NO: 207.

58. The multispecific binding polypeptide of any one of claims 1 to 57, wherein the multispecific antigen binding polypeptide binds to VEGF-A with an ECso of about 70 picomolar or less.

59. The multispecific binding polypeptide of any one of claims 1 to 58, wherein the multispecific antigen binding polypeptide binds to complement C5 with an ECso of about 180 picomolar or less.

60. The multispecific binding polypeptide of any one of claims 1 to 59, wherein the multispecific antigen binding polypeptide binds to VEGF-A with an ECso of about 70 picomolar or less, and wherein the multispecific antigen binding polypeptide binds to complement C5 with an ECso of about 180 picomolar or less.

61. The multispecific binding polypeptide of any one of claims 1 to 60, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF- A receptor with an IC50 of about 32 nanomolar or less.

62. The multispecific binding polypeptide of any one of claims 1 to 61, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF- A receptor with an IC50 of about 15 nanomolar or less in the presence of complement C5.

63. The multispecific binding polypeptide of any one of claims 1 to 62, wherein the multispecific antigen binding polypeptide inhibits the interaction of VEGF-A and a VEGF- A receptor with an IC50 of about 29 nanomolar or less in the presence of VEGF-A.

64. The multispecific binding polypeptide of any one of claims 1 to 63, wherein the multispecific antigen binding polypeptide inhibits red blood cell hemolysis with an IC50 of about 28 nanomolar or less.

65. The multispecific binding polypeptide of any one of claims 1 to 63, wherein the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide is at least 1 :600.

66. The multispecific binding polypeptide of any one of claims 1 to 65, wherein the pre-existing anti-drug antibodies (AD As) titer in the pooled human serum against the multispecific binding polypeptide ranges from 1 :300 to 1 :600.Atorney Docket No. 63209-724.60167. A pharmaceutical composition comprising the multispecific antigen binding polypeptide of any one of claims 1 to 66 and a pharmaceutically acceptable carrier, excipient, or diluent.

68. The pharmaceutical composition of claim 67 formulated for intravenous administration.

69. The pharmaceutical composition of claim 67 formulated for intravitreal administration.

70. A nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of any one of claims 1 to 66.

71. An expression vector comprising the nucleic acid of claim 70.

72. The expression vector of claim 71, wherein the expression vector is an RNA.

73. The expression vector of claim 71, wherein the expression vector is plasmid or linearized DNA.

74. The expression vector of claim 71, wherein the expression vector is a viral vector.

75. The viral vector of claim 74, wherein the viral vector is an adenovirus.

76. The viral vector of claim 74, wherein the viral vector is an adeno-associated virus.

77. The viral vector of claim 74, wherein the viral vector is a lentivirus virus.

78. A pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of claim 70 and a pharmaceutically acceptable carrier, excipient, or diluent.

79. The pharmaceutical composition of claim 78 formulated for intravenous administration.

80. The pharmaceutical composition of claim 78 formulated for intravitreal administration.

81. A method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition of any one of the preceding claims, thereby treating the disease.

82. The method of claim 81, wherein the disease is an ocular disease.

83. The method of claim 82, wherein the ocular disease comprises macular degeneration.

84. The method of claim 83, wherein the macular degeneration is age-related macular degeneration.

85. The method of claim 82, wherein the ocular disease comprises geographic atrophy.

86. The method of claim 82, wherein the ocular disease comprises diabetic macular edema.

87. The method of claim 82, wherein the ocular disease comprises retinal vein occlusion.

88. A multispecific binding polypeptide comprising a VEGF-A binding moiety and aComplement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a) an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b) an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c) an HCDR3 amino acid sequence set forth in SEQ ID NO: 5;Attomey Docket No. 63209-724.601 d) an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e) an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f) an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the heavy chain first constant region of the Fab.

89. A multispecific binding polypeptide comprising a VEGF-A binding moiety and a Complement C5 binding moiety; wherein the VEGF-A binding moiety comprises: a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 3; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 4; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 5; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 104; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 105; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 106; and wherein the Complement C5 binding moiety comprises; a. an HCDR1 amino acid sequence set forth in SEQ ID NO: 13; b. an HCDR2 amino acid sequence set forth in SEQ ID NO: 14; c. an HCDR3 amino acid sequence set forth in SEQ ID NO: 15; d. an LCDR1 amino acid sequence set forth in SEQ ID NO: 16; e. an LCDR2 amino acid sequence set forth in SEQ ID NO: 17; and / or f. an LCDR3 amino acid sequence set forth in SEQ ID NO: 18, wherein the VEGF-A binding moiety comprises a scFv, wherein the Complement C5 binding moiety comprises a Fab, wherein the Fab comprises a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain first constant region (CHI), and a light chain constant domain (CL), and wherein the N-terminus of the scFv is covalently coupled to the C-terminus of the light chain constant region of the Fab.Attorney Docket No. 63209-724.60190. The multispecific binding polypeptide of claim 88 or 89, wherein the scFv comprises a light chain variable region or fragment thereof and a heavy chain variable region or fragment thereof, and wherein the light chain variable region is a surrogate light chain variable region.

91. The multispecific binding polypeptide of any one of claims 88 to 90 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 206.

92. The multispecific binding polypeptide of any one of claims 88 to 90 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 205, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 206.

93. The multispecific binding polypeptide of any one of claims 88 to 90 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to that set forth in SEQ ID NO: 207.

94. The multispecific binding polypeptide of any one of claims 88 to 90 wherein the multispecific binding polypeptide comprises a complex of a first polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 208, and a second polypeptide comprising an amino acid sequence identical to that set forth in SEQ ID NO: 207.

95. A pharmaceutical composition comprising the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide of any one of claims 88 to 94 and a pharmaceutically acceptable carrier, excipient, or diluent.

96. The pharmaceutical composition of claim 95 formulated for intravenous administration.

97. The pharmaceutical composition of claim 95 formulated for intravitreal administration.

98. A method of treating a disease in an individual comprising administering to the individual the multispecific antigen binding polypeptide, the nucleic acid or plurality of nucleic acids encoding the multispecific antigen binding polypeptide, or pharmaceutical composition of any one of the preceding claims, thereby treating the disease.

99. The method of claim 98, wherein the disease is an ocular disease.

100. The method of claim 99, wherein the ocular disease comprises macular degeneration.

101. The method of claim 100, wherein the macular degeneration is age-related macular degeneration.

102. The method of claim 99, wherein the ocular disease comprises geographic atrophy.Attomey Docket No. 63209-724.601103. The method of claim 99, wherein the ocular disease comprises diabetic macular edema.

104. The method of claim 99, wherein the ocular disease comprises retinal vein occlusion.

105. The multispecific binding polypeptide of any one of claims 14-17 and 23-27, wherein the VEGF-A binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Bevacizumab, Ranibizumab, Aflibercept, Faricimab, and Brolucizumab.

106. The multispecific binding polypeptide of any one of claims 14-22, wherein the Complement C5 binding moiety comprises complementary determining regions (CDRs) derived from the group consisting of Eculizumab, Ravulizumab, Crovalimab, Pozelimab, and IM-101.

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