Compositions and methods

Abstract

Provided herein are macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand, compositions comprising the same, and methods of using the same.

Description

[0001] PATENT

[0002] Attorney Docket No. 51661 -008W06

[0003] Flagship Reference: VL75014-W1

[0004] COMPOSITIONS AND METHODS

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 8, 2025, is named 51661 -008W06_Sequence_Listing_10_8_25 and is 63,094 bytes in size.

[0007] CROSS REFERENCE TO RELATED APPLICATIONS

[0008] This application claims priority to U.S. Provisional Patent Application No. 63 / 707,910, filed on October 16, 2024; U.S. Provisional Patent Application No. 63 / 724,716, filed on November 25, 2024; U.S. Provisional Patent Application No. 63 / 745,11 1 , filed on January 14, 2025; U.S. Provisional Patent Application No. 63 / 779,667, filed on March 28, 2025; and U.S. Provisional Patent Application No. 63 / 789,552, filed on April 16, 2025, the contents of which are hereby incorporated by reference in their entirety.

[0009] BACKGROUND

[0010] There is a need in the art for macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand and for methods of using the same.

[0011] SUMMARY OF THE INVENTION

[0012] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) complementarity-determining regions (CDRs) of SEQ ID NOs: 4, 5, and 6, and further comprising an additional polypeptide.

[0013] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12, and further comprising an additional polypeptide.

[0014] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and heavy chain constant region 1 (CH1) of SEQ ID NO: 2, and further comprising an additional polypeptide.

[0015] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and heavy chain constant region 1 (CH1) of SEQ ID NO: 44, and further comprising an additional polypeptide.

[0016] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) of SEQ ID NO: 3, and further comprising an additional polypeptide. PATENT

[0017] Attorney Docket No. 51661 -008W06

[0018] Flagship Reference: VL75014-W1

[0019] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) of SEQ ID NO: 3 and the LO variable domain (VL) of SEQ ID NO: 9, and further comprising an additional polypeptide.

[0020] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 2 and the LC variable domain (VL) of SEQ ID NO: 9, and further comprising an additional polypeptide.

[0021] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) of SEQ ID NO: 9, and further comprising an additional polypeptide.

[0022] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 2 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 8, and further comprising an additional polypeptide.

[0023] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 8, and further comprising an additional polypeptide.

[0024] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) of SEQ ID NO: 3 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 8, and further comprising an additional polypeptide.

[0025] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 2 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide.

[0026] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide.

[0027] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) of SEQ ID NO: 3 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide.

[0028] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC of SEQ ID NO: 1 , and further comprising an additional polypeptide. PATENT

[0029] Attorney Docket No. 51661 -008W06

[0030] Flagship Reference: VL75014-W1

[0031] In some aspects, the disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC of SEQ ID NO: 1 and the LC of SEQ ID NO: 7, and further comprising an additional polypeptide.

[0032] In some aspects, the disclosure provides a macromolecule wherein the antibody or antigen binding portion thereof (e.g., a HC, a VH, a VH + CH1 , or HC CDRs 1 , 2, or 3, a LC, a VL, a VL + CL, or LC CDRs 1 , 2, or 3) comprises at least 90% identity to a sequence provided herein (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence), and e.g., binds to human 4-1 BB, wherein the macromolecule further comprises an additional polypeptide.

[0033] In some aspects, the disclosure provides a macromolecule wherein the antibody or antigen binding portion thereof comprises the CDR sequences (e.g., 1 , 2 or 3 CDRs, or the HC CDRs and / or LC CDRs) of an antibody provided herein and comprises at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the antibody or antigen binding portion thereof (e.g., a HC, a VH, a VH + CH1 , a LC, a VL + CL, or a VL), and, e.g., binds to human 4-1 BB, wherein the macromolecule further comprises an additional polypeptide.

[0034] In some embodiments of the macromolecules provided herein, the additional polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof. In some embodiments of the macromolecules provided herein, the additional polypeptide is a human VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., human VEGF, e.g., in an assay provided herein. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., mouse VEGF, e.g., in an assay provided herein.

[0035] As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948). For example, “Gen 1 Detuned L221 W” indicates that this variant has the 221stresidue derived from the full length VEGFR1 mutated from leucine to tryptophan. For example, for an VEGF-R polypeptide or fragment or variant thereof that comprises an amino acid substitution mutation of one or more of F172, Y199, L221 , H223, and R224 (e.g., comprises a substitution of the wild-type amino acid residue at one or more of these positions with a different amino acid residue), the numbering of these amino acid residues refers to the sequence of Uniprot ID 17948.

[0036] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form thereof. For example, the VEGF-R polypeptide comprises SEQ ID NO: 46 or 54. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 46 or 54 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0037] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 38 or 53. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 38 or 53 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. PATENT

[0038] Attorney Docket No. 51661 -008W06

[0039] Flagship Reference: VL75014-W1

[0040] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a variant of VEGF-R (e.g., a variant of a wild-type VEGF-R, e.g., a variant in which one or more amino acid residues differ from a wild-type residue). For example, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0041] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R that also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, in some embodiments, the VEGF-R polypeptide is equal in length to the full form, and also comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the full form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the full form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0042] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form, which also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the abbreviated form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0043] In some embodiments, the variant of the VEGF-R polypeptide comprises any one of SEQ ID NOs: 47 to 52 and 55 to 61 . PATENT

[0044] Attorney Docket No. 51661 -008W06

[0045] Flagship Reference: VL75014-W1

[0046] In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 47 to 52 and 55 to 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) thereto, and, e.g., binds to human VEGF.

[0047] In some embodiments, the variant VEGF-R polypeptide (which can be of the full-length VEGF-R polypeptide or fragment thereof) comprises an amino acid substitution mutation of one or more of F172,

[0048] Y199, L221 , H223, and R224 (e.g., comprises a substitution of the wild-type amino acid residue at one or more of these positions with a different amino acid residue). In some embodiments, the VEGF-R polypeptide comprises a F172 amino acid substitution mutation, In some embodiments, the VEGF-R polypeptide comprises a Y199 amino acid substitution mutation, In some embodiments, the VEGF-R polypeptide comprises a L221 amino acid substitution mutation, In some embodiments, the VEGF-R polypeptide comprises a H223 amino acid substitution mutation, In some embodiments, the VEGF-R polypeptide comprises a R224 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises H223A and R224A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises Y199A and F172A amino acid substitution mutations.

[0049] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 47 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47).

[0050] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 47.

[0051] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0052] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 48 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48).

[0053] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224Q amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 48.

[0054] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0055] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 49 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49).

[0056] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 49. PATENT

[0057] Attorney Docket No. 51661 -008W06

[0058] Flagship Reference: VL75014-W1

[0059] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 49.

[0060] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 50 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50).

[0061] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 50.

[0062] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.

[0063] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 51 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 51).

[0064] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199F amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 51 .

[0065] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0066] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 52 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52).

[0067] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199L amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 52.

[0068] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52.

[0069] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 55 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 55).

[0070] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises H223A and R224A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 55.

[0071] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55. PATENT

[0072] Attorney Docket No. 51661 -008W06

[0073] Flagship Reference: VL75014-W1

[0074] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 56 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56).

[0075] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 56.

[0076] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56.

[0077] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 57 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 57).

[0078] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 57.

[0079] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0080] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 58 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 58).

[0081] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 58.

[0082] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58.

[0083] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 59 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 59).

[0084] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 W amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 59.

[0085] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0086] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 60 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 60). PATENT

[0087] Attorney Docket No. 51661 -008W06

[0088] Flagship Reference: VL75014-W1

[0089] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a F172A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 60.

[0090] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 60.

[0091] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 61).

[0092] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises Y199A and F172A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 61 .

[0093] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[0094] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 38.

[0095] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 46.

[0096] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 47.

[0097] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs :4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 48.

[0098] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 49.

[0099] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and further comprising an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 50. PATENT

[0100] Attorney Docket No. 51661 -008W06

[0101] Flagship Reference: VL75014-W1

[0102] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 51 .

[0103] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 52.

[0104] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 55.

[0105] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 56.

[0106] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 57.

[0107] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 58.

[0108] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 59.

[0109] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 60.

[0110] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 61 .

[0111] In some aspects, the disclosure provides a macromolecule comprising (i) an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain constant region PATENT

[0112] Attorney Docket No. 51661 -008W06

[0113] Flagship Reference: VL75014-W1

[0114] (CL) of SEQ ID NO: 45, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 38. In some embodiments, the macromolecule comprises SEQ ID NOs: 38, 44, and 45.

[0115] In some embodiments, the macromolecule comprises SEQ ID NO: 40 and SEQ ID NO: 45.

[0116] In some embodiments, the macromolecule comprises SEQ ID NO: 41 and SEQ ID NO: 45.

[0117] In some embodiments, the macromolecule comprises SEQ ID NO: 53 and SEQ ID NO: 45.

[0118] In some embodiments, the macromolecule comprises SEQ ID NO: 54 and SEQ ID NO: 45.

[0119] In some embodiments, the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[0120] In some embodiments, the macromolecule comprises SEQ ID NO: 63 and SEQ ID NO: 45.

[0121] In some embodiments, the macromolecule comprises SEQ ID NO: 64 and SEQ ID NO: 45.

[0122] In some embodiments, the macromolecule comprises a signal peptide.

[0123] In some embodiments, the macromolecule comprises an Fc region.

[0124] In some embodiments, the macromolecule comprises one or more copies of a linker.

[0125] In some embodiments, the macromolecule comprises one to four copies of a GGGGS linker (SEQ ID NO: 13). In some embodiments, the macromolecule comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker in one location of the macromolecule. In some embodiments, the macromolecule comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker in two locations of the macromolecule.

[0126] In some embodiments, the macromolecule comprises one to four copies of a GGGGT linker (SEQ ID NO:14).

[0127] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a linker.

[0128] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker.

[0129] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO: 14) linker.

[0130] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker and one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO: 14) linker.

[0131] In some embodiments, the macromolecule comprises an Fc region at the carboxy terminus of the macromolecule.

[0132] In some embodiments, the macromolecule comprises a linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide. In some embodiments, the linker comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker.

[0133] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide.

[0134] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO:13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ PATENT

[0135] Attorney Docket No. 51661 -008W06

[0136] Flagship Reference: VL75014-W1

[0137] ID NOs: 4, 5, and 6. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LO) CDRs of SEQ ID NOs: 10, 11 , and 12. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 38, 46-52, and 55-61 .

[0138] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide; and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker and / or one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO: 14) linker between the polypeptide and the Fc region. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 1 1 , and 12. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 38, 46-52, and 55-61 .

[0139] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO:13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide; and one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO: 14) linker between the polypeptide and the Fc region. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 11 , and 12. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 38, 46- 52, and 55-61 .

[0140] In some embodiments, the macromolecule comprises a bivalent structure.

[0141] In some embodiments, the macromolecule comprises a structure shown in Fig. 8A.

[0142] In some embodiments, the macromolecule comprises a structure shown in Figs. 8A and 8B.

[0143] In some embodiments, the macromolecule comprises a structure shown in Fig. 8C.

[0144] In some embodiments, the macromolecule comprises a structure shown in Fig. 9.

[0145] In some embodiments, the macromolecule comprises a structure shown in Fig. 10A.

[0146] In some embodiments, the macromolecule comprises a signal peptide.

[0147] In some embodiments, the macromolecule does not comprise a signal peptide.

[0148] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an IgG isotype.

[0149] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG1 isotype.

[0150] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG2 isotype.

[0151] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG3 isotype.

[0152] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG4 isotype. PATENT

[0153] Attorney Docket No. 51661 -008W06

[0154] Flagship Reference: VL75014-W1

[0155] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a LALAPG mutation.

[0156] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a kappa light chain.

[0157] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a lambda light chain.

[0158] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB.

[0159] In some aspects, the disclosure provides a VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the polypeptide is a human VEGF-R polypeptide or a fragment or variant thereof.

[0160] As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948). For example, “Gen 1 Detuned L221 W” indicates that this variant has the 221stresidue derived from the full length VEGFR1 mutated from leucine to tryptophan. For example, for an VEGF-R polypeptide or fragment or variant thereof that comprises an amino acid substitution mutation at one or more of F172, Y199, L221 , H223, and R224, the numbering of these amino acid residues refers to the sequence of Uniprot ID 17948.

[0161] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form thereof. For example, the VEGF-R polypeptide comprises SEQ ID NO: 46. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 46 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0162] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form thereof. For example, the VEGF-R polypeptide comprises SEQ ID NO: 54. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 54 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0163] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 38. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0164] In some embodiments in which the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 53. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 53 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0165] In some embodiments in which the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 62. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 62 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. PATENT

[0166] Attorney Docket No. 51661 -008W06

[0167] Flagship Reference: VL75014-W1

[0168] In some embodiments in which the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 63. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 63 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0169] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 64. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 64 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0170] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a variant of VEGF-R (e.g., a variant of a wild-type VEGF-R, e.g., a variant in which one or more amino acid residues differ from a wild-type residue). For example, in some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0171] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R that also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, in some embodiments, the VEGF-R polypeptide is equal in length to the full form, and also comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the full form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the full form which also comprises a variant comprises one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0172] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form, which also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the abbreviated form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least PATENT

[0173] Attorney Docket No. 51661 -008W06

[0174] Flagship Reference: VL75014-W1

[0175] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0176] In some embodiments, the variant of the VEGF-R polypeptide comprises any one of SEQ ID NOs: 47 to 52 and 55 to 61 .

[0177] In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 47 to 52 and 55 to 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0178] In some embodiments, the variant thereof (which can be of the VEGF-R polypeptide or fragment thereof) comprises an amino acid substitution mutation of one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the VEGF-R polypeptide comprises a F172 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a L221 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a H223 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a R224 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises H223A and R224A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises Y199A and F172A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises a Y199A amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199F amino acid substitution mutation.

[0179] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 47 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47).

[0180] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 47.

[0181] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0182] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 48 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48).

[0183] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224Q amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 48. PATENT

[0184] Attorney Docket No. 51661 -008W06

[0185] Flagship Reference: VL75014-W1

[0186] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0187] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 49 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49).

[0188] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 49.

[0189] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 49.

[0190] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 50 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50).

[0191] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 50.

[0192] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.

[0193] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 51 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 51).

[0194] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199F amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 51 .

[0195] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0196] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 52 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52).

[0197] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199L amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 52.

[0198] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52. PATENT

[0199] Attorney Docket No. 51661 -008W06

[0200] Flagship Reference: VL75014-W1

[0201] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 55 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 55).

[0202] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises H223A and R224A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 55.

[0203] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55.

[0204] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 56 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56).

[0205] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 56.

[0206] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56.

[0207] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 57 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 57).

[0208] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 57.

[0209] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0210] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 58 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 58).

[0211] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 58.

[0212] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58.

[0213] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 59 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 59). PATENT

[0214] Attorney Docket No. 51661 -008W06

[0215] Flagship Reference: VL75014-W1

[0216] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 W amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 59.

[0217] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0218] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 60 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 60).

[0219] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a F172A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 60.

[0220] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 60.

[0221] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 61).

[0222] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises Y199A and F172A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 61 .

[0223] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[0224] In some aspects, the disclosure provides a polypeptide comprising a VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the polypeptide comprises a human VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof described herein. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., human VEGF, e.g., in an assay provided herein. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., mouse VEGF, e.g., in an assay provided herein. In some embodiments, the polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof described herein.

[0225] In some aspects, the disclosure provides a polypeptide that comprises a VEGF-R polypeptide or a fragment or variant thereof described herein.

[0226] In some aspects, the disclosure provides a composition comprising a polypeptide described herein.

[0227] In some aspects, the disclosure provides a pharmaceutical composition comprising a polypeptide described herein, or a composition described herein, and a pharmaceutically acceptable excipient.

[0228] In some aspects, the disclosure provides nucleic acid or set of nucleic acids encoding a polypeptide described herein. PATENT

[0229] Attorney Docket No. 51661 -008W06

[0230] Flagship Reference: VL75014-W1

[0231] In some aspects, the disclosure provides a method of treatment comprising administering a polypeptide, or a composition, or a pharmaceutical composition or nucleic acid described herein (e.g., a therapeutically effective amount thereof) to a subject, e.g., a subject in need thereof.

[0232] In some aspects, the disclosure provides a composition comprising a macromolecule described herein.

[0233] In some aspects, the disclosure provides a pharmaceutical composition comprising a macromolecule described herein, or a composition described herein, and a pharmaceutically acceptable excipient.

[0234] In some aspects, the disclosure provides a method of treatment comprising administering a macromolecule comprising an antibody or antigen binding portion thereof, a polypeptide, or a composition, or a pharmaceutical composition described herein (e.g., a therapeutically effective amount thereof) to a subject, e.g., a subject in need thereof.

[0235] In some embodiments, the subject has a tumor e.g., a solid tumor.

[0236] In some embodiments, the subject has a cancer, e.g., a colorectal cancer or a melanoma.

[0237] In some embodiments, the method comprises administering an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0238] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0239] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand, wherein the two macromolecules are identical.

[0240] In one aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand PATENT

[0241] Attorney Docket No. 51661 -008W06

[0242] Flagship Reference: VL75014-W1 and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0243] In one aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0244] In some embodiments, the conjugation is covalent. In some embodiments, the covalent conjugation comprises a chemical linker or a polypeptide linker. In some embodiments, the covalent conjugation comprises disulfide linkages between the macromolecules.

[0245] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary moieties is biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides. In some embodiments, the pair of complementary polypeptides is a pair of engineered Fc fragments, e.g., a knob-into-hole pair.

[0246] In some embodiments, the conjugation sterically orients the two macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0247] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0248] In some embodiments, the conjugation sterically orients the first member of the pair of macromolecules and the second member of the pair of macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0249] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0250] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the PATENT

[0251] Attorney Docket No. 51661 -008W06

[0252] Flagship Reference: VL75014-W1 effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0253] In one aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0254] In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0255] In one aspect, provided herein is a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0256] In one aspect, provided herein is a macromolecule comprising an FBD1 and an FBD2 linked to two SBDs by linker domains, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0257] In one aspect, provided herein is a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease PATENT

[0258] Attorney Docket No. 51661 -008W06

[0259] Flagship Reference: VL75014-W1 signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0260] In one aspect, provided herein is macromolecule comprising an FBD1 and an FBD2 linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; (c) the SBD1 specifically binds a first effector ligand in the biological sample; and (d) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0261] In certain of the foregoing aspects, the first binding domain and second binding domain are linked covalently (e.g., by small molecule, peptide (e.g., as a single polypeptide chain comprising the first binding domain and second binding domain), or a combination thereof).

[0262] In some embodiments of any of the above aspects, the disease signature ligand comprises VEGF.

[0263] In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 comprises a polypeptide that specifically binds the disease signature ligand.

[0264] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[0265] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0266] In some embodiments, the polypeptide comprises an endogenous binding domain. In some embodiments, the endogenous binding domain comprises a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligandbinding domain, or a DNA-binding domain. In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain.

[0267] In some embodiments, the polypeptide comprises a vascular endothelial growth factor receptor (VEGF-R) polypeptide or fragment or variant thereof. PATENT

[0268] Attorney Docket No. 51661 -008W06

[0269] Flagship Reference: VL75014-W1

[0270] In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 has affinity to two or more disease signature moieties.

[0271] In some embodiments of any of the above aspects, the effector ligand comprises 4-1 BB.

[0272] In some embodiments, the SBD comprises an agonist of the effector ligand. In some embodiments, the SBD is an agonist of the effector ligand. In some embodiments, the effector ligand is homodimerized to exert a cellular effector function. In some embodiments, the effector ligand is homodimerized in the presence of the macromolecule complex or macromolecule and the disease signature ligand. In some embodiments, the effector ligand is homotrimerized to exert a cellular effector function. In some embodiments, the effector ligand is homotrimerized in the presence of the macromolecule complex or macromolecule and the disease signature ligand.

[0273] In some embodiments, the first effector ligand and the second effector ligand must be associated to exert a cellular effector function. In some embodiments, the first effector ligand and the second effector ligand are associated in the presence of the macromolecule complex or macromolecule and the disease signature ligand. In some embodiments, the association is heterodimerization.

[0274] In some embodiments of any of the above aspects, the cellular effector function comprises a biological activity. In some embodiments, the cellular effector function comprises a therapeutic activity. In some embodiments, the cellular effector function comprises a disease activity. In some embodiments of any of the above aspects, the cellular effector function is a biological activity. In some embodiments, the cellular effector function is a therapeutic activity. In some embodiments, the cellular effector function is a disease activity.

[0275] In some embodiments of any of the above aspects, the SBD, SBD1 , or SBD2 comprises a polypeptide that specifically binds the effector ligand.

[0276] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises an scFv, a monospecific taFv, a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a DART. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific taFv, a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a DART.

[0277] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the antibody mimetic comprises an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a FN3-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a FN3-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0278] In some embodiments, the polypeptide comprises an endogenous binding domain. In some embodiments, the endogenous binding domain comprises a ligand of the effector ligand or a fragment PATENT

[0279] Attorney Docket No. 51661 -008W06

[0280] Flagship Reference: VL75014-W1 thereof. In some embodiments, the endogenous binding domain comprises a viral binding protein or a fragment thereof. In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[0281] In some embodiments, the SBD1 comprises a first portion of a binding moiety and the SBD2 comprises a second portion of the binding moiety. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first portion of a binding moiety and the SBD2 is a second portion of the binding moiety.

[0282] In some embodiments, the SBD1 specifically binds to a first component of a heterodimeric receptor and the SBD2 specifically binds to a second component of the heterodimeric receptor.

[0283] In some embodiments, the SBD1 comprises a first component of a dimeric moiety and the SBD2 comprises a second component of a dimeric moiety. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first component of a dimeric moiety and the SBD2 is a second component of a dimeric moiety.

[0284] In some embodiments, the SBD1 comprises a first fragment of a polypeptide chain and the SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain comprises a hormone, a cytokine, or a growth factor. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first fragment of a polypeptide chain and the SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, a cytokine, or a growth factor.

[0285] In some embodiments, the SBD1 and the SBD2 have been engineered to have reduced affinity for one another.

[0286] In some embodiments of any of the above aspects, the macromolecule comprises a reporter moiety. In some embodiments, the reporter moiety comprises an affinity tag, a fluorescent marker, a radioactive marker, or a chromogenic marker. In some embodiments, the affinity tag is a FLAG affinity tag or the chromogenic marker is luciferase or beta-lactamase.

[0287] In some embodiments of any of the above aspects, the macromolecule complex or macromolecule comprises one or more linker domains. In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linkers comprise one or more GS linkers. In some embodiments, the GS linkers comprise one or more GS(GnS)m linkers or one or more (GnS)m linkers. In some embodiments, the GS linkers comprise one or more (G4S)m linkers. In some embodiments, the peptide linkers comprise one or more GT linkers. In some embodiments, the GT linkers comprise one or more GT(GnT)mlinkers or one or more (GnT)mlinkers. In some embodiments, the GT linkers comprise one or more (G4T)mlinkers. In some embodiments, n equals four. In some embodiments, m equals one, two, three, or four.

[0288] In some embodiments of any of the above aspects, the macromolecule comprises a polypeptide.

[0289] In some embodiments of any of the above aspects, the macromolecule is a polypeptide.

[0290] In some embodiments of any of the above aspects, the biological sample comprises an extract, fluid, fraction, cell, tissue, or subject. In some embodiments of any of the above aspects, the biological sample is an extract, fluid, fraction, cell, tissue, or subject. PATENT

[0291] Attorney Docket No. 51661 -008W06

[0292] Flagship Reference: VL75014-W1

[0293] In some embodiments of any of the above aspects, the macromolecule or one or both members of pair of macromolecules comprises a leader sequence. In some embodiments, the leader sequence comprises a secretion signal.

[0294] In some embodiments of any of the above aspects, the macromolecule complex or one or both macromolecules of the macromolecule complex comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0295] In another aspect, provided herein is a nucleic acid encoding the macromolecule of any one of the aspects.

[0296] In another aspect, provided herein is a pair of nucleic acids encoding the pair of macromolecules of an aspect.

[0297] In some embodiments, the nucleic acid is an RNA or a DNA.

[0298] In some embodiments, the nucleic acid is formulated with a delivery platform.

[0299] In some embodiments, the delivery platform is a lipid-based carrier or a vector delivery system. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP). In some embodiments, the vector delivery system comprises or is derived from an adenovirus, an anellovirus, an AAV, or a lentivirus.

[0300] In another aspect, provided herein is a nucleic acid encoding a macromolecule according to any aspect, wherein the nucleic acid is formulated with a carrier.

[0301] In another aspect, provided herein is a pair of nucleic acids encoding a pair of macromolecules according to an aspect, wherein the pair of nucleic acids is formulated with a carrier.

[0302] In some embodiments, the nucleic acid is an RNA or a DNA.

[0303] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a LNP.

[0304] In another aspect, the disclosure provides a vector comprising the nucleic acid of any of the above aspects.

[0305] In another aspect, the disclosure provides a vector or pair of vectors comprising the pair of nucleic acids of any of the above aspects.

[0306] In some embodiments, the vector or pair of vectors is formulated with a carrier.

[0307] In another aspect, the disclosure provides a host cell comprising the nucleic acid, pair of nucleic acids, vector or pair of vectors of any one of the above aspects.

[0308] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure. In some embodiments, the macromolecule complex or macromolecule is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure, e.g., as determined by the percentage thereof in the peak of interest (POI), e.g., as described herein. In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure after one purification step, e.g., purification using 1 -step Protein A (such as MabSelect PRISMA™) affinity chromatography. In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure after two PATENT

[0309] Attorney Docket No. 51661 -008W06

[0310] Flagship Reference: VL75014-W1 purification steps, e.g., purification using Protein A affinity chromatography followed by polishing using cation exchange chromatography.

[0311] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is manufactured according to the U.S. Food and Drug Administration (FDA)’s Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0312] In some embodiments, the macromolecule complex or the macromolecule has a melting temperature (Tm) above about 50 degrees Celsius (C), above about 55 degrees C, above about 60 degrees C, above about 65 degrees C, wherein Tm is determined as described herein, e.g., using nano- DSF (differential scanning fluorimetry), followed by quantifying spectral shift via the barycentric mean (BCM) calculation, wherein the BCM data was calculated as a function of temperature and the first derivative (dBCM) was determined from the BCM melt curve, as detailed herein.

[0313] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4- 1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF. See, e.g., the drawings and examples herein.

[0314] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0315] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4- 1 BB; wherein the two macromolecules are not conjugated to each other in the absence of VEGF; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding VEGF. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of VEGF, and the non-covalent conjugation is mediated by VEGF. See, e.g., the drawings and examples herein.

[0316] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0317] In some embodiments, the VEGF binding domain is an anti-VEGF scFv. In some embodiments, the VEGF binding domain is a VEGF receptor TRAP (also referred to as a “VEGF TRAP”). In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0318] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0319] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab. PATENT

[0320] Attorney Docket No. 51661 -008W06

[0321] Flagship Reference: VL75014-W1

[0322] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-TRAP. In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0323] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV.

[0324] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-TRAP. In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0325] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-scFV. In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 38.

[0326] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 38.

[0327] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 39.

[0328] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 39.

[0329] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 38.

[0330] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 38.

[0331] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 39.

[0332] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 39.

[0333] In some embodiments, the macromolecule comprises SEQ ID NO: 40 and SEQ ID NO: 45.

[0334] In some embodiments, the macromolecule comprises SEQ ID NO: 41 and SEQ ID NO: 45.

[0335] In some embodiments, the macromolecule comprises SEQ ID NO: 53 and SEQ ID NO: 45.

[0336] In some embodiments, the macromolecule comprises SEQ ID NO: 54 and SEQ ID NO: 45.

[0337] In some embodiments, the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[0338] In some embodiments, the macromolecule comprises SEQ ID NO: 63 and SEQ ID NO: 45.

[0339] In some embodiments, the macromolecule comprises SEQ ID NO: 64 and SEQ ID NO: 45.

[0340] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0341] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0342] In another aspect, the disclosure provides a macromolecule comprising a VEGF binding domain linked to a 4-1 BB binding domain. See, e.g., the drawings and examples herein.

[0343] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0344] In some embodiments, the VEGF binding domain is an anti-VEGF scFv. In some embodiments, the VEGF binding domain is a VEGF receptor

[0345] TRAP (also referred to as a “VEGF TRAP”). In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0346] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv. PATENT

[0347] Attorney Docket No. 51661 -008W06

[0348] Flagship Reference: VL75014-W1

[0349] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab.

[0350] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-TRAP. In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0351] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV.

[0352] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-TRAP. In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP.

[0353] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-scFV.

[0354] In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 38.

[0355] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 38.

[0356] In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 39.

[0357] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 39.

[0358] In some embodiments, the macromolecule comprises SEQ ID NO: 40 and SEQ ID NO: 45.

[0359] In some embodiments, the macromolecule comprises SEQ ID NO: 41 and SEQ ID NO: 45.

[0360] In some embodiments, the macromolecule comprises SEQ ID NO: 53 and SEQ ID NO: 45.

[0361] In some embodiments, the macromolecule comprises SEQ ID NO: 54 and SEQ ID NO: 45.

[0362] In some embodiments, the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[0363] In some embodiments, the macromolecule comprises SEQ ID NO: 63 and SEQ ID NO: 45.

[0364] In some embodiments, the macromolecule comprises SEQ ID NO: 64 and SEQ ID NO: 45.

[0365] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0366] In some aspects, the disclosure provides a composition, wherein the composition comprises a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein.

[0367] In some embodiments, the composition comprises a macromolecule complex described herein.

[0368] In some embodiments, the composition comprises a macromolecule described herein.

[0369] In some aspects, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein and a pharmaceutically acceptable excipient.

[0370] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient.

[0371] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0372] In some aspects, the disclosure provides method for preparing a pharmaceutical composition, the method comprising: combining a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein with a pharmaceutically acceptable excipient, thereby preparing the pharmaceutical composition.

[0373] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient. PATENT

[0374] Attorney Docket No. 51661 -008W06

[0375] Flagship Reference: VL75014-W1

[0376] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0377] In another aspect, the disclosure provides a method comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to a cell.

[0378] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0379] In some embodiments, no significant dermatitis is observed in the subject.

[0380] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for providing access to a cell.

[0381] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0382] In some embodiments, no significant dermatitis is observed in the subject.

[0383] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in providing access to a cell.

[0384] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0385] In some embodiments, no significant dermatitis is observed in the subject.

[0386] In another aspect, the disclosure provides a method of modulating the state of a cell, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby modulating the state of the cell.

[0387] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for modulating the state of a cell.

[0388] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in modulating the state of a cell.

[0389] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby inducing the cellular effector function in the cell.

[0390] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0391] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in inducing a cellular effector function in a cell. PATENT

[0392] Attorney Docket No. 51661 -008W06

[0393] Flagship Reference: VL75014-W1

[0394] In some embodiments, the cell is in a subject and the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition is administered in a therapeutically effective amount.

[0395] In some embodiments, the subject has, or is suspected of having, a disease or disorder characterized by abnormal levels of the disease signature target, optionally wherein the subject was previously determined to have abnormal levels of the disease signature target.

[0396] In another aspect, the disclosure provides a method of determining the state of a cell, comprising providing the macromolecule complex, or macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0397] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for determining the state of a cell.

[0398] In another aspect, the disclosure provides a method of treating a cancer in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby treating the cancer.

[0399] In some embodiments, the method comprises administering an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0400] In some embodiments, the cancer comprises a solid tumor.

[0401] In some embodiments, the cancer is resistant to PD-1 blockade.

[0402] In some embodiments, the cancer comprises a colorectal cancer or a melanoma.

[0403] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0404] In some embodiments, hepatomegaly is not induced in the subject.

[0405] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0406] In some embodiments, no significant dermatitis is observed in the subject. In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a cancer in a subject.

[0407] In some embodiments, the use comprises administration of an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0408] In some embodiments, the cancer comprises a solid tumor.

[0409] In some embodiments, the cancer is resistant to PD-1 blockade.

[0410] In some embodiments, the cancer comprises a colorectal cancer or a melanoma.

[0411] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0412] In some embodiments, hepatomegaly is not induced in the subject.

[0413] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration). PATENT

[0414] Attorney Docket No. 51661 -008W06

[0415] Flagship Reference: VL75014-W1

[0416] In some embodiments, no significant dermatitis is observed in the subject.

[0417] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in treating a cancer in a subject.

[0418] In some embodiments, the use comprises administration of an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0419] In some embodiments, the cancer comprises a solid tumor.

[0420] In some embodiments, the cancer is resistant to PD-1 blockade.

[0421] In some embodiments, the cancer comprises a colorectal cancer or a melanoma.

[0422] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0423] In some embodiments, hepatomegaly is not induced in the subject.

[0424] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0425] In some embodiments, no significant dermatitis is observed in the subject.

[0426] In another aspect, the disclosure provides a method of suppressing tumor growth in a subject (e.g., a subject with a tumor), comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby suppressing tumor growth in the subject.

[0427] In some embodiments, the method comprises administering an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0428] In some embodiments, the tumor is resistant to PD-1 blockade.

[0429] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0430] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0431] In some embodiments, hepatomegaly is not induced in the subject.

[0432] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0433] In some embodiments, no significant dermatitis is observed in the subject.

[0434] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for suppressing tumor growth in a subject (e.g., a subject with a tumor).

[0435] In some embodiments, the use comprises administration of an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0436] In some embodiments, the tumor is resistant to PD-1 blockade.

[0437] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0438] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0439] In some embodiments, hepatomegaly is not induced in the subject. PATENT

[0440] Attorney Docket No. 51661 -008W06

[0441] Flagship Reference: VL75014-W1

[0442] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0443] In some embodiments, no significant dermatitis is observed in the subject.

[0444] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in suppressing tumor growth in a subject (e.g., a subject with a tumor).

[0445] In some embodiments, the use comprises administration of an additional therapy to the subject. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[0446] In some embodiments, the tumor is resistant to PD-1 blockade.

[0447] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0448] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0449] In some embodiments, hepatomegaly is not induced in the subject.

[0450] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0451] In some embodiments, no significant dermatitis is observed in the subject.

[0452] In another aspect, the disclosure provides a method of increasing tumor-infiltrating lymphocytes (TILs) and / or immune cell density in a subject (e.g., a subject with a tumor), comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing TILs and / or immune cell density in the subject.

[0453] In some embodiments, the tumor is resistant to PD-1 blockade.

[0454] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0455] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0456] In some embodiments, hepatomegaly is not induced in the subject.

[0457] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0458] In some embodiments, no significant dermatitis is observed in the subject.

[0459] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for increasing TILs and / or immune cell density in a subject (e.g., a subject with a tumor).

[0460] In some embodiments, the tumor is resistant to PD-1 blockade.

[0461] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0462] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0463] In some embodiments, hepatomegaly is not induced in the subject.

[0464] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0465] In some embodiments, no significant dermatitis is observed in the subject. PATENT

[0466] Attorney Docket No. 51661 -008W06

[0467] Flagship Reference: VL75014-W1

[0468] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in increasing TILs and / or immune cell density in a subject (e.g., a subject with a tumor).

[0469] In some embodiments, the tumor is resistant to PD-1 blockade.

[0470] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0471] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0472] In some embodiments, hepatomegaly is not induced in the subject.

[0473] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0474] In some embodiments, no significant dermatitis is observed in the subject.

[0475] In another aspect, the disclosure provides a method of promoting monocyte differentiation into M1 and H7Rh'9hM1 macrophages in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby promoting monocyte differentiation into M1 and H7Rh'9hM1 macrophages in the subject.

[0476] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for promoting monocyte differentiation into M1 and H7Rh'9hM1 macrophages in a subject.

[0477] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in promoting monocyte differentiation into M1 and H7Rh'9hM1 macrophages in a subject.

[0478] In another aspect, the disclosure provides a method of enhancing innate immune cell-mediated anti-tumor immunity in a subject (e.g., a subject with a tumor), comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby enhancing innate immune cell-mediated antitumor immunity in the subject.

[0479] In some embodiments, the tumor is resistant to PD-1 blockade.

[0480] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0481] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0482] In some embodiments, hepatomegaly is not induced in the subject.

[0483] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0484] In some embodiments, no significant dermatitis is observed in the subject.

[0485] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for enhancing innate immune cell- mediated anti-tumor immunity in a subject (e.g., a subject with a tumor).

[0486] In some embodiments, the tumor is resistant to PD-1 blockade.

[0487] In some embodiments, the tumor comprises a colorectal cancer or a melanoma. PATENT

[0488] Attorney Docket No. 51661 -008W06

[0489] Flagship Reference: VL75014-W1

[0490] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0491] In some embodiments, hepatomegaly is not induced in the subject.

[0492] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0493] In some embodiments, no significant dermatitis is observed in the subject.

[0494] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in enhancing innate immune cell-mediated anti-tumor immunity in a subject (e.g., a subject with a tumor).

[0495] In some embodiments, the tumor is resistant to PD-1 blockade.

[0496] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0497] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0498] In some embodiments, hepatomegaly is not induced in the subject.

[0499] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0500] In some embodiments, no significant dermatitis is observed in the subject.

[0501] In another aspect, the disclosure provides a method of reducing red blood cell and hemoglobin accumulation within a tumor microenvironment (TME) in a subject (e.g., a subject with a tumor), comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby reducing red blood cell and hemoglobin accumulation within the TME in the subject.

[0502] In some embodiments, the tumor is resistant to PD-1 blockade.

[0503] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0504] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0505] In some embodiments, hepatomegaly is not induced in the subject.

[0506] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0507] In some embodiments, no significant dermatitis is observed in the subject.

[0508] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing red blood cell and hemoglobin accumulation within a tumor microenvironment in a subject (e.g., a subject with a tumor).

[0509] In some embodiments, the tumor is resistant to PD-1 blockade.

[0510] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0511] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0512] In some embodiments, hepatomegaly is not induced in the subject.

[0513] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0514] In some embodiments, no significant dermatitis is observed in the subject. PATENT

[0515] Attorney Docket No. 51661 -008W06

[0516] Flagship Reference: VL75014-W1

[0517] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in reducing red blood cell and hemoglobin accumulation within a tumor microenvironment in a subject (e.g., a subject with a tumor).

[0518] In some embodiments, the tumor is resistant to PD-1 blockade.

[0519] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0520] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0521] In some embodiments, hepatomegaly is not induced in the subject.

[0522] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0523] In some embodiments, no significant dermatitis is observed in the subject.

[0524] In another aspect, the disclosure provides a method of increasing glycolysis in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing glycolysis in the subject.

[0525] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for increasing glycolysis in a subject.

[0526] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in increasing glycolysis in a subject.

[0527] In another aspect, the disclosure provides a method of reducing LDHA expression in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby reducing LDHA expression in the subject.

[0528] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing LDHA expression in a subject.

[0529] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in reducing LDHA expression in a subject.

[0530] In another aspect, the disclosure provides a method of elevating MPC1 / MPC2 levels in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby elevating MPC1 / MPC2 levels in the subject.

[0531] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for elevating MPC1 / MPC2 levels in a subject. PATENT

[0532] Attorney Docket No. 51661 -008W06

[0533] Flagship Reference: VL75014-W1

[0534] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in elevating MPC1 / MPC2 levels in a subject.

[0535] In another aspect, the disclosure provides a method of supporting expansion of progenitor- exhausted CD8+T cells (CD8+PD1 +TIM3 , Pex) in a tumor microenvironment (TME) in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby supporting expansion of progenitor-exhausted CD8+T cells (CD8+PD1 +TIM3-, Pex) in the TME in the subject.

[0536] In some embodiments, the tumor is resistant to PD-1 blockade.

[0537] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0538] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0539] In some embodiments, hepatomegaly is not induced in the subject.

[0540] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0541] In some embodiments, no significant dermatitis is observed in the subject.

[0542] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for supporting expansion of progenitor- exhausted CD8+T cells (CD8+PD1+TIM3‘, Pex) in a tumor microenvironment (TME) in a subject.

[0543] In some embodiments, the tumor is resistant to PD-1 blockade.

[0544] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0545] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0546] In some embodiments, hepatomegaly is not induced in the subject.

[0547] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0548] In some embodiments, no significant dermatitis is observed in the subject.

[0549] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in supporting expansion of progenitor-exhausted CD8+T cells (CD8+PD1+TIM3‘, Pex) in a tumor microenvironment (TME) in a subject.

[0550] In some embodiments, the tumor is resistant to PD-1 blockade.

[0551] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0552] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0553] In some embodiments, hepatomegaly is not induced in the subject.

[0554] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0555] In some embodiments, no significant dermatitis is observed in the subject.

[0556] In another aspect, the disclosure provides a method of increasing the fraction of progenitor exhausted T cells (e.g., in a tumor) in a subject, comprising providing the macromolecule complex, PATENT

[0557] Attorney Docket No. 51661 -008W06

[0558] Flagship Reference: VL75014-W1 macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing the fraction of progenitor exhausted T cells in the subject.

[0559] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for increasing the fraction of progenitor exhausted T cells (e.g., in a tumor) in a subject.

[0560] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in increasing the fraction of progenitor exhausted T cells (e.g., in a tumor) in a subject.

[0561] In another aspect, the disclosure provides a method of reducing the proportion of terminally exhausted T cells (e.g., in a tumor) in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby reducing the proportion of terminally exhausted T cells in the subject.

[0562] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing the proportion of terminally exhausted T cells (e.g., in a tumor) in a subject.

[0563] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in reducing the proportion of terminally exhausted T cells (e.g., in a tumor) in a subject.

[0564] In another aspect, the disclosure provides a method of increasing long-term immunity in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing long-term immunity in the subject.

[0565] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for increasing long-term immunity in a subject.

[0566] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in increasing long-term immunity in a subject.

[0567] In another aspect, the disclosure provides a method of promoting an expanded fraction of CD8+ T cells within a tumor in a subject, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby promoting an expanded fraction of CD8+ T cells within the tumor.

[0568] In some embodiments, the tumor is resistant to PD-1 blockade.

[0569] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0570] In some embodiments, no significant hepatotoxicity is observed in the subject. PATENT

[0571] Attorney Docket No. 51661 -008W06

[0572] Flagship Reference: VL75014-W1

[0573] In some embodiments, hepatomegaly is not induced in the subject.

[0574] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0575] In some embodiments, no significant dermatitis is observed in the subject.

[0576] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for promoting an expanded fraction of CD8+ T cells within the tumor.

[0577] In some embodiments, the tumor is resistant to PD-1 blockade.

[0578] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0579] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0580] In some embodiments, hepatomegaly is not induced in the subject.

[0581] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0582] In some embodiments, no significant dermatitis is observed in the subject.

[0583] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in increasing long-term immunity within a tumor.

[0584] In some embodiments, the tumor is resistant to PD-1 blockade.

[0585] In some embodiments, the tumor comprises a colorectal cancer or a melanoma.

[0586] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0587] In some embodiments, hepatomegaly is not induced in the subject.

[0588] In some embodiments, CD8 T cell inflammation is not induced in the subject (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration).

[0589] In some embodiments, no significant dermatitis is observed in the subject.

[0590] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in determining the state of a cell.

[0591] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand.

[0592] In another aspect, the disclosure provides the use of a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a PATENT

[0593] Attorney Docket No. 51661 -008W06

[0594] Flagship Reference: VL75014-W1 cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0595] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0596] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0597] In another aspect, the disclosure provides the use of a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0598] In another aspect, the disclosure provides a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds PATENT

[0599] Attorney Docket No. 51661 -008W06

[0600] Flagship Reference: VL75014-W1 a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0601] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0602] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0603] In another aspect, the disclosure provides a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0604] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows PATENT

[0605] Attorney Docket No. 51661 -008W06

[0606] Flagship Reference: VL75014-W1 binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0607] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0608] In another aspect, the disclosure provides a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0609] Other features and advantages of the invention will be apparent from the following Detailed Description and the Claims.

[0610] Definitions

[0611] As used herein, the term “macromolecule” refers to a large molecule (e.g., a molecule with a size greater than 1000 Daltons (1 kDa)) comprising one or more polypeptide, oligonucleotide, chemical, lipid, and / or carbohydrate moieties. In some embodiments, the macromolecule is a recombinant protein (e.g., a fusion protein).

[0612] As used herein, the term “multimer” refers to a molecule made up of at least two subunits (e.g., at least two subunits comprising one or more polypeptide, oligonucleotide, chemical, lipid, and / or carbohydrate moieties). Multimers include homomultimers and heteromultimers. A “homomultimer” is a multimer consisting of two or more identical or substantially identical subunits (e.g., two or more macromolecules). Homomultimers include homodimers (comprising two identical or substantially identical subunits), homotrimers (comprising three identical or substantially identical subunits), and homotetramers (comprising four identical or substantially identical subunits). “Substantially identical subunits” include subunits having differences in amino acid sequences that do not significantly affect the PATENT

[0613] Attorney Docket No. 51661 -008W06

[0614] Flagship Reference: VL75014-W1 function of the subunit, e.g., that do not significantly affect the affinity of the subunit for one or more ligands. A “heteromultimer” is a multimer consisting of two or more non-identical subunits.

[0615] Heteromultimers include heterodimers (comprising a first and a second subunit, e.g., comprising a pair of non-identical macromolecules), heterotrimers (comprising one copy of a first subunit and two copies of a second subunit), and homotetramers (comprising two copies or versions of each of a first and a second subunit). Multimers further include higher-order multimers, e.g., hexamers, heptamers, octamers, nonamers, and decamers.

[0616] As used herein, the term “binding domain” refers to any domain that has specific affinity for a ligand. Binding domains include, without limitation, polypeptides (e.g., an antibody or a fragment thereof (e.g., an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART)), an antibody mimetic (e.g., an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR)), an endogenous binding domain or a variant or derivative thereof (e.g., a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, a DNA-binding domain, or a receptor trap))), oligonucleotides (e.g., a nucleic acid aptamer (e.g., a DNA aptamer)), and chemical molecules, as well as combinations thereof.

[0617] As used herein, the term “ligand” refers to any moiety for which a binding domain as described herein may have affinity. Ligands include, without limitation, a chemical moiety, a portion of a molecule, a molecule (e.g., an allergen or a toxin), a macromolecule (e.g., a polypeptide, a nucleic acid, or carbohydrate), a post-translational modification state of a macromolecule (e.g., a macromolecule that is phosphorylated, glycosylated, acylated, alkylated, and the like), a higher-order macromolecular structure (e.g., a complex of two or more polypeptides), a cell (e.g., a cancer cell), a portion of a cell (e.g., a tumor antigen), a receptor on the surface of a cell, a pathogen (e.g., a virus or a portion or a virus; a bacterium or a portion of a bacterium; a fungus or a portion of a fungus; or a parasite or a portion of a parasite), or a tissue-type.

[0618] The term “disease signature ligand” refers to a ligand that is associated with a disease state or a disorder of a cell, tissue, or subject (e.g., mammal, e.g., human). Disease signature ligands may be proteins, e.g., soluble proteins, insoluble proteins, monomeric proteins, and multimeric proteins. Disease signature ligands include, without limitation, cell surface receptors, cell surface antigens, membranebound proteins, extracellular matrix components, integrins, cytokines, neurotransmitters, anti-drug antibodies (ADAs), autoantibodies, nucleic acids, carbohydrates, lipids, peptides, nucleosides, hormones, viruses, bacteria, fungi, or a fragment or antigen thereof.

[0619] The term “effector ligand” refers to a ligand that is capable of effecting a cellular effector function upon being bound by a binding domain, e.g., a multimer of the invention. Disease signature ligands include proteins and peptides, e.g., cell-surface receptors (e.g., catalytic receptors, e.g., a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 PATENT

[0620] Attorney Docket No. 51661 -008W06

[0621] Flagship Reference: VL75014-W1 cytokine receptor, a tumor necrosis factor (TNF) superfamily receptor (e.g., TNFR2 or 4-1 BB), or a nuclear hormone receptor).

[0622] As used herein, the term “associated with” a disease, disorder, or condition refers to a relationship, either causative or correlative, between an entity and the occurrence or severity of a disease, disorder, or condition in a subject. For example, if a target is associated with a disease, disorder, or condition, the target may be the causative agent of the disease, disorder, or condition. For example, a virus may be the causative agent in a viral infection, bacteria may be the causative agent in a bacterial infection, a fungus may be the causative agent in a fungal infection, a parasite may be the causative agent in a parasitic infection, a cancer cell may be the causative agent of a cancer, a toxin may be the causative agent of toxicity, or an allergen may be the causative agent of an allergic reaction. The target associated with a disease, disorder, or condition may also or alternately be correlated with an increased likelihood of occurrence or an increased severity of a disease disorder, or condition.

[0623] As used herein, the term “carrier” means a compound, composition, reagent, or molecule that facilitates the stability, transport or delivery of a composition (e.g., a macromolecule or pair or macromolecules as described herein) into a subject, a tissue, or a cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular or linear polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the polyribonucleotide), and cationic carriers (e.g., a cationic lipopolymer or transfection reagent).

[0624] As used herein, the terms “disease,” “disorder,” and “condition” each refer to a state of sub- optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professional.

[0625] The term “pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids for medicinal or therapeutic use.

[0626] The term “polynucleotide” as used herein means a molecule comprising one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide”. A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five- carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic acids, or RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. The polynucleotides provided herein may include one or more modified nucleotides.

[0627] Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, means macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide means a PATENT

[0628] Attorney Docket No. 51661 -008W06 Flagship Reference: VL75014-W1 deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, and may include detectable tags, such as luminescent tags or markers (e.g., fluorophores). One or more of the nucleotides may be a modified nucleotide. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e. , C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as singlestranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, a polynucleotide molecule is circular (e.g., a circular RNA). A polynucleotide can have various lengths. A nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. As embodied herein, the polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0629] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural, including D, L, or a combination thereof) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or may be a multi- molecular complex such as a dimer, trimer, or tetramer. They can also comprise single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0630] As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) 18 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be PATENT

[0631] Attorney Docket No. 51661 -008W06

[0632] Flagship Reference: VL75014-W1 determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively or additionally, percent identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.

[0633] A “signal sequence” or “leader sequence” refers to a polypeptide sequence, e.g., between 10 and 30 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein and targets the polypeptide sequence to the secretory pathway.

[0634] As used herein, the term “specifically binds” refers to a preferential interaction between a binding domain and its target or ligand (such as binding between an antibody and an antigen or epitope) that may be determinative of the presence of the target or ligand in the presence of a heterogeneous population of molecules including biological molecules. For example, a binding domain that specifically binds to a ligand (e.g., an antibody that specifically binds to an antigen or epitope) may be a binding domain that binds this ligand with greater affinity, avidity, more readily, and / or with greater duration than it binds to other ligands. In one aspect, the extent of binding of a binding domain to an unrelated molecule (nonligand) is less than about 10% of the binding of the binding domain to the ligand as measured using an appropriate assay. In certain aspects, a binding domain that specifically binds to a ligand has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. Specific binding can include, but does not require exclusive binding. In one aspect, “specific binding” refers to binding wherein a binding domain binds to a particular ligand (e.g., a polypeptide or antigen or epitope on a particular polypeptide) without substantially binding to any other ligand (e.g., polypeptide or polypeptide antigen or epitope).

[0635] The term “cancer,” as used herein, refers to a disease caused by an uncontrolled division of abnormal cells in a part of the body. The cancer may comprise a solid tumor. In one instance, the cancer is a colorectal cancer (CRC). CRC includes cancers of the colon and / or rectum, e.g., adenocarcinoma of the colon or rectum. In another instance, the cancer is a melanoma. In another instance, the cancer is a lung cancer. Lung cancer includes, e.g., small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The cancer (e.g., CRC, lung cancer, or melanoma) may be locally advanced or metastatic, e.g., may be a Stage I, Stage II, Stage III, or Stage IV cancer.

[0636] As used herein, the term “treat,” or “treating” refers to a therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic reaction) in a subject. The effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e. , not worsening) the state of the disease or disorder, and / or preventing the spread of the disease or disorder as compared to the state and / or the condition of the disease or disorder in the absence of the therapeutic treatment.

[0637] BRIEF DESCRIPTION OF THE DRAWINGS

[0638] Fig. 1 A is a schematic diagram showing the domain structure of a fusion protein that modulates 4-1 BB activity conditional on the presence of VEGF (VEGF binder / 4-1 BB binder fusion protein PATENT

[0639] Attorney Docket No. 51661 -008W06

[0640] Flagship Reference: VL75014-W1 constructs), in which from N- to C-terminus, the fusion protein includes a leader polypeptide; an anti-4- 1 BB scFv; a (G4S)n linker; an anti-IL-8 scFv; a VEGF binder; and an Fc region.

[0641] Fig. 1 B is a schematic diagram showing the fusion proteins of Fig. 13A in unbound format and bound to VEGF and to a 4-1 BB receptor on the surface of a T cell. Left: monovalent construct in which the fusion protein comprises a monomeric Fc region. Right: bivalent construct in which the fusion protein comprises an Fc region capable of dimerization.

[0642] Fig. 2A is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF binder 14-1 BB binder fusion protein construct (“fusion protein construct”) comprising a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a monovalent Fc region alone or in the presence of human VEGF-A, mouse VEGF-A, or mouse VEGF-B. A urelumab reference monoclonal antibody (mAb) is shown as a control.

[0643] Fig. 2B is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF binder 14-1 BB binder fusion protein construct comprising a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a divalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A urelumab reference monoclonal antibody (mAb) is shown as a control.

[0644] Fig. 3A is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF binder 14-1 BB binder fusion protein construct comprising a utomilumab-based effector domain, a VEGF binding domain (anti-VEGF scFv), and a monovalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A utomilumab reference mAb is shown as a control.

[0645] Fig. 3B is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF binder 14-1 BB binder fusion protein construct comprising a utomilumab-based effector domain, a VEGF binding domain (receptor trap; “VEGF-trap"), and a divalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A utomilumab reference mAb is shown as a control.

[0646] Fig. 3C is a set of graphs showing fold induction of luminescence in Jurkat T cells that express 4- 1 BB and a NFkB-driven luciferase reporter gene that were treated with constructs comprising an anti-4- 1 BB scFv-based effector domain and a VEGF trap domain comprising the indicated amino acid substitutions (SEQ ID NOs: 55, 56, and 61).

[0647] Fig. 3D is a set of graphs showing fold induction of luminescence in Jurkat T cells that express 4- 1 BB and a NFkB-driven luciferase reporter gene that were treated with constructs comprising an anti-4- 1 BB scFv-based effector domain and a VEGF trap domain comprising the indicated amino acid substitutions (SEQ ID NOs: 50, 58, and 59).

[0648] Fig. 3E is a set of graphs showing fold induction of luminescence in Jurkat T cells that express 4- 1 BB and a NFkB-driven luciferase reporter gene that were treated with constructs comprising an anti-4- 1 BB scFv-based effector domain and a VEGF trap domain comprising the indicated amino acid substitutions (SEQ ID NO: 58). PATENT

[0649] Attorney Docket No. 51661 -008W06

[0650] Flagship Reference: VL75014-W1

[0651] Fig. 4A is a schematic diagram showing the design of a lymphopenia-induced proliferation study designed to assess T cell expansion in vivo following intravenous (IV) administration of a VEGF binder I 4-1 BB binder fusion protein construct (“4-1 BB SB”) in the presence or absence of VEGF. CFSE: carboxyfluorescein succinimidyl ester.

[0652] Fig. 4B is a plot showing the percentage of T cells in each of the indicated treatment groups (see Table 4) that were undergoing fast lymphopenia-induced proliferation (LIP) at Day 7, as measured by dilution of CFSE dye.

[0653] Fig. 4C is a plot showing the percentage of T cells derived from the donor population that were effector T cells following treatment in each of the indicated treatment groups (see Table 4).

[0654] Fig. 4D is a plot showing the percentage of donor-derived T cells that were naive T cells in each of the indicated treatment groups (see Table 4).

[0655] Fig. 5A is a schematic diagram showing the design of a tumor efficacy study designed to assess the anti-tumor activity of the VEGF binder 1 4-1 BB binder fusion protein construct anti-4-1 BBAx VEGF- Trap (Bi) (“VEGF-4-1 bb SB”) at doses of 10, 3, 1 , and 0.5 milligrams per kilogram of body weight (MPK) in mice having MC38 tumors.

[0656] Fig. 5B is a plot showing tumor size (in mm3) over time in mice having MC38 tumors that were treated with various doses of anti-4-1 BBAx VEGF-Trap (Bi), a urelumab comparator, or a hlgG4 isotype control.

[0657] Fig. 5C is a set of stacked bar graphs showing the proportion of mice that experienced a partial response (PR) (black bar; tumor size was smaller than average tumor size in the isotype control group) or a complete response (OR) (gray bar; mouse became tumor-free after treatment) at 17 days after treatment with anti-4-1 BBAx VEGF-Trap (Bi) (“SB”), a urelumab comparator, PD1 blockade (“PD1 ”), or PD1 blockade in combination with anti-4-1 BBAx VEGF-Trap (Bi) (low dose) at the indicated doses.

[0658] Fig. 6A is a plot showing tumor size (in mm3) over time in mice that had had MC38 tumors, became tumor-free following treatment with anti-4-1 BBAx VEGF-Trap (Bi) or a urelumab comparator, and were re-challenged with a lethal dose of MC38 tumor cells as compared to treatment-naive mice.

[0659] Fig. 6B is a series of three graphs showing that treatment with a urelumab biosimilar results in hepatomegaly (increased liver weight) and increased number of infiltrating CD8+ T cells relative to the anti-4-1 BBAx VEGF (Bi) construct. Liver weight, CD8+, and CD4+T cell counts were measured 7 days post-treatment.

[0660] Fig. 7A is a pair of graphs showing the results of dimeric anti-4-1 BBAx VEGF-trap (Bi) construct purification. The top graph shows that the protein product from the first peak was 96.5% monodisperse and eluted at a size consistent with dimeric (-130 kDa) stoichiometry. The bottom graph shows the migration of gel filtration standards run to generate elution time estimates for various protein sizes. POI: Peak of interest.

[0661] Fig. 7B is an image showing dimeric stoichiometry of dimeric anti-4-1 BBAx VEGF-trap (Bi) construct confirmed by SDS-PAGE. Under non-reducing conditions (lane “NR”), the macromolecule complex runs as a dimer. Under reducing conditions (lane “R”), the macromolecule complex collapses to its constituent monomeric subunits, as expected. Estimated monomer size is ~65kDa, and dimer is -130 kDa. The molecular weight standard is in the left-hand lane. PATENT

[0662] Attorney Docket No. 51661 -008W06

[0663] Flagship Reference: VL75014-W1

[0664] Fig. 8A is a linear schematic of a first member of a construct pair encoding a VEGF binder 14- 1 BB binder fusion protein construct. The second member is shown in Fig. 8B.

[0665] Fig. 8B is a linear schematic of a second member of a construct pair encoding a VEGF binder I 4-1 BB binder fusion protein construct. The first member is shown in Fig. 8A.

[0666] Fig. 8C is a schematic diagram showing the chain composition of a bivalent VEGF binder I 4-1 BB binder fusion protein construct.

[0667] Fig. 9 is a chart showing improved developability, simplified purification methods (as shown by analytical size exclusion chromatography (aSEC) evaluation), and improved production yields (mg / L) in Generation 2 (Gen 2) molecules (comprising a Fab-based anti-4-1 BB effector domain) as compared to Generation 1 (Gen 1) molecules (which comprise an scFv-based anti-4-1 BB effector domain).

[0668] Fig. 10A is a series showing improved thermal stability of Gen 2 VEGF binder I 4-1 BB binder fusion proteins (PRO677 and PRO679) as compared to a Gen 1 scFv-based construct (PRO312), a VEGF trap benchmark (aflibercecpt), and urelumab.

[0669] Fig. 10B is a series of graphs showing that anti-4-1 BBAx VEGF (Bi) constructs bind to 4-1 BB in a manner that does not preclude binding of the native ligand 4-1 BBL; as assessed by biolayer interferometry (BLI).

[0670] Fig. 11 is a series of graphs showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with constructs comprising VEGF binder 14-1 BB binder fusion protein constructs comprising a Fab-based effector domain and a VEGF binding domain, that neutralizes both human and mouse VEGF-A.

[0671] Fig. 12 is a series of graphs showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with detuned VEGF binder 14-1 BB binder fusion protein constructs comprising the indicated amino acid substitutions in the VEGF trap domain.

[0672] Fig. 13 is a graph showing the degree of potency loss for mouse (m) and human (h) VEGF in the constructs comprising a detuned VEGF trap domain, expressed as the ratio of EGso for the mutant construct over that of the native (wild-type (WT)) construct.

[0673] Fig. 14A is a schematic showing the design of a mouse study and a tumor growth curve evaluating Gen 2 constructs comprising an anti-4-1 BB Fab-based effector domain and a VEGF trap domain in the MC38 tumor model.

[0674] Fig. 14B is a pair of graphs showing that the Gen 2 MiniTrap construct comprising an anti-4-1 BB Fab-based effector domain and a VEGF-binding domain (“PRO677”) had significantly extended pharmacokinetics (PK) in serum relative to other constructs (left panel); resulting in greater systemic VEGF target engagement, as assessed by elevation of total circulating VEGF (right panel).

[0675] Fig. 15A is a series of graphs showing the efficacy of the Gen 2 anti-4-1 BBAx VEGF MiniTrap (Bi) molecule in the B16F10 murine melanoma model, as both tumor growth curves (top panels) and survival curves (lower panels), each at three concentrations of the molecule (expressed as milligrams per kilogram of body weight (MPK)). PATENT

[0676] Attorney Docket No. 51661 -008W06

[0677] Flagship Reference: VL75014-W1

[0678] Fig. 15B is a schematic showing the design of a mouse study (top) testing Gen 2 molecules at doses of 0.5, 3, and 10 mg / kg, and graphs showing T cell counts and CD4 and CD8 T cell counts in the draining lymph node in B16F10-bearing mice treated with the molecules (bottom panels).

[0679] Fig. 16 is a schematic showing the design of a mouse study and graphs showing the efficacy of a Gen 2 anti-4-1 BBAx VEGF MiniTrap (Bi) molecule together with PD-1 antagonism as both tumor growth curves (top panels) and survival curves (lower panels), each at two concentrations of the molecule.

[0680] Fig. 17A is a schematic diagram showing the design of a study in MC38 tumor-bearing b-h4-1 BB transgenic mice designed to assess the effects of a single 10 MPK dose of hlgG4, urelumab, or a VEGF binder 14-1 BB binder fusion protein.

[0681] Fig. 17B is plot showing tumor size (in mm3) over time in mice from the study described in Fig. 17A.

[0682] Figs. 18A and 18B provide a second MC38 tumor efficacy study assessing efficacy of Gen 1 VEGF binder 14-1 BB binder construct (SEQ ID NO: 41 ), performed independently from the study described in Fig. 5A. In this study, h4-1 bb transgenic mice were inoculated with 1 million MC38 cells, and following tumor outgrowth, administered three times I.V. with the indicated amounts of Gen 1 VEGF binder 14-1 BB binder construct (AND-Body), Urelumab, Aflibercept, or hlgG4 control antibody (Fig. 18A). Fig. 18B shows a series of tumor growth curves, with each thin line representing the growth of an individual tumor, and thick lines indicating the median growth kinetics of the treatment group. The VEGF binder 14-1 BB binder fusion construct had high tumor efficacy. Compared to Urelumab and Aflibercept, the VEGF binder I 4-1 BB binder fusion construct had greater complete response rate (CRR), with 8 of 10 mice becoming tumor-free post treatment.

[0683] Figs. 19A and 19B provide a MC38 study in human 4-1 BB transgenic mice. The mice were inoculated with one million MC38 tumor cells on day 0, and following tumor outgrowth, administered I.V. with three doses of the indicated amounts of protein at 10 mg / kg (Fig. 19A). Fig. 19B is a series of graphs profiling tumor infiltrating lymphocytes from mice treated with the AND-Body comprising SEQ ID NO: 41 , Urelumab (Ure), or lgG4 isotype control. Seven days post-treatment, tumors (if still present) were collected and tumor infiltrating lymphocytes were analyzed via flow cytometry for the fraction of total CD8+ T cells, CD8+ progenitor exhausted (Pex) T cells (PD-1 + Tim-), and CD8+ terminally exhausted (Tex) T cells (PD-1 + Tim3+). Relative to Urelumab, the macromolecule comprising SEQ ID: 41 promoted an expanded fraction of CD8+ T cells within the tumor, increased fraction of progenitor exhausted T cells, and reduced proportion of terminally exhausted T cells (Fig. 19B).

[0684] Figs. 20A and 20B provide an efficacy study in a PD-1 resistant B16F10 melanoma model. The study schematic is shown in Fig. 20A, and the data here is from the same study described in Fig. 15A. Fig. 20B is a series of graphs illustrating the tumor growth trajectories of individual mice inoculated with B16F10 tumor cells, and treated with a VEGF binder 14-1 BB binder fusion construct (comprised of SEQ ID NO: 53 and SEQ ID NO: 45) (AND-Body), urelumab, anti-PD1 , or hlgG4 isotype control antibody. Individual tumor growth curves are rendered in thin lines, and the group median is shown in thick dotted lines. Overall, treatment with the VEGF binder 14-1 BB binder fusion construct was more effective at suppressing B16F10 tumor growth than treatment with either Urelumab comparator or PD-1 blockade. PATENT

[0685] Attorney Docket No. 51661 -008W06

[0686] Flagship Reference: VL75014-W1

[0687] Additionally, a higher proportion of mice (20%) had a complete response to the VEGF binder I 4-1 BB binder fusion construct as compared with Urelumab (10%) or anti-PD1 (0%).

[0688] Figs. 21 A-21D provide SPR sensograms of a VEGF binder 14-1 BB binder fusion construct (“Construct”; comprised of SEQ ID NO: 62 and SEQ ID NO: 45) binding to 4-1 BB in the presence and absence of human VEGF. (A, B). At lower 4-1 BB surface densities, the construct showed a 4-fold enhanced apparent affinity to 4-1 BB in the presence of VEGF. (C) Construct binding to 4-1 BB at higher 4- 1 BB surface density. (D) Binding parameters at different 4-1 BB concentrations (corresponding to varying 4-1 BB surface densities).

[0689] Fig. 22 is a plot showing tumor size (in mm3) over time (days after tumor inoculation) in hA549 tumor-bearing hu-NSG mice treated with the indicated amounts of the VEGF binder 14-1 BB binder fusion construct (AND-Body) or a VEGF-PD-1 bispecific antibody, or with a h IgG 1 negative control.

[0690] Figs. 23A and 23B provide an efficacy study in a A549 non-small cell lung cancer (NSCLC) cell line-derived xenograft (CDX) model. The study schematic is shown in Fig. 23A. Fig. 23B is a graph illustrating tumor size (mm3) over time (days after inoculation). Mice were treated with the indicated amounts of a VEGF binder 14-1 BB binder fusion construct (AND-Body) or a VEGF-PD-1 bispecific antibody (bsAb), or with a h IgG 1 negative control.

[0691] Fig. 24 is a graph illustrating the probability of survival over time (days after inoculation) for mice treated according to the study depicted in Fig. 23A.

[0692] Fig. 25 is a graph illustrating the clinical score over time (days after inoculation) for mice treated according to the study depicted in Fig. 23A.

[0693] Fig. 26 is a graph illustrating human interferon-y (hlFN-y) levels (pg / ml) over time (days) in mice treated according to the study depicted in Fig. 23A.

[0694] Figs. 27A and 27B are graphs illustrating serum PK and target engagement for the bivalent VEGF binder 14-1 BB binder fusion protein (AND-Body), Mut_1 , Mut_2, and bevacizumab. Serum PK (as compound concentration in nM over time in hours (h)) is shown in Fig. 27A. Target engagement (as VEGF (pM) over time in hours (h)) is shown in Fig. 27B.

[0695] Figs. 28A and 28B provide a study in MC38 tumor-bearing B-h4-1 BB mice examining later-stage tumor progression. Fig. 28A shows a treatment schematic in which MC38 tumor-bearing B-h4-1 BB mice treated with two doses of a control IgG (10 mpk), urelumab (10 mpk), or the VEGF binder I 4-1 BB binder fusion protein (AND-Body) (10 mpk) on days 14 and 18, with tumors harvested on day 25 for scRNA-seq analysis. Fig. 28B is a graph illustrating tumor size (RTV) over time (days after inoculation). Relative tumor volume (RTV): tumor size over time, expressed as the ratio of tumor volume at each time point to its initial volume on the pre-treatment day (“day 0”— in this case, Day 14).

[0696] Fig. 29 shows a UMAP of later-stage scRNA-seq analysis of CD45+ tumor-infiltrating immune cells following VEGF binder 14-1 BB binder fusion protein (AND-Body) treatment for mice treated according to the study depicted in Fig. 28A.

[0697] Fig. 30 provides stacked bar plots showing proportions of the clusters shown in Fig. 29 within CD45+ tumor-infiltrating cells across treatments for mice treated according to the study depicted in Fig. 28A. PATENT

[0698] Attorney Docket No. 51661 -008W06

[0699] Flagship Reference: VL75014-W1

[0700] Fig. 31 provides a bar graph showing tumor-microenvironment immune subset composition (% of each cell type) after urelumab or VEGF binder 14-1 BB binder fusion protein (AND-Body) treatment for mice treated according to the study depicted in Fig. 28A. Tex: terminally exhausted CD8 T cells; Pex: progenitor-exhausted CD8 T cells; NK / NKT: innate-adaptive bridging NK / NKT cells; M2: M2-like macrophages; M1 : M1 -like macrophages.

[0701] DETAILED DESCRIPTION

[0702] Featured herein are macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand, multimers thereof, compositions comprising the same, and methods of using the same.

[0703] Antibodies and Antigen Binding Portions of the Macromolecules

[0704] The term "antibody" as referred to herein includes whole antibodies and any antigen binding portion (i.e., "antigen-binding portion" or "antigen-binding fragment") or single chain thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0705] The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to a full-length antibody or one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). In some embodiments, the antibody or antigen-binding portion thereof comprises an scFv, BsIgG, a BsAb portion, a BiTE, a dual-affinity re-targeting protein (DART), a tandem diabody (TandAb), a diabody, an Fab2, a di-scFv, chemically linked F(ab’)2, an Ig molecule with 2, 3 or 4 different antigen binding sites, a DVI-IgG four-in-one, an ImmTac, an HSAbody, an IgG-IgG, a Cov-X- PATENT

[0706] Attorney Docket No. 51661 -008W06

[0707] Flagship Reference: VL75014-W1

[0708] Body, an scFv1 -PEG-scFv2, an appended IgG, an DVD-IgG, an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a monobody, a nanoCLAMP, a bis-Fab, an Fv, a Fab, a Fab’-SH, a linear antibody, an scFv, an antibody with only a heavy chain (Humabody), an ScFab, an IgG antibody portion, a single-chain variable region antibody, a single-domain heavy chain antibody, a bispecific triplebody, a BiKE, a CrossMAb, a dsDb, an scDb, tandem a dAb I VHH, a triple dAb VHH, a tetravalent dAb I VHH, a Fab-scFv, a Fab-Fv, or a DART-Fc, an adnectin, a Kunitz-type inhibitor, or a receptor decoy.

[0709] Provided herein are macromolecules that comprise antibodies or antigen binding portions thereof, e.g., that bind to human 4-1 BB (e.g., CD137), e.g., for the macromolecules provided herein. For example, the antibodies and antigen binding portions hereof bind human 4-1 BB, for example, as described in published PCT applications WO 95 / 07984 and / or WO 96 / 29348.

[0710] As used herein, "isotype" refers to the antibody class (e.g., IgM, IgE, IgG such as IgG 1 or lgG2) that is provided by the heavy chain constant region genes.

[0711] In some embodiments, the antibody or antigen binding portion thereof is an IgG isotype.

[0712] In some embodiments, the antibody or antigen binding portion thereof is an IgG 1 isotype.

[0713] In some embodiments, the antibody or antigen binding portion thereof is an lgG2 isotype.

[0714] In some embodiments, the antibody or antigen binding portion thereof is an lgG3 isotype.

[0715] In some embodiments, the antibody or antigen binding portion thereof is an lgG4 isotype.

[0716] In some embodiments, the antibody or antigen binding portion thereof comprises a LALAPG mutation.

[0717] In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain constant region 1 (CH1).

[0718] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a kappa light chain.

[0719] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a lambda light chain.

[0720] In some embodiments, the antibody or antigen binding portion thereof comprises a light chain constant region (CL).

[0721] In some embodiments, the antibody or antigen binding portion thereof binds to human CDH17. For example, the antibodies and antigen binding portions thereof bind human 4-1 BB as described in published PCT applications WO 95 / 07984 and / or WO 96 / 29348.

[0722] In some embodiments, the antibody or antigen binding portion thereof (e.g., a HC, a VH, VH+CH1 , or HC CDRs 1 , 2, or 3, a LC, a VL, VL+CL, or LC CDRs 1 , 2, or 3) has at least 90% identity to a sequence provided herein (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and e.g., binds to human 4-1 BB.

[0723] In some embodiments, the antibody or antigen binding portion thereof comprises one or more (e.g., 1 , 2, 3, 4, 5, or 6) CDR sequences provided herein (e.g., comprises all six CDRs of an anti-4-1 BB antibody provided herein) and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the antibody or antigen binding portion thereof (e.g., a HC, a VH+CH1 , a VH, a LC, a VL+CL, or a VL), and, e.g., binds to human 4-1 BB. PATENT

[0724] Attorney Docket No. 51661 -008W06

[0725] Flagship Reference: VL75014-W1

[0726] In some embodiments, the antibody or antigen binding portion thereof of the disclosure includes Antibody 1 ; the HC, VH, VH+CH1 , HC CDRs 1 , 2, and / or 3; and / or the LC, VL, VL+CL, and / or LC CDRs 1 , 2, and / or 3 of Antibody 1 , and / or or the antigen binding portion thereof.

[0727] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the HC or LC of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the HC and LC of Antibody 1 .

[0728] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 or VL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 and VL of Antibody 1 .

[0729] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH or VL + CL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH and VL + CL of Antibody 1 .

[0730] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 or VL + CL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 and VL + CL of Antibody 1 .

[0731] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH or VL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH and VL of Antibody 1 .

[0732] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the HC CDRs 1 , 2, and 3 or the LC CDRs 1 , 2, and 3 of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the HC CDRs 1 , 2, and 3 and the LC CDRs 1 , 2, and 3 of Antibody 1 .

[0733] Antibody 1

[0734] SEQ ID NO: 1- Antibody HC:

[0735] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0736] SEQ ID NO: 2- VH+CH1 :

[0737] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYG

[0738] SEQ ID NO: 3- VH:

[0739] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS PATENT

[0740] Attorney Docket No. 51661 -008W06

[0741] Flagship Reference: VL75014-W1

[0742] SEQ ID NO: 4- HC CDR1 : GYYWS

[0743] SEQ ID NO: 5- HC CDR2: EINHGGYVTYNPSLES

[0744] SEQ ID NO: 6- HC CDR3: DYGPGNYDWYFDL

[0745] SEQ ID NO: 7- Antibody LC:

[0746] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0747] SEQ ID NO:8- VL+CL:

[0748] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0749] SEQ ID NO: 9- VL:

[0750] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK

[0751] SEQ ID NO: 10- LC CDR1 : RASQSVSSYLA

[0752] SEQ ID NO: 11- LC CDR2: DASNRAT

[0753] SEQ ID NO: 12- LC CDR3: QQRSNWPPALT

[0754] See also published PCT application WO 2005 / 035584.

[0755] VEGF-R Polypeptides and Fragments and Variants Thereof

[0756] In some aspects, the disclosure provides VEGF-R polypeptides or a fragments or variant thereof.

[0757] In some embodiments, the disclosure provides a polypeptide comprising a VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the polypeptide comprises a human VEGF-R polypeptide or a fragment or variant thereof. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., human VEGF, e.g., in an assay provided herein. In some embodiments, the VEGF-R polypeptide or a fragment or variant thereof binds to VEGF, e.g., mouse VEGF, e.g., in an assay provided herein. In some embodiments, the polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof described herein.

[0758] As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948), e.g., a full form of VEGFR1 . For example, “Gen 1 Detuned L221 W” indicates that this variant has the 221stresidue derived from the full length VEGFR1 mutated from leucine to tryptophan. For example, for an VEGF-R polypeptide or fragment or variant thereof that comprises an amino acid substitution mutation of one or more of F172, Y199, L221 , H223, and R224, the numbering of these amino acid residues refers to the sequence of Uniprot ID 17948.

[0759] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R. For example, the VEGF-R polypeptide comprises SEQ ID NO: 46. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID PATENT

[0760] Attorney Docket No. 51661 -008W06

[0761] Flagship Reference: VL75014-W1

[0762] NO: 46 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0763] In some embodiments in which the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R, the VEGF-R polypeptide comprises SEQ ID NO: 54. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 54 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0764] In some embodiments, the VEGF-R polypeptide or variant thereof comprises a fragment thereof, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 38. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0765] In some embodiments, the VEGF-R polypeptide or variant thereof comprises a fragment of a full form of VEGF-R, e.g., an abbreviated form thereof. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 53. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 53 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0766] In some embodiments in which the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form thereof, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 62. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 62 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0767] In some embodiments in which the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form thereof, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 63. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 63 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0768] In some embodiments in which the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form thereof, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 64. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 64 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0769] In some embodiments, the VEGF-R polypeptide or fragment thereof comprises a variant of VEGF-R (e.g., a variant of a wild-type VEGF-R, e.g., a variant in which one or more amino acid residues differ from a wild-type residue). For example, in some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to such a variant (e.g., a variant presented in any one of SEQ ID NOs: 38, 46-52, and 55-61) (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at PATENT

[0770] Attorney Docket No. 51661 -008W06

[0771] Flagship Reference: VL75014-W1 least 90% identity to such a variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0772] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R that also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, in some embodiments, the VEGF-R polypeptide is equal in length to the full form, and also comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the full form which also comprises a variant comprises a sequence having at least 90% identity to such a variant (e.g., a variant presented in any one of SEQ ID NOs: 38, 46-52, and 55-61) (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the full form which also comprises a variant comprises one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such a variant (e.g., has at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0773] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form, which also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, the abbreviated form, which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the abbreviated form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0774] In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 47 to 52 and 55 to 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0775] In some embodiments, the variant of VEGF-R (which can be of the VEGF-R polypeptide or fragment thereof) comprises an amino acid substitution mutation at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the VEGF-R polypeptide comprises a F172 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a L221 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a H223 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a R224 amino acid PATENT

[0776] Attorney Docket No. 51661 -008W06

[0777] Flagship Reference: VL75014-W1 substitution mutation. In some embodiments, the VEGF-R polypeptide comprises H223A and R224A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises Y199A and F172A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises a Y199A amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199F amino acid substitution mutation.

[0778] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 47 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47).

[0779] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 47.

[0780] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0781] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 48 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48).

[0782] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224Q amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 48.

[0783] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0784] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 49 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49).

[0785] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 49.

[0786] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 49.

[0787] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 50 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50).

[0788] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 50. PATENT

[0789] Attorney Docket No. 51661 -008W06

[0790] Flagship Reference: VL75014-W1

[0791] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.

[0792] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 51 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 51).

[0793] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199F amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 51 .

[0794] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0795] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 52 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52).

[0796] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199L amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 52.

[0797] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52.

[0798] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 55 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 55).

[0799] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises H223A and R224A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 55.

[0800] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55.

[0801] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 56 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56).

[0802] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 56.

[0803] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56. PATENT

[0804] Attorney Docket No. 51661 -008W06

[0805] Flagship Reference: VL75014-W1

[0806] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 57 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 57).

[0807] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 57.

[0808] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0809] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 58 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 58).

[0810] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 58.

[0811] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58.

[0812] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 59 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 59).

[0813] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 W amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 59.

[0814] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0815] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 60 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 60).

[0816] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a F172A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 60.

[0817] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 60.

[0818] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 61). PATENT

[0819] Attorney Docket No. 51661 -008W06

[0820] Flagship Reference: VL75014-W1

[0821] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises Y199A and F172A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 61 .

[0822] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[0823] In some aspects, the disclosure provides a polypeptide that comprises a polypeptide described herein. E.g., the polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof described herein.

[0824] In some aspects, the disclosure provides nucleic acid encoding a polypeptide described herein.

[0825] In some aspects, the disclosure provides a macromolecule that comprises a VEGF-R polypeptide or a fragment or variant thereof.

[0826] In some embodiments of the macromolecules provided herein, the polypeptide comprises a VEGF-R polypeptide or a fragment or variant thereof. In some embodiments of the macromolecules provided herein, the polypeptide is a human VEGF-R polypeptide or a fragment or variant thereof.

[0827] As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948). For example, “Gen 1 Detuned L221 W” indicates that this variant has the 221stresidue derived from the full length VEGFR1 mutated from leucine to tryptophan. For example, for an VEGF-R polypeptide or fragment or variant thereof that comprises an amino acid substitution mutation of one or more of F172, Y199, L221 , H223, and R224, the numbering of these amino acid residues refers to the sequence of Uniprot ID 17948.

[0828] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form thereof. For example, the VEGF-R polypeptide comprises SEQ ID NO: 46. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 46 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0829] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R. For example, the VEGF-R polypeptide comprises SEQ ID NO: 54. In some embodiments, the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 54 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0830] In some embodiments, the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 38. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0831] In some embodiments, the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 53. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 53 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. PATENT

[0832] Attorney Docket No. 51661 -008W06 Flagship Reference: VL75014-W1

[0833] In some embodiments, the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form. For example, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 62. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 62 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0834] In some embodiments in which the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form thereof, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 63. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 63 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0835] In some embodiments in which the VEGF-R polypeptide or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form thereof, the abbreviated form of the VEGF-R polypeptide comprises SEQ ID NO: 64. In some embodiments, the abbreviated form of the VEGF-R polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 64 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0836] In some embodiments, the VEGF-R polypeptide or fragment thereof comprises a variant of VEGF-R (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to such a variant (e.g., a variant presented in any one of SEQ ID NOs: 38, 46-52, and 55-61) (e.g., has at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the variant of the VEGF-R polypeptide comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such a variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT 1 , Uniprot ID 17948).

[0837] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form of VEGF-R that also comprises a variant (e.g., comprises a difference in one or more amino acid residues relative to a reference wild-type sequence). For example, in some embodiments, the VEGF-R polypeptide is equal in length to the full form and also comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the full form which also comprises a variant comprises a sequence having at least 90% identity to such a variant (e.g., a variant presented in any one of SEQ ID NOs: 38, 46-52, and 55-61) (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the full form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such a variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948). PATENT

[0838] Attorney Docket No. 51661 -008W06

[0839] Flagship Reference: VL75014-W1

[0840] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of VEGF-R, e.g., an abbreviated form, which also comprises a variant. For example, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the abbreviated form which also comprises a variant comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF. In some embodiments, the abbreviated form which also comprises a variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224 and comprises a sequence having at least 90% identity to such variant (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence, and, e.g., binds to human VEGF. As used herein, numbering of amino acid residues in the VEGF-R polypeptide are with reference to full length VEGFR1 (also referred to as FLT1 , Uniprot ID 17948).

[0841] In some embodiments, the variant of the VEGF-R polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 47 to 52 and 55 to 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and, e.g., binds to human VEGF.

[0842] In some embodiments, the variant of VEGF-R (which can be of the VEGF-R polypeptide or fragment thereof) comprises an amino acid substitution mutation of one or more of F172, Y199, L221 , H223, and R224. In some embodiments, the VEGF-R polypeptide comprises a F172 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a L221 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a H223 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a R224 amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises H223A and R224A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises Y199A and F172A amino acid substitution mutations. In some embodiments, the VEGF-R polypeptide comprises a Y199A amino acid substitution mutation. In some embodiments, the VEGF-R polypeptide comprises a Y199F amino acid substitution mutation.

[0843] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 47 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47).

[0844] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 47.

[0845] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0846] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 48 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48). PATENT

[0847] Attorney Docket No. 51661 -008W06

[0848] Flagship Reference: VL75014-W1

[0849] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224Q amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 48.

[0850] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0851] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 49 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 49).

[0852] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 49.

[0853] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 49.

[0854] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 50 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50).

[0855] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 50.

[0856] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.

[0857] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 51 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 51).

[0858] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199F amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 51 .

[0859] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0860] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 52 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52).

[0861] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a Y199L amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 52. PATENT

[0862] Attorney Docket No. 51661 -008W06

[0863] Flagship Reference: VL75014-W1

[0864] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52.

[0865] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 55 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 55).

[0866] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises H223A and R224A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 55.

[0867] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55.

[0868] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 56 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 56).

[0869] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a R224D amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 56.

[0870] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56.

[0871] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 57 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 57).

[0872] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 57.

[0873] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0874] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 58 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 58).

[0875] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 S amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 58.

[0876] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58. PATENT

[0877] Attorney Docket No. 51661 -008W06

[0878] Flagship Reference: VL75014-W1

[0879] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 59 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 59).

[0880] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221 W amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 59.

[0881] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0882] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 60 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 60).

[0883] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a F172A amino acid substitution mutation and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF-R polypeptide or fragment or variant thereof to SEQ ID NO: 60.

[0884] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 60.

[0885] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 61 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 61).

[0886] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises Y199A and F172A amino acid substitution mutations and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the VEGF- R polypeptide or fragment or variant thereof to SEQ ID NO: 61 .

[0887] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[0888] Macromolecules:

[0889] The disclosure provides a macromolecule that includes a heavy chain (HC) or HC variable region (VH) and / or the three HC CDRs of an antibody described herein, such as Antibody 1 , wherein the macromolecule further includes a polypeptide (such as VEGF-R polypeptide or a fragment or variant thereof) in addition to the HC or VH or VH+CH1 and / or the three HC CDRs of the antibody. In some embodiments, the macromolecule comprises a light chain (LC) or LC variable region (VL) or the three LC CDRs of an antibody described herein, such as Antibody 1 .

[0890] The macromolecule can further comprise a signal peptide, an Fc region, and / or one or more (e.g., four) copies of a linker, such as a GGGGS linker (SEQ ID NO: 13) and / or a GGGGT linker (SEQ ID NO: 14). For example, the macromolecule can comprise an Fc region and / or one or more (e.g., four) copies of a linker, such as a GGGGS (SEQ ID NO: 13) linker. For example, the macromolecule can comprise an Fc region and / or one or more (e.g., four) copies of a linker, such as a GGGGT (SEQ ID NO: PATENT

[0891] Attorney Docket No. 51661 -008W06

[0892] Flagship Reference: VL75014-W1

[0893] 14) linker. For example, the macromolecule can comprise an Fc region and / or one or more (e.g., four) copies of a GGGGS (SEQ ID NO: 13) linker and of a GGGGT (SEQ ID NO: 14) linker. For example, the macromolecule comprises, in order: a VH domain, a CH1 domain, zero to four copies of a GGGGS (SEQ ID NO: 13) linker, a VEGF-R polypeptide or fragment or variant thereof, zero to four copies of a GGGGS (SEQ ID NO: 13) linker, zero to four copies of a GGGGT (SEQ ID NO: 14) linker, and an Fc region. For example, the macromolecule comprises, in order: a VH domain, a CH1 domain, two copies of a GGGGS (SEQ ID NO: 13) linker, a VEGF-R polypeptide or fragment or variant thereof, zero copies of a GGGGS (SEQ ID NO: 13) linker, one copy of a GGGGT (SEQ ID NO:14) linker, and an Fc region. In some embodiments, the macromolecule further comprises a VL. In some embodiments, the macromolecule further comprises a VL and a CL domain. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6. In some embodiments, the macromolecule comprises the heavy chain (HC) CDRs of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 11 , and 12.

[0894] For example, the disclosure provides the macromolecules of SEQ ID NOs: 40, 41 , 53, 54, 62, 63 and 64.

[0895] The disclosure provides a macromolecule that includes a heavy chain (HC) or HC variable region (VH) of an antibody described herein, wherein the macromolecule further includes a polypeptide in addition to the HC or VH or the three HC CDRs, and further includes a light chain (LC) or LC variable region (VL) or the three LC CDRs of an antibody described herein.

[0896] The macromolecules can be bivalent.

[0897] For example, the disclosure provides macromolecules comprising (i) any one of SEQ ID NOs: 40, 41 , 53, 54, 62, 63, and 64 and (ii) SEQ ID NO: 45.

[0898] For example, a macromolecule can include SEQ ID NO: 40 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 41 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 53 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 54 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 62 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 63 and SEQ ID NO: 45. For example, a macromolecule can include SEQ ID NO: 64 and SEQ ID NO: 45.

[0899] The disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO:38. The disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NQ:50. The disclosure provides a macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the HC variable domain (VH) and CH1 of SEQ ID NO: 44 and the LC variable domain (VL) and light chain PATENT

[0900] Attorney Docket No. 51661 -008W06

[0901] Flagship Reference: VL75014-W1 constant region (CL) of SEQ ID NO: 45, and further comprising an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO:51 .

[0902] In some embodiments, a signal peptide is not included in the macromolecule.

[0903] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an IgG isotype.

[0904] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG1 isotype.

[0905] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG2 isotype.

[0906] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG3 isotype.

[0907] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG4 isotype.

[0908] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a LALAPG mutation.

[0909] In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain CH1.

[0910] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a kappa light chain.

[0911] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a lambda light chain.

[0912] In some embodiments, the antibody or antigen binding portion thereof comprises a light chain CL.

[0913] In some embodiments, the macromolecule comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker. In some embodiments, the macromolecule comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker in one or two locations of the macromolecule.

[0914] In some embodiments, the macromolecule comprises an Fc region.

[0915] In some embodiments, the macromolecule comprises one to four copies of a GGGGT (SEQ ID NO: 14) linker.

[0916] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a linker.

[0917] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker.

[0918] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO: 14) linker.

[0919] In some embodiments, the macromolecule comprises an Fc region and / or one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker and one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO:14) linker.

[0920] In some embodiments, the macromolecule comprises an Fc region at the carboxy terminus of the macromolecule. PATENT

[0921] Attorney Docket No. 51661 -008W06

[0922] Flagship Reference: VL75014-W1

[0923] In some embodiments, the macromolecule comprises a linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide. In some embodiments, the linker comprises one to four copies of a GGGGS (SEQ ID NO: 13) linker.

[0924] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide.

[0925] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide.

[0926] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide; and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker and / or one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO:14) linker between the polypeptide and the Fc region.

[0927] In some embodiments, the macromolecule comprises an Fc region, e.g., at the carboxy terminus, and one or more (e.g., one, two, three, or four) copies of a GGGGS (SEQ ID NO: 13) linker between the heavy chain (HC) or HC variable region (VH) or VH+CH1 or the three HC CDRs of an antibody and the polypeptide; and one or more (e.g., one, two, three, or four) copies of a GGGGT (SEQ ID NO:14) linker between the polypeptide and the Fc region.

[0928] In some embodiments, the macromolecule comprises a bivalent structure.

[0929] In some embodiments, the macromolecule comprises a structure shown in Fig. 8C.

[0930] In some embodiments, the macromolecule comprises a structure shown in Fig. 9.

[0931] In some embodiments, the macromolecule comprises a structure shown in Fig. 10A.

[0932] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB. For example, the antibodies and antigen binding portions thereof bind human 4- 1 BB described in published PCT applications WO 95 / 07984 and / or WO 96 / 29348.

[0933] In some embodiments, the antibody or antigen binding portion thereof (e.g., a HC, a VH, or HC CDRs 1 , 2, or 3, a LC, a VL, or LC CDRs 1 , 2, or 3) of the macromolecule has at least 90% identity to a sequence provided herein (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), and e.g., binds to human 4-1 BB.

[0934] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises the (e.g., 1 , 2 or 3) CDR sequences provided herein and has at least 90% identity (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over the remaining sequence of the antibody or antigen binding portion thereof (e.g., a HC, a VH, a LC, or a VL), and, e.g., binds to human 4- 1 BB. PATENT

[0935] Attorney Docket No. 51661 -008W06

[0936] Flagship Reference: VL75014-W1

[0937] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises Antibody 1 ; or the HC, VH, VH+CH1 , and / or HC CDRs 1 , 2, and / or 3; and / or the LC, VL+CL, VL, and / or LC CDRs 1 , 2, and / or 3 of Antibody 1 , or the antigen binding portion thereof.

[0938] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises the HC or LC of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises the HC and LC of Antibody 1 .

[0939] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 or VL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 and VL of Antibody 1 .

[0940] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH or VL + CL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH and VL + CL of Antibody 1 .

[0941] In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 or VL + CL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the disclosure comprises the VH + CH1 and VL + CL of Antibody 1 .

[0942] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises the VH or VL of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises the VH and VL of Antibody 1 .

[0943] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises HC CDRs 1 , 2, and 3, or LC CDRs 1 , 2, and 3 of Antibody 1 . In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises HC CDRs 1 , 2, and 3, and LC CDRs 1 , 2, and 3 of Antibody 1 .

[0944] Methods of production: VEGF binder 14-1 BB binder fusion protein constructs can expressed as secreted proteins in EXPI293F™ cells via transient transfection and purified via Protein A affinity chromatography. In vitro activity can be tested using a commercial 4-1 BB luciferase reporter assay using Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene.

[0945] Binding assays. Binding of each molecule (e.g., antibody or macromolecule, e.g., macromolecule that comprises a polypeptide, e.g., a VEGF-R polypeptide or fragment or variant thereof (e.g., comprising an Fc)) to a target, e.g., human VEGF, can be evaluated using via biolayer interferometry (BLI), e.g., as described herein.

[0946] I. Compositions

[0947] Also provided herein is a composition comprising a macromolecule described herein, e.g., a composition comprising (i) a macromolecule comprising an antibody or antigen binding portion thereof and further comprising an additional polypeptide, and (ii) an additional component.

[0948] A composition can be prepared, for example, by combining a macromolecule described herein, e.g., a macromolecule comprising an antibody or antigen binding portion thereof and further comprising an additional polypeptide, with an additional component. PATENT

[0949] Attorney Docket No. 51661 -008W06 Flagship Reference: VL75014-W1

[0950] A. Macromolecules and macromolecule complexes i. Homomultimeric macromolecule complexes

[0951] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0952] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0953] In some embodiments, the conjugation between the two macromolecules is covalent. For example, in some embodiments, the two macromolecules are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between the two macromolecules is non- covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, wherein each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The pair of complementary moieties may be, e.g., biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides, (e.g., a pair of Fc fragments (e.g., a pair of engineered Fc fragments). For example, in some embodiments, the pair of complementary moieties is a knob-into-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “knob” mutations and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “hole” mutations. Knob-into-hole Fc pairs are described, e.g., in Xu et al., mAbs, 7(1): 231 -242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) facilitates concurrent binding of the disease signal and effector ligands conditional on the presence of the disease signature ligands, e.g., the linker or pair of complementary moieties is configured to sterically orient the two copies of the macromolecule to permit conditional concurrent binding of disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules are conjugated only in the presence of the disease signature ligand).

[0954] In another aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature PATENT

[0955] Attorney Docket No. 51661 -008W06

[0956] Flagship Reference: VL75014-W1 ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand). In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0957] In some embodiments, the two macromolecules are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two macromolecules may be non-identical in sequence, but comprise substantially the same first binding domain and second binding domain. For example, the two macromolecules may comprise binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence but have substantially the same affinity for the disease signature ligand or the effector ligand.

[0958] In some embodiments of the invention, the second binding domain is an antibody or antibody fragment that does not bind the effector ligand when the two macromolecules are not bound to the disease signature ligand. For example, in some embodiments, the second binding domain comprises heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody.

[0959] In one exemplary embodiment, each of the two macromolecules is a fusion protein comprising, from N- to C-terminus, a leader polypeptide; a first binding domain comprising a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a (648)3 linker, wherein the VH and VL domains have affinity for the disease signal ligand; a (G4S)n linker; a second binding domain comprising VH and VL domains having affinity for the effector ligand, wherein the VH and VL domains are connected by a G4S linker; and a FLAG affinity tag.

[0960] In another aspect of the invention, provided herein is a complex comprising a macromolecule complex as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0961] In some embodiments, the macromolecules are polypeptides. In other embodiments, the macromolecules comprise one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0962] In some embodiments, the macromolecules further comprise one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described below.

[0963] Further provided herein are macromolecule complexes comprising three or more (e.g., three, four, five, or more than five) macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the three or more macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the three or more macromolecules binding the PATENT

[0964] Attorney Docket No. 51661 -008W06

[0965] Flagship Reference: VL75014-W1 disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0966] / / . Heteromultimeric macromolecule complexes

[0967] In another aspect, provided herein is a macromolecule complex providing a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule is conditional upon each member of the pair of macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0968] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided herein.

[0969] In some embodiments, the conjugation between the two macromolecules is covalent. For example, in some embodiments, the two macromolecules are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between the two macromolecules is non- covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, wherein each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The pair of complementary moieties may be, e.g., biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides, (e.g., a pair of Fc fragments (e.g., a pair of engineered Fc fragments). For example, in some embodiments, the pair of complementary moieties is a knob-into-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “knob” mutations and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “hole” mutations. Knob-into-hole Fc pairs are described, e.g., in Xu et al., mAbs, 7(1): 231 -242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) facilitates concurrent binding of the disease signal and effector ligands conditional on the presence of the disease signature ligands, e.g., the linker or pair of complementary moieties is configured to sterically orient the two copies of the macromolecule to permit conditional concurrent binding of disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules are conjugated only in the presence of the disease signature ligand). PATENT

[0970] Attorney Docket No. 51661 -008W06

[0971] Flagship Reference: VL75014-W1

[0972] In another aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand). In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0973] In another aspect of the invention, provided herein is a complex comprising a macromolecule complex as described herein in complex with one, two, or all three of the disease signature ligand, the first effector ligand, and the second effector ligand.

[0974] In some embodiments, each member of the pair of macromolecules is a polypeptide. In other embodiments, one or both members of the pair of macromolecules comprises one or more nonpolypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0975] In another aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, each comprising a FBD linked to a SBD, wherein (a) the FBD of a first member of the pair of macromolecules specifically binds a first moiety of a disease signature ligand in a biological sample; (b) the FBD of a second member of the pair of macromolecules specifically binds a second moiety of the disease signature ligand in the biological sample; and (c) the SBD of each macromolecule specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule is conditional upon each member of the macromolecule complex binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0976] In another aspect of the invention, provided herein is a macromolecule complex comprising a set of three macromolecules, each comprising a FBD linked to a SBD, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the set of macromolecules comprises a SBD1 that specifically binds a first effector ligand in the biological PATENT

[0977] Attorney Docket No. 51661 -008W06

[0978] Flagship Reference: VL75014-W1 sample; (c) the second member of the set of macromolecules comprises a SBD2 that specifically binds a second effector ligand in the biological sample; and (d) the third member of the set of macromolecules comprises a second binding domain 3 (SBD3) that specifically binds a third effector ligand in the biological sample; wherein the SBD1 , SBD2, and SBD3 induce a cellular effector function upon binding to the first, second, and third effector ligands; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each member of the set of three macromolecules binding the disease signature ligand. In some embodiments, the disease signature ligand is trimeric.

[0979] In some embodiments, one or both members of the pair of macromolecules, or one, two, or all three of the set of three macromolecules, further comprise one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. iii. Macromolecules comprising two FBDs and two SBDs

[0980] In another aspect, provided herein is a macromolecule comprising two first binding domains (FBDs) linked to two second binding domains (SBDs), wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand (e.g., the macromolecule does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0981] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0982] In some embodiments, the two FBDs are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two FBDs may differ in sequence, but have substantially the same affinity for the disease signature ligand.

[0983] In some embodiments, the two SBDs are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two SBDs may differ in sequence, but have substantially the same affinity for the effector ligand.

[0984] In some embodiments of the invention, the SBDs are antibodies or antibody fragments that do not bind the effector ligand when the two FBDs of the macromolecule are not bound to the disease signature ligand. For example, in some embodiments, the SBD comprises heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody. PATENT

[0985] Attorney Docket No. 51661 -008W06

[0986] Flagship Reference: VL75014-W1

[0987] In another aspect of the invention, provided herein is a complex comprising a macromolecule as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0988] In some embodiments, the macromolecule is a polypeptide (e.g., comprises a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0989] In some embodiments, the macromolecule further comprises one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. iv. Macromolecules comprising two FBDs, an SBD1, and an SBD2

[0990] In another aspect, provided herein is a macromolecule comprising two first binding domains (FBDs) linked to two non-identical second binding domains (SBDs), e.g., a first second binding domain (SBD1) and a second second binding domain (SBD2), wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand (e.g., the macromolecule does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0991] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0992] In some embodiments of the invention, the SBDs (e.g., SBD1 and / or SBD2) are antibodies or antibody fragments that do not bind the effector ligand when the two FBDs of the macromolecule are not bound to the disease signature ligand. For example, in some embodiments, the SBDs (e.g., SBD1 and / or SBD2) comprise heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the SBDs (e.g., SBD1 and / or SBD2) are diabodies.

[0993] In another aspect of the invention, provided herein is a complex comprising a macromolecule as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0994] In some embodiments, the macromolecule is a polypeptide (e.g., comprises a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0995] In some embodiments, the macromolecule further comprises one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. PATENT

[0996] Attorney Docket No. 51661 -008W06 Flagship Reference: VL75014-W1 v. Nucleic acids, vectors, and host cells

[0997] In another aspect of the invention, provided herein are one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules) encoding one or more of any of the macromolecules described above (e.g., a pair of nucleic acids (e.g., a pair of RNA molecules or a pair of DNA molecules) encoding any of the pairs of macromolecules described above). The one or more nucleic acids may be circular or linear. The one or more nucleic acids may be formulated with a carrier and / or a delivery platform, e.g., a lipid-based carrier (e.g., a lipid nanoparticle (LNP)) and / or a vector delivery system (e.g., an adenovirus, an adeno-associated virus (AAV), an anellovirus, or a lentivirus). Further examples of lipid-based carriers that may be used in the invention are provided in Section l(l) herein. For example, in some aspects, provided herein are one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules, e.g., circular or linear RNA molecules or DNA molecules) encoding one or more of any of the macromolecules described above, wherein the one or more nucleic acids are formulated with a carrier, e.g., a lipid carrier, e.g., a LNP. In some aspects, the nucleic acids include one or more modified nucleotides.

[0998] Further provided herein are vectors (e.g., plasmids or viral vectors) comprising or encoding any of the above-described nucleic acids. The vector may be formulated with a carrier, e.g., a carrier appropriate for delivery to a target cell (e.g., a mammalian cell), such as, for example, a lipid-containing carrier, such as an LNP-containing formulation.

[0999] Further provided herein are host cells that have been modified to comprise the above-described nucleic acids or vectors. Suitable host cells include bacterial and eukaryotic cells (e.g., mammalian cells). In some embodiments, a nucleic acid or vector as described herein is manufactured in and isolated from a host cell.

[1000] B. Disease signature ligands and first binding domains

[1001] I. Disease signature ligands

[1002] The disease signature ligand bound by the macromolecule, pair of macromolecules, or macromolecule complex may be any moiety (e.g., protein, peptide, or small molecule) associated with a disease state or a disorder of a cell, tissue, or subject (e.g., mammal, e.g., human).

[1003] In some embodiments, the disease signature ligand is a protein. In some embodiments, the protein is a soluble protein or an insoluble protein. For example, the disease signature ligand may be present in solution in the biological sample (e.g., may be present in the extracellular space) or may be embedded in a membrane present in the biological sample (e.g., may be embedded in a cell membrane).

[1004] In some embodiments, the disease signature ligand is VEGF (e.g., VEGF-A or VEGF-B).

[1005] / / . Disease signature ligand binding domains (first binding domains)

[1006] Each of the macromolecules provided herein comprises at least one first binding domain (FBD) that specifically binds to the disease signature ligand (e.g., binds to a disease signature ligand as described in Section IB(i), above). In some embodiments, the first binding domain permits binding of an additional binding domain to the disease signature ligand (e.g., is designed or selected such that at least two copies of the first binding domain can bind to the disease signature ligand and / or such that the first binding domain of each member of a pair of macromolecules can bind to the disease signature ligand). PATENT

[1007] Attorney Docket No. 51661 -008W06

[1008] Flagship Reference: VL75014-W1

[1009] In some embodiments, the first binding domain comprises a polypeptide that specifically binds the disease signature ligand.

[1010] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[1011] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[1012] In some embodiments, the polypeptide is an endogenous binding domain of an organism from which the biological sample and / or the disease signature ligand is derived, e.g., a binding domain that is naturally produced by the organism. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain. For example, in some embodiments, the first binding domain is a polypeptide that comprises or consists of a receptor for a cytokine (e.g., a multimerized cytokine, e.g., a dimeric, trimeric, or tetrameric cytokine) or is a polypeptide that comprises or consists of a receptor for an immunologically active multimer (e.g., an immunologically active dimer, trimer, or tetramer). In some embodiments, the disease signature ligand is VEGF and the first binding domain comprises a receptor TRAP derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept). In some embodiments, the VEGF TRAP is a full-length (“full”) TRAP. In some embodiments, the VEGF TRAP is a minimal (“mini”) TRAP.

[1013] In some embodiments, the disease signature ligand-binding domain has an affinity (KD value) for the disease signature ligand of >10 to >100 pM, <10 nM, >10 nM, or >100 nM or has micromolar affinity for the disease signature ligand (e.g., KD of < 1 pM). In some aspects, the disease signature ligandbinding domain binds the disease signature ligand with a KD of 1 nM or lower.

[1014] In some embodiments of any of the macromolecules provided herein, the first binding domain has affinity to two or more disease signature moieties. For example, the first binding domain may comprise at least two binding moieties as described above, wherein the at least two binding moieties specifically bind to at least two different disease signature moieties.

[1015] In some embodiments comprising pairs of macromolecules (e.g., embodiments as described in Sections I A(i) and IA(ii) herein), the first and second members of the pair of macromolecules comprise first binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence, but have substantially the same affinity for the disease signature ligand or the effector ligand. Alternatively, in other embodiments, the first and second members of the pair of macromolecules comprise different first binding domains. For example, in some embodiments, the first binding domain of a first member of the pair of macromolecules specifically binds a first epitope or moiety of a disease signature ligand in a biological sample, and the second binding domain of a second PATENT

[1016] Attorney Docket No. 51661 -008W06

[1017] Flagship Reference: VL75014-W1 member of the pair of macromolecules specifically binds a second epitope or moiety of the disease signature ligand in the biological sample.

[1018] For example, in some embodiments, the disclosure provides a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein: (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[1019] In a further example, in some embodiments, the disclosure provides a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; (c) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds a first effector ligand in the biological sample; and (d) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[1020] Similarly, in some embodiments comprising macromolecules comprising two FBDs (e.g., embodiments as described in Sections IA(iii) and IA(iv) herein), the two FBDs are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence, but have substantially the same affinity for the disease signature ligand or the effector ligand. Alternatively, in other embodiments, the macromolecule comprises two different FBDs. For example, in some embodiments, the macromolecule comprises a first FBD that specifically binds a first epitope or moiety of a disease signature ligand in a biological sample and a second FBD that specifically binds a second epitope or moiety of the disease signature ligand in the biological sample.

[1021] For example, in some embodiments, the disclosure provides a macromolecule comprising an FBD1 and an FBD2 linked to two SBDs, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the PATENT

[1022] Attorney Docket No. 51661 -008W06

[1023] Flagship Reference: VL75014-W1 effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[1024] In a further example, in some embodiments, the disclosure provides a macromolecule comprising an FBD1 and an FBD2 linked to an SBD1 and an SBD2, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; (c) the SBD1 specifically binds a first effector ligand in the biological sample; and (d) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs (FBD1 and FBD2) binding the disease signature ligand.

[1025] In embodiments comprising non-identical first binding domains (e.g., a first binding domain 1 (FBD1) and a first binding domain 2 (FBD2)), the first binding domains may bind to different epitopes of the disease signature ligand, e.g., partially overlapping epitopes or non-overlapping epitopes, e.g., such that the two first binding domains do not sterically hinder each other from binding their respective epitopes on the disease signature ligand; only partially sterically hinder each other from binding their respective epitopes on the disease signature ligand; or do not completely hinder each other from binding their respective epitopes on the disease signature ligand. Accordingly, in some aspects, the first binding domains are able to concurrently bind the disease signature ligand.

[1026] In some embodiments comprising non-identical first binding domains (e.g., an FBD1 and an FBD2), the two first binding domains have substantially similar affinities for their respective epitopes (e.g., affinities that differ by less than 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%). In other embodiments, the FBD1 and FBD2 have substantially different affinities for their respective epitopes (e.g., affinities that differ by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2500%, 5000%, 7500%, or 10000%).

[1027] In some embodiments in which the disease signature ligand is a polypeptide, the first binding domain binds to the disease signature ligand at a binding site that contains an Arg, Lys, Asp, His or Glu amino acid residue (or a combination thereof) (e.g., a binding site that is enriched for one or more of these residues).

[1028] In one embodiment, the disease signature ligand is VEGF.

[1029] In one embodiment, the disease signature ligand is VEGF and the first binding domain comprises (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor TRAP derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept). In some embodiments, the VEGF TRAP is a full TRAP. In some embodiments, the VEGF TRAP is a mini TRAP. PATENT

[1030] Attorney Docket No. 51661 -008W06

[1031] Flagship Reference: VL75014-W1

[1032] C. Effector ligands and second binding domains

[1033] / . Effector ligands

[1034] The effector ligand bound by the macromolecule, pair of macromolecules, or macromolecule complex may be any moiety (e.g., protein or peptide) that is present in the biological sample and is capable of effecting a cellular effector function upon being bound by a multimer of the invention.

[1035] In some embodiments, the effector ligand is a protein or a peptide.

[1036] In some embodiments, the effector ligand is a cell-surface receptor.

[1037] In some embodiments, the effector ligand is 4-1 BB.

[1038] / / . Mechanisms of effector ligand activation

[1039] In some embodiments, the second binding domain (SBD) (e.g., SBD1 , SBD2, and / or SBD3) is an agonist of the effector ligand. In other embodiments, the second binding domain is an antagonist of the effector ligand.

[1040] In some embodiments, the effector ligand must be homodimerized to exert a cellular effector function. In some embodiments, the effector ligand is capable of homodimerization and exerts a cellular effector function with at least 2-fold, 5-fold, 10-fold, 10O-fold, or 10OO-fold greater strength in the homodimerized form as compared to a monomeric form. In some embodiments, the effector ligand is homodimerized in the presence of the macromolecule or macromolecule complex. In some embodiments, the effector ligand is activated by binding of the macromolecule or macromolecule complex in the absence of its endogenous ligand.

[1041] In some embodiments, the macromolecule or macromolecule complex exhibits conditional avidity, triggered avidity, and / or dimerization avidity. For example, in some aspects, a plurality of macromolecules or macromolecule complexes bind to a disease signature ligand (e.g., a tumor antigen or a pathogen surface marker), and the proximity results in increased avidity which then activates a potent downstream effect.

[1042] In some embodiments comprising non-identical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must be associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) to exert a cellular effector function. In some embodiments, the effector ligand is capable of heterodimerization and exerts a cellular effector function with at least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater strength in the heterodimerized form as compared to a monomeric form. Accordingly, in some embodiments, the first effector ligand and the second effector ligand are associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) in the presence of the macromolecule or macromolecule complex.

[1043] In other embodiments comprising non-identical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must be associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) and must further be associated with one or more additional moieties to exert a cellular effector function. For example, in some embodiments, the first effector ligand and the second effector ligand are members of a receptor complex comprising at least three members (e.g., a PATENT

[1044] Attorney Docket No. 51661 -008W06

[1045] Flagship Reference: VL75014-W1 homotrimeric receptor complex, a heterotrimeric receptor complex, a homotetrameric receptor complex, or a heterotetrameric receptor complex).

[1046] In some embodiments of any of the macromolecules, pairs of macromolecules, and macromolecule complexes provided herein, the disease signature ligand is a soluble protein (e.g., a cytokine) and the effector ligand is a catalytic receptor (e.g., a catalytic receptor that exerts a cellular function upon multimerization (e.g., homomultimerization or heteromultimerization)).

[1047] Hi. Cellular effector functions

[1048] In some embodiments, the cellular effector function of the disease signature ligand is a biological activity.

[1049] In some embodiments, the cellular effector function of the disease signature ligand is a therapeutic activity.

[1050] In some embodiments, the cellular effector function of the disease signature ligand is a disease activity (e.g., an aberrant activity associated with a disease state), and the cellular effector function is repressed by binding of the multimerized macromolecule or pair of macromolecules to the effector ligand. iv. Effector ligand binding domains (second binding domains)

[1051] In some embodiments, the second binding domain (SBD) (e.g., SBD1 , SBD2, or SBD3) comprises a polypeptide that specifically binds the effector ligand. In some embodiments, the first binding domain permits binding of an additional binding domain to the disease signature ligand (e.g., is designed or selected such that at least two copies of the second binding domain can bind to the effector ligand).

[1052] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[1053] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[1054] In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[1055] In some embodiments, the second binding domain comprises an oligonucleotide that specifically binds the effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (e.g., a DNA aptamer).

[1056] Additional binding domains that may be used in the invention are described, e.g., in Zhong and D’Antona, Antibodies, 10(2): 13, 2021.

[1057] In some embodiments, the effector ligand-binding domain has an affinity for the effector ligand that is similar to that of a native ligand of the effector ligand. In some embodiments, the effector ligand- PATENT

[1058] Attorney Docket No. 51661 -008W06

[1059] Flagship Reference: VL75014-W1 binding domain has an affinity (KD value) for the effector ligand that is in the picomolar (pM) range or is <1 pM. In some embodiments, the effector ligand-binding domain has an affinity for the effector ligand that is 500 nM or lower. In some embodiments in which the effector ligand binding domain comprises two monomers, the affinity of each of the monomers for the effector ligand is 1 -2 orders of magnitude higher than the affinity of the effector ligand binding domain monomers for one another.

[1060] In some embodiments of any of the macromolecules provided herein, the second binding domain has affinity to two or more effector ligands. For example, the second binding domain may comprise at least two binding moieties as described above, wherein the at least two binding moieties specifically bind to at least two different effector moieties.

[1061] Pairs of second binding domains

[1062] In some embodiments comprising macromolecules or pairs of macromolecules comprising nonidentical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), one of the two SBDs (e.g., the SBD1) specifically binds a first effector ligand in a biological sample, and the other SBD (e.g., SBD2) specifically binds a second effector ligand in the biological sample.

[1063] For example, in some embodiments, the SBD1 is a first portion of a binding moiety and the SBD2 is a second portion of the binding moiety.

[1064] In another example, one of the two SBDs (e.g., the SBD1) specifically binds to a first component of a heteromultimeric (e.g., heterodimeric) receptor and the other SBD (e.g., the SBD2) specifically binds to a second component of the heteromultimeric (e.g., heterodimeric) receptor.

[1065] In another example, one of the two SBDs (e.g., the SBD1) is a first component of a dimeric moiety and the other SBD (e.g., the SBD2) is a second component of a dimeric moiety.

[1066] In another example, one of the two SBDs (e.g., the SBD1) is a first fragment of a polypeptide chain and the other SBD (e.g., the SBD2) is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, a cytokine, or a growth factor.

[1067] In some embodiments of any of the above examples, the non-identical second binding domains (e.g., SBD1 and an SBD2) have been engineered to have reduced affinity for one another. In some embodiments, the non-identical second binding domains (e.g., SBD1 and an SBD2) have an affinity (KD) for one another of >1 pM, e.g., >5-10 pM, but less than 1 mM (e.g., 10-200 pM).

[1068] In some embodiments of any of the macromolecules or macromolecule complexes described herein, the second binding domain comprises a conditional effector domain.

[1069] D. Pairs of first and second binding domains

[1070] In some embodiments, the disease signature ligand is VEGF and the effector ligand is 4-1 BB. For example, in some embodiments, the first binding domain comprises a VEGF binding domain (e.g., (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. afl ibercept)) and the second binding domain comprises a 4-1 BB binding domain (e.g., an anti-4-1 BB antibody or antibody fragment, e.g., an anti-4-1 BB scFv). In some embodiments of either of the above aspects, the VEGF binding domain is an anti-VEGF scFv (e.g., as PATENT

[1071] Attorney Docket No. 51661 -008W06

[1072] Flagship Reference: VL75014-W1 provided in SEQ ID NO: 39). In some embodiments, the VEGF binding domain is a VEGF receptor trap (e.g., as provided in any one of SEQ ID NOs: 38, 46-52, and 55-61 ). In some embodiments, the VEGF binding domain is (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept). In some embodiments, the 4-1 BB binding domain is a scFv reformatted from a full-length agonistic mAb, such as urelumab or utomilumab (e.g., as shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively). In some embodiments, the 4-1 BB binding domain is a Fab (e.g., containing any one of SEQ ID NOs: 2 and 3 and / or all of SEQ ID NOs: 4-6, in combination with any one of SEQ ID NOs: 8 and 9 and / or all of SEQ ID NOs: 10-12).

[1073] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF. In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[1074] In another aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding VEGF. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, wherein the non-covalent conjugation is mediated by the disease signature ligand. In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety (e.g., an Fc domain or a fragment thereof).

[1075] In some embodiments, the VEGF binding domain is an anti-VEGF scFv (e.g., as provided in SEQ ID NO: 39). In some embodiments, the VEGF binding domain is a VEGF receptor trap (e.g., as provided in any one of SEQ ID NOs: 38, 46-52, and 55-61 ). In some embodiments, the VEGF binding domain is (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept).

[1076] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv. In some embodiments, the 4-1 BB binding domain is a scFv reformatted from a full-length agonistic mAb, such as urelumab or utomilumab (e.g., as shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively). In some embodiments, the 4-1 BB binding domain is a Fab (e.g., containing any one of SEQ ID NOs: 2 and 3 and / or all of SEQ ID NOs: 4-6, in combination with any one of SEQ ID NOs: 8 and 9, and / or all of SEQ ID NOs: 10-12).

[1077] Exemplary reference polypeptides (e.g., antibodies and antigen-binding fragments thereof) can be employed in the invention, including variants of a reference sequence. For example, a “variant of a 4- PATENT

[1078] Attorney Docket No. 51661 -008W06

[1079] Flagship Reference: VL75014-W1

[1080] 1 BB-binding polypeptide,” (e.g., a “variant” in reference to a polypeptide provided by the invention), and the like, comprises an amino acid sequence with, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or more amino acid substitutions relative to the reference sequence. In some embodiments, the variant retains a function of the disclosed polypeptide, e.g., in an assay provided herein, e.g., in an example provided herein.

[1081] In certain embodiments, a variant of a polypeptide provided by the invention includes up to 1 , 2, 3, 4, 5, 6, or 7 amino acid substitutions, e.g., 1 -3 amino acid substitutions. A variety of amino acid substitutions for variants of a polypeptide provided by the invention are possible, including substitution with non-canonical amino acids. In some embodiments, the variant of a polypeptide provided by the invention retains a function of the disclosed polypeptide, e.g., in an assay provided herein, e.g., in an example provided herein.

[1082] In some embodiments, a variant of a polypeptide comprises conservative substitutions or highly conservative substitutions, relative to the reference sequence. “Conservative substitutions” relative to a reference sequence means a given amino acid substitution has a value of 0 or greater in BLOSUM62.

[1083] “Highly conservative substitutions” relative to a reference sequence means a given amino acid substitution has a value of 1 or greater (e.g., in some embodiments, 2, or more) in BLOSUM62.

[1084] In certain embodiments, a variant of a polypeptide provided by the invention comprises the paratope of a polypeptide provided by the invention (e.g., an antibody or fragment thereof provided by the invention), where only highly conservative substitutions are made in the residues of the paratope (e.g., up to 1 , 2, 3, 4, or 5 substitutions in the paratope), while in certain embodiments, no substitutions are made in the paratope, and any substitutions are outside of the paratope and, in various embodiments, these non-paratope residues may be either highly conservative substitutions, conservative substitutions, or nonconservative substitutions, or a combination thereof. For example, in certain embodiments, any substitutions of non-paratope residues that are in the CDRs of a variable region are either conservative substitutions or, in some embodiments, highly conservative substitutions — e.g., in certain embodiments, the CDRs of a variant of a polypeptide may have up to 1 , 2, 3, 4, or 5 substitutions per CDR in residues other than those that make up the paratope, wherein the substitutions are conservative substitutions or, in more particular embodiments, highly conservative substitutions. In more particular embodiments, nonconservative substitutions can be made outside of the CDRs, although the skilled artisan will appreciate that substitutions outside of the CDRs may, in some embodiments, be conservative or highly conservative. In certain embodiments, a variant polypeptide provided by the invention, relative to a reference immunoglobulin light chain variable region or immunoglobulin heavy chain variable region is at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, or more, identical, with due regard for the various substitution criteria described above (e.g., substitutions relative to the paratope, CDRs, non-paratope residues, and non-CDRs residues).

[1085] E. Leader, reporter and linker moieties Leaders

[1086] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises a leader peptide, e.g., a leader peptide that targets the macromolecule for secretion. The leader peptide may be cleaved from the macromolecule prior to formation of the multimer. PATENT

[1087] Attorney Docket No. 51661 -008W06

[1088] Flagship Reference: VL75014-W1

[1089] In some embodiments, the leader peptide is a mouse immunoglobulin kappa variable 3 (IgKVIll) leader peptide (e.g., UniProt ID A0A140T8P0 positions M1 to G20). Further exemplary leader sequences are provided in Table 1 (SEQ ID NOs: 15-31). Table 1. Leader sequences

[1090] Reporters

[1091] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises a reporter moiety. For example, in embodiments comprising a pair of macromolecules, one or both members of the pair of macromolecules may comprise the reporter moiety, or the members of the pair of macromolecules may each comprise different reporter moieties.

[1092] Exemplary reporter moieties include, without limitation, affinity tags (e.g., FLAG affinity tags), fluorescent markers, and chromogenic markers. For example, in some embodiments, the reporter moiety is a near-infrared probe (e.g., indocyanine green (ICG) or methylene blue (MB)) or a near-infrared fluorescent protein or a fragment thereof. In other embodiments, the reporter moiety comprises a fragment of a bait protein and is detected by adding an exogenous dye that detects the bait protein. PATENT

[1093] Attorney Docket No. 51661 -008W06

[1094] Flagship Reference: VL75014-W1

[1095] In some embodiments comprising a pair of macromolecules, the first and second members of the pair of macromolecules comprise complementary reporter moieties, e.g., reporter moieties that are detectable (e.g., produce a fluorescent signal) when the first and second members of the pair of macromolecules form a multimer. For example, the first and second members of the pair of macromolecules may comprise members of a fluorescence resonance energy transfer (FRET) pair (e.g., a near-infrared FRET pair), e.g., a peptide-based or protein-based FRET pair.

[1096] Linkers

[1097] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises one or more linker domains, e.g., linker domains that connect the first binding domain to the second binding domain; connect one or more sub-domains within the first binding domain or the second binding domain; and / or connect the first binding domain or the second binding domain to a leader peptide or a reporter moiety.

[1098] In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linkers are GS linkers. In some embodiments, the peptide linkers are glycineserine (GS) linkers, e.g., GS linkers having the format GS(GnS)mor GS linkers having the format (GnS)m, e.g., wherein n = 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1 -5 or 5-10, e.g., n = 4) and m = 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 (e.g., m = 1 -5, 5-10, or 10-15, e.g., m = 5). In some embodiments, the peptide linkers are GS linkers having the format (G4S)n, e.g., wherein n = 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1 -5 or 5-10).

[1099] F. Half-life extension moieties

[1100] Any of the macromolecules (e.g., polypeptides) provided herein may be modified to alter (e.g., extend) their half-life (e.g., to alter (e.g., extend) their half-life (e.g., half, life in circulation (e.g., in serum)) and / or to elicit a desired effector function. For example, in some embodiments, any of the macromolecules provided herein may include a moiety (e.g., a heterologous moiety) that extends the halflife of the macromolecule. Exemplary half-life extension moieties include polypeptides (e.g., a fragment crystallizable region (Fc region) or a fragment or variant thereof or an albumin domain or a fragment or variant thereof) and non-polypeptide moieties (e.g., polyethylene glycol (PEG) or a modified derivative thereof).

[1101] In some aspects, a polypeptide provided herein is modified to include a Fc region that extends the half-life of the polypeptide relative to a version of the polypeptide not comprising the Fc region. In some embodiments, the Fc region is an IgG isotype Fc region, e.g., an lgG1 , lgG2, or lgG4 subtype Fc region (e.g., such an Fc region from a human, a mouse, or a non-human primate (NHP)). In some embodiments, the Fc region comprises one or more Fc effector function-silencing mutations (e.g., LALA or LALAPG mutations (mutations in lgG1 at positions L234, L235, G236, N297, or P329)); in other aspects, the Fc region is capable of eliciting one or more Fc effector functions. The Fc region may be modified to extend half-life using one or more mutations that enhance neonatal Fc receptor (FcRn)-based recycling. Further Fc variants that may be used in the invention include mutated Fc variants previously described to alter Fc gamma receptor binding or Fc neonatal receptor binding and recycling and Fc variants comprising glycosylation modifications. Variant Fc regions that may be used in the invention PATENT

[1102] Attorney Docket No. 51661 -008W06

[1103] Flagship Reference: VL75014-W1 include those provided in Saunders, Frontiers in Immunology, 10: Article 1296, 2019; Delidakis et al., Annual Review of Biomedical Engineering, 24: 249-274, 2022; and Wilkinson et al., PLoS ONE, 16(12): e0260954, 2021.

[1104] In some aspects, a polypeptide provided herein is modified to include an Fc region that alters Fc gamma receptor binding and / or effector function or Fc neonatal receptor binding and / or recycling. In some aspects, a polypeptide provided herein is modified to include an Fc region that comprises one or more glycosylation modifications.

[1105] In some aspects, a macromolecule (e.g., polypeptide) provided herein is modified to include a human serum albumin (HSA) or binder thereof that extends the half-life (e.g., half-life in circulation) of the polypeptide relative to a version of the polypeptide not comprising the HSA or binder thereof. For example, in some embodiments, the polypeptide is directly fused to HSA. In other embodiments, the polypeptide is fused to a HSA binder, e.g., a short peptide sequence, a VHH, or any other antibody or natural scaffold that targets HSA.

[1106] In some aspects, the modification (e.g., heterologous moiety) increases the half-life of the macromolecule (e.g., polypeptide) by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (e.g., 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65- 70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%) relative to a control macromolecule, e.g., a version of the macromolecule not comprising the modification.

[1107] G. Manufacturing and purity

[1108] In some embodiments of any of the compositions and methods provided herein, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is at least 95% pure (e.g., at least 95% free of any impurity or undesired substance). In some embodiments, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is more than 95% pure, e.g., is at least 96%, 97%, 98%, or 99% pure or is 100% pure.

[1109] In some embodiments of any of the compositions and methods provided herein, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same are manufactured in accordance with one or more International Organization for Standardization (ISO) standards.

[1110] In some embodiments, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is manufactured according to the U.S. Food and Drug Administration (FDA)’s Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[1111] H. Biological samples

[1112] In some embodiments of any of the compositions and methods provided herein, the biological sample is an extract, fluid, or fraction (e.g., an extract, fluid, or fraction derived from a subject and comprising a cell); a cell; a tissue; or a subject (e.g., a mammalian subject, e.g., a human subject). PATENT

[1113] Attorney...

Claims

PATENTAttorney Docket No. 51661 -008W06Flagship Reference: VL75014-W1WHAT IS CLAIMED IS:1 . A macromolecule comprising an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof comprises the heavy chain (HC) complementarity-determining regions (CDRs) of SEQ ID NOs: 4, 5, and 6 and the light chain (LC) CDRs of SEQ ID NOs: 10, 11 , and 12 and further comprises an additional polypeptide, wherein the additional polypeptide comprises a vascular endothelial growth factor receptor (VEGF-R) polypeptide or a fragment or variant thereof.

2. The macromolecule of claim 1 , wherein the VEGF-R polypeptide or a fragment or variant thereof comprises SEQ ID NO: 38.

3. The macromolecule of claim 2, wherein the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

4. The macromolecule of claim 1 , wherein the VEGF-R polypeptide or a fragment or variant thereof comprises SEQ ID NO: 50.

5. The macromolecule of claim 4, wherein the macromolecule comprises SEQ ID NO: 64 and SEQ ID NO: 45.

6. The macromolecule of claim 1 , wherein the VEGF-R polypeptide or a fragment or variant thereof comprises SEQ ID NO: 51 .

7. The macromolecule of claim 6, wherein the macromolecule comprises SEQ ID NO: 63 and SEQ ID NO: 45.

8. The macromolecule of any one of claims 1 to 7, wherein the macromolecule is bivalent.

9. The macromolecule of any one of claims 1 to 8, wherein the macromolecule comprises a structure shown in Fig. 8C.

10. A polypeptide comprising a vascular endothelial growth factor receptor (VEGF-R) fragment or variant, wherein the VEGF-R fragment or variant comprises an amino acid substitution at one or more of F172, Y199, L221 , H223, and R224.11 . The polypeptide of claim 10, wherein the VEGF-R fragment or variant comprises one of SEQ ID NOs: 47 to 52 or 55 to 61 .

12. The polypeptide of claim 10 or 11 , wherein the polypeptide comprises a second polypeptide, and the second polypeptide comprises an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof binds to human 4-1 BB.PATENTAttorney Docket No. 51661 -008W06Flagship Reference: VL75014-W113. The polypeptide of any one of claims 10 to 12, wherein the VEGF-R fragment or variant comprises SEQ ID NO: 50.

14. The polypeptide of any one of claims 10 to 12, wherein the VEGF-R fragment or variant comprises SEQ ID NO: 51 .

15. A composition comprising a macromolecule of any one of claims 1 to 9 or a polypeptide of any one of claims 10 to 14.

16. A pharmaceutical composition comprising a macromolecule of any one of claims 1 to 9, a polypeptide of any one of claims 10 to 14, or a composition of claim 15, and a pharmaceutically acceptable excipient.

17. A method of treatment, the method comprising administering a macromolecule of any one of claims 1 to 9, a polypeptide of any one of claims 10 to 14, or a composition of claim 15, or a pharmaceutical composition of claim 16 to a subject.

18. The method of claim 17, wherein the subject has a tumor.

19. The method of claim 17 or 18, wherein the method further comprises administering an additional therapy to the subject.

20. The method of claim 19, wherein the additional therapy comprises an anti-PD1 therapy.

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