Compositions and methods

Abstract

Provided herein are methods of using macromolecules comprising an antibody or antigen binding portion thereof and an additional polypeptide, and compositions comprising the macromolecules.

Description

[0001] PATENT

[0002] Flagship Reference: VL75015-W1

[0003] Attorney Docket No. 51661-009W07

[0004] COMPOSITIONS AND METHODS

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims priority to U.S. Patent Application No. 63 / 833,903, filed on January 24, 2025; U.S. Patent Application No. 63 / 750,148, filed on January 27, 2025; U.S. Patent Application No. 63 / 779,684, filed on March 28, 2025; U.S. Patent Application No. 63 / 786,709, filed on April 10, 2025; U.S. Patent Application No. 63 / 789,537, filed on April 16, 2025; and U.S. Patent Application No. 63 / 884,612, filed on September 19, 2025, the contents of which are incorporated herein by reference in their entirety.

[0007] SEQUENCE LISTING

[0008] 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 January 22, 2026, is named 51661-009WO7_Sequence_Listing_1_22_26 and is 63,061 bytes in size.

[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, 11, 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.

[0017] 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 variablePATENT

[0018] Flagship Reference: VL75015-W1

[0019] Attorney Docket No. 51661-009W07

[0020] domain (VH) of SEQ ID NO: 3 and the LO 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: 2 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: 44 and the LC variable domain (VL) of SEQ ID NO: 9, 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: 2 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) 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.

[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) 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.

[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: 2 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) 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.

[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 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.

[0029] 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.

[0030] 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 a polypeptide.PATENT

[0031] Flagship Reference: VL75015-W1

[0032] Attorney Docket No. 51661-009W07

[0033] 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0034] 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 . In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment of a full form of VGEF-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.

[0039] 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 acidPATENT

[0040] Flagship Reference: VL75015-W1

[0041] Attorney Docket No. 51661-009W07

[0042] 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).

[0043] 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 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).

[0044] 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 thereto), 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).

[0045] In some embodiments, the variant of the VEGF-R polypeptide comprises any one of SEQ ID NOs: 47 to 52 and 55 to 61.

[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), and, e.g., binds to human VEGF.

[0047] In some embodiments, the variant of the 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 morePATENT

[0048] Flagship Reference: VL75015-W1

[0049] Attorney Docket No. 51661-009W07

[0050] 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). 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.

[0051] 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).

[0052] 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.

[0053] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0054] 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).

[0055] 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.

[0056] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0057] 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).

[0058] 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.

[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).PATENT

[0061] Flagship Reference: VL75015-W1

[0062] Attorney Docket No. 51661-009W07

[0063] 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.

[0064] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.

[0065] 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).

[0066] 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.

[0067] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0068] 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).

[0069] 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.

[0070] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52.

[0071] 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).

[0072] 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.

[0073] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55.

[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.PATENT

[0076] Flagship Reference: VL75015-W1

[0077] Attorney Docket No. 51661-009W07

[0078] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56.

[0079] 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).

[0080] 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.

[0081] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0082] 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).

[0083] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221S 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.

[0084] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58.

[0085] 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).

[0086] 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.

[0087] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0088] 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).

[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.PATENT

[0091] Flagship Reference: VL75015-W1

[0092] Attorney Docket No. 51661-009W07

[0093] 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).

[0094] 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.

[0095] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[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, 11, and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 38.

[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, 11, and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 46.

[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, 11, and 12, and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 47.

[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, 11, and12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 48.

[0100] 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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 49. 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, 11, and 12 and further comprising an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 50.

[0101] 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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 51. 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 heavyPATENT

[0102] Flagship Reference: VL75015-W1

[0103] Attorney Docket No. 51661-009W07

[0104] 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 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 52.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 55.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 56.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 57.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 58.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 59.

[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, 11, and 12 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 60.

[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 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 and (ii) an additional polypeptide, wherein the additional polypeptide comprises SEQ ID NO: 61.

[0112] 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 (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.

[0113] In some embodiments, the macromolecule comprises SEQ ID NO: 40 and SEQ ID NO: 45. In some embodiments, the macromolecule comprises SEQ ID NO: 41 and SEQ ID NO: 45. In some embodiments, the macromolecule comprises SEQ ID NO: 53 and SEQ ID NO: 45. In some embodiments, the macromolecule comprises SEQ ID NO: 54 and SEQ ID NO: 45. In some embodiments, the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.PATENT

[0114] Flagship Reference: VL75015-W1

[0115] Attorney Docket No. 51661-009W07

[0116] In some embodiments, the macromolecule comprises SEQ ID NO: 63 and SEQ ID NO: 45.

[0117] In some embodiments, the macromolecule comprises SEQ ID NO: 64 and SEQ ID NO: 45.

[0118] In some embodiments, the macromolecule comprises a signal peptide.

[0119] In some embodiments, the macromolecule comprises an Fc region.

[0120] In some embodiments, the macromolecule comprises one or more copies of a linker.

[0121] 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.

[0122] In some embodiments, the macromolecule comprises one to four copies of a GGGGT linker (SEQ ID NO:14).

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] In some embodiments, the macromolecule comprises an Fc region at the carboxy terminus of the macromolecule.

[0128] 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.

[0129] 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.

[0130] 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 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.

[0131] 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) linkerPATENT

[0132] Flagship Reference: VL75015-W1

[0133] Attorney Docket No. 51661-009W07

[0134] 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, 11, and 12. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 38, 46-52, and 55-61.

[0135] 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 .

[0136] In some embodiments, the macromolecule comprises a bivalent structure.

[0137] In some embodiments, the macromolecule comprises a structure shown in Fig. 1 A.

[0138] In some embodiments, the macromolecule comprises a structure shown in Fig. 8C.

[0139] In some embodiments, the macromolecule comprises a signal peptide.

[0140] In some embodiments, the macromolecule does not comprise a signal peptide.

[0141] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an IgG isotype.

[0142] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG1 isotype.

[0143] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG2 isotype.

[0144] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG3 isotype.

[0145] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG4 isotype.

[0146] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a LALAPG mutation.

[0147] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a kappa light chain.

[0148] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a lambda light chain.

[0149] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB.

[0150] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.PATENT

[0151] Flagship Reference: VL75015-W1

[0152] Attorney Docket No. 51661-009W07

[0153] In some aspects, the disclosure provides a composition comprising a macromolecule described herein.

[0154] In some aspects, the disclosure provides a pharmaceutical composition comprising a macromolecule described herein, or a composition described herein, and a pharmaceutically acceptable excipient.

[0155] In some aspects, the disclosure provides a method of treatment comprising administering a macromolecule comprising an antibody or antigen binding portion thereof, 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.

[0156] In some embodiments, the subject has a tumor e.g., a solid tumor.

[0157] In some embodiments, the subject has a cancer, e.g., a colorectal cancer or a melanoma.

[0158] 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.

[0159] 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).

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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, aPATENT

[0164] Flagship Reference: VL75015-W1

[0165] Attorney Docket No. 51661-009W07

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] In another aspect, provided herein is a nucleic acid encoding the macromolecule of any one of the aspects.

[0171] In another aspect, provided herein is a pair of nucleic acids encoding the pair of macromolecules of an aspect.

[0172] In some embodiments, the nucleic acid is an RNA or a DNA.

[0173] In some embodiments, the nucleic acid is formulated with a delivery platform.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] In some embodiments, the nucleic acid is an RNA or a DNA.

[0178] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a LNP.PATENT

[0179] Flagship Reference: VL75015-W1

[0180] Attorney Docket No. 51661-009W07

[0181] In another aspect, the disclosure provides a vector comprising the nucleic acid of any of the above aspects.

[0182] In another aspect, the disclosure provides a vector or pair of vectors comprising the pair of nucleic acids of any of the above aspects.

[0183] In some embodiments, the vector or pair of vectors is formulated with a carrier.

[0184] 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.

[0185] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors 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, pair of nucleic acids, vector, or pair of vectors 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, pair of nucleic acids, vector, or pair of vectors is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure after two purification steps, e.g., purification using Protein A affinity chromatography followed by polishing using cation exchange chromatography.

[0186] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors 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.

[0187] 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.

[0188] 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.

[0189] 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 ofPATENT

[0190] Flagship Reference: VL75015-W1

[0191] Attorney Docket No. 51661-009W07

[0192] 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.

[0193] 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.

[0194] In some embodiments of either of the above two aspects, 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.

[0195] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0196] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab.

[0197] 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.

[0198] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV. 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.

[0199] 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.

[0200] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 38.

[0201] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 39.

[0202] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 39.

[0203] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 38.

[0204] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 38.

[0205] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 39.

[0206] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 39.

[0207] In some embodiments, the macromolecule comprises SEQ ID NQ:40 and SEQ ID NO:45.

[0208] In some embodiments, the macromolecule comprises SEQ ID NO:41 and SEQ ID NO:45.

[0209] In some embodiments, the macromolecule comprises SEQ ID NO:53 and SEQ ID NO:45.

[0210] In some embodiments, the macromolecule comprises SEQ ID NO:54 and SEQ ID NO:45.

[0211] In some embodiments, the macromolecule comprises SEQ ID NO:62 and SEQ ID NO:45.

[0212] In some embodiments, a macromolecule comprises SEQ ID NOs: 38, 44, and 45.

[0213] 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.

[0214] 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 FcPATENT

[0215] Flagship Reference: VL75015-W1

[0216] Attorney Docket No. 51661-009W07

[0217] fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0222] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab.

[0223] 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.

[0224] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV. 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.

[0225] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-scFV. In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 38.

[0226] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 38.

[0227] In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 39.

[0228] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 39.

[0229] In some embodiments, the macromolecule comprises SEQ ID NQ:40 and SEQ ID NO:45.

[0230] In some embodiments, the macromolecule comprises SEQ ID NO:41 and SEQ ID NO:45.

[0231] In some embodiments, the macromolecule comprises SEQ ID NO:53 and SEQ ID NO:45.

[0232] In some embodiments, the macromolecule comprises SEQ ID NO:54 and SEQ ID NO:45.

[0233] In some embodiments, the macromolecule comprises SEQ ID NO:62 and SEQ ID NO:45.

[0234] In some embodiments, a macromolecule comprises SEQ ID NOs: 38, 44, and 45.

[0235] 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.

[0236] 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.

[0237] In some embodiments, the composition comprises a macromolecule complex described herein. In some embodiments, the composition comprises a macromolecule described herein.

[0238] In some aspects, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors provided herein and a pharmaceutically acceptable excipient.

[0239] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient.PATENT

[0240] Flagship Reference: VL75015-W1

[0241] Attorney Docket No. 51661-009W07

[0242] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0243] In some aspects, the disclosure provides method for preparing a pharmaceutical composition, the method comprising: combining a macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors provided herein with a pharmaceutically acceptable excipient, thereby preparing the pharmaceutical composition.

[0244] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient.

[0245] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0246] In another aspect, the disclosure provides a method comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects access to a cell.

[0247] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0248] In some embodiments, no significant dermatitis is observed in the subject.

[0249] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for providing access to a cell.

[0250] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0251] In some embodiments, no significant dermatitis is observed in the subject.

[0252] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in providing access to a cell.

[0253] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0254] In some embodiments, no significant dermatitis is observed in the subject.

[0255] 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, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby modulating the state of the cell.

[0256] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for modulating the state of a cell.

[0257] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in modulating the state of a cell.

[0258] 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,PATENT

[0259] Flagship Reference: VL75015-W1

[0260] Attorney Docket No. 51661-009W07

[0261] vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby inducing the cellular effector function in the cell.

[0262] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0263] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in inducing a cellular effector function in a cell.

[0264] In some embodiments, the cell is in a subject and the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition is administered in a therapeutically effective amount.

[0265] 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.

[0266] 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, vector, or pair of vectors, 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.

[0267] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for determining the state of a cell.

[0268] In another aspect, the disclosure provides a method of treating a cancer in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby treating the cancer.

[0269] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a cancer in a subject.

[0270] 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.

[0271] In some embodiments, the cancer comprises a solid tumor.

[0272] In some embodiments, the cancer is resistant to PD-1 blockade.

[0273] In some embodiments, the cancer comprises a colorectal cancer or a melanoma.

[0274] In some embodiments, no significant hepatotoxicity is observed in the subject.

[0275] In some embodiments, hepatomegaly is not induced in the subject.PATENT

[0276] Flagship Reference: VL75015-W1

[0277] Attorney Docket No. 51661-009W07

[0278] 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).

[0279] In some embodiments, no significant dermatitis is observed in the subject.

[0280] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in treating a cancer in a subject.

[0281] In another aspect, the disclosure provides a method of suppressing tumor growth in a subject (e.g., a subject with a tumor), comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby suppressing tumor growth in the subject.

[0282] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0283] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0284] In another aspect, the disclosure provides a method of increasing tumor-infiltrating leukocytes (TILs) and / or immune cell density in a subject (e.g., a subject with a tumor), comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0285] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0286] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0287] In another aspect, the disclosure provides a method of promoting monocyte differentiation into M1 and H7Rh'9hM1 macrophages in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0288] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.PATENT

[0289] Flagship Reference: VL75015-W1

[0290] Attorney Docket No. 51661-009W07

[0291] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0292] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby enhancing innate immune cell-mediated anti-tumor immunity in the subject.

[0293] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0294] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0295] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0296] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0297] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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).

[0298] In another aspect, the disclosure provides a method of increasing glycolysis in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing glycolysis in the subject.

[0299] 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.PATENT

[0300] Flagship Reference: VL75015-W1

[0301] Attorney Docket No. 51661-009W07

[0302] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in increasing glycolysis in a subject.

[0303] In another aspect, the disclosure provides a method of reducing LDHA expression in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby reducing LDHA expression in the subject.

[0304] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing LDHA expression in a subject.

[0305] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in reducing LDHA expression in a subject.

[0306] In another aspect, the disclosure provides a method of elevating MPC1 / MPC2 levels in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby elevating MPC1 / MPC2 levels in the subject.

[0307] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0308] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in elevating MPC1 / MPC2 levels in a subject.

[0309] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0310] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0311] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.PATENT

[0312] Flagship Reference: VL75015-W1

[0313] Attorney Docket No. 51661-009W07

[0314] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0315] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0316] 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.

[0317] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0318] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0319] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0320] In another aspect, the disclosure provides a method of increasing long-term immunity in a subject, comprising providing (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects to the subject, thereby increasing long-term immunity in the subject.

[0321] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0322] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in increasing long-term immunity in a subject.

[0323] 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 (e.g., administering) the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, orPATENT

[0324] Flagship Reference: VL75015-W1

[0325] Attorney Docket No. 51661-009W07

[0326] pharmaceutical composition of any one of the above aspects to the subject, thereby promoting an expanded fraction of CD8+ T cells within the tumor.

[0327] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, 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.

[0328] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in increasing long-term immunity within a tumor.

[0329] In another aspect, the disclosure provides a method of treating a subject with a tumor, the method comprising: (i) administering to the subject a composition (e.g., a pharmaceutical composition) comprising macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects; and (ii) measuring serum interferon gamma (I FNg) levels (e.g., human IFNg) (e.g., protein levels) (e.g., about 21 , 25, 29, 32, or 35 days after administering), e.g., before and / or after the administering (e.g., wherein levels are measured (e.g., are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are measured about 1-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are measured about 21 , 25, 29, 32, or 35 days after administering / treating; and / or are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before administering / treating) (e.g., wherein levels are measured as described herein, e.g., by ELISA) in a sample (e.g., blood or serum sample) from the subject as described herein, e.g., by ELISA) in a sample (e.g., blood or serum sample) from the subject.

[0330] In another aspect, the disclosure provides a method of treating a tumor in a subject; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) do not significantly increase after treating (e.g., are not increased about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are not increased about 1 -5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are not increased about 21 , 25, 29, 32, or 35 days after administering / treating) (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[0331] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in treating a tumor in a subject; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg levels) (e.g., protein levels) are measured before and / or after administration of or treatment with the macromolecule (e.g., are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are measured about 1 -5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are measured about 21 , 25, 29, 32, or 35 days after administering / treating; and / or are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before administering / treating), (e.g., wherein levels are measured as described herein, e.g., by ELISA) in a sample (e.g., blood or serum sample) from the subject.PATENT

[0332] Flagship Reference: VL75015-W1

[0333] Attorney Docket No. 51661-009W07

[0334] In some aspects, the disclosure provides methods comprising administering the macromolecule to a subject having a tumor, measuring serum I FNg levels in the subject following the treatment, and continuing to treat the subject with the macromolecule if serum I FNg levels are not increased following the treatment.

[0335] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a tumor in a subject; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg levels) (e.g., protein levels) are measured before and / or after administration of or treatment with the macromolecule (e.g., are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are measured about 1-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are measured about 21 , 25, 29, 32, or 35 days after administering / treating; and / or are measured about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before administering / treating) (e.g., wherein levels are measured as described herein, e.g., by ELISA) in a sample (e.g., blood or serum sample) from the subject.

[0336] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in treating a tumor in a subject; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) do not significantly increase after treating (e.g., are not increased about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are not increased about 1-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are not increased about 21 , 25, 29, 32, or 35 days after administering / treating) (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[0337] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a tumor in a subject; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg levels) (e.g., protein levels) do not significantly increase after treating (e.g., are not increased about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after administering / treating; are not increased about 1-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, 30-35 days; or 35-40 days after administering / treating; or are not increased about 21 , 25, 29, 32, or 35 days after administering / treating) (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[0338] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects for use in treating a tumor in a subject; wherein tertiary lymphoid structure (TLS) formation in the tumor increases after treating (e.g., about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 days after administering / treating or about 68 days after administering / treating) (e.g., wherein TLS formation is measured as described herein).PATENT

[0339] Flagship Reference: VL75015-W1

[0340] Attorney Docket No. 51661-009W07

[0341] In some aspects, the disclosure provides methods comprising administering the macromolecule to a subject having a tumor, measuring TLS formation in the tumor following the treatment, and continuing to treat the subject with the macromolecule if TLS formation in the tumor is increased following the treatment.

[0342] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, vector, or pair of vectors, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a tumor in a subject; wherein tertiary lymphoid structure (TLS) formation in the tumor increases after treating (e.g., about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 days after administering / treating or about 68 days after administering / treating) (e.g., wherein TLS formation is measured as described herein).

[0343] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the macromolecule comprises SEQ ID NOs: 38, 44 and 45.

[0344] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[0345] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the subject has a solid tumor.

[0346] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the subject has a cancer.

[0347] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the cancer comprises colorectal cancer.

[0348] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the cancer comprises melanoma.

[0349] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0350] Flagship Reference: VL75015-W1

[0351] Attorney Docket No. 51661-009W07

[0352] pharmaceutical compositions) for use provided herein, the method further comprises administering an additional therapy to the subject.

[0353] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the additional therapy comprises an anti-PD1 therapy.

[0354] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the additional therapy comprises pembrolizumab.

[0355] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the composition is administered intravenously.

[0356] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the composition is administered subcutaneously.

[0357] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the cancer is resistant to PD-1 blockade.

[0358] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the cancer comprises a colorectal cancer.

[0359] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the cancer comprises a melanoma.

[0360] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, no significant hepatotoxicity (e.g., as evaluated by a method provided herein) is observed in the subject after administration.

[0361] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0362] Flagship Reference: VL75015-W1

[0363] Attorney Docket No. 51661-009W07

[0364] pharmaceutical compositions) for use provided herein, hepatomegaly (e.g., as evaluated by a method provided herein) is not induced in the subject after administration.

[0365] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, CD8 T cell inflammation is not induced in the subject after administration (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 not increasing after administration) (e.g., as evaluated by a method provided herein).

[0366] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, no significant dermatitis (e.g., as evaluated by a method provided herein) is observed in the subject after administration.

[0367] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, serum I FNg levels (e.g., protein levels) are not significantly increased in the subject after administration (e.g., as evaluated by a method provided herein, e.g., as evaluated by ELISA, e.g., as described herein).

[0368] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, Kupffer cell and CD8+ T cell-mediated liver inflammation is reduced in the subject after administration (e.g., as evaluated by a method provided herein) as compared to after urelumab treatment.

[0369] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, tumor growth is suppressed in the subject after administration (e.g., as evaluated by a method provided herein).

[0370] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, tumor-infiltrating leukocytes (TILs) increase in the subject after administration (e.g., as evaluated by a method provided herein).

[0371] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, immune cell density increases in the subject after administration (e.g., as evaluated by a method provided herein).PATENT

[0372] Flagship Reference: VL75015-W1

[0373] Attorney Docket No. 51661-009W07

[0374] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, monocyte differentiation into M1 macrophages is promoted in the subject after administration (e.g., as evaluated by a method provided herein).

[0375] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, monocyte differentiation into H7Rhigh M1 macrophages is promoted in the subject after administration (e.g., as evaluated by a method provided herein).

[0376] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, innate immune cell-mediated anti-tumor immunity in the subject is enhanced after administration (e.g., as evaluated by a method provided herein).

[0377] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, glycolysis increases in the subject after administration (e.g., as evaluated by a method provided herein).

[0378] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, a metabolic shift from anaerobic glycolysis toward oxidative phosphorylation (OXPHOS) is promoted in the subject after administration, e.g., in the tumor microenvironment (TME) (e.g., as evaluated by a method provided herein).

[0379] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, LDHA expression is reduced in the subject after administration (e.g., as evaluated by a method provided herein).

[0380] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, MPC1 / MPC2 levels are elevated in the subject after administration (e.g., as evaluated by a method provided herein).

[0381] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0382] Flagship Reference: VL75015-W1

[0383] Attorney Docket No. 51661-009W07

[0384] pharmaceutical compositions) for use provided herein, the fraction of progenitor exhausted T cells (e.g., in a tumor) increases in the subject after administration (e.g., as evaluated by a method provided herein).

[0385] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the proportion of terminally exhausted T cells (e.g., in a tumor) is reduced in the subject after administration (e.g., as evaluated by a method provided herein).

[0386] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, long-term immunity increases in the subject after administration (e.g., as evaluated by a method provided herein).

[0387] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, an expanded fraction of CD8+ T cells within a tumor is promoted in the subject after administration (e.g., as evaluated by a method provided herein).

[0388] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, M1 macrophage expansion is promoted in the subject after administration (e.g., as evaluated by a method provided herein).

[0389] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, conventional dendritic cells (eDCs) programs is promoted in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3), Entpdl (CD39), Nt5e (CD73), and / or Tox after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0390] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, antigen presentation and T-cell costimulation (e.g., as assessed by increased expression of Cd80, Cd86, Cd40, and Icaml) are promoted in the subject after administration (e.g., as evaluated by a method provided herein).

[0391] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, CD8+T cells expressed lower levels of terminal exhaustion markers (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3),PATENT

[0392] Flagship Reference: VL75015-W1

[0393] Attorney Docket No. 51661-009W07

[0394] Entpdl (CD39), Nt5e (CD73), and / or Tox)) is promoted in the subject after administration; e.g., in a tumor microenvironment (TME) (e.g., as evaluated by a method provided herein).

[0395] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, hypoxia is normalized in the subject after administration (e.g., as evaluated by a method provided herein).

[0396] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, inflammatory signatures in CD8+T cells are reduced in the subject (e.g., as assessed by increased expression of Batf3 (cDC1 lineage marker), 1112b (IL-12 production), Ccr7 (migration to lymph nodes), and Ly 75 (antigen uptake receptor) in the subject after administration (e.g., as evaluated by a method provided herein).

[0397] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, inflammatory signatures in Kupffer cells are reduced in the subject after administration (e.g., as assessed by levels of 111 b, Tnf (TNFa , 116, Cxcl2, and / or Tgfbl decreasing in the subject after administration; e.g., in the liver) (e.g., as evaluated by a method provided herein).

[0398] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, one or more terminal exhaustion markers are reduced in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3), Entpdl (CD39), Nt5e (CD73), and / or Tox) after administration; e.g., in a tumor microenvironment (TME) (e.g., as evaluated by a method provided herein).

[0399] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, a progenitor exhausted T cell signature is promoted in the subject after administration (e.g., as assessed by expression of Lef1, Tcf7 (TCF1), Slamf6, Ccr7, and / or Cd44 after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0400] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the expansion of PD1+TIM3’ progenitor exhaustedPATENT

[0401] Flagship Reference: VL75015-W1

[0402] Attorney Docket No. 51661-009W07

[0403] T cells (Pex) is promoted in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0404] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) is reduced in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0405] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, tertiary lymphoid structure (TLS) formation is promoted in a subject after administration (e.g., within a tumor) (e.g., as evaluated by a method provided herein).

[0406] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, a tumor microenvironment (TME) is altered in the subject after administration (e.g., as evaluated by a method provided herein).

[0407] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, red blood cell and hemoglobin accumulation within a tumor microenvironment (TME) is reduced in the subject after administration (e.g., as evaluated by a method provided herein).

[0408] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, progenitor-exhausted CD8+ T cells (CD8+PD1 +TIM3-, Pex) are expanded in a tumor microenvironment (TME) in the subject after administration (e.g., as evaluated by a method provided herein).

[0409] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, Tnfrsf9 (4-1BB) is upregulated in the subject after administration, e.g., on eDCs (e.g., cCD1 cells) (e.g., as evaluated by a method provided herein).

[0410] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, Tnfrsf9 expression is upregulated in the subjectPATENT

[0411] Flagship Reference: VL75015-W1

[0412] Attorney Docket No. 51661-009W07

[0413] after administration (e.g., as assessed by levels of Tnfrsf9 increasing after administration, e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0414] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, / / / rexpression is upregulated in the subject after administration (e.g., as assessed by levels of H7r increasing after administration, e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0415] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, 4-1 BB receptor expression on M1 macrophages is increased in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0416] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the tumor microenvironment (TME) is normalized in the subject after administration (e.g., as evaluated by a method provided herein).

[0417] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, lactate dehydrogenase A (LDHA) expression is reduced in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0418] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, MPC1 / 2 (subunits of mitochondrial pyruvate carrier (MPC)) expression is enhanced in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0419] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, glycolysis increases in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0420] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0421] Flagship Reference: VL75015-W1

[0422] Attorney Docket No. 51661-009W07

[0423] pharmaceutical compositions) for use provided herein, aerobic respiration is promoted in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0424] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, expansion of progenitor exhausted T cells is promoted in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0425] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, one or more terminal exhaustion markers is reduced in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1 ), Lag3, Ha vcr2 (Tl M3), Entpdl (CD39), Nt5e (CD73), and / or Tox after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0426] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, a progenitor exhausted T cell signature is promoted in the subject after administration (e.g., as assessed by expression of Lef1, Tcf7 (TCF1), Slamf6, Ccr7, and / or Cd44 after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0427] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the expansion of PD1+TIM3' progenitor exhausted T cells (Pex) is promoted in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0428] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) is reduced in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0429] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating hepatotoxicity (e.g., as evaluated by a method provided herein) in the subject after administration.PATENT

[0430] Flagship Reference: VL75015-W1

[0431] Attorney Docket No. 51661-009W07

[0432] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating hepatomegaly (e.g., as evaluated by a method provided herein) in the subject after administration.

[0433] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating CD8 T cell inflammation in the subject after administration (e.g., as assessed by levels of Fas1, Prf1, Gzma, Gzmb, Ifng, and / or Ccl5 after administration) (e.g., as evaluated by a method provided herein).

[0434] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating dermatitis (e.g., as evaluated by a method provided herein) in the subject after administration.

[0435] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating serum I FNg levels (e.g., protein levels) in the subject after administration (e.g., as evaluated by a method provided herein, e.g., as evaluated by ELISA, e.g., as described herein).

[0436] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating Kupffer cell and CD8+ T cell-mediated liver inflammation in the subject after administration (e.g., as evaluated by a method provided herein).

[0437] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating tumor growth in the subject after administration (e.g., as evaluated by a method provided herein).

[0438] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating tumor-infiltrating leukocytes (TILs) in the subject after administration (e.g., as evaluated by a method provided herein).

[0439] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes,PATENT

[0440] Flagship Reference: VL75015-W1

[0441] Attorney Docket No. 51661-009W07

[0442] macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating immune cell density in the subject after administration (e.g., as evaluated by a method provided herein).

[0443] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating monocyte differentiation into M1 macrophages in the subject after administration (e.g., as evaluated by a method provided herein).

[0444] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating monocyte differentiation into ll7Rhigh M1 macrophages in the subject after administration (e.g., as evaluated by a method provided herein).

[0445] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating innate immune cell-mediated anti-tumor immunity in the subject after administration (e.g., as evaluated by a method provided herein).

[0446] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating glycolysis in the subject after administration (e.g., as evaluated by a method provided herein).

[0447] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating a metabolic shift from anaerobic glycolysis toward oxidative phosphorylation (OXPHOS) in the subject after administration, e.g., in the tumor microenvironment (TME) (e.g., as evaluated by a method provided herein).

[0448] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating LDHA expression in the subject after administration (e.g., as evaluated by a method provided herein).

[0449] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0450] Flagship Reference: VL75015-W1

[0451] Attorney Docket No. 51661-009W07

[0452] pharmaceutical compositions) for use provided herein, it further comprises evaluating MPC1 / MPC2 levels in the subject after administration (e.g., as evaluated by a method provided herein).

[0453] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the fraction of progenitor exhausted T cells (e.g., in a tumor) in the subject after administration (e.g., as evaluated by a method provided herein).

[0454] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the proportion of terminally exhausted T cells (e.g., in a tumor) in the subject after administration (e.g., as evaluated by a method provided herein).

[0455] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating long-term immunity in the subject after administration (e.g., as evaluated by a method provided herein).

[0456] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating an expanded fraction of CD8+ T cells within a tumor in the subject after administration (e.g., as evaluated by a method provided herein).

[0457] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating M1 macrophage expansion in the subject after administration (e.g., as evaluated by a method provided herein).

[0458] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating conventional dendritic cells (eDCs) programs in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIMS), Entpdl (CD39), Nt5e (CD73), and / or Tox after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0459] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes,PATENT

[0460] Flagship Reference: VL75015-W1

[0461] Attorney Docket No. 51661-009W07

[0462] macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating antigen presentation and T-cell costimulation (e.g., as assessed by increased expression of Cd80, Cd86, Cd40, and Icaml) in the subject after administration (e.g., as evaluated by a method provided herein).

[0463] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating CD8+T cells expression of terminal exhaustion markers (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3), Entpdl (CD39), Nt5e (CD73), and / or Tox)) in the subject after administration; e.g., in a tumor microenvironment (TME) (e.g., as evaluated by a method provided herein).

[0464] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating hypoxia in the subject after administration (e.g., as evaluated by a method provided herein).

[0465] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating inflammatory signatures in CD8+T cells in the subject (e.g., as assessed by increased expression of Batf3 (cDC1 lineage marker), 1112b (IL-12 production), Ccr7 (migration to lymph nodes), and Ly 75 (antigen uptake receptor) in the subject after administration (e.g., as evaluated by a method provided herein).

[0466] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating inflammatory signatures in Kupffer cells in the subject after administration (e.g., as assessed by levels of 111 b, Tnf (TNFa , 116, Cxcl2, and / or Tgfbl decreasing in the subject after administration; e.g., in the liver) (e.g., as evaluated by a method provided herein).

[0467] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating one or more terminal exhaustion markers in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3), Entpdl (CD39), Nt5e (CD73), and / or Tox after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0468] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes,PATENT

[0469] Flagship Reference: VL75015-W1

[0470] Attorney Docket No. 51661-009W07

[0471] macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating a progenitor exhausted T cell signature in the subject after administration (e.g., as assessed by expression of Lef1, Tcf7 (TCF1), Slamf6, Ccr7, and / or Cd44 after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0472] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the expansion of PD1+TIM3’ progenitor exhausted T cells (Pex) in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0473] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0474] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating tertiary lymphoid structure (TLS) formation in a subject after administration (e.g., within a tumor) (e.g., as evaluated by a method provided herein).

[0475] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating a tumor microenvironment (TME) in the subject after administration (e.g., as evaluated by a method provided herein).

[0476] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating red blood cell and hemoglobin accumulation within a tumor microenvironment (TME) in the subject after administration (e.g., as evaluated by a method provided herein).

[0477] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating progenitor-PATENT

[0478] Flagship Reference: VL75015-W1

[0479] Attorney Docket No. 51661-009W07

[0480] exhausted CD8+ T cells (CD8+PD1 +TIM3-, Pex) in a tumor microenvironment (TME) in the subject after administration (e.g., as evaluated by a method provided herein).

[0481] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating Tnfrsf9 (4-1 BB) in the subject after administration, e.g., on eDCs (e.g., cCD1 cells) (e.g., as evaluated by a method provided herein).

[0482] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating Tnfrsf9 expression in the subject after administration (e.g., as assessed by levels of Tnfrsf9 increasing after administration, e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0483] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating / / / rexpression in the subject after administration (e.g., as assessed by levels of Il7r increasing after administration, e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0484] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating 4-1 BB receptor expression on M1 macrophages in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0485] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the tumor microenvironment (TME) in the subject after administration (e.g., as evaluated by a method provided herein).

[0486] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating lactate dehydrogenase A (LDHA) expression in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0487] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes,PATENT

[0488] Flagship Reference: VL75015-W1

[0489] Attorney Docket No. 51661-009W07

[0490] macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating MPC1 / 2 (subunits of mitochondrial pyruvate carrier (MPC)) expression in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0491] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating glycolysis in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0492] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating aerobic respiration in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0493] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating expansion of progenitor exhausted T cells in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0494] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating one or more terminal exhaustion markers in the subject after administration (e.g., as assessed by decreased expression of Pdcdl (PD1), Lag3, Havcr2 (TIM3), Entpdl (CD39), Nt5e (CD73), and / or Tox after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0495] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating a progenitor exhausted T cell signature in the subject after administration (e.g., as assessed by expression of Lef1, Tcf7 (TCF1), Slamf6, Ccr7, and / or Cd44 after administration; e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0496] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, orPATENT

[0497] Flagship Reference: VL75015-W1

[0498] Attorney Docket No. 51661-009W07

[0499] pharmaceutical compositions) for use provided herein, it further comprises evaluating the expansion of PD1+TIM3’ progenitor exhausted T cells (Pex) in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0500] In some embodiments of the methods of administering or providing, of the uses in the manufacture of a medicament, and / or of the compositions (e.g., macromolecule complexes, macromolecules, nucleic acids, pair of nucleic acids, vector, pair of vectors, compositions, or pharmaceutical compositions) for use provided herein, it further comprises evaluating the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) in the subject after administration (e.g., in a tumor microenvironment (TME)) (e.g., as evaluated by a method provided herein).

[0501] As used herein, the recitation of one or more active steps in a method, e.g., administering, measuring, or evaluating, also refers to the performance of the step by another party on behalf of and under direction of a party, e.g., a party that is performing one or more other steps of the method. For example, the recitation of “evaluating the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) in the subject after administration” also refers to “having evaluated the population of PD1+TIM3+ terminally exhausted CD8+ T cells (Tex) in the subject after administration”. As additional examples, a step of “administering” also refers to the step of “having administered”; a step of “measuring” also refers to the step of “having measured.”

[0502] Other features and advantages of the invention will be apparent from the following Detailed Description and the Claims.

[0503] Definitions

[0504] 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).

[0505] 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 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.

[0506] 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 secondPATENT

[0507] Flagship Reference: VL75015-W1

[0508] Attorney Docket No. 51661-009W07

[0509] subunit). Multimers further include higher-order multimers, e.g., hexamers, heptamers, octamers, nonamers, and decamers.

[0510] 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.

[0511] 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.

[0512] 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, or 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 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.

[0513] 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 carriersPATENT

[0514] Flagship Reference: VL75015-W1

[0515] Attorney Docket No. 51661-009W07

[0516] (e.g., a protein covalently linked to the polyribonucleotide), and cationic carriers (e.g., a cationic lipopolymer or transfection reagent).

[0517] 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.

[0518] 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.

[0519] 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.

[0520] 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 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 sequencesPATENT

[0521] Flagship Reference: VL75015-W1

[0522] Attorney Docket No. 51661-009W07

[0523] may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0524] 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.

[0525] 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 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.

[0526] 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 which targets the polypeptide sequence to the secretory pathway.

[0527] 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 canPATENT

[0528] Flagship Reference: VL75015-W1

[0529] Attorney Docket No. 51661-009W07

[0530] 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).

[0531] 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.

[0532] 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.

[0533] As used herein (other than tumor clinical score evaluations which were compared between groups using two-way ANOVA test), “significantly” and / or “significant” refer to a degree of statistical significance using a two-tailed unpaired t-test in Prism software. P-values less than 0.05 are considered significant, with levels of significance denoted as *P < 0.05, **P < 0.01 , ***P < 0.001 , and ****P < 0.0001 .

[0534] BRIEF DESCRIPTION OF THE DRAWINGS

[0535] Figs. 1A-1D. Macromolecule A activates 4-1 BB signaling upon binding to VEGF. (Fig. 1A) Domain schematic of Macromolecule A (MmA), with bivalent binding to human / mouse (h / m) VEGF and human 4-1 BB (h4-1 BB). (Fig. 1 B) Kinetics and affinity of Macromolecule A binding to h / m VEGF-165 (VEGF-A) and to h4-1BB ectodomain, as assessed by SPR. (Fig. 1C) Schematic depicting mechanism of action for Macromolecule A: conditional 4-1 BB activation by VEGF binding. (Fig. 1 D) 4-1 BB signaling and VEGF-dependence was evaluated by NF-kB luciferase assay. For dose-response curves, Macromolecule A was pre-complexed ± equimolar ratio of VEGF-165 dimer. Activity was normalized to that of unstimulated reporter cells (“cells alone”).

[0536] Figs.2A-2H. Macromolecule A markedly suppresses tumor growth and induces durable anti-tumor immunity in MC38 model, and in the PD-1 -refractory B16F10 model both Macromolecule A monotherapy and combination with anti-PD-1 achieve strong tumor control and extend survival. (Fig. 2A) Schematic of the MC38 tumor efficacy study corresponding to tumor growth curves presented in (Fig.2B) and (Fig. 2C). MC38 tumor-bearing B-h4-1BB mice were randomly assigned to different treatment groups: control group (hlgG4, 10 mpk, n=5), aflibercept group (10 mpk, n=5), urelumab groups (10, 3, 0.5 mpk, n=10 per dose level), and ScFv group (10, 3, 0.5 mpk, n=10 per dose level). All groups received treatment every 4 days for a total of 3 administrations. Primary anti-tumorPATENT

[0537] Flagship Reference: VL75015-W1

[0538] Attorney Docket No. 51661-009W07

[0539] efficacy at each dose level is plotted in (Fig. 2B). For anti-tumor long-term memory immunity, we obtained tumor-free mice resulting from urelumab treatment group (all dose levels combined, n=9) and the ScFv treatment group (all dose levels combined, n=17), along with naive mice without prior MC38 tumor exposure (n=10). These mice were then inoculated with 5x106MC38 tumor cells (a lethal dose) to assess long-term anti-tumor immunity (Fig.2C). (Fig.2D) Schematic representation of B16F10 efficacy study. (Fig.2E) B16F10 tumor-bearing B-h4-1 BB mice were randomly assigned to groups of n= (5, 10, 10, 10, 8) and treated with hlgG4, aflibercept, urelumab, Macromolecule A, or anti-PD-1 , respectively. Tumor growth was monitored to assess primary anti-tumor efficacy following the two administrations at each dose level (Fig.2D), and corresponding survival curves are displayed in (Fig.2F). (Fig.2G) Tumor growth curve of B16F10 tumor-bearing B-h4-1 BB mice receiving combination therapy with anti-PD-1 and the indicated reagents according to the dosing schedule in (Fig.2D). Mice were randomly assigned to treatment groups: hlgG4 (n=5), anti-PD-1 (n=8), Macromolecule A (n=10), anti-PD-1 +aflibercept (n=10), and anti-PD-1 +Macromolecule A (n=10). For combination therapy, the two drugs were mixed together for IV administration. (Fig.2H) Survival curve of B16F10 tumor-bearing mice from (Fig.2G) following combination therapy. Summary graphs are shown as mean ± s.e.m.

[0540] Figs. 3A-3I. Macromolecule A drives tumor suppression and reshapes the TME toward proinflammatory macrophage, migratory cDc1s, and functional CDS T cells. (Fig. 3A) Treatment schematic for MC38 tumor-bearing B-h4-1 BB mice receiving a single dose of control hlgG4 (10 mpk), urelumab (10 mpk), or Macromolecule A (10 mpk), followed by tumor harvest for scRNA-seq. (Fig. 3B) Tumor growth curves after one treatment (n=4 for hlgG4, n=8 for urelumab and Macromolecule A). To ensure sufficient tissue for immune profiling, 2x106MC38 cells were implanted per mouse and treatment initiated at tumor size >100 mm3. Data shown as relative tumor volume (RTV). (Fig. 3C) UMAP of scRNA-seq from CD45+tumor-infiltrating immune cells, split by treatment group, identifying 14 immune clusters.

[0541] (Fig. 3D) Stacked bar plots of cluster proportions across groups, highlighting clusters 2 (M1 TAMs) and 3 (IL7Rh'9hM1 TAMs). (Fig. 3E) KEGG pathway enrichment in cluster 2 (M1 TAMs). (Fig. 3F) KEGG pathway enrichment in cluster 3 (IL7Rh'9hM1 TAMs). (Fig. 3G) Dot plot of glycolysis-related gene expression in cluster 3. Dot size = % cells expressing; color = average expression. (Fig. 3H) Dot plot of migration- and antigen-presentation-related genes in cDC. (Fig. 31) Dot plot of exhaustion-related genes in cluster 5 (CD8+T cells and minor Th1 CD4+cells), showing distribution of progenitor-exhausted (Pex) and terminally exhausted (Tex) populations across treatment groups.

[0542] Figs.4A-4L. Macromolecule A treatment in advanced MC38 tumors sustains antitumor macrophage activity and expands NK / NKTs and progenitor-exhausted CD8+ T cells. (Fig.4A) Treatment schematic for MC38 tumor-bearing B-h4-1 BB mice treated with two doses of control IgG (10 mpk), urelumab (10 mpk), or Macromolecule A (10 mpk) on days 14 and 18, with tumors harvested on day 25 for scRNA-seq. (Fig.4B) Tumor growth curves after two treatments (n=3 for hlgG4, n=6 for urelumab and n=8 for Macromolecule A). To ensure sufficient tissue for immune profiling, 3x106MC38 cells were implanted per mouse and treatment was initiated at tumor size >150 mm3. Data shown as relative tumor volume (RTV). (Fig.4C) UMAP of scRNA-seq from CD45+tumor-infiltrating immune cells focusing on the TAM compartment, with pseudotime trajectory analysis combining all treatment groups.

[0543] (Fig.4D) Stacked bar plot of TAM cluster proportions across treatment groups. (Figs.4E-4H) Dot plotsPATENT

[0544] Flagship Reference: VL75015-W1

[0545] Attorney Docket No. 51661-009W07

[0546] showing signature gene expression for TAM clusters 0, 11, 1, and 8. Dot size = % cells expressing; color = average expression. (Fig. 4I) UMAP highlighting treatment-dominated regions within TAM clusters. (Fig.4J) UMAP of NK / NKT and CD8+T-cell populations. (Fig.4K) Stacked bar plot of NK / NKT and CD8+T-cell cluster proportions by treatment group. (Fig. 4L) Feature plots of signature gene expression in NK / NKT and CD8+T cells.

[0547] Figs. 5A-5I. Macromolecule A minimally perturbs dLNs compared to urelumab while maintaining antitumor activity (Fig. 5A) Treatment schematic for MC38 tumor-bearing B-h4-1 BB mice receiving three doses of control hlgG4 (10 mpk), aflibercept (6.6 mpk), urelumab (10 mpk), ScFv (10 mpk), or Macromolecule A (10 mpk). (Fig. 5B) Total cell counts in draining lymph nodes (dLNs) at day 22 post-treatment. (Fig. 5C) CD8+T-cell counts per dLN determined by flow cytometry. (Fig. 5D) Schematic of TOR repertoire diversity metrics, showing richness (breadth) and clonal evenness (depth). (Fig. 5E) CD8+TCRp CDR3 diversity in tumors and dLNs across treatment groups. (Fig. 5F) Clonality index (1 -normalized Shannon entropy; 0 = polyclonal, 1 = monoclonal). (Fig. 5G) D50 values, defined as the number of clonotypes comprising 50% of the repertoire (higher values indicate greater diversity). (Fig. 5H) TOR overlap between tumor and dLN repertoires, where higher values indicate greater clonal sharing. (Fig. 5I) Cumulative frequency curves of CD8+TCRp repertoires, plotting clone rank (x-axis, descending abundance) versus cumulative repertoire coverage (y-axis). Steeper curves indicate oligoclonal expansion, while flatter curves indicate broader clonal diversity. Dashed vertical lines mark the top 10, 50, and 100 clones. Summary graphs are shown as mean ± s.e.m.

[0548] Figs.6A-6F. Macromolecule A demonstrates a non-inflamed liver profile in comparison to urelumab. (Fig. 6A) Liver weight is normal in tumor-bearing mice treated with Macromolecule A. Mice were randomized into control (no treatment, n = 5), hlgG4 (10 mpk, n = 10), urelumab (8.4 mpk, n = 10), or Macromolecule A (10 mpk, n = 10) groups. Treatments were administered on days 9 and 13 (two doses, every 4 days), and samples were collected on day 20. (Fig. 6B) Quantification of Kupffer cells (liver-resident macrophages, CD45+CD68+) and (Fig.6C) pan-CD8+T cells in the liver, determined via flow cytometry under the study conditions described in (Fig.6A). (Fig.6D) UMAP plot showing Kupffer cell and CD8+T cell subsets from MC38 tumor-bearing mice treated with 10 mpk of hlgG4, urelumab, or Macromolecule A every 4 days for a total of three administrations, based on scRNA sequencing of liver CD45+immune cells. (Fig. 6E) Dot plot illustrating the expression patterns of inflammatory signature genes in effector CD8+T cells, (Fig.6F) Dot plot illustrating the expression patterns of inflammatory signature genes in activated Kupffer cells. Summary graphs are shown as mean ± s.e.m.

[0549] Figs.7A-7H. Macromolecule A demonstrates superior antitumor activity in a humanized NSCLC model and high VEGF level in human tumor intestinal fluid support translational potential. (Fig.7A) Treatment schematic for A549 human NSCLC tumor-bearing hu-NCG mice pre-conditioned with five plasmids to boost myeloid and NK cell populations 7 days prior to tumor inoculation. Mice received three doses of control hlgG1 (8.8 mpk, n=6), the VEGF-PD-1 bispecific antibody (bsAb, 3.5 mpk, n=6), or Macromolecule A (3.0 mpk, n=6). Tumor size was measured 2-3 times per week, and blood samples collected as indicated. (Fig.7B) Tumor growth curves. (Fig.7C) Kaplan-Meier survival curves. (Fig.7D) Clinical score of tumor-bearing mice. (Fig.7E) Human IL-8 serum levels. (Fig.7F) Human IFN-y serum levels. (Fig.7G) VEGF concentrations measured in interstitial fluid (ISF) andPATENT

[0550] Flagship Reference: VL75015-W1

[0551] Attorney Docket No. 51661-009W07

[0552] matched serum from four types of human tumors (lung, colorectal, head & neck, ovarian). (Fig. 7H) Ratio of VEGF concentration in tumor ISF versus matched serum. Summary graphs are shown as mean ± s.e.m.

[0553] Figs. 8A-8E. Macromolecule A activates 4-1 BB signaling upon binding to VEGF.

[0554] (Fig. 8A) SPR sensorgrams of Macromolecule A binding to hVEGF-165 (left), mVEGF-165 (middle), and h4-1 BB ectodomain (right). Real time binding data are represented as black and the overlaid red lines represent a global fit of the entire data set using a 1 :1 binding model. Labels report the highest concentrations of VEGF and 4-1 BB used to fit the data. (Fig. 8B) Macromolecule A binding to 4-1 BB is non-competitive with 4-1 BBL. Macromolecule A (blue), urelumab (gray dotted line), or a competitive binding control were captured onto BLI sensor probes via anti-human Fc. Binding response to 4-1 BB was assessed, followed by application of 4-1 BBL in the presence of 4-1 BB, as indicated by labeled bars. Urelumab served as a control for non-competitive binding. Sensorgrams are normalized to baseline immediately prior to h4-1 BB association. (Fig. 8C) Schematic illustrating Macromolecule A scFv (“ScFv”) as a precursor molecule to Macromolecule A. The scFv utilizes an scFv 4-1 BB agonist domain format, whereas the agonist domain is reformatted to a Fab in Macromolecule A. The CDR loops of the 4-1 BB agonist, VEGF Trap, and Fc domains are identical between ScFv and Macromolecule A. (Fig. 8D) 4-1 BB signaling and VEGF-dependence of ScFv were evaluated by NF-KB luciferase assay. ScFv was precomplexed ± equimolar ratio of VEGF dimer, and activity was normalized to that of unstimulated reporter cells. Apparent EC50values are summarized in (Fig. 8E).

[0555] Figs. 9A-9H. Macromolecule A promotes in vivo activation of human 4-1 BB+CD8+T cells. (Fig. 9A) Schematic representation showing the replacement of the mouse Tnfrsf9 gene ectodomain genomic region with the human TNFRSF9 gene region. (Fig. 9B) Schematic depicting the replacement of the mouse 4-1 BB ectodomain (m ecto, left) with the human 4-1 BB ectodomain (h ecto, right). Endo, endodomain. (Fig. 9C) Naive mice received an intraperitoneal (IP) injection of 7.5 pg anti-mouse CD3e. After 24 hours, mice were sacrificed, and 4-1 BB receptor expression was evaluated on CD8+and conventional CD4+T cells. (Fig. 9D) Schematic of the proliferation study to evaluate in vivo VEGF-dependence of ScFv activity. CFSE-labeled pan-T cells (CD45.2+) from B-h4-1 BB transgenic mice were adoptively transferred into sublethally irradiated B6.SJL (CD45.1) mice. The recipients were administered 2 mpk each of ScFv ± mVEGF-A or comparators on days 1 and 4, as indicated in (Fig. 9D). (Fig. 9E) Representative histograms showing Fast Lymphopenia-Induced Proliferation (Fast LIP) induced by different treatments, as measured by CFSE dilution in CD45.2+CD8+transferred cells. Fast LIP group averages are summarized in (Fig. 9F). Adoptively transferred B-h4-1 BB CD8+T cells were profiled by flow cytometry to categorize them into effector (CD44+CD62L“), central memory (CD44+CD62L+), and naive (CD44“CD62L+) subsets. Representative plots are shown in (Fig. 9G) and expansion of the effector CD8+T population is summarized in (Fig. 9H). Summary graphs are shown as mean ± s.e.m.

[0556] Figs. 10A-10H. Conditional activity of ScFv linked to tumor-compartment VEGF enrichment, demonstrates strong combination therapy efficacy in anti-tumor immunity. (Fig. 10A) Tumor interstitial fluid was collected from B6 mice inoculated subcutaneously in the flanks with 1 x 106MC38 , 0.2 x 106B16F10, or 0.5 x 106LLC1 tumor cells. For each tumor model, three mice per time pointPATENT

[0557] Flagship Reference: VL75015-W1

[0558] Attorney Docket No. 51661-009W07

[0559] were sacrificed at days 7, 14, and 21 post-tumor inoculation. Tumors, liver, and blood were harvested and analyzed for VEGF detection, as detailed in the methods section. VEGF level was enriched 100-1000-fold in the tumor compartment with healthy tissue; N.D., not detected. (Fig. 10B) 4-1 BB signaling and VEGF-dependence of ScFv (wild-type and two attenuated VEGF-binding variants) were evaluated by NF-KB luciferase assay. ScFvs were pre-complexed ± equimolar ratio of VEGF-165 dimer, and activity was normalized to that of unstimulated reporter cells. Apparent EC50values are summarized in Fig. 10C. Potency of ScFv_mut1 and ScFv_mut2 were ~10X and ~65X weakened relative to wild-type ScFv, respectively. (Fig. 10D) Conditional activity of ScFv required VEGF binding in vivo. MC38 tumor-bearing B-h4-1 BB mice were randomly assigned treatment groups (n = 5 per group): PBS control (PBS), ScFv (10 mpk), ScFv _mut1 (10 mpk), and ScFv _mut2 (10 mpk). All groups received administrations every 4 days for a total of 3 administrations. Summary graphs are shown as mean ± s.e.m. (Fig. 10E) Experimental design for hlgG4 (10 mpk, n=5) or ScFv (0.5 mpk, n=5) treatment of MC38 tumor-bearing B-h4-1 BB mice, dosed every 4 days for three injections. (Fig. 10F) Design of anti-PD-1 (12.5 mpk, n=5) versus combination therapy (anti-PD-1 , 12.5 mpk x3; plus a single low dose of ScFv, 0.5 mpk, with the third injection; n=5). (Fig. 10G) Tumor growth curves corresponding to the treatments in (Fig. 10E) and (Fig.

[0560] 10F). (Fig. 10H) Rechallenge of tumor-free mice from the anti-PD-1 + ScFv group (n=5, from G) and naive controls (n=5) with a lethal dose of MC38 cells (5x106) to test long-term antitumor immunity.

[0561] Figs. 11 A-11O. scRNA-seq analysis of tumor-infiltrating immune cells reveals treatmentdependent remodeling of macrophage and dendritic cell populations. (Fig. 11 A) UMAP of scRNA-seq from CD45+tumor-infiltrating immune cells, identifying 14 immune clusters. (Fig. 11 B) Table summarizing the percentage of each cluster per treatment, annotated with identified cell populations and representative signature genes. (Fig. 11 C) Dot plot of signature gene expression across all clusters. (Fig.

[0562] 11 D) UMAP feature plot showing Il7r expression in tumor-infiltrating immune cells. The red circle highlights cluster 3, identified as M1 macrophages with high Il7r expression. (Fig. 11 E) Dot plot of Tnfrsf9 expression in Il7rh'9hM1 macrophages (cluster 3) across treatments. (Fig. 11 F) Schematic of the glycolysis pathway branching into anaerobic (left) and aerobic (right) respiration. (Fig. 11 G) Schematic of the dendritic cell (DC) axis: tumor antigen-bearing DCs migrate from tumor to dLNs, prime CD8+T cells, which then return to the TME for antigen-specific selection. (Fig. 11 H) UMAP of reclustered eDCs identifying three eDC subsets. (Fig. 111) Stacked bar plot showing relative proportions of eDC clusters across treatments. (Fig. 11 J) Table summarizing eDC clusters, proportions per treatment, annotations, and representative signature genes. (Fig. 11 K) Dot plot of signature gene expression across DC clusters.

[0563] (Fig. 11 L) UMAP feature plot of Tnfrsf9 expression in DCs within the TME. (Fig. 11 M) Dot plot of Tnfrsf9 expression in cDC1s (cluster 0) across treatments. (Fig. 11 N) Evaluation of the VEGF arm in Macromolecule A-mediated antitumor immunity. MC38 tumor-bearing B6 mice were randomized (n = 10 per group) to isotype control (hlgG 1 , 10 mpk), aflibercept high dose (6.6 mpk, equimolar to Macromolecule A 10 mpk), aflibercept low dose (1 .98 mpk, equimolar to Macromolecule A 3 mpk), Macromolecule A high dose (10 mpk), or Macromolecule A low dose (3 mpk). All groups received two administrations as indicated. (Fig. 110) MC38 tumor-bearing B-h4-1 BB mice (n = 5 per group) treated with 10 mpk hlgG1 isotype control, aflibercept, or Macromolecule A, each receiving two administrations as shown on the tumor growth curves.PATENT

[0564] Flagship Reference: VL75015-W1

[0565] Attorney Docket No. 51661-009W07

[0566] Figs. 12A-12E. Later-stage scRNA-seq analysis of CD45+tumor-infiltrating immune cells following Macromolecule A treatment. (Fig. 12A) UMAP of CD45+immune cells, identifying 16 clusters. (Fig. 12B) Dot plot of representative signature gene expression across all clusters. (Fig. 12C) Table summarizing cluster frequencies per treatment, annotated with identified cell populations and representative signature genes. (Fig. 12D) UMAP of CD45+immune cells, showing treatment-specific distributions. (Fig. 12E) Stacked bar plot showing cluster proportions across treatments.

[0567] Figs 13A-13G. Draining lymph nodes (dLNs) serve as hubs linking the TME with systemic immunity and enable TCR repertoire analysis. (Fig. 13A) Schematic depicting dLNs as functional hubs connecting the tumor microenvironment (TME) with the peripheral immune system. (Fig. 13B) UMAP of CD45+immune cells from dLNs identifying 15 clusters. (Fig. 13C) Dot plot of representative signature gene expression across all clusters. (Fig. 13D) Table summarizing cluster frequencies per treatment, annotated with identified cell populations and representative signature genes. (Fig. 13E) Stacked bar plot showing cluster proportions across treatments. (Fig. 13F) Workflow schematic of scRNA-seq and scTCR-seq analysis. CD45+TILs were profiled by scRNA-seq and scTCR-seq using shared barcodes. Among T cells, only CD8+, CD4+, and NKT subsets carry ap TCRs; CD8+T-cell TCRs were selected for downstream analysis, focusing on the TCRp CDR3 region to avoid bias introduced by dual TCRa chains (present in -15% of mouse and -33% of human T cells). The same analysis was performed in dLNs, and CD8+TCRp CDR3 repertoires were compared between TME and dLNs across treatments. (Fig. 13G) Table summarizing the number of unique CD8+TCRp CDR3 clonotypes detected in the TME and dLNs across treatments.

[0568] Figs. 14A-14G. scRNA-seq analysis of liver-infiltrating immune cells following treatment. (Fig. 14A) Kupffer cells and total CD8+T cells were quantified from liver immune cells (CD45+) by flow cytometry, gating on CD45+CD68+(Kupffer) and CD45+CD8+(CD8) populations (n=10),and normalized to total liver immune cells per mouse. Ratios were expressed relative to urelumab. (Fig. 14B) Schematic showing MC38 tumor-bearing mice randomized into treatment groups: hlgG4 control (10 mpk), urelumab (10 mpk), and Macromolecule A (10 mpk). All groups received three administrations every 4 days prior to liver harvest for CD45+immune cell isolation. (Fig. 14C) UMAP of CD45+liver immune cells identifying 13 clusters. (Fig. 14D) Stacked bar plot showing cluster proportions across treatments. (Fig. 14E) Table summarizing cluster frequencies per treatment, annotated with identified cell populations and representative signature genes. (Fig. 14F) Dot plot of representative signature gene expression across all clusters. (Fig. 14G) CD8+T-cell subsets from liver scRNA-seq were enumerated by multiplying subset percentages by average CD8+T-cell counts from (Fig. 14A); effector T-cell ratios were calculated as Macromolecule A relative to urelumab.

[0569] Figs. 15A-15D. Humanization efficiency, clinical monitoring, major human and mouse components in tumor bearing mice , and human tumor sample summary. (Fig. 15A) Dot plot showing the percentage of human CD45+cells among total CD45+cells at 14 weeks after hCD34+HSC transfer. Each dot represents an individual mouse, color-coded by donor origin. (Fig. 15B) Clinical score monitoring matrix for humanized mice during the study. (Fig. 15C) Summary of major human and mouse components in A549 tumor-bearing hu-NCG mice which contribute to hlL-8 and hlFNg production. (Fig.PATENT

[0570] Flagship Reference: VL75015-W1

[0571] Attorney Docket No. 51661-009W07

[0572] 15D) Summary table of human tumor sample collection, including four cancer types (lung, colorectal, head & neck, and ovarian).

[0573] Figs. 16A-16D. Macromolecule A demonstrates superior antitumor activity in a humanized NSCLC model. (Fig. 16A) Treatment schematic for A549 human NSCLC tumor-bearing hu-NCG mice pre-conditioned with five plasmids to boost myeloid and NK cell populations 7 days prior to tumor inoculation. Mice received three doses of control hlgG 1 (8.8 mpk, n=6), the VEGF-PD-1 bispecific antibody (bsAb, 3.5 mpk, n=6), Macromolecule A (Mm A) (3.0 mpk, n=6), or the anti-PD1 antibody (10mpk, n=6). Tumor size was measured 2-3 times per week, and blood samples collected as indicated.

[0574] (Fig. 16B) Tumor growth curves. (Fig. 16C) Kaplan-Meier survival curves. (Fig. 16D) Human IFN-y serum levels. Summary graphs are shown as mean ± s.e.m.

[0575] Figs. 17A-17D. Macromolecule A demonstrates equivalent efficacy and full bioavailability following subcutaneous (subQ) administration as with intravenous (IV) administration. (Fig. 17A) Treatment schematic for MC38 tumor-bearing female B-h4-1 BB mice. Mice received two doses of control hlgG 1 (2.64 mpk, n=3), or Macromolecule A (Mm A) (3 mpk, n=6). Tumor size was measured 2-3 times per week. (Fig. 17B) Tumor growth curves. (Fig. 17C) Treatment schematic for female B-h4-1 BB mice. Mice received one dose of Macromolecule A (3 mpk) via intravenous (IV) (n=6) or subcutaneous (subQ) (n=3) administration. (Fig. 17D) Serum Macromolecule A concentrations (nM) over time (h). Summary graphs are shown as mean ± s.e.m.

[0576] DETAILED DESCRIPTION

[0577] 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.

[0578] Antibodies and Antigen Binding Portions of the Macromolecules

[0579] 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.PATENT

[0580] Flagship Reference: VL75015-W1

[0581] Attorney Docket No. 51661-009W07

[0582] 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-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.

[0583] 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is noncompetitive with binding of the native ligand 4-1 BBL.

[0584] 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.

[0585] In some embodiments, the antibody or antigen binding portion thereof is an IgG isotype.

[0586] In some embodiments, the antibody or antigen binding portion thereof is an IgG 1 isotype.

[0587] In some embodiments, the antibody or antigen binding portion thereof is an lgG2 isotype.

[0588] In some embodiments, the antibody or antigen binding portion thereof is an lgG3 isotype.

[0589] In some embodiments, the antibody or antigen binding portion thereof is an lgG4 isotype.

[0590] In some embodiments, the antibody or antigen binding portion thereof comprises a LALAPG mutation.

[0591] In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain constant region 1 (CH1).

[0592] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a kappa light chain.PATENT

[0593] Flagship Reference: VL75015-W1

[0594] Attorney Docket No. 51661-009W07

[0595] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a lambda light chain.

[0596] In some embodiments, the antibody or antigen binding portion thereof comprises a light chain constant region (CL).

[0597] In some embodiments, the antibody or antigen binding portion thereof binds to human 4-1 BB. 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0598] 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0599] 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 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0600] 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.

[0601] 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 .

[0602] 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 .

[0603] 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 .

[0604] 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 .

[0605] 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 .PATENT

[0606] Flagship Reference: VL75015-W1

[0607] Attorney Docket No. 51661-009W07

[0608] 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.

[0609] Antibody 1

[0610] SEQ ID NO: 1- Antibody HC:

[0611] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 2- VH+CH1:

[0612] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYG SEQ ID NO: 3- VH:

[0613] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS SEQ ID NO: 4- HC CDR1 : GYYWS

[0614] SEQ ID NO: 5- HC CDR2: EINHGGYVTYNPSLES

[0615] SEQ ID NO: 6- HC CDR3: DYGPGNYDWYFDL

[0616] SEQ ID NO: 7- Antibody LC:

[0617] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO:8- VL+CL:

[0618] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID NO: 9- VL:

[0619] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK SEQ ID NO: 10- LC CDR1: RASQSVSSYLA

[0620] SEQ ID NO: 11- LC CDR2: DASNRATPATENT

[0621] Flagship Reference: VL75015-W1

[0622] Attorney Docket No. 51661-009W07

[0623] SEQ ID NO: 12- LC CDR3: QQRSNWPPALT

[0624] See also published POT application WO 2005 / 035584.

[0625] VEGF-R Polypeptides and Fragments and Variants Thereof of the Macromolecules

[0626] 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.

[0627] 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.

[0628] 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.

[0629] 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.

[0630] 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.

[0631] 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: 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.

[0632] 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: 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.

[0633] In some embodiments, the VEGF-R polypeptide or fragment thereof comprises a variant thereof. For example, the variant of the VEGF-R polypeptide comprises 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,PATENT

[0634] Flagship Reference: VL75015-W1

[0635] Attorney Docket No. 51661-009W07

[0636] 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 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).

[0637] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a full form thereof which also comprises a variant. For example, the full form which also comprises a variant comprises 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).

[0638] In some embodiments, the VEGF-R polypeptide or fragment or variant thereof comprises a fragment thereof, e.g., an abbreviated form, which also comprises a variant. For example, the abbreviated form, which also comprises a variant comprises 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 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).

[0639] 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.

[0640] 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 acidPATENT

[0641] Flagship Reference: VL75015-W1

[0642] Attorney Docket No. 51661-009W07

[0643] substitution mutations. In some embodiments, the VEGF-R polypeptide comprises Y199A and F172A amino acid substitution mutations.

[0644] 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).

[0645] 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.

[0646] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 47.

[0647] 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).

[0648] 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.

[0649] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 48.

[0650] 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).

[0651] 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.

[0652] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 49.

[0653] 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).

[0654] 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.

[0655] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 50.PATENT

[0656] Flagship Reference: VL75015-W1

[0657] Attorney Docket No. 51661-009W07

[0658] 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).

[0659] 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.

[0660] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 51.

[0661] 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).

[0662] 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.

[0663] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 52.

[0664] 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).

[0665] 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.

[0666] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 55.

[0667] 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).

[0668] 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.

[0669] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 56.

[0670] 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).PATENT

[0671] Flagship Reference: VL75015-W1

[0672] Attorney Docket No. 51661-009W07

[0673] 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.

[0674] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 57.

[0675] 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).

[0676] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises a L221S 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.

[0677] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 58.

[0678] 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).

[0679] 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.

[0680] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 59.

[0681] 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).

[0682] 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.

[0683] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 60.

[0684] 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).

[0685] 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.PATENT

[0686] Flagship Reference: VL75015-W1

[0687] Attorney Docket No. 51661-009W07

[0688] In some embodiments, the variant of the VEGF-R polypeptide or fragment thereof comprises SEQ ID NO: 61.

[0689] Macromolecules:

[0690] The disclosure provides a macromolecule that includes a heavy chain (HC) or HC variable region (VH) and / or the three HO 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.

[0691] 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: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 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 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.

[0692] For example, the disclosure provides the macromolecules of SEQ ID NOs: 40, 41 , 53, 54, and 62.

[0693] 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.

[0694] The macromolecules can be bivalent.

[0695] For example, the macromolecule comprises a structure shown in Fig. 1 A.

[0696] For example, the macromolecule comprises a structure shown in Fig. 8C.

[0697] For example, the disclosure provides macromolecules comprising any one of SEQ ID NOs: 40, 41 , 53, 54 and 62 and SEQ ID NO: 45.PATENT

[0698] Flagship Reference: VL75015-W1

[0699] Attorney Docket No. 51661-009W07

[0700] 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.

[0701] For example, a macromolecule can include SEQ ID NO: 62 and SEQ ID NO: 45.

[0702] 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 a polypeptide, wherein the polypeptide comprises SEQ ID NO:38. In some embodiments, a macromolecule comprises SEQ ID NOs: 38, 44, and 45.

[0703] In some embodiments, a signal peptide is not included in the macromolecule.

[0704] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an IgG isotype.

[0705] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG1 isotype.

[0706] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG2 isotype.

[0707] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG3 isotype.

[0708] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule is an lgG4 isotype.

[0709] In some embodiments, the antibody or antigen binding portion thereof of the macromolecule comprises a LALAPG mutation.

[0710] In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain CH1.

[0711] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a kappa light chain.

[0712] In some embodiments, the light chain (LC) of the antibody or antigen binding portion thereof comprises a lambda light chain.

[0713] In some embodiments, the antibody or antigen binding portion thereof comprises a light chain CL. 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.

[0714] In some embodiments, the macromolecule comprises an Fc region.

[0715] In some embodiments, the macromolecule comprises one to four copies of a GGGGT (SEQ ID NO:14) linker.

[0716] 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.

[0717] 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.PATENT

[0718] Flagship Reference: VL75015-W1

[0719] Attorney Docket No. 51661-009W07

[0720] 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.

[0721] 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.

[0722] In some embodiments, the macromolecule comprises an Fc region at the carboxy terminus of the macromolecule.

[0723] 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.

[0724] 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.

[0725] 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.

[0726] 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.

[0727] 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.

[0728] In some embodiments, the macromolecule comprises a bivalent structure.

[0729] In some embodiments, the macromolecule comprises a structure shown in Fig. 1 A.

[0730] In some embodiments, the macromolecule comprises a structure shown in Fig. 8C.

[0731] 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 4-1BB described in published PCT applications WO 95 / 07984 and / or WO 96 / 29348. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is noncompetitive with binding of the native ligand 4-1 BBL.

[0732] 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%PATENT

[0733] Flagship Reference: VL75015-W1

[0734] Attorney Docket No. 51661-009W07

[0735] identity), and e.g., binds to human 4-1 BB. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0736] 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. In some embodiments, the antibody or antigen binding portion thereof of the macromolecule binds to human 4-1 BB and is non-competitive with binding of the native ligand 4-1 BBL.

[0737] 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.

[0738] 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.

[0739] 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 .

[0740] 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.

[0741] 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.

[0742] 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.

[0743] 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.

[0744] Methods of production: VEGF 14-1 BB 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.

[0745] 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.PATENT

[0746] Flagship Reference: VL75015-W1

[0747] Attorney Docket No. 51661-009W07

[0748] Compositions

[0749] Also provided herein is a composition comprising a macromolecule described herein, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, and an additional component.

[0750] A composition can be prepared, for example, by combining a macromolecule described herein, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, with an additional component.

[0751] Nucleic acids, vectors, and host cells

[0752] 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.

[0753] In some aspects, the nucleic acids include one or more modified nucleotides.

[0754] 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.

[0755] 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.

[0756] Leader, reporter and linker moieties

[0757] Leaders

[0758] 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. 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).PATENT

[0759] Flagship Reference: VL75015-W1

[0760] Attorney Docket No. 51661-009W07

[0761] Table 1. Leader sequences

[0762]

[0763] Reporters

[0764] 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.

[0765] 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.

[0766] 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 ofPATENT

[0767] Flagship Reference: VL75015-W1

[0768] Attorney Docket No. 51661-009W07

[0769] 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.

[0770] Linkers

[0771] 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.

[0772] 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, 11 , 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).

[0773] Half-life extension moieties

[0774] 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).

[0775] 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 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.PATENT

[0776] Flagship Reference: VL75015-W1

[0777] Attorney Docket No. 51661-009W07

[0778] 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.

[0779] In some aspects, a polypeptide provided herein is modified to include an Fc region that comprises one or more glycosylation modifications.

[0780] 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.

[0781] 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.

[0782] Manufacturing and purity

[0783] 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.

[0784] 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.

[0785] 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.

[0786] Lipid nanoparticles

[0787] The compositions (e.g., macromolecules, pairs of macromolecules, macromolecule complexes, polypeptides, nucleic acids, and compositions comprising the same), methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery modality described herein, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers describedPATENT

[0788] Flagship Reference: VL75015-W1

[0789] Attorney Docket No. 51661-009W07

[0790] in Table 5 of WG2019217941 ; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).

[0791] Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941 , which is incorporated by reference — e.g., a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941 , incorporated by reference.

[0792] In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2, 3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WG2019051289 (incorporated by reference), and combinations of the foregoing.

[0793] In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0794] In some embodiments, the lipid particle includes an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle includes an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0795] In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 :1 to about 25:1 , from about 10:1 to about 14:1 , from about 3:1 to about 15:1 , from about 4:1 to about 10:1, from about 5:1 to about 9:1 , or about 6:1 to about 9:1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0796] Some non-limiting example of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein includes,PATENT

[0797] Flagship Reference: VL75015-W1

[0798] Attorney Docket No. 51661-009W07

[0799]

[0800] In some embodiments an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0801]

[0802] In some embodiments an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0803]

[0804] In some embodiments an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0805]

[0806] In some embodiments an LNP including Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.PATENT

[0807] Flagship Reference: VL75015-W1

[0808] Attorney Docket No. 51661-009W07

[0809]

[0810] In some embodiments an LNP including Formula (vi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0811]

[0812] In some embodiments an LNP including Formula (viii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0813]

[0814] In some embodiments an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0815]

[0816] wherein

[0817] X1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1-3 alkyl, R2is C1-3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogenPATENT

[0818] Flagship Reference: VL75015-W1

[0819] Attorney Docket No. 51661-009W07

[0820] atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from

[0821]

[0822] (in either orientation), (in either orientation), (in either orientation),

[0823] n is 0 to 3, R4is C1-15 alkyl, Z1is C1-6 alkylene or a direct bond,

[0824]

[0825] (in either orientation) or absent, provided that if Z1is a direct bond, Z2is absent;

[0826] R5is C5-9 alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2 alkyls, X1is O, X2is linear C3 alkylene, X3is C(=0), Y1is linear Ce alkylene, (Y2)n-R4is

[0827]

[0828] , R4is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not Cx alkoxy.

[0829] In some embodiments an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0830]

[0831] In some embodiments an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0832]

[0833] PATENT

[0834] Flagship Reference: VL75015-W1

[0835] Attorney Docket No. 51661-009W07

[0836] > > &

[0837]

[0838] In some embodiments an LNP includes a compound of Formula (xiii) and a compound of Formula (xiv).

[0839]

[0840] In some embodiments an LNP including Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0841]

[0842] In some embodiments an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0843]

[0844] (xviii)(a)PATENT

[0845] Flagship Reference: VL75015-W1

[0846] Attorney Docket No. 51661-009W07

[0847]

[0848] (xix)

[0849] In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein is made by one of the following reactions:

[0850]

[0851] In some embodiments an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxi) is an LNP described by WO2021113777 (e.g., a lipid of Formula (1) such as a lipid of Table 1 of WO2021113777).

[0852]

[0853] wherein

[0854] each n is independently an integer from 2-15; Li and L3 are each independently -OC(O)-* or - C(O)O-*, wherein indicates the attachment point to R1 or R3;

[0855] R1 and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl,PATENT

[0856] Flagship Reference: VL75015-W1

[0857] Attorney Docket No. 51661-009W07

[0858] aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl sulfonyl, and alkyl sulfonealkyl; and

[0859] R2 is selected from a group consisting of:

[0860]

[0861] In some embodiments an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021113777).

[0862]

[0863] wherein

[0864] each n is independently an integer from 1-15;

[0865] R1 and R2 are each independently selected from a group consisting of:PATENT

[0866] Flagship Reference: VL75015-W1

[0867] Attorney Docket No. 51661-009W07

[0868]

[0869] Rs is selected from a group consisting of:PATENT

[0870] Flagship Reference: VL75015-W1

[0871] Attorney Docket No. 51661-009W07

[0872]

[0873] In some embodiments an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxiii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO2021113777).

[0874]

[0875] (xxiii)

[0876] wherein

[0877] X is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to Ri;

[0878] Ri is selected from a group consisting of:

[0879]

[0880] and R2 is selected from a group consisting of:PATENT

[0881] Flagship Reference: VL75015-W1

[0882] Attorney Docket No. 51661-009W07

[0883]

[0884] In some embodiments, a composition described herein (e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein) is provided in an LNP that includes an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), e.g., as synthesized in Example 13 of W02015 / 095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of US2012 / 0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1 ,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010 / 053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro-IH-cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020 / 106946 (incorporated by reference herein in its entirety).

[0885] In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle includes a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKaPATENT

[0886] Flagship Reference: VL75015-W1

[0887] Attorney Docket No. 51661-009W07

[0888] over 6.0. In embodiments, a lipid nanoparticle may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may include between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the lipid nanoparticle may include a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle including one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and / or (ix) encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule.

[0889] Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 0311759; I of US20150376115 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or HA of

[0890] US20170210967; l-c of US20150140070; A of US2013 / 0178541 ; I of US2013 / 0303587 or

[0891] US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 0119904; I or II of WO2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058 ; A of W02012 / 162210 ; I of US2008 / 042973 ; 1 , 11 , 111 , or I V of US2012 / 01287670 ; I or 11 of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, HA, IIB, IIC, HD, or lll-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131 ; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of

[0892] US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871 ; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of

[0893] US2010 / 0062967; l-X of US2013 / 0189351 ; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221 , 127; HI-3 of WO2018 / 081480; I-5 or I-8 of W02020 / 081938; 18 or 25 of US9, 867, 888; A of US2019 / 0136231 ; II of W02020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; CKK-E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of US9,708,628; I of W02020 / 106946; I of W02020 / 106946; and (1), (2), (3), or (4) of WO2021 / 113777. Exemplary lipids further include a lipid of any one of Tables 1-16 of WO2021 / 113777.

[0894] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 IZ)-heptatriaconta- 6,9,28,3 I-tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289 A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizablePATENT

[0895] Flagship Reference: VL75015-W1

[0896] Attorney Docket No. 51661-009W07

[0897] lipid is the lipid ATX-002, e.g., as described in Example 10 of WG2019051289 A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289 A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289 A9 (incorporated by reference herein in its entirety).

[0898] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-l-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).

[0899] Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety.

[0900] In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety. The non-cationic lipid can include, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the noncationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. InPATENT

[0901] Flagship Reference: VL75015-W1

[0902] Attorney Docket No. 51661-009W07

[0903] embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , or 8:1).

[0904] In some embodiments, the lipid nanoparticles do not include any phospholipids.

[0905] In some aspects, the lipid nanoparticle can further include a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4 '-hydroxy)-buty1 ether. Exemplary cholesterol derivatives are described in PCT publication W02009 / 127060 and US patent publication

[0906] US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0907] In some embodiments, the component providing membrane integrity, such as a sterol, can include 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0908] In some embodiments, the lipid nanoparticle can include a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.

[0909] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5, 885,613, US6,287,59I, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058,

[0910] US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, lll-a-l, lll-a-2, lll-b-1 , lll-b-2, or V of US2018 / 0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-PATENT

[0911] Flagship Reference: VL75015-W1

[0912] Attorney Docket No. 51661-009W07

[0913] cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes a structure selected from:

[0914]

[0915] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0916] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WG2019051289A9, the contents of all of which are incorporated herein by reference in their entirety.

[0917] In some embodiments, the PEG or the conjugated lipid can include 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG / conjugated lipid can be varied as needed. For example, the lipid particle can include 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition includes 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of thePATENT

[0918] Flagship Reference: VL75015-W1

[0919] Attorney Docket No. 51661-009W07

[0920] composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example including 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation includes ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1.5. In some other embodiments, the lipid particle formulation includes ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1.5.

[0921] In some embodiments, the lipid particle includes ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.

[0922] In some embodiments, the lipid particle includes ionizable lipid I non-cationic- lipid I sterol I conjugated lipid at a molar ratio of 50:10:38.5: 1.5.

[0923] In an aspect, the disclosure provides a lipid nanoparticle formulation including phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[0924] In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.

[0925] In some embodiments, the LNPs include biodegradable, ionizable lipids. In some embodiments, the LNPs include (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-PATENT

[0926] Flagship Reference: VL75015-W1

[0927] Attorney Docket No. 51661-009W07

[0928] (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g., lipids of WO2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.

[0929] In some embodiments, the average LNP diameter of the LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about I mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.

[0930] A LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.

[0931] The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV,PATENT

[0932] Flagship Reference: VL75015-W1

[0933] Attorney Docket No. 51661-009W07

[0934] from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0935] The efficiency of encapsulation of a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0936] A LNP may optionally include one or more coatings. In some embodiments, a LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0937] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020 / 061457 and WO2021 / 113777, each of which is incorporated herein by reference in its entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021). doi.org / 10.1038 / S41578-021 -00358-0, which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.).

[0938] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoyJLM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51 (34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0939] LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference, and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides described herein.

[0940] Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name

[0941] ONPATTRO.PATENT

[0942] Flagship Reference: VL75015-W1

[0943] Attorney Docket No. 51661-009W07

[0944] Exemplary closing of polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) LNP may include about 0.1 , 0.25, 0.3, 0.5, 1 , 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). Exemplary dosing of AAV including a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) may include an MOI of about 1011, 1012, 1013, and 1014vg / kg.

[0945] Compositions

[0946] In one aspect, provided herein is a composition comprising a macromolecule complex (e.g., a macromolecule described herein), wherein the macromolecule complex comprises a dimer, and wherein at least about 90% of the dimer is monodisperse (i.e., has at least about 90% monodispersity).

[0947] In some embodiments, at least about 92% of the dimer is monodisperse. In some embodiments, at least about 93% of the dimer is monodisperse. In some embodiments, at least about 94% of the dimer is monodisperse. In some embodiments, at least about 95% of the dimer is monodisperse. In some embodiments, at least about 96% of the dimer is monodisperse. In some embodiments, at least about 97% of the dimer is monodisperse. In some embodiments, at least about 98% of the dimer is monodisperse. In some embodiments, at least about 99% of the dimer is monodisperse.

[0948] In another aspect, provided herein is a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein), wherein the macromolecule complex comprises two macromolecules that are capable of dimerization, and wherein the composition is at least about 90% monodisperse at a size consistent with the dimerized macromolecule complex.

[0949] In some embodiments, the composition is at least about 92% monodisperse, at least about 93% monodisperse, at least about 94% monodisperse, at least about 95% monodisperse, at least about 96% monodisperse, at least about 97% monodisperse, at least about 98% monodisperse, or at least about 99% monodisperse (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodisperse) at a size consistent with the dimerized macromolecule complex.

[0950] The composition may be, e.g., a composition produced from one or more cell lines that produce one or more components of the macromolecule complex (e.g., a cell line that produces a single macromolecule that forms a homodimer, a cell line that expresses two macromolecules that form a heterodimer, or two cell lines that each express one macromolecule of a pair that forms a heterodimer). For example, the cell line may be transiently or stably transfected with a plasmid encoding a secreted form of the macromolecule or macromolecules. The composition may be purified before monodispersity is assessed. The purification may include, e.g., clarifying culture supernatant of the cell line(s) (e.g., via a two-step purification scheme using Protein A affinity chromatography followed by polishing using cation exchange chromatography). The purification may include, e.g., clarifying culture supernatant of the cell line(s) (e.g., via one-step purification scheme using Protein A affinity (such as MabSelect PRISMA™) affinity chromatography.

[0951] In some embodiments, the percent monodispersity is determined by size exclusion chromatography (SEC), e.g., as described in the examples provided herein. In some embodiments, the percent monodispersity is determined by analytical SEC. In some embodiments, the percent monodispersity is determined by analytical SEC using a SUPERDEX® 200 column.PATENT

[0952] Flagship Reference: VL75015-W1

[0953] Attorney Docket No. 51661-009W07

[0954] In some embodiments, the size of the macromolecule complex is consistent with the size of a dimer, e.g., as described in the examples provided herein.

[0955] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain.

[0956] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0957] 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.

[0958] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0959] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab.

[0960] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-trap. In some embodiments, the VEGF trap is a full-length (“full”) trap. In some embodiments, the VEGF trap is a minimal (“mini”) trap.

[0961] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV. 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.

[0962] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-scFV. In some embodiments, the macromolecule complex comprises SEQ ID NOs: 38, 44 and 45. In some embodiments, the macromolecule complex comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[0963] In some embodiments, the macromolecule complex is used in a method of administering or providing, is of a use in the manufacture of a medicament, and / or is of a composition (e.g., pharmaceutical composition) for use provided herein.

[0964] Methods

[0965] In one aspect, the present disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0966] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);

[0967] (b) measuring or having measured the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed; and

[0968] (c) formulating or having formulated the composition as a pharmaceutical composition if the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed in the sample is equal to or greater than a pre-determined threshold.

[0969] In another aspect, the present disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0970] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);PATENT

[0971] Flagship Reference: VL75015-W1

[0972] Attorney Docket No. 51661-009W07

[0973] (b) measuring or having measured the percent monodispersity of the macromolecule in the composition (or the sample thereof); and

[0974] (c) formulating or having formulated the composition as a pharmaceutical composition if the percent monodispersity of the macromolecule in the composition (or the sample thereof) is equal to or greater than a pre-determined threshold.

[0975] In some embodiments of the above aspects, formulating or having formulated the composition as a pharmaceutical composition comprises combining the composition with one or more pharmaceutically acceptable excipients.

[0976] In one aspect, the present disclosure provides a method for selecting a pharmaceutical composition, the method comprising:

[0977] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);

[0978] (b) measuring or having measured the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed; and

[0979] (c) selecting or having selected the composition (e.g., for use) if the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed in the sample is equal to or greater than a pre-determined threshold.

[0980] In another aspect, the present disclosure provides a method for selecting a pharmaceutical composition, the method comprising:

[0981] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);

[0982] (b) measuring or having measured the percent monodispersity of the macromolecule in the composition (or the sample thereof); and

[0983] (c) selecting or having selected the composition (e.g., for use) as a pharmaceutical composition if the percent monodispersity of the macromolecule in the composition (or the sample thereof) is equal to or greater than a pre-determined threshold.

[0984] In some embodiments of the above aspects, the macromolecule is a dimer.

[0985] In some embodiments, the pre-determined threshold is at least about 90% of the dimer is monodisperse (i.e., has at least about 90% monodispersity). In some embodiments, the pre-determined threshold is at least about 92% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 93% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 94% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 95% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 96% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 97% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 98% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 99% of the dimer is monodisperse.

[0986] In some embodiments, the pre-determined threshold is at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at leastPATENT

[0987] Flagship Reference: VL75015-W1

[0988] Attorney Docket No. 51661-009W07

[0989] about 98%, or at least about 99% monodispersity (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodispersity) at a size consistent with the dimerized macromolecule complex.

[0990] In some embodiments, the percent monodispersity is determined by size exclusion chromatography (SEC), e.g., as described in the examples provided herein. In some embodiments, the percent monodispersity is determined by analytical SEC. In some embodiments, the percent monodispersity is determined by analytical SEC using a SUPERDEX® 200 column.

[0991] In some embodiments, the size of the macromolecule complex is consistent with the size of a dimer, e.g., as described in the examples provided herein.

[0992] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain.

[0993] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0994] In some embodiments, the VEGF binding domain is a VEGF receptor trap. In some embodiments, the VEGF TRAP is a full trap. In some embodiments, the VEGF TRAP is a mini trap.

[0995] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0996] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB Fab.

[0997] 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.

[0998] In some embodiments, the macromolecule complex comprises a 4-1 BB Fab X VEGF-scFV. 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.

[0999] In some embodiments, the macromolecule complex comprises a 4-1 BB scFv X VEGF-scFV. In some embodiments, the macromolecule complex comprises SEQ ID NOs: 38, 44 and 45. In some embodiments, the macromolecule complex comprises SEQ ID NO: 62 and SEQ ID NO: 45.

[1000] In some embodiments, the macromolecule complex (e.g., the prepared or selected macromolecule complex) is used in a method of administering or providing, is of a use in the manufacture of a medicament, and / or is of a composition (e.g., pharmaceutical composition) for use provided herein.

[1001] Pharmaceutical Compositions

[1002] Also provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a macromolecule described herein, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition comprising a macromolecule described herein, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide. 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 donor cell or a composition comprising a donor cell for medicinal or therapeutic use.

[1003] A pharmaceutical composition can be prepared, for example, by combining a macromolecule described herein, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition provided herein with a pharmaceutically acceptable excipient.PATENT

[1004] Flagship Reference: VL75015-W1

[1005] Attorney Docket No. 51661-009W07

[1006] In some embodiments, the pharmaceutical composition is a unit dosage form suitable for administration to a subject, e.g., a human subject (e.g., intravenous, oral, or subcutaneous administration).

[1007] Methods of Use

[1008] A macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, can administered to a subject, e.g., a subject in need of such treatment.

[1009] The disclosure provides a method of administration, the method comprising administering a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) to a subject, e.g., a subject in need thereof.

[1010] The disclosure provides a method of treating a subject, the method comprising administering a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) to a subject, e.g., a subject in need thereof.

[1011] The disclosure also provides use of a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) for the preparation of a medicament for administering to a subject, e.g., a subject in need thereof.

[1012] The disclosure also provides use of a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) for the preparation of a medicament for treating a subject, e.g., a subject in need thereof.

[1013] The disclosure also provides a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) for use in administration to a subject, e.g., a subject in need thereof.

[1014] The disclosure also provides a macromolecule, e.g., comprising an antibody or antigen binding portion thereof and further comprising a polypeptide, or a composition, or a pharmaceutical composition, each as described herein, (e.g., a therapeutically effective amount thereof) for use in treating a subject, e.g., a subject in need thereof.

[1015] The methods, uses and compositions can be, e.g., in combination with an additional therapy. The additional therapy can be, e.g., a treatment for a cancer. The additional therapy can be, e.g., a treatment for a tumor, e.g., a solid tumor. In some embodiments, the additional therapy comprises an anti-PD1 therapy, such as a PD1 inhibitor. In some embodiments, the PD1 inhibitor comprises pembrolizumab.

[1016] In some embodiments, the subject has a tumor e.g., a solid tumor.

[1017] In some embodiments, the subject has a cancer, e.g., a colorectal cancer or a melanoma.PATENT

[1018] Flagship Reference: VL75015-W1

[1019] Attorney Docket No. 51661-009W07

[1020] Exemplary Embodiments

[1021] 1. A method of treating a subject with a tumor, the method comprising:

[1022] (i) administering to the subject a composition (e.g., a pharmaceutical composition) comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; and

[1023] (ii) measuring serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) (e.g., about 21 , 25, 29, 32, or 35 days after administering), e.g., before and / or after the administering (e.g., wherein levels are measured as described herein, e.g., by ELISA) in a sample (e.g., blood or serum sample) from the subject.

[1024] 2. The method of embodiment 1 , wherein the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO:45.

[1025] 3. The method of embodiment 1 or 2, wherein serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) do not significantly increase after the administering (e.g., about 21 , 25, 29, 32, or 35 days after administering) (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[1026] 4. The method of any one of embodiments 1 to 3, wherein the subject has a solid tumor.

[1027] 5. The method of any one of embodiments 1 to 3, wherein the subject has a cancer.

[1028] 6. The method of embodiment 5, wherein the cancer comprises colorectal cancer.

[1029] 7. The method of embodiment 5, wherein the cancer comprises melanoma.

[1030] 8. The method of any one of embodiments 1 to 7, wherein the method further comprises administering an additional therapy to the subject.

[1031] 9. The method of embodiment 8, wherein the additional therapy comprises an anti-PD1 therapy. 10. The method of embodiment 8, wherein the additional therapy comprises pembrolizumab.

[1032] 11. The method of any one of embodiments 1 to 10, wherein the composition is administered intravenously.

[1033] 12. The method of any one of embodiments 1 to 10, wherein the composition is administered subcutaneously.

[1034] 13. A method of treating a subject with a tumor, the method comprising:

[1035] administering to the subject a composition (e.g., a pharmaceutical composition) comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; wherein serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) do not significantly increase after the administering (e.g., about 21 , 25, 29, 32, or 35 days after administering) (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[1036] 14. The method of embodiment 13, wherein the macromolecule comprises SEQ ID NO:62 and SEQ ID NO:45.

[1037] 15. The method of embodiment 13 or 14, wherein the method further comprises measuring serum interferon gamma (IFNg) levels (e.g., human IFNg) (e.g., protein levels) (e.g., about 21 , 25, 29, 32, or 35 days after administering), e.g., before and / or after the administering (e.g., wherein levels are measured as described herein, e.g., by ELISA).

[1038] 16. The method of any one of embodiments 13 to 15, wherein the subject has a solid tumor.

[1039] 17. The method of any one of embodiments 13 to 15, wherein the subject has a cancer.

[1040] 18. The method of embodiment 17, wherein the cancer comprises colorectal cancer.PATENT

[1041] Flagship Reference: VL75015-W1

[1042] Attorney Docket No. 51661-009W07

[1043] 19. The method of embodiment 17, wherein the cancer comprises melanoma.

[1044] 20. The method of any one of embodiments 13 to 19, wherein the method further comprises administering an additional therapy to the subject.

[1045] 21. The method of embodiment 20, wherein the additional therapy comprises an anti-PD1 therapy.

[1046] 22. The method of embodiment 20, wherein the additional therapy comprises pembrolizumab. 23. The method of any one of embodiments 13 to 22, wherein the composition is administered intravenously.

[1047] 24. The method of any one of embodiments 13 to 22, wherein the composition is administered subcutaneously.

[1048] 25. A method of treating a subject with a tumor, the method comprising:

[1049] administering to the subject a composition (e.g., a pharmaceutical composition) comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; wherein tertiary lymphoid structure (TLS) formation in the tumor increases after the administering (e.g., about 68 days after administering) (e.g., wherein TLS formation is measured as described herein).

[1050] 26. The method of embodiment 25, wherein the macromolecule comprises SEQ ID NO:62 and SEQ ID NO:45.

[1051] 27. The method of embodiment 25 or 26, wherein the method further comprises measuring tertiary lymphoid structure (TLS) formation in the tumor (e.g., before and / or after the administering e.g., about 68 days after administering) (e.g., wherein TLS formation is measured as described herein).

[1052] 28. The method of any one of embodiments 25 to 27 wherein the subject has a solid tumor.

[1053] 29. The method of any one of embodiments 25 to 27, wherein the subject has a cancer.

[1054] 30. The method of embodiment 29, wherein the cancer comprises colorectal cancer.

[1055] 31. The method of embodiment 29, wherein the cancer comprises melanoma.

[1056] 32. The method of any one of embodiments 25 to 31 , wherein the method further comprises administering an additional therapy to the subject.

[1057] 33. The method of embodiment 32, wherein the additional therapy comprises an anti-PD1 therapy.

[1058] 34. The method of embodiment 32, wherein the additional therapy comprises pembrolizumab. 35. The method of any one of embodiments 25 to 34, wherein the composition is administered intravenously.

[1059] 36. The method of any one of embodiments 25 to 34, wherein the composition is administered subcutaneously.

[1060] EXAMPLES

[1061] The following are examples of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.PATENT

[1062] Flagship Reference: VL75015-W1

[1063] Attorney Docket No. 51661-009W07

[1064] Example 1. VEGF-4-1 BB conditional bifunctional antibody drives tumor-localized immunity through sustained macrophage and T cell activation

[1065] Table 2. Additional Sequences

[1066]

[1067] PATENT

[1068] Flagship Reference: VL75015-W1

[1069] Attorney Docket No. 51661-009W07

[1070]

[1071] PATENT

[1072] Flagship Reference: VL75015-W1

[1073] Attorney Docket No. 51661-009W07

[1074]

[1075] PATENT

[1076] Flagship Reference: VL75015-W1

[1077] Attorney Docket No. 51661-009W07

[1078]

[1079] PATENT

[1080] Flagship Reference: VL75015-W1

[1081] Attorney Docket No. 51661-009W07

[1082]

[1083] “Macromolecule A” (“MmA”) is a macromolecule of SEQ ID NO: 53 and SEQ ID NO: 45 or a macromolecule of SEQ ID NO: 62 and SEQ ID NO: 45, the difference being the presence or absence, respectively, of a six-histidine tag.

[1084] “ScFv”, as used in the examples and the figures, is a macromolecule that contains an scFv derived from urelumab and a VEGF MiniTrap.

[1085] As described in detail below, Macromolecule A achieved potent anti-tumor efficacy with high therapeutic index and favorable tolerability. It is a conditional bifunctional VEGF-4-1 BB biologic designed to simultaneously neutralize VEGF and activate 4-1 BB signaling, delivering a localized and synergistic approach to cancer immunotherapy. By leveraging VEGF-dependent activation of 4-1 BB, Macromolecule A preferentially enhanced immune responses within the tumor microenvironment (TME), minimizing systemic toxicity while amplifying both innate and adaptive immunity.

[1086] Macromolecule A promoted rapid M1 macrophage differentiation and enhanced progenitor exhausted (Pex) T cell signatures, suggesting coordinated activation of innate and adaptive compartments. At later stages, Macromolecule A sustained macrophage anti-tumor activity and a continued expansion of Pex cells, consistent with long-term immune engagement. Localized activity was indicated by clonal T cell expansion within tumors with minimal draining lymph node (dLN) exchange.PATENT

[1087] Flagship Reference: VL75015-W1

[1088] Attorney Docket No. 51661-009W07

[1089] Compared with the systemic 4-1 BB agonist urelumab, Macromolecule A provided superior longterm protection with reduced exhaustion and improved hepatic tolerability. In head-to-head testing against a VEGF-PD-1 bispecific antibody, Macromolecule A achieved robust tumor suppression and survival benefits without systemic IFN-y surges.

[1090] To establish translational relevance, VEGF concentrations were measured in tumor interstitial fluid from patients with four cancer types. VEGF levels were consistently 2-4 orders of magnitude higher in tumors than in matched serum, mirroring gradients observed in mice and supporting the clinical opportunity of VEGF-restricted 4-1 BB activation.

[1091] Together, these studies support the use of Macromolecule A as a promising next-generation immunotherapy for both monotherapy and combination settings, with the potential to overcome key limitations of existing checkpoint- and 4-1 BB-based approaches.

[1092] Here, we report the development of Macromolecule A, a conditional VEGF-4-1 BB bifunctional antibody targeting 4-1 BB. Macromolecule A combines VEGF antagonism with conditionally activated and localized 4-1 BB agonism, exploiting VEGF enrichment in the TME to selecti...

Claims

1. PATENTFlagship Reference: VL75015-W1Attorney Docket No. 51661-009W07WHAT IS CLAIMED IS:1 . A method of treating a subject with a tumor, the method comprising:(i) administering to the subject a composition comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; and(ii) measuring serum interferon gamma (IFNg) levels in a sample from the subject.

2. The method of claim 1 , wherein:(i) a serum IFNg level in the sample from the subject is measured before the administering; and / or (ii) a serum IFNg level in the sample from the subject is measured after the administering, optionally wherein the serum IFNg level is measured about 21 , 25, 29, 32, or 35 days after the administering.

3. A method of treating a subject with a tumor, the method comprising:administering to the subject a composition comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; wherein serum interferon gamma (IFNg) levels do not significantly increase after the administering.

4. A method of treating a subject with a tumor, the method comprising:administering to the subject a composition comprising a macromolecule that comprises SEQ ID NOs: 38, 44 and 45; wherein tertiary lymphoid structure (TLS) formation in the tumor increases after the administering.

5. The method of any one of claims 1 to 4, wherein the macromolecule comprises SEQ ID NO: 62 and SEQ ID NO: 45.

6. The method of any one of claims 1 to 5, wherein the method further comprises measuring tertiary lymphoid structure (TLS) formation in the tumor.

7. The method of any one of claims 1 , 2, and 4 to 6, wherein serum interferon gamma (IFNg) levels do not significantly increase after the administering.

8. The method of any one of claims 1-3 and 5-7, wherein tertiary lymphoid structure (TLS) formation in the tumor increases after the administering.

9. The method of any one of claims 3 to 8, wherein the method further comprises measuring serum interferon gamma (IFNg) levels.

10. The method of any one of claims 1 to 9, wherein the subject has a solid tumor.11 . The method of any one of claims 1 to 10, wherein the subject has a cancer.PATENTFlagship Reference: VL75015-W1Attorney Docket No. 51661-009W0712. The method of claim 11 , wherein the cancer comprises colorectal cancer.

13. The method of claim 11 , wherein the cancer comprises melanoma.

14. The method of any one of claims 1 to 13, wherein the method further comprises administering an additional therapy to the subject.

15. The method of claim 14, wherein the additional therapy comprises an anti-PD1 therapy.

16. The method of claim 15, wherein the additional therapy comprises pembrolizumab.

17. The method of any one of claims 1 to 16, wherein the composition is administered intravenously.

18. The method of any one of claims 1 to 16, wherein the composition is administeredsubcutaneously.

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