Anti-nectin-4 antibodies and uses thereof

Abstract

This disclosure provides novel heavy chain-only antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins thereof that bind to Nectin Cell Adhesion Molecule 4 (Nectin-4), related antibody-drug conjugates, related nucleic acids, related cells, related kits, related compositions, and related methods or uses.

Description

[0001] ANTI-NECTIN-4 ANTIBODIES AND USES THEREOF

[0002] REFERENCE TO SEQUENCE LISTING

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SequenceListing_35472800201.xml, created on July 10, 2025, which is 130,399 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 670,507, filed July 12, 2024. The foregoing application is incorporated by reference herein in its entirety.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to heavy chain-only antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins thereof that bind to Nectin Cell Adhesion Molecule 4 (Nectin-4), and related antibody-drug conjugates.

[0008] BACKGROUND

[0009] Heavy-chain only antibodies (HCAbs) of Carnelidae have a unique structure consisting of a single variable domain (VHH: Variable domain of the Heavy chain of Heavy-chain antibodies), a hinge region, and two constant domains (CH2 and CH3), which are highly homologous to the CH2 and CH3 domains of classical antibodies. These HCAbs lack the first domain of the constant region (CHI), which is present in the genome, but is spliced out during mRNA processing. The absence of the CHI domain explains the absence of the light chain in the HCAbs, since this domain is the anchoring place for the constant domain of the light chain. Such HCAbs naturally evolved to confer antigen-binding specificity and high affinity by three CDRs from conventional antibodies or fragments thereof. Heavy chain antibodies with high specificity and affinity can be generated against various antigens through immunization, and the VHH portion can be readily cloned and expressed in yeast. Their expression levels, solubility, and stability are significantly higher than those of classical F(ab) or Fv fragments. Nectin Cell Adhesion Molecule 4 (Nectin-4) is a type I transmembrane glycoprotein encoded by the PVRL4 (poliovirus receptor-related 4) gene and is involved in the formation of adherens junctions through homophilic and heterophilic interactions with other nectins and cell adhesion molecules. Nectin-4 plays an essential role in cellular processes such as proliferation, differentiation, and migration, and has been identified as a tumor-associated antigen overexpressed in various human malignancies, including breast, bladder, lung, and ovarian cancers.

[0010] SUMMARY

[0011] In one aspect, this disclosure provides an anti-Nectin-4 heavy chain-only antibody that binds to a Nectin-4. In some embodiments, the anti-Nectin-4 heavy chain-only antibody comprises a heavy chain variable region that comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (h) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; or (iv) SEQ ID NOs: 2, 3, and 19; (v) SEQ ID NOs: 22, 3, and 19.

[0012] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18, or 21.

[0013] In some embodiments, the anti-Nectin-4 antibody comprises a Fc region comprising human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, the anti-Nectin-4 antibody is a bispecific or multi-specific antibody. In some embodiments, the anti-Nectin-4 antibody binds to two different epitopes on the same Nectin-4 protein or two different Nectin-4 proteins.

[0015] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2) or (ii) a human epidermal growth factor 2 (Her2). In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety fused to the N-terminus of a first Fc chain that comprises a T336Y substitution (Knob) according to EU numbering or a Y407T substitution (Hole) according to EU numbering; wherein the second polypeptide chain comprises a Trop2 binding moiety or a Her2 binding moiety fused to the N- terminus of a second Fc chain that comprises a Y407T substitution according to EU numbering or a T336Y substitution according to EU numbering; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0016] In some embodiments, the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (h) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0017] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0018] In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0019] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0020] In some embodiments, the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0021] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0022] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trop2 or (ii) a Her2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a first Trop2 or Her2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the first Trop2 or Her2 binding moiety is fused to the N-terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a second Trop2 or Her2 binding moiety, and the N-terminus of a second Fc chain, and wherein the second Nectin-4 binding moiety or the second Trop2 or Her2 binding moiety is fused to the N- terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0023] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Nectin-4 binding moiety; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

[0024] In some embodiments, the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 or Her2 binding moiety; and the second Trop2 or Her2 binding moiety is fused to the N- terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0025] In some embodiments, the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain; and the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain.

[0026] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the C-terminus of the first Fc chain; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the C-terminus of the second Fc chain.

[0027] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0028] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21. In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0029] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0030] In some embodiments, the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0031] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0032] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0033] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0034] In some embodiments, the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N- terminus of the first Nectin-4, Trop2 or Her2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Nectin- 4, Trop2 or Her2 binding moiety through a linker.

[0035] In some embodiments, the first Nectin-4, Trop2, or Her2 binding moiety is fused to the C- terminus of the first Fc chain through a linker; and / or wherein the second Nectin-4, Trop2, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

[0036] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58-65, or comprises an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58- 65. In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0037] In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or

[0038] 39.

[0039] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0040] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 38,

[0041] 40, 66, or 67, or comprises an amino acid sequence of SEQ ID NOs: 38, 40, 66, or 67.

[0042] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, a Trop2, and a Her2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0043] In some embodiments, the first Her2 binding moiety is fused to the N-terminus of the first Fc chain, the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Her2 binding moiety; and the second Her2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin- 4 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Her2 binding moiety. In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Nectin-4 binding moiety; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

[0044] In some embodiments, the first Trop2 binding moiety is fused to the N-terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety; and the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0045] In some embodiments, the first Trop2 binding moiety is fused to the N-terminus of the first Fc chain, the first Nectin-4 or Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Her2 or Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety; and the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin-4 or Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Her2 or Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0046] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0047] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0048] In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27. In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0049] In some embodiments, the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0050] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0051] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0052] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0053] In some embodiments, the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N- terminus of the first Her2, Nectin-4, or Trop2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Her2, Nectin-4, or Trop2 binding moiety through a linker.

[0054] In some embodiments, the first Nectin-4, or Her2 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and / or wherein the second Nectin-4, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

[0055] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, a Trop2, and a Her2. The fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a Trop2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the Trop2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the second Nectin-4 binding moiety or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0056] In some embodiments, the Trop2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the Trop2 binding moiety; and the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the Her2 binding moiety.

[0057] In some embodiments, the Trop2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain; and the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin- 4 binding moiety is fused to the C-terminus of the second Fc chain.

[0058] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0059] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0060] In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0061] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0062] In some embodiments, the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (ii) SEQ ID NOs: 55, 56, and 57.

[0063] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54. In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0064] In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

[0065] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0066] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety through a linker; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Her2 binding moiety through a linker.

[0067] In some embodiments, the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

[0068] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 90- 94, or comprises an amino acid sequence of SEQ ID NOs: 90-94.

[0069] In one aspect, this disclosure provides a fusion protein that binds a human CD3 and a Nectin-4, or a Trop2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 or Trop2 binding moiety, a first CD3 binding moiety, and a first Fc chain, and wherein the first Nectin-4, or Trop2 binding moiety is fused to the N-terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C-terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4, or Trop2 binding moiety, a second CD3 binding moiety, and a second Fc chain, and wherein the second Nectin-4, or Trop2 binding moiety is fused to the N- terminus of a second Fc chain, and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0070] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0071] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0072] In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0073] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0074] In some embodiments, the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98, and the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

[0075] In some embodiments, the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100- 102, and the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

[0076] In some embodiments, the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 99, or comprises an amino acid sequence of SEQ ID NO: 99; and / or the CD3 binding light chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95.

[0077] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NOs: 104 and 106.

[0078] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0079] In some embodiments, the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminal of the first Fc chain through a linker; and / or the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminal of the second Fc chain through a linker.

[0080] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

[0081] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, a Trop2, and a human CD3. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a first CD3 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety is fused to the N-terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C-terminus of a first Fc chain, wherein the second polypeptide chain comprises a Trop2 binding moiety; a second CD3 binding moiety; and a second Fc chain, and wherein the Trop2 binding moiety is fused to the N-terminus of a second Fc chain; and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain, and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0082] In some embodiments, the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (n) SEQ ID NOs: 9, 10, and 11; (m) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0083] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0084] In some embodiments, the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98; and the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

[0085] In some embodiments, the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100- 102; and the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

[0086] In some embodiments, the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 99, or comprises an amino acid sequence of SEQ ID NO: 99; and / or the CD3 binding light chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95.

[0087] In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0088] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NOs: 104 and 106.

[0089] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0090] In some embodiments, the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminus of the first Fc chain through a linker; and / or the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminus of the second Fc chain through a linker.

[0091] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

[0092] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0093] In some embodiments, the first and second Fc chains comprise a N297G substitution according to EU numbering. In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 104 or 106, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 106 or 104.

[0094] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2), (ii) a human epidermal growth factor 2 (Her2), or (iii) an epidermal growth factor receptor (EGFR), wherein the fusion protein comprises: a first Nectin- 4 binding moiety fused to the N-terminus of a first heavy chain variable region of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the N-terminus of a second heavy chain variable region of the anti-Trop2, anti-Her2, or anti-EGFR antibody; or wherein the fusion protein comprises: a first Nectin-4 binding moiety fused to the C-terminus of a first Fc chain of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the C-terminus of a second Fc chain of the anti-Trop2, anti-Her2, or anti-EGFR antibody.

[0095] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0096] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0097] In some embodiments, the anti-Trop2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43, or comprises an amino acid sequence of SEQ ID NO: 43.

[0098] In some embodiments, the anti-Trop2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 45, or comprises an amino acid sequence of SEQ ID NO: 45.

[0099] In some embodiments, the anti-Trop2 antibody is hRS7.

[0100] In some embodiments, the anti-Her2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 76, or comprises an amino acid sequence of SEQ ID NO: 76.

[0101] In some embodiments, the anti-Her2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 77, or comprises an amino acid sequence of SEQ ID NO: 77.

[0102] In some embodiments, the anti-Her2 antibody is Herceptin.

[0103] In some embodiments, the anti-EGFR antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 85, or comprises an amino acid sequence of SEQ ID NO: 85. In some embodiments, the anti-EGFR antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 86, or comprises an amino acid sequence of SEQ ID NO: 86.

[0104] In some embodiments, the anti-EGFR antibody is Cetuximab.

[0105] In another aspect, this disclosure provides an antibody-drug conjugate comprising the heavy chain-only antibody described herein or the fusion protein described herein that is connected to a cytotoxic agent through a linker.

[0106] In some embodiments, the cytotoxic agent is selected from monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), Trastuzumab emtansine (T-DM1), Ravtansine (DM4), an auristatin, a maytansinoid, a tubulysin, an amberstatin269, an anthracycline, an Exatecan, a DXd, an SN-38, a camptothecin, a pyrrolobenzodiazepine, an indolinobenzodiazepine, a calicheamicin, a duocarmycin, a doxorubicin, an antibiotic, a spliceostatin, a thailanstatin, a derivative thereof, an analog thereof, an isomer thereof, a prodrug thereof, a radioisotope agent, and a pharmaceutically acceptable salt.

[0107] In some embodiments, the cytotoxic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0108] In some embodiments, the linker is a non-cleavable linker or a cleavable linker selected from an acid cleavable linker, a disulfide cleavable linker, a protease cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.

[0109] In some embodiments, the linker is cathepsin B, hydrazone, succinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), maleimidocaproic acid (me), valinecitrulline (vc), N-hydroxy succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N- hydroxy succinimidyl 4-(2-pyridydithio)butanoate (SPDB), N-succinimidyl 4-(2- pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethylene glycol 8-valine- citrulline (PEG8-va), mb-vc, CL2A, a cleavable vc-based linker, mc-Gly-Gly-Phe-Gly (mc-GGFG), mc- Gly-Gly-Phe-Gly-P AB-OH (mc-GGFG-P AB-OH), or a fleximer polymer linker.

[0110] In some embodiments, a linker-cytotoxic agent pair in the antibody-drug conjugate is mc- vc-MMAE, mc-MMAF, SMCC-DM1, sulfo-SPDB-DM4, SPDB-DM4, SPP-DM1, va-SGD1882, polyethylene glycol 8 (PEG8)-va-SG3199, sulfo-SPDB-DGN462, hydrazone-CMl, vc-seco- DUB A, mb-vc-MGBA, CL2A-SN38, mc-GGFG-Exatecan, peptide linker with DX-8951 derivative, hydrazone-doxorubicin, cleavable vc-based linker with AurO 101, vc-PF06380101, fleximer polymer linker with auristatin F, cleavable linker-tubulin inhibitor, or vc-rifalogue.

[0111] In another aspect, this disclosure provides a pharmaceutical composition comprising: the heavy chain-only antibody, the fusion protein, or the antibody-drug conjugate, as described herein.

[0112] In another aspect, this disclosure provides a polynucleotide encoding the heavy chain-only antibody described herein; or the first polypeptide chain and / or the second polypeptide chain of the fusion protein described herein.

[0113] Also within the scope of this disclosure is a vector comprising the polynucleotide described herein or a cell comprising the vector described herein.

[0114] In another aspect, this disclosure provides a method of producing the heavy chain-only antibody or the fusion protein as described herein. In some embodiments, the method comprises growing the cell described herein under conditions permissive for expressing the heavy chain-only antibody or the fusion protein, and isolating the heavy chain-only antibody or the fusion protein from the cells.

[0115] In another aspect, this disclosure provides a kit comprising the heavy chain-only antibody, the fusion protein, the antibody-drug conjugate, or the pharmaceutical composition, as described herein.

[0116] In another aspect, this disclosure further provides a method of treating a disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the heavy chain-only antibody, the fusion protein, the antibody-drug conjugate, or the pharmaceutical composition, as described herein.

[0117] In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is characterized by expression of one or more of Nectin-4, Trop2, Her2, and CD3.

[0118] In some embodiments, the cancer is epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharynx cancer, cancer of the nose, pancreatic cancer, skin cancer, oral cancer, cancer of the tongue, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0119] In another aspect, this disclosure further provides the heavy chain-only antibody, the fusion protein, the antibody-drug conjugate, or the pharmaceutical composition, as described herein, for use in the treatment of cancer.

[0120] In another aspect, this disclosure further provides use of the heavy chain-only antibody, the fusion protein, the antibody-drug conjugate, or the pharmaceutical composition, as described herein, in the treatment of cancer.

[0121] In another aspect, this disclosure additionally provides a method for diagnosing a disease or disorder in a subject or monitoring progression of the disease or disorder in the subject. In some embodiments, the method comprises detecting binding of the heavy chain-only antibody, the fusion protein, or the antibody-drug conjugate, as described herein, to one or more of one or more of Nectin-4, Trop2, Her2, and CD3 in a biological sample of the subject.

[0122] In another aspect, this disclosure also provides an engineered T or NK cell comprising the heavy chain-only antibody or the fusion protein, as described herein.

[0123] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0124] BRIEF DESCRIPTION OF THE DRAWINGS

[0125] FIGs. 1A, IB, and 1C show whole-cell binding (WCB) of top anti-Nectin-4 VHH-Fc clones to human Nectin-4 stably transfected mouse 4T1 cells (4Tl-h-Nectin-4 stable cells, FIG. 1A), OE19 cancer cells (FIG. IB), and HT-1376 cancer cells (FIG. 1C), determined by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. 2A9_Fc, lE3_Fc, and 1C1 l_Fc are lead anti-Nectin-4 VHH-Fc clones.

[0126] FIG. 2 shows whole-cell binding of top anti-Nectin-4 VHH-Fc clones to mouse Nectin-4 stably transfected 293T cells (293T-m-Nectin-4 stable cells), determined by flow cytometry assays with AlexaFluor488 conjugated anti-human IgG Fc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. 2A9_Fc, lE3_Fc, and 1C1 l_Fc are anti-Nectin-4 VHH-Fc clones.

[0127] FIGs. 3A and 3B show competition of lead anti-Nectin-4 VHH-Fc clones to Enfortumab in whole-cell binding. 293T-h-Nectin-4 stable cells (FIG. 3A) or 4Tl-h-Nectin-4 stable cells (FIG. 3B) were pre-incubated with anti-Nectin-4 VHH-Fc antibodies at 500 nM for 30 min. After washing, Enfortumab was added at different concentrations and incubated for 30 min. Binding was detected by adding AlexaFluor 488 conjugated goat anti-human IgG F(ab)2 fragment specific secondary antibody. X axis is values of Enfortumab concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. 2A9_Fc, lE3_Fc, andlCH Fc are anti-Nectin-4 VHH-Fc antibodies. Negative control (Ctrl) is an anti-human PD-L1 VHH-Fc antibody.

[0128] FIGs. 4A and 4B show whole-cell binding of high affinity anti-Nectin-4 VHH-Fc clones to human Nectin-4 stably transfected 4T1 cells (4Tl-h-Nectin-4 stable cells FIG. 4A) and human cancer cells MDA-MB453 (FIG.4B), determined by flow cytometry assays with AlexaFluor488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. 2A9_Fc, lD5_Fc, and 3F4_Fc are anti-Nectin-4 VHH-Fc clones.

[0129] FIGs. 5A, 5B, 5C, 5D, and 5E show schematic diagrams of BsAbs constructed by anti- Nectin-4 (2A9, 1C11 or 1E3) and anti-Trop2 (lHl l-hv8) in different formats: bivalent tandem format (FIG. 5A and FIG. 5C); bivalent N, C-terminal format (FIG. 5B and FIG. 5D); and monovalent format with knob (K)-into-hole (H) (FIG. 5E). FIGs. 6A and 6B show whole-cell binding of anti-Nectin-4 / anti-Trop2 BsAbs in different formats to 4Tl-h-Nectin-4 stable cells (FIG. 6A) or CHO-h-Trop2 stable cells (FIG. 6B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. hRS7 is a reference anti-Trop2 antibody.

[0130] FIG. 7 shows the killing effects of MMAE conjugated anti-Nectin-4 / anti-Trop2 BsAbs in different formats in 4Tl-h-Nectin-4 stable cells. The viable cells were measured by incubating cells with cell counting-8 (CCK-8) reagent and reading at OD 450 nm. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0131] FIG. 8 shows the killing effects of MMAE-conjugated anti-Nectin-2 / anti-Trop2 BsAbs in NCI-H1975 cancer cells. The viable cells were measured by the CCK-8 assay. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0132] FIG. 9 shows killing effects of MMAE conjugated anti-Nectin-2 / anti-Trop2 BsAbs in HT- 1376 cells. The viable cells were measured by the CCK8 assay. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0133] FIG. 10 shows killing effects of MMAE conjugated anti-Nectin-2 / anti-Trop2 BsAbs in SW-780 cells. The viable cells were measured by the CCK-8 assay. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0134] FIGs. 11 A and 11B show the tumor volume data from the in vivo efficacy study. FIG.11A shows the tumor volume data from the in vivo efficacy study of the OE19 tumors in Nu / Nu nude mice. Treatment groups are indicated in the figure legends, with N=8 mice per group. FIG.11B shows the tumor volume data from the in vivo efficacy study of the HT-1376 tumors in Nu / Nu nude mice. Treatment groups are indicated in the figure legends, with N=8 mice per group. FIGs. 12A and 12B show schematic diagrams of BsAbs constructed by anti-Nectin-4 (2A9, 1D5, 3F4, 1C11 or 1E3) and an IgGl anti-Trop2 (hRS7) in two different formats: VHH anti- Nectin-4 fused to the N-terminal of Heavy Chain (FIG. 12A) and the VHH anti-Nectin-4 fused to the C-terminal of Fc region (FIG. 12B).

[0135] FIGs. 13A and 13B show whole-cell binding of anti-Nectin-4 / anti-Trop2 BsAbs to 4T1- h-Nectin-4 stable cells (FIG. 13A) or CHO-h-Trop2 stable cells (FIG. 13B), by flow cytometry assays with AlexaFluor488 conjugated anti-human IgG Fc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. hRS7 is a reference anti- Trop2 antibody.

[0136] FIGs. 14A and 14B show killing effects of MMAE conjugated anti-Nectin-2 / anti-Trop2 BsAbs in 4Tl-h-Nectin-4 (FIG. 14A) and 293T-h-Trop2 (FIG. 14B) stable cells. The viable cells were measured by the CCK-8 assay. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0137] FIGs. 15A, 15B, 15C, 15D, and 15E show schematic diagrams of BsAbs constructed by anti-Nectin-4 (2A9, 1D5, 3F4, 1C11 or 1E3) and anti-Her2 (3E12-hv5 or 5E4-hv2) in different formats: bivalent tandem format (FIGs. 15A and 15C); bivalent N, C-terminal format (FIGs. 15B and 15D); and monovalent format with knob (K)-into-hole (H) (FIG. 15E).

[0138] FIGs. 16A and 16B show whole-cell binding of anti-Nectin-4 / anti-Her2 BsAbs in different formats to 4Tl-h-Nectin-4 stable cells (FIG. 16A) or CHO-h-Her2 stable cells (FIG. 16B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. Herceptin is a reference anti-Her2 antibody.

[0139] FIGs. 17A and 17B show the killing effects of MMAE conjugated anti-Nectin-4 / anti-Her2 BsAbs in OE19 cancer cells (FIG. 17A) and 293T-h-Nectin-4 stable cells (FIG. 17B). The viable cells were measured by the CCK-8 assay. X axis is values of antibody concentration in nanomolar. Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends. FIGs. 18A and 18B show whole-cell binding of anti-Nectin-4 / anti-Her2 BsAbs with biparatopic Her2 binders to CHO-h-Her2 stable cells (FIG. 18A) or 4Tl-h-Nectin-4 stable cells (FIG. 18B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgG Fc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Herceptin is a reference anti-Her2 antibody. Enfortumab is a reference anti-Nectin-4 antibody.

[0140] FIGs. 19A and 19B show the tumor volume data from the in vivo efficacy study. FIG. 19A shows the tumor volume data from the in vivo efficacy study of the OE19 tumors in Nu / Nu nude mice. Treatment groups are indicated in the figure legends, with N=8 mice per group. FIG. 19B shows the tumor volume data from the in vivo efficacy study of the HT-1376 tumors in Nu / Nu nude mice. Treatment groups are indicated in the figure legends, with N=8 mice per group.

[0141] FIGs. 20A and 20B show schematic diagrams of BsAbs constructed by the VHH anti- Nectin-4 (2A9, 1D5, 3F4, 1C11 or 1E3) and the IgGl anti-Her2 (Herceptin) in two different formats: the VHH anti-Nectin-4 fused at the N-terminal of the Heavy Chain of Herceptin (FIG. 20A) and the VHH anti-Nectin-4 fused to the C-terminal of Fc region (FIG. 20B).

[0142] FIGs. 21A and 21B show whole-cell binding of anti-Nectin-4 / anti-Her2 BsAbs in different formats to 4Tl-h-Nectin-4 stable cells (FIG. 21A) or CHO-h-Her2 stable cells (FIG. 21B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab was used as a reference anti-Nectin-4 antibody. Herceptin was used as a reference anti-Her2 antibody.

[0143] FIGs. 22A and 22B show whole-cell binding of different clones of anti-Nectin-4 / anti-Her2 BsAbs to 4Tl-h-Nectin-4 stable cells (FIG. 22A) or CHO-h-Her2 stable cells (FIG. 22B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. Herceptin is a reference anti-Her2 antibody.

[0144] FIGs. 23A and 23B show the killing effects of MMAE conjugated anti-Nectin-4 / anti-Her2 BsAbs in 4Tl-h-Nectin-4 stable cells (FIG. 23A) and OE19 cancer cells (FIG. 23B). The viable cells were measured by the CCK-8 assay. X axis is values of antibody concentration in nanomolar. 1 Y axis is values of OD at 450 nm. Names of MMAE conjugated antibodies are shown in figure legends.

[0145] FIGs. 24A and 24B show schematic diagrams of BsAbs constructed by the VHH anti- Nectin-4 (2A9, 1D5, 3F4, 1C11 or 1E3) and the IgGl anti-EGFR (Cetuximab) in two different formats: the VHH anti-Nectin-4 fused in the N-terminal of the Heavy Chain (FIG. 24A) and the VHH anti-Nectin-4 fused to the C-terminal of Fc region (FIG. 24B).

[0146] FIGs. 25A and 25B show whole-cell binding of anti-Nectin-4 / anti-EGFR BsAbs in different formats to 4Tl-h-Nectin-4 stable cells (FIG. 25A) or 293T-h-EGFR stable cells (FIG. 25B), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Cetuximab is a reference anti-EGFR antibody. Enfortumab is a reference anti-Nectin-4 antibody.

[0147] FIGs. 26A and 26B show schematic diagrams of Trispecific Antibodies constructed by the VHH anti-Nectin-4 (2A9, 1D5, 3F4, 1C11 or 1E3), anti-Her2 (3E12-hv5 or 5E4-hv2) and anti- Trop2 (1H1 l-hv8) in two different formats: TriAbs constructed with bivalent VHH anti-Nectin-4 fused at the N-terminal of VHH (FIG. 26A) or the C-terminal of Fc (FIG. 26B) and monovalent VHH anti-Trop2 and anti-Her2 fused at the N-terminal of hinge region, all with mutation of knob- into-hole in Fc.

[0148] FIGs. 27A and 27B show schematic diagram of anti-Her2 / anti-CD3 or anti-Trop2 / anti- CD3 BiTEs (FIG. 27 A) or anti-Nectin-4 / anti-Trop2 / anti-CD3 TriTEs (FIG. 27B) in different formats with anti-CD3 at the C-terminal of Fc. K stands for a mutation of knob. H stands for a mutation of hole. G stands for a mutation of N297G.

[0149] FIGs. 28A, 28B, and 28C show whole-cell binding of anti-Nectin-4 / anti-Her2 / anti-CD3 TriTEs, or anti-Nectin-4 / anti-CD3 BiTE, or anti-Trop2 / anti-CD3 BiTE to 4Tl-h-Nectin-4 stable cells (FIG. 28A), CHO-h-Trop2 stable cells (FIG. 28B), or Jurkat cells (FIG. 28C), by flow cytometry assays with AlexaFluor 488 conjugated anti-human IgGFc as a secondary antibody. X axis is values of antibody concentration in nanomolar. Y axis is values of Median Fluorescence Intensity of AlexaFluor 488. Enfortumab is a reference anti-Nectin-4 antibody. hRS7 is a reference anti-Trop2 antibody. Mosunetuzumab is a reference anti-CD20 / anti-CD3 BiTE. FIGs. 29A and 29B show TriTEs and BiTEs induced activation of human CD3 in Jurkat cells stably transfected with NF AT luciferase reporter gene in the presence of 4T-h-Nectin-4 stable cells (FIG. 29A) or 293T-h-Trop2 stable cells (FIG. 29B). X axis is values of antibody concentration in nanomolar. Y axis is values of NF AT activity in Relative Light Unit (RLU). Names of antibodies are shown in figure legends.

[0150] DETAILED DESCRIPTION OF THE INVENTION

[0151] This disclosure provides novel heavy chain-only antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins thereof that bind to Nectin Cell Adhesion Molecule 4 (Nectin-4), related antibody-drug conjugates, related nucleic acids, related cells, related kits, related compositions, and related methods or uses.

[0152] Heavy Chain-Only Antibodies (HCAb)

[0153] In another aspect, this disclosure provides an anti-Nectin-4 heavy chain-only antibody that binds to a Nectin-4 (e.g., human Nectin-4).

[0154] As used herein, “Nectin-4,” also known as poliovirus receptor-related protein 4 (PVRL4), LNIR, PRIM, and EDSS1, is a member of the nectin subfamily of immunoglobulin-like adhesion molecules that participate in Ca2+-independent cell-cell adhesion. Nectins bind to the actin cytoskeleton through the adaptor protein afadin (AFDN) and are key components of adherens junctions. Nectin-4 contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It may be a single pass type I membrane protein or a soluble form is produced by proteolytic cleavage at the cell surface by the metalloproteinase ADAMI 7 / T ACE. Nectin-4 is overexpressed in multiple human cancers, including but not limited to triple negative breast cancer, bladder cancer, urothelial cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, skin cancer, and esophageal cancer. Its abnormal expression may be associated with cancer progression and poor prognosis. Nectin-4 includes mammalian Nectin-4 proteins, e.g., mouse, rat, rabbit, guinea pig, pig, sheep, dog, non-human primate, and human. In some embodiments, Nectin- 4 is a human Nectin-4 (encoded by GenBank accession number AF426163) or a mouse Nectin-4 (see Reymond et al., Journal of Biological Chemistry 276:43205-15, 2001).

[0155] In one aspect, this disclosure provides an anti-Nectin-4 heavy chain-only antibody that binds to a Nectin-4. In some embodiments, the anti-Nectin-4 heavy chain-only antibody comprises a heavy chain variable region that comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (n) SEQ ID NOs: 9, 10, and 11; (hi) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0156] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1 , 8, or 13, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18, or 21.

[0157] In some embodiments, the anti-Nectin-4 antibody is a bispecific or multi-specific antibody. In some embodiments, the anti-Nectin-4 antibody binds to two different epitopes on the same Nectin-4 protein or two different Nectin-4 proteins.

[0158] The terms “heavy chain-only antibody,” “heavy-chain antibody,” and “HCAb” are used interchangeably, and refer, in the broadest sense, to antibodies lacking the light chain of a conventional antibody. Since the homodimeric HCAbs lack a light chain and thus a VL domain, the antigen is recognized by one single domain, i.e., the variable domain of the heavy chain of a heavy-chain antibody (VHH). The term specifically includes, without limitation, homodimeric antibodies comprising the VHH antigen-binding domain and the CH2 and CH3 constant domains, in the absence of the CHI domain; functional (antigen-binding) variants of such antibodies, soluble VHH variants, Tg-NAR comprising a homodimer of one variable domain (V-NAR) and five C- like constant domains (C-NAR) and functional fragments thereof; and soluble single domain antibodies (sdAbs). In one embodiment, the heavy chain-only antibody comprises the variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In one embodiment, the heavy chain-only antibody comprises an antigen-binding domain, at least part of a hinge region, and CH2 and CH3 domains. In another embodiment, the heavy chain-only antibody comprises an antigen-binding domain, at least part of a hinge region, and a CH2 domain. In a further embodiment, the heavy chain- only antibody comprises an antigen-binding domain, at least part of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domain is truncated are also included herein. In some embodiments, the heavy chain includes an antigen binding domain and at least one CH (CHI, CH2, CH3, or CH4) domain but no hinge region. The heavy chain-only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise covalently or non-covalently attached to each other. The heavy chain-only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD, and IgE subclass, are also included herein. In a particular embodiment, the heavy chain antibody is of the IgGl , IgG2, IgG3, or IgG4 subtype, particularly the IgGl subtype. In one embodiment, the heavy chain-only antibodies herein are used as a binding (targeting) domain of a chimeric antigen receptor (CAR).

[0159] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0160] The term “variable,” as used in connection with antibodies, refers to the fact that certain portions of the antibody variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen but exhibit various effector functions, such as the participation of the antibody in antibody- dependent cellular cytotoxicity (ADCC). The term “hypervariable region,” when used herein, refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 31-35 (Hl), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a “hypervariable loop” residues 26-32 (Hl), 53- 55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework Region” or “FR” residues are variable domain residues other than the hypervariable region residues as defined herein.

[0161] Exemplary CDR designations are shown herein; however, one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge-based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010; 47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877-883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001; 309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008; 181 :6230- 6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004; 17: 132-143; and Padlan et al. “Identification of specificitydetermining residues in antibodies.” Faseb J. 1995; 9: 133-139., each of which is herein specifically incorporated by reference.

[0162] An “isolated” antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0163] In some embodiments, the CH3 domains of the multispecific (e.g., bispecific) antibody can be altered by the “knob-into-hole” technology, described in detail with several examples in, e.g., WO 96027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681; W098 / 050431. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the “knob,” while the other is the “hole.” Thus in one embodiment of the invention the antibody according to the invention (comprises a CH3 domain in each heavy chain and) is further characterized in that the first CH3 domain of the first heavy chain of the antibody under a) and the second CH3 domain of the second heavy chain of the antibody under b) each meet at an interface which comprises an original interface between the antibody CH3 domains, wherein the interface is altered to promote the formation of the antibody according to the invention, wherein the alteration is characterized in that: i) the CH3 domain of one heavy chain is altered, so that within the original interface of the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the antibody according to the invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and ii) the CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the antibody according to the invention an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.

[0164] In some embodiments, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W). In some embodiments, both CH3 domains are further altered by introducing cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.

[0165] Other techniques for CH3 -modifications to enforcing the heterodimerization are contemplated as alternatives of the invention and described, e.g., in WO96 / 27011, W098 / 050431, EP1870459, W02007 / 110205, W02007 / 147901, W02009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 157953, and WO2013 / 096291, the disclosures of which are incorporated herein by reference.

[0166] In some embodiments, the anti-Nectin-4 antibody comprises a Fc region comprising human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0167] In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering, or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0168] In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

[0169] In some embodiments, the first and second Fc chains comprise a N297G substitution according to EU numbering.

[0170] Fragment

[0171] In some embodiments, an antibody or a fusion protein provided herein can be an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and single-chain Fv (scFv) fragments, and other fragments described below, e.g., diabodies, triabodies tetrabodies, and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0172] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0173] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (DOMANHS, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

[0174] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0175] Chimeric and Humanized Antibodies

[0176] In some embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g, in U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a nonhuman variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or nonhuman primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0177] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g, CDRs (or portions thereof), are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non- human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0178] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann etal., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25- 34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0179] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g, Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g, Carter etal. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611-22618 (1996)).

[0180] Human Antibodies

[0181] In some embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art or using techniques described herein. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0182] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g, U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE technology; U.S. Pat. No. 5,770,429 describing HUMAB technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0183] Human antibodies can also be made using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li etal., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0184] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0185] Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0186] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter etal., Ann. Rev. Immunol., 12: 433- 455 (1994). Phage typically displays antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725- 734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V- gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0187] Variants

[0188] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. Substitution, Insertion, and Deletion Variants

[0189] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are defined herein. Amino acid substitutions may be introduced into an antibody of interest, and the products are screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0190] Accordingly, an antibody of this disclosure can comprise one or more conservative modifications of the CDRs, heavy chain variable region, or light variable regions described herein. A conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed in this invention refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It substantially retains the activity of the parent peptide, polypeptide, or protein (such as those disclosed in this invention). In general, a conservative modification or functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to a parent. Accordingly, within the scope of this invention are heavy chain variable regions or light variable regions having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof, as well as antibodies having the variant regions.

[0191] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0192] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0193] Additionally or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).

[0194] As used herein, the term “conservative modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: (i) amino acids with basic side chains (e.g., lysine, arginine, histidine), (ii) acidic side chains (e.g., aspartic acid, glutamic acid), (iii) uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), (iv) nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), (v) beta-branched side chains (e.g., threonine, valine, isoleucine), and (vi) aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0195] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described in, e.g., Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, N.J., (2001). Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C- terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide, which increases the serum half-life of the antibody.

[0196] Glycosylation Variants

[0197] In some embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0198] For example, an agly coslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0199] Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g, 298, to thereby reduce glycosylation on N297.

[0200] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art. They can be used as host cells to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant Chinese Hamster Ovary cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which result in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0201] Fc Region Variants

[0202] The variable regions of the antibody described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgGl, IgG2, IgG3 or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgGl: Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); forIgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(gl), G3m28(g5), G3ml l(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Kml, Km2, Km3 (see, e.g., Jefferies etal. (2009) mAbs 1 : 1). In some embodiments, the antibodies variable regions described herein are linked to an Fc that binds to one or more activating Fc receptors (Fcyl, Fcylla, or Fcyllla), and thereby stimulate ADCC and may cause T cell depletion. In some embodiments, the antibody variable regions described herein are linked to an Fc that causes depletion.

[0203] In some embodiments, the antibody variable regions described herein may be linked to an Fc comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigendependent cellular cytotoxicity. Furthermore, an antibody described herein may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to

[0204] 31 alter its glycosylation, to alter one or more functional properties of the antibody. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0205] The Fc region encompasses domains derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, including a fragment, analog, variant, mutant, or derivative of the constant region. Suitable immunoglobulins include IgGl, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide homologous to the immunoglobulin C-terminal region. It can include a CHI domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination. In some embodiments, an antibody of this invention has an Fc region other than that of a wild type IgAl. The antibody can have an Fc region from that of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) or other classes such as IgA2, IgD, IgE, and IgM. The Fc can be a mutant form of IgAl .

[0206] The constant region of an immunoglobulin is responsible for many important antibody functions, including Fc receptor (FcR) binding and complement fixation. There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses: IgGl, IgG2, IgG3, and IgG4.

[0207] Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibodies, namely FcyRI, FcyRII, and FcyRIIL. The important sequences for binding IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains. The serum half- life of an antibody is influenced by the ability of that antibody to bind to an FcR.

[0208] In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and / or biological activity. For example, one may make modifications in the Fc region to generate an Fc variant that (a) has increased or decreased ADCC, (b) increased or decreased CDC, (c) has increased or decreased affinity for Clq and / or (d) has increased or decreased affinity for an Fc receptor relative to the parent Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region may include two, three, four, five, etc., substitutions therein, e.g., of the specific Fc region positions identified herein.

[0209] A variant Fc region may also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain held together non-covalently. In other embodiments, the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the Clq binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgGl. In some embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites. In other embodiments, an Fc region may be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) about ADCC sites in IgGl. Specific examples of variant Fc domains are disclosed, for example, in WO 97 / 34631 and WO 96 / 32478.

[0210] In one embodiment, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of Fc is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward etal. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter etal.

[0211] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished CDC. This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[0212] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94 / 29351 by Bodmer et al.

[0213] In yet another example, the Fc region may be modified to increase ADCC and / or to increase the affinity for an Fey receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263,

[0214] 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296,

[0215] 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332,

[0216] 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434,

[0217] 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0218] Fc modifications that increase binding to a Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (WO00 / 42072).

[0219] Other Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and / or complement proteins, thereby decreasing or ablating Fc-mediated effector functions such as ADCC, antibody-dependent cellular phagocytosis (ADCP), and CDC. Exemplary modifications include but are not limited to substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index. Exemplary substitutions include but are not limited to 234G, 235 G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index. An Fc variant may comprise 236R / 328R. Other modifications for reducing FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 33 IS, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0220] Optionally, the Fc region may comprise a non-naturally occurring amino acid residue at additional and / or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; WO00 / 42072; WOOl / 58957; W002 / 06919; W004 / 016750; W004 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; W004 / 063351; W005 / 070963; W005 / 040217, WO05 / 092925 and W006 / 020114).

[0221] In some embodiments, Fc variants that enhance affinity for the inhibitory Fc gamma receptor FcyRIIb may be employed. Such Fc variants may confer immune-modulatory activity on an Fc fusion protein through interactions with FcyRIIb-expressing immune cells, including, for example, B cells and monocytes. In some embodiments, the Fc variants exhibit selectively increased binding affinity to FcyRIIb relative to one or more activating Fc gamma receptors (e.g., FcyRIIIa). Modifications that alter binding affinity to FcyRIIb include one or more amino acid substitutions at positions selected from the group consisting of residues 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, based on EU numbering. Exemplary amino acid substitutions that enhance FcyRIIb binding include, but are not limited to: 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. In some embodiments, substitutions for enhancing FcyRIIb binding include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. In further embodiments, combinations of amino acid substitutions may be used to enhance FcyRIIb binding. Exemplary combination variants include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F..

[0222] The affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological-based assays) known in the art, including but not limited to equilibrium methods (e.g., ELISA or radioimmunoassay), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the examined components and / or employ various detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0223] In some embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this may be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton etal. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton etal. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305 A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591- 6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 IS, 433R, 433 S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 31 IS (Dall Acqua etal. Journal of Immunology, 2002, 169:5171-5180, Dall’Acqua et al., 2006, Journal ofBiological Chemistry 281:23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671. In some embodiments, hybrid IgG isotypes with particular biological characteristics may be used. For example, an IgGl / IgG3 hybrid variant may be constructed by substituting IgG 1 positions in the CH2 and / or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments described herein, an IgGl / IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and / or CH3 region with amino acids from IgGl at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to an insertion of a glycine at position 236), and 321 h.

[0224] Moreover, the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to FcyRIII. Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which has been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al., 2001). Other IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52- specific), trastuzumab (HER2 / neu- specific), rituximab (CD20- specific), and cetuximab (EGFR- specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgGl mutants containing L235V, F243L, R292P, Y300L, and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that may be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0225] In some embodiments, an Fc is chosen that has reduced binding to FcyRs. An exemplary Fc, e.g., IgGl Fc, with decreased FcyR binding, comprises the following three amino acid substitutions: L234A, L235E, and G237A.

[0226] In some embodiments, a Fc that has reduced complement fixation is chosen. An exemplary Fc, e.g, IgGl Fc, with reduced complement fixation, has the following two amino acid substitutions: A330S and P331S.

[0227] In some embodiments, an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation. An exemplary Fc, e.g, IgGl Fc, that is effectorless, comprises the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0228] When using an IgG4 constant domain, it is usually preferable to include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules.

[0229] Antibody Derivatives

[0230] An antibody provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water-soluble polymers.

[0231] Non-limiting examples of water-soluble polymers include, but are not limited to, PEG, copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3 -di oxolane, poly-l,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, poly oxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. The number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0232] In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al. , Proc. Natl. Acad. Sci. USA 102: 11600- 11605 (2005)). The radiation may be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

[0233] Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with PEG, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In some embodiments, pegylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0 154 316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0234] The present invention also encompasses a human monoclonal antibody described herein conjugated to a therapeutic agent, a polymer, a detectable label, or an enzyme. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the polymer is PEG.

[0235] Multivalent Antibodies

[0236] In one embodiment, the antibodies or fusion proteins comprising the antibodies or antigen binding fragments thereof as disclosed on may be monovalent or multivalent (e.g., bivalent, trivalent, etc.). As used herein, the term “valent” refers to a specified number of binding sites in an antibody molecule.

[0237] A “multi-valent” antibody has two or more binding sites. Thus, the terms “bivalent,” “trivalent,” and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, an antibody disclosed herein can be bivalent, trivalent, tetravalent, or multi- valent.

[0238] Various methods and protein configurations are known and used for the preparation of bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, and the like.

[0239] As used herein, the term “valency” refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds one target molecule or a specific position or locus on a target molecule. When an antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may specifically bind to the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen). See, for example, U.S.P.N. 2009 / 0130105. In each case, at least one of the binding sites will comprise an epitope, motif, or domain associated with a DLL3 isoform.

[0240] Antibodies of the invention include multi-specific antibodies. Multi-specific antibodies have more than one binding specificity. The term “multi-specific” specifically includes “bispecific” and “trispecific,” as well as higher-order independent specific binding affinities, such as higher- order polyepitopic specificity, as well as tetravalent antibodies and antibody fragments. “Multispecific” antibodies specifically include antibodies comprising a combination of different binding entities as well as antibodies comprising more than one of the same binding entity. The terms “multi-specific antibody,” multi-specific single chain-only antibody,” and “multi-specific HCAb” are used herein in the broadest sense and cover all antibodies with more than one binding specificity.

[0241] In one embodiment, the antibodies are bispecific antibodies in which the two chains have different specificities, as described in Millstein et al., 1983, Nature, 305:537-539. Other embodiments include antibodies with additional specificities, such as trispecific antibodies. Other more sophisticated compatible multispecific constructs and methods of their fabrication are set forth in U.S.P.N. 2009 / 0155255, as well as WO 94 / 04690; Suresh et al., 1986, Methods in Enzymology, 121:210; and WO96 / 27011.

[0242] As stated above, multivalent antibodies may immunospecifically bind to different epitopes of the desired target molecule or may immunospecifically bind to both the target molecule as well as a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, the multivalent antibodies may include bispecific or trispecific antibodies. Bispecific antibodies also include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0243] In some embodiments, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences, such as an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and / or CH3 regions, using methods well known to those of ordinary skill in the art.

[0244] Anti-Nectin-4 / Trop2 (or Herl) Bispecific Antibodies

[0245] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2) or (ii) a human epidermal growth factor 2 (Her2). In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety fused to the N-terminus of a first Fc chain that comprises a T336Y substitution (Knob) according to EU numbering or a Y407T substitution (Hole) according to EU numbering; wherein the second polypeptide chain comprises a Trop2 binding moiety or a Her2 binding moiety fused to the N- terminus of a second Fc chain that comprises a Y407T substitution according to EU numbering or a T336Y substitution according to EU numbering; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region. In some embodiments, the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (h) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0246] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, or 13, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0247] In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0248] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 18, or comprises an amino acid sequence of SEQ ID NO: 24.

[0249] In some embodiments, the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0250] Her2, or human epidermal growth factor receptor 2, is a protein that helps regulate how cells grow and divide. When cancer cells, such as those in breast, ovarian, bladder, pancreatic, stomach, or esophageal cancers, produce higher than normal amounts of Her2, they can grow and spread more quickly. This can lead to tumors and uncontrolled growth.

[0251] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54. In some embodiments, the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein with hinge of SEQ ID NO: 6 may form two disulfide-bonds (the critical structure for vc-MMAE conjugation), leading to DAR4 being generated by MMAE conjugation. In some embodiments, the fusion protein with a hinge may form three disulfide-bonds, leading to DAR6 being generated by MMAE conjugation.

[0252] As used herein, the term “linker” refers to any means, entity, or moiety used to join two or more entities. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked. The linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center, such as a platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea, and the like. To provide for linking, the domains can be modified by oxidation, hydroxylation, substitution, reduction, etc., to provide a site for coupling. Methods for conjugation are well-known by persons skilled in the art and are encompassed for use in the present disclosure. Linker moieties include, but are not limited to, chemical linker moieties, or for example, a peptide linker moiety (a linker sequence).

[0253] A peptide linker can range from 2 amino acids to 60 or more amino acids. In some embodiments, a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length. In some embodiments, a peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length. In some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length. In some embodiments, the linker comprises polar (e.g., serine (S)) or charged (e.g., lysine (K)) residues. In some embodiments, the linker is a flexible linker, e.g., comprising one or more glycine (G) or serine (S) residues. Examples of flexible linkers that can be used in the fusion protein of the disclosure include those disclosed by Chen etal., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines, e.g., represented by [Ser]m[Gly]n, where m or n is an integer from 1 to 20 (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Polyglycine linkers can suitably be used in the fusion protein of the disclosure. In some embodiments, a peptide linker comprises two or more consecutive glycines, represented by [Gly]n where n is an integer from 1 to 20 (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0254] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trop2 or (ii) a Her2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a first Trop2 or Her2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the first Trop2 or Her2 binding moiety is fused to the N-terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a second Trop2 or Her2 binding moiety, and the N-terminus of a second Fc chain, and wherein the second Nectin-4 binding moiety or the second Trop2 or Her2 binding moiety is fused to the N- terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0255] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Nectin-4 binding moiety; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

[0256] In some embodiments, the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 or Her2 binding moiety; and the second Trop2 or Her2 binding moiety is fused to the N- terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0257] In some embodiments, the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain; and the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain.

[0258] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the C-terminus of the first Fc chain; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the C-terminus of the second Fc chain.

[0259] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0260] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, or 13, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0261] In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0262] Trop2 is a transmembrane protein that is highly expressed in many malignant tumors, but has limited expression in normal human tissues. It is also known as tumor-associated calcium signal transducer 2 (Trop-2), epithelial glycoprotein- 1 antigen, and pancreatic carcinoma marker protein GA733-1 / GA733. Trop2 is encoded by the TACSTD2 gene.

[0263] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0264] In some embodiments, the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0265] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 44 or 48, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0266] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0267] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0268] In some embodiments, the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N- terminus of the first Nectin-4, Trop2 or Her2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Nectin- 4, Trop2 or Her2 binding moiety through a linker.

[0269] In some embodiments, the first Nectin-4, Trop2, or Her2 binding moiety is fused to the C- terminus of the first Fc chain through a linker; and / or wherein the second Nectin-4, Trop2, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

[0270] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58-65, or comprises an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58-65.

[0271] In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0272] In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

[0273] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0274] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 38, 40, 66, or 67, or comprises an amino acid sequence of SEQ ID NOs: 38, 40, 66, or 67.

[0275] Anti-Trop2 / Her2 / Nectin-4 Tri-Specific Antibodies

[0276] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, a Trop2, and a Her2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0277] In some embodiments, the first Her2 binding moiety is fused to the N-terminus of the first Fc chain, the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Her2 binding moiety; and the second Her2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin- 4 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Her2 binding moiety.

[0278] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Nectin-4 binding moiety; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

[0279] In some embodiments, the first Trop2 binding moiety is fused to the N-terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety; and the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0280] In some embodiments, the first Trop2 binding moiety is fused to the N-terminus of the first Fc chain, the first Nectin-4 or Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Her2 or Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety; and the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin-4 or Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Her2 or Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

[0281] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0282] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21. In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0283] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0284] In some embodiments, the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0285] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0286] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

[0287] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0288] In some embodiments, the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N- terminus of the first Her2, Nectin-4, or Trop2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Her2, Nectin-4, or Trop2 binding moiety through a linker.

[0289] In some embodiments, the first Nectin-4, or Her2 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and / or wherein the second Nectin-4, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker. In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, a Trop2, and a Her2. The fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a Trop2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the Trop2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the second Nectin-4 binding moiety or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0290] In some embodiments, the Trop2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the Trop2 binding moiety; and the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the Her2 binding moiety.

[0291] In some embodiments, the Trop2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain; and the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin- 4 binding moiety is fused to the C-terminus of the second Fc chain.

[0292] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0293] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0294] In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27. In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0295] In some embodiments, the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0296] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0297] In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0298] In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

[0299] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0300] In some embodiments, the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety through a linker; and the second Nectin-4 binding moiety is fused to the N-terminus of the second Her2 binding moiety through a linker.

[0301] In some embodiments, the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain through a linker. In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 79-83, or comprises an amino acid sequence of SEQ ID NOs: 90-94.

[0302] Anti-Nectin-4 / CD3 and Anti-Trop2 / CD3 BITEs

[0303] In one aspect, this disclosure provides a fusion protein that binds a human CD3 and (i) a Nectin-4, or (ii) a Trop2. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin- 4, Trop2, or Her2 binding moiety, a first CD3 binding moiety, and a first Fc chain, and wherein the first Nectin-4, or Trop2binding moiety is fused to the N-terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C-terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4, or Trop2 binding moiety, a second CD3 binding moiety, and a second Fc chain, and wherein the second Nectin-4, Trop2, or Her2 binding moiety is fused to the N-terminus of a second Fc chain, and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

[0304] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0305] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18, or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0306] In some embodiments, the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27. In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0307] In some embodiments, the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98, and the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

[0308] Cluster of differentiation 3 (CD3) is a protein complex that activates T cells. It’s a defining feature of T cells and can be used as a marker for them. Anti-CD3 antibodies are used to characterize T cells. CD3 is made up of four polypeptide chains: epsilon (s), gamma (y), delta (8), and zeta (Q. These chains assemble into three pairs of dimers: sy, s8, and CD3 is a common T- lymphocyte antigen, found on the surface of all T-lymphocytes. A low absolute CD3 cell count may indicate immunodeficiency or compromised immune function. High counts may suggest conditions like infections or autoimmune disorders.

[0309] In some embodiments, the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100- 102; and the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

[0310] In some embodiments, the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 94, or comprises an amino acid sequence of SEQ ID NO: 94; and / or the CD3 binding light chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95.

[0311] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NOs: 104-106.

[0312] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0313] In some embodiments, the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminal of the first Fc chain through a linker; and / or the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminal of the second Fc chain through a linker.

[0314] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

[0315] Anti-Nectin-4 / CD3 / Trop2 TriTEs

[0316] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4, and (i) a Trophoblast cell surface antigen-2 (Trop2), and a human CD3. In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a first CD3 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety is fused to the N-terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C-terminus of a first Fc chain, wherein the second polypeptide chain comprises a Trop2 binding moiety; a second CD3 binding moiety; and a second Fc chain, and wherein the Trop2 binding moiety is fused to the N-terminus of a second Fc chain; and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain, and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region. In some embodiments, the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (h) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0317] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

[0318] In some embodiments, the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98; and the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

[0319] In some embodiments, the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100- 102; and the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

[0320] In some embodiments, the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 99, or comprises an amino acid sequence of SEQ ID NO: 99; and / or the CD3 binding light chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95. In some embodiments, the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

[0321] In some embodiments, the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

[0322] In some embodiments, the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

[0323] In some embodiments, the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 44 or 48, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

[0324] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the Fc region comprises an amino acid sequence of SEQ ID NOs: 104 and 106.

[0325] In some embodiments, the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering. In some embodiments, the N- terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

[0326] In some embodiments, the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminus of the first Fc chain through a linker; and / or the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminus of the second Fc chain through a linker.

[0327] In some embodiments, the first or second polypeptide chain comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

[0328] In some embodiments, the Fc region comprises a human IgGl. In some embodiments, the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

[0329] In some embodiments, the first and second Fc chains comprise a N297G substitution according to EU numbering. In some embodiments, the first Fc chain comprises the amino acid sequence of SEQ ID NO: 104 and 106, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 106-104.

[0330] Anti-Nectin-4 / (hRS7, Herceptin, or Cetuximab) Bispecific Antibodies

[0331] In one aspect, this disclosure provides a fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2), (ii) a human epidermal growth factor 2 (Her2), or (iii) an epidermal growth factor receptor (EGFR), wherein the fusion protein comprises: a first Nectin- 4 binding moiety fused to the N-terminus of a first heavy chain variable region of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the N-terminus of a second heavy chain variable region of the anti-Trop2, anti-Her2, or anti-EGFR antibody; or wherein the fusion protein comprises: a first Nectin-4 binding moiety fused to the C-terminus of a first Fc chain of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the C-terminus of a second Fc chain of the anti-Trop2, anti-Her2, or anti-EGFR antibody.

[0332] In some embodiments, the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising CDR1, CDR2, and CDR3 having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

[0333] In some embodiments, the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18, or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18, or 21.

[0334] In some embodiments, the anti-Trop2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 43, or comprises an amino acid sequence of SEQ ID NO: 43.

[0335] In some embodiments, the anti-Trop2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NOs: 45, or comprises an amino acid sequence of SEQ ID NOs: 45.

[0336] In some embodiments, the anti-Trop2 antibody is hRS7. hRS7 (also referred to as Sacituzumab) is an anti-Trop2 recombinant monoclonal antibody to Trop2 (EGP-1).

[0337] In some embodiments, the anti-Her2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 76, or comprises an amino acid sequence of SEQ ID NO: 76.

[0338] In some embodiments, the anti-Her2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 77, or comprises an amino acid sequence of SEQ ID NO: 77.

[0339] In some embodiments, the anti-Her2 antibody is Herceptin (Trastuzumab). Herceptin is a monoclonal antibody used to treat breast cancer and stomach cancer. It is used for cancer that is HER2 receptor positive.

[0340] In some embodiments, the anti-EGFR antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 85, or comprises an amino acid sequence of SEQ ID NO: 85.

[0341] In some embodiments, the anti-EGFR antibody comprises a light chain variable region that comprises an amino acid sequence having at least 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of SEQ ID NO: 86, or comprises an amino acid sequence of SEQ ID NO: 86.

[0342] In some embodiments, the anti-EGFR antibody is Cetuximab. Cetuximab is a chimeric monoclonal antibody consisting of human IgGl kappa constant regions and murine variable regions. It specifically targets the extracellular domain of the epidermal growth factor receptor (EGFR), thereby inhibiting tumor growth

[0120] , Upon binding to the EGFR, cetuximab blocks ligand-induced dimerization and signaling, which are essential for cell growth and tumor survival.

[0343] Antibody-Drug Conjugates (ADCs)

[0344] In another aspect, this disclosure provides an antibody-drug conjugate comprising the heavy chain-only antibody (VHH) described herein or the fusion protein described herein that is connected to a cytotoxic agent through a linker.

[0345] As used herein, the term “antibody-drug-conjugate” or “ADC” refers to a binding protein, such as an antibody or antigen binding fragment thereof (or a fusion protein) as disclosed, chemically linked to one or more chemical drug(s) (also referred to herein as agent(s)) that may optionally be therapeutic or cytotoxic agents. In some embodiments, an ADC includes an antibody, a cytotoxic or therapeutic drug, and a linker that enables attachment or conjugation of the drug to the antibody. An ADC typically has anywhere from 1 to 8 drugs conjugated to the antibody, including drug-loaded species of 2, 4, 6, or 8.

[0346] In some embodiments, the ADC has the following formula (formula I): Ab-(L-D), (I) wherein Ab is the antibody and (L-D) is a Linker-Drug moiety. The Linker-Drug moiety is made of L- which is a Linker, and -D, which is a drug moiety having, for example, cytostatic, cytotoxic, or otherwise therapeutic activity against a target cell, e.g., a cell expressing Her2; and n is an integer from 1 to 20. In some embodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is 1. The DAR of an ADC is equivalent to the “n” referred to in Formula I. In one embodiment, the ADC has a formula of Ab-(L-D), wherein Ab is an anti-Nectin-4 antibody, L is a linker, e.g., valine citrulline (vc), D is a drug, e.g., an auristatin such as MMAF or MMAE, and n is 2 to 4 (equivalent to a DAR of 2-4). Additional details regarding drugs (D of Formula I) and linkers (L of Fornula I) that may be used in the ADCs of the invention, as well as alternative ADC structures, are described below.

[0347] As used herein, the terms “drug,” “agent,” and “drug moiety” are used interchangeably herein. The terms “linked” and “conjugated” are also used interchangeably herein and indicate that the antibody and moiety are covalently linked.

[0348] Non-limiting examples of drugs that may be included in the ADCs are mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.

[0349] The term “drug-to-antibody ratio” or “DAR” refers to the number of drugs, e.g., auristatin, attached to the antibody of the ADC. The DAR of an ADC can range from 1 to 8, although higher loads, e.g., 10, are also possible depending on the number of linkage sites on an antibody. The term DAR may be used in reference to the number of drugs loaded onto an individual antibody, or, alternatively, may be used in reference to the average or mean DAR of a group of ADCs.

[0350] The term “undesired ADC species,” as used herein, refers to any drug-loaded species to be separated from an ADC species with a different drug load. In one embodiment, the term undesired ADC species may refer to drug-loaded species of 6 or more, i.e., ADCs with a DAR of 6 or more, including DAR6, DAR7, DAR8, and DAR greater than 8 (i.e., drug-loaded species of 6, 7, 8, or greater than 8). In a separate embodiment, the term undesired ADC species may refer to drug- loaded species of 8 or more, i.e., ADCs with a DAR of 8 or more, including DAR8, and DAR greater than 8 (i.e., drug-loaded species of 8 or greater than 8).

[0351] The term “ADC mixture,” as used herein, refers to a composition containing a heterogeneous DAR distribution of ADCs. In one embodiment, an ADC mixture contains ADCs having a distribution of DARs of 1 to 8, e.g., 2, 4, 6, and 8 (i.e., drug-loaded species of 2, 4, 6, and 8). Notably, degradation products may result such that DARs of 1, 3, 5, and 7 may also be included in the mixture. Further, ADCs within the mixture may also have DARs greater than 8. The ADC mixture results from interchain disulfide reduction followed by conjugation. In one embodiment, the ADC mixture comprises both ADCs with a DAR of 4 or less (i.e., a drug-loaded species of 4 or less) and ADCs with a DAR of 6 or more (i.e., a drug-loaded species of 6 or more).

[0352] In some embodiments, the cytotoxic agent is an auristatin (such as monomethyl auristatin E (MMAE), monomethyl auristatinF (MMAF), AurOlOl, PF06380101, Auristatin W, or auristatin F) or derivative thereof, a maytansinoid (such as DM1 or DM4), a pyrrolobenzodiazepine (PBD) (such as SGD1882 or SG3199), an indolinobenzodiazepine (such as DGN462 or DGN549), a calicheamicin (ozogamicin) (such as CM1), a camptothecin analog (such as SN38, DX-8951f, or DX- 895If derivative), a duocarmycin (such as seco-duocarmycin-hydroxy-benzamide-azaindole (seco-DUB A), minor groove-binding alkylating agent (MGB A), or MED-2460), a tubulin inhibitor (such as cryptophycin), a tubulysin or tubulysin analog (such as AZ 13599185), amberstatin269, doxorubicin, an antibiotic (such as rifalogue), an anthracycline (such as PNU- 159682), a microtubule inhibitor (such as rhizoxin), a spliceostatin, or a thailanstatin.

[0353] In some embodiments, the cytotoxic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0354] An ADC comprises an antibody (or fusion protein) and at least one drug(s), whereby the antibody and at least one drug are conjugated by a linker. The term “linker,” as used herein, refers to a chemical moiety that may be bifunctional or multifunctional, and is used to attach an antibody to a drug moiety. A linker may include one conjugating component or may include multiple components.

[0355] For example, the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of the antibody or improving the solubility of the ADC. Other examples of components of linkers include a stretcher unit and an amino acid unit.

[0356] At least two methods are commonly used for conjugating drugs to antibodies: alkylation of reduced interchain cysteine disulfides through an enzymatically non-cleavable maleimide or simple and cleavable disulfide linker and acylation of lysines by cleavable linear amino acids. In some embodiments, a linker covalently attaches an antibody to a drug moiety. An ADC is prepared using a linker having reactive functionality for binding to the antibody and the drug. For example, a cysteine thiol, or an amine, e.g., N-terminus or amino acid side chain such as lysine, of the antibody may form a bond with a functional group of the linker.

[0357] In one aspect, a linker has a functionality that can react with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, oc-haloacetyl, activated esters such as succinimide esters, 4- nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 ofKlussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773.

[0358] In some embodiments, a linker has a functionality that can react with an electrophilic group present on an antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0359] Exemplary linker components include 6-maleimidocaproyl, maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p- aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N- maleimidomethyl)cyclohexane-l carboxylate (“MCC”).

[0360] In one aspect, an anti-EGFR antibody is conjugated to an auristatin, e.g., MMAE, via a linker comprising maleimidocaproyl (“me”), valine citrulline (val-cit or “vc”), and PAB A (referred to as a “mc-vc-PABA linker”). Maleimidocaproyl acts as a linker to the anti-EGFR antibody and is not cleavable. Val-cit is a dipeptide that is an amino acid unit of the linker and allows for cleavage of the linker by a protease, specifically the protease cathepsin B. Thus, the val- cit component of the linker provides a means for releasing the auristatin from the ADC upon exposure to the intracellular environment. Within the linker, p-aminobenzylalcohol (PAB A) acts as a spacer and is self-immolative, allowing for the release of the MMAE. The structure of the mc- vc-PABA-MMAE linker is provided in Figure 11. Suitable linkers include, for example, cleavable and non-cleavable linkers. A linker may be a “cleavable linker,” facilitating drug release. Nonlimiting exemplary cleavable linkers include acid- labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127- 131 (1992); U.S. Pat. No. 5,208,020). A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as a lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.

[0361] In some embodiments, linkers are stable extracellularly in a sufficient manner to be therapeutically effective. Before transport or delivery into a cell, the ADC is preferably stable and remains intact, i.e., the antibody remains conjugated to the drug moiety. Linkers that are stable outside the target cell may be cleaved at some efficacious rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g, cytotoxic effect, of a drug moiety.

[0362] In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the drug from the antibody in the intracellular environment to be therapeutically effective. In some embodiments, the cleavable linker is pH- sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acyl hydrazone bond (see, e.g, U.S. Pat. No. 5,622,929).

[0363] In other embodiments, the linker is cleavable under reducing conditions (e.g, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2- pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N- succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.).

[0364] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, e.g, a. peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes that are present in EGFR-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stabilities of the conjugates are typically high.

[0365] In other embodiments, the linker is a mal onate linker (Johnson et al. , 1995, Anti cancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12), or a 3’-N-amide analog thereof.

[0366] In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. See U.S. Publication No. 20050238649 incorporated by reference herein in its entirety. An ADC comprising a non-cleavable linker may be designed such that the ADC remains substantially outside the cell and interacts with certain receptors on a target cell surface such that the binding of the ADC initiates (or prevents) a particular cellular signaling pathway. In some embodiments, the linker is substantially hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cancer cells through MDR (multiple drug resistance) or functionally similar transporters.

[0367] In some embodiments, the linker directly or indirectly inhibits cell growth and / or cell proliferation upon cleavage. For example, in some embodiments, the linker, upon cleavage, can function as an intercalating agent, thereby inhibiting macromolecular biosynthesis (e.g., DNA replication, RNA transcription, and / or protein synthesis).

[0368] In some embodiments, the linker is designed to facilitate bystander killing (killing neighboring cells) through diffusion of the linker-drug and / or the drug alone to neighboring cells. In some embodiments, the linker promotes cellular internalization.

[0369] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond, enhancing the potency of the ADC. Thus, in one embodiment, the linker includes a sterically hindered disulfide linkage. A sterically hindered disulfide refers to a disulfide bond present within a particular molecular environment, wherein the environment is characterized by a particular spatial arrangement or orientation of atoms, typically within the same molecule or compound, which prevents or at least partially inhibits the reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains proximal to the disulfide bond prevents or at least partially inhibits the disulfide bond from potential interactions that would result in the reduction of the disulfide bond.

[0370] The aforementioned linker types are not mutually exclusive. For example, in one embodiment, the linker used in the ADCs described herein is a non-cleavable linker that promotes cellular internalization.

[0371] In some embodiments, the linker is a non-cleavable linker or a cleavable linker selected from an acid cleavable linker, a disulfide cleavable linker, a protease cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.

[0372] In some embodiments, the linker is cathepsin B, hydrazone, succinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), maleimidocaproic acid (me), valinecitrulline (vc), N-hydroxy succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N- hydroxy succinimidyl 4-(2-pyridydithio)butanoate (SPDB), N-succinimidyl 4-(2- pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethylene glycol 8-valine- citrulline (PEG8-va), mb-vc, CL2A, a cleavable vc-based linker, a fleximer polymer linker, or mc-Gly-Gly- Phe-Gly (mc-GGFG), mc-Gly-Gly-Phe-Gly-PAB-OH (mc-GGFG-PAB-OH).

[0373] In some embodiments, a linker-cytotoxic agent pair in the antibody-drug conjugate is vc- MMAE, mc-MMAF, SMCC-DM1, sulfo- SPDB -DM4, SPDB-DM4, SPP-DM1, va-SGD1882, polyethylene glycol 8 (PEG8)-va-SG3199, sulfo- SPDB-DGN462, hydrazone-CMl, vc-seco- DUBA, mb-vc-MGBA, CL2A-SN38, peptide linker with DX-8951 derivative, mc-GGFG- Exatecan, hydrazone-doxorubicin, cleavable vc-based linker with AurO 101, vc-PF06380101, fleximer polymer linker with auristatin F, cleavable linker-tubulin inhibitor, or vc-rifalogue.

[0374] Nucleic Acids, Expression Cassettes, and Vectors

[0375] The present invention provides isolated nucleic acid segments that encode the polypeptides, peptide fragments, antibodies, and fusion proteins. The nucleic acid segments of the invention also include segments that encode the same amino acids due to the degeneracy of the genetic code. For example, the amino acid threonine is encoded by ACU, ACC, ACA, and ACG and is, therefore, degenerate. It is intended that the invention includes all variations of the polynucleotide segments that encode for the same amino acids. Such mutations are known in the art (Watson el al., Molecular Biology of the Gene, Benjamin Cummings 1987). Mutations also include alteration of a nucleic acid segment to encode for conservative amino acid changes, for example, the substitution of leucine for isoleucine and so forth. Such mutations are also known in the art. Thus, the genes and nucleotide sequences of the invention include both naturally occurring sequences and mutant forms.

[0376] The nucleic acid segments of the invention may be contained within a vector. A vector may include, but is not limited to, any plasmid, phagemid, F-factor, virus, cosmid, or phage in a double- or single-stranded linear or circular form, which may or may not be self-transmissible or mobilizable. The vector can also transform a prokaryotic or eukaryotic host either by integration into the cellular genome or existing extra-chromosomally (e.g., an autonomously replicating plasmid with an origin of replication).

[0377] The nucleic acid segment in the vector can be under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in vitro or in a host cell, such as a eukaryotic cell, or a microbe, e.g., bacteria. The vector may be a shuttle vector that functions in multiple hosts. The vector may also be a cloning vector that typically contains one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion. Such insertion can occur without loss of essential biological function of the cloning vector. A cloning vector may also contain a marker gene suitable for the identification and selection of cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (Stratagene, New England Biolabs, Clonetech).

[0378] The nucleic acid segments of the invention may also be inserted into an expression vector. Typically, an expression vector contains prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the amplification and selection of the expression vector in a bacterial host; regulatory elements that control initiation of transcription such as a promoter; and DNA elements that control the processing of transcripts such as introns, or a transcription termination / polyadenylation sequence.

[0379] Methods to introduce nucleic acid segments into a vector are available in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, a vector into which a nucleic acid segment is to be inserted is treated with one or more restriction enzymes (restriction endonuclease) to produce a linearized vector having a blunt end, a “sticky” end with a 5' or a 3' overhang, or any combination of the above. The vector may also be treated with a restriction enzyme and subsequently treated with another modifying enzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase, to create a linearized vector that has characteristics useful for ligation of a nucleic acid segment into the vector. The nucleic acid segment to be inserted into the vector is treated with one or more restriction enzymes to create a linearized segment with a blunt end, a “sticky” end with a 5' or a 3' overhang, or any combination of the above. The nucleic acid segment may also be treated with a restriction enzyme and subsequently treated with another DNA-modifying enzyme. Such DNA modifying enzymes include, but are not limited to, polymerase, exonuclease, phosphatase, or a kinase to create a nucleic acid segment with characteristics useful for ligating a nucleic acid segment into the vector.

[0380] 1 The treated vector and nucleic acid segment are then ligated together to form a construct containing a nucleic acid segment according to methods available in the art (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001). Briefly, the treated nucleic acid fragment, and the treated vector are combined in the presence of a suitable buffer and ligase. The mixture is then incubated under appropriate conditions, allowing the ligase to ligate the nucleic acid fragment into the vector.

[0381] The invention also provides an expression cassette containing a nucleic acid sequence capable of directing expression of a particular nucleic acid segment, either in vitro or in a host cell. Also, a nucleic acid segment of the invention may be inserted into the expression cassette to produce an anti-sense message. The expression cassette is an isolatable unit such that the expression cassette may be in linear form and functional for in vitro transcription and translation assays. The materials and procedures to conduct these assays are commercially available from Promega Corp. (Madison, Wis.). For example, an in vitro transcript may be produced by placing a nucleic acid sequence under the control of a T7 promoter and then using T7 RNA polymerase to produce an in vitro transcript. This transcript may then be translated in vitro using a rabbit reticulocyte lysate. Alternatively, the expression cassette can be incorporated into a vector, allowing for replication and amplification of the expression cassette within a host cell or also in vitro transcription and translation of a nucleic acid segment.

[0382] Such an expression cassette may contain one or a plurality of restriction sites, allowing for the nucleic acid segment to be placed under the regulation of a regulatory sequence. The expression cassette can also contain a termination signal operably linked to the nucleic acid segment as well as regulatory sequences required for proper translation of the nucleic acid segment. The expression cassette containing the nucleic acid segment may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Expression of the nucleic acid segment in the expression cassette may be controlled by a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.

[0383] The expression cassette may include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a nucleic acid segment, and a transcriptional and translational termination region functional in vivo and / or in vitro. The termination region may be native to the transcriptional initiation region, may be native to the nucleic acid segment, or may be derived from another source.

[0384] The regulatory sequence can be a polynucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, which influences the transcription, RNA processing, or stability, or translation of the associated coding sequence. Regulatory sequences can include, but are not limited to, enhancers, promoters, repressor binding sites, translation leader sequences, introns, and polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. While regulatory sequences are not limited to promoters, some useful regulatory sequences include constitutive promoters, inducible promoters, regulated promoters, tissue-specific promoters, viral promoters, and synthetic promoters.

[0385] A promoter is a nucleotide sequence that controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. A promoter includes a minimal promoter, consisting only of all basal elements needed for transcription initiation, such as a TATA-box and / or initiator that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A promoter may be derived entirely from a native gene, composed of elements derived from different promoters found in nature, or even composed of synthetic DNA segments. A promoter may contain DNA sequences involved in binding protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0386] The invention also provides a construct containing a vector and an expression cassette. The vector may be selected from, but not limited to, any vector previously described. Into this vector may be inserted an expression cassette through methods known in the art and previously described (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). In one embodiment, the regulatory sequences of the expression cassette may be derived from a source other than the vector into which the expression cassette is inserted. In another embodiment, a construct containing a vector and an expression cassette is formed upon inserting a nucleic acid segment of the invention into a vector containing regulatory sequences. Thus, an expression cassete is formed upon inserting the nucleic acid segment into the vector. Vectors containing regulatory sequences are available commercially, and methods for their use are known in the art (Clonetech, Promega, Stratagene).

[0387] In another aspect, this disclosure also provides (i) a nucleic acid molecule encoding a polypeptide chain of the antibody or antigen-binding fragment thereof or the fusion protein described herein, (ii) a vector comprising the nucleic acid molecule as described, and (iii) a cultured host cell comprising the vector as described. Also provided is a method for producing a polypeptide, comprising: (a) obtaining the cultured host cell as described; (b) culturing the cultured host cell in a medium under conditions permiting expression of a polypeptide encoded by the vector and assembling of an antibody or fragment thereof, and (c) purifying the antibody or fragment from the cultured cell or the medium of the cell.

[0388] Methods of Production

[0389] Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody (or a fusion protein) described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) or lymphoid cell (e.g, YO, NSO, Sp20 cell). In one embodiment, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of an antibody, a nucleic acid encoding an antibody, e.g., as described herein, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0390] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are unnecessary. For expression of antibody fragments and polypeptides in bacteria, see, e.g, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N. J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0391] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

[0392] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified, which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0393] Plant cell cultures can also be utilized as hosts. See, e.g, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES technology for producing antibodies in transgenic plants).

[0394] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g, in Mather, Biol. Reprod. 23:243- 251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include CHO cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0395] CAR-T or CAR-NKT

[0396] In another aspect, this disclosure also provides an engineered T or NK cell comprising the heavy chain-only antibody (VHH) or the fusion protein, as described herein.

[0397] Also provided is a method for preparing an engineered T or NK cell comprising the heavy chain-only antibody or the fusion protein, as described herein. In some embodiments, the method may additionally include expanding the first plurality of lymphocytes in a cell culture medium following the step of introducing the first nucleic acid or expanding the second plurality of lymphocytes in a cell culture medium following the step of introducing the second nucleic acid.

[0398] As used herein, the term “culturing” or “expanding” refers to maintaining or cultivating cells under conditions in which they can proliferate and avoid senescence. For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include neoantigen peptides. Stable cell lines may be established to allow for the continued propagation of cells.

[0399] Prior to the expansion and genetic modification of the lymphocytes described herein, a source of lymphocytes from a subject is obtained. Lymphocytes can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. As described herein, any number of lymphocyte lines available in the art can be used. Lymphocytes can be obtained from a unit of blood collected from a subject using any number of techniques known to the person skilled in the art, such as the Ficoll™ separation. Circulating blood cells of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated white blood cells, red blood cells, and platelets. The cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for the subsequent processing steps. The cells may be washed with phosphate-buffered saline (PBS). Alternatively, the wash solution may lack calcium and may lack magnesium or may lack many, if not all, divalent cations. As those of ordinary skill in the art would readily appreciate, a washing step can be achieved by methods known to those skilled in the art, such as using a semiautomatic continuous flow centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CytoMate, or elHaemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+ free, PBS free Mg2+, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells resuspended directly in a culture medium.

[0400] Lymphocytes may be isolated from peripheral blood by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugal elutriation. If needed, specific subpopulation lymphocytes, such as T lymphocytes (i.e., Cd3 +, CD28 +, CD4 +, CD8 +, CD45RA + or CD45RO + T lymphocytes) can be further isolated by positive or negative selection techniques. For example, T lymphocytes may be isolated by incubation with conjugated anti-CD3 / anti-CD28 beads (i.e., 3x28), such as DYNABEADS® M-450 CD3 / CD28 T, for a sufficient period of time i.e., 30 minutes to 24 hours) for positive selection of the desired T lymphocytes. For the isolation of T lymphocytes from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cellular performance. Longer incubation times can be used to isolate T lymphocytes in any situation where there are few T lymphocytes compared to other cell types, such as isolating tumor- infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. The person skilled in the art will recognize that multiple rounds of selection may also be used. It may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also undergo new rounds of selection.

[0401] Enrichment of a population of lymphocytes (e.g., T lymphocytes) by negative selection can be performed with a combination of antibodies directed to unique surface markers for the negatively selected cells. One method is the sorting and / or selection of cells by negative magnetic immune adherence or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present in the negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody typically includes antibodies against CD 14, CD20, GDI lb, CD16, HLA-DR, and CD8. Alternatively, the regulatory T lymphocytes are depleted by anti-C25 conjugate beads or other similar selection method.

[0402] Lymphocytes for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, freezing and the following thawing step provide a more uniform product by eliminating granulocytes and, to some extent, monocytes in the cell population. After the washing step that removes the plasma and platelets, the cells can be suspended in a freezing solution. Although many solutions and freezing parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose human albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% of dextrose, 20% serum of human albumin and 7.5% of DMSO or other suitable cell freezing medium containing for example Hespan and PlasmaLyte A. The cells may then be frozen at -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0403] The cryopreserved cells may be thawed and washed as described herein and allowed to stand for one hour at room temperature before activation using the methods of the present invention. As described herein, lymphocytes can be expanded, frozen, and used later. As described herein, samples may be collected from a patient shortly after the diagnosis of a particular disease as described herein, but before any treatment. The cells may be isolated from a blood sample or an apheresis of a subject before any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablatories such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit calcium-dependent calcineurin phosphatase (e.g., ciclosporin and FK506) or inhibit p70S6 kinase that is important for signaling induced by the growth factor (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316- 321, 1991, Bierer et al., Curr. Opin. Immun., 5: 763-773, 1993). The cells may be isolated from a patient and frozen for later use together with (e.g., before, simultaneously or after) bone marrow or stem cell transplant, therapy with T lymphocyte ablation using chemotherapeutic agents such as fludarabine, radiotherapy external beam (XRT), cyclophosphamide, or antibodies such as 0KT3 or CAMPATH. As described herein, the cells may be isolated before and can be frozen for later use in the treatment after therapy with ablation of B lymphocytes, such as agents that react with CD20, for example, Rituxan.

[0404] Either before or after the genetic modification of lymphocytes (e.g., T lymphocytes) to express a desirable transgene, lymphocytes can be activated and expanded generally using methods such as those described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and the publication of US patent application. No. 20060121005.

[0405] Compositions and Kits

[0406] In another aspect, this disclosure provides a pharmaceutical composition comprising the antibodies or fusion proteins described herein formulated together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a therapeutic agent.

[0407] In some embodiments, the pharmaceutical comprises two or more of the antibody or antigen-binding fragments thereof described herein, such as any combinations of the antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain that comprise the respective amino acid sequences described herein.

[0408] The pharmaceutical compositions also can be administered in a combination therapy with, for example, another immune- stimulatory agent, an antiviral agent, a vaccine, etc. In some embodiments, a composition comprises an antibody of this invention at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100- 300 mg / ml.

[0409] In some embodiments, the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound. In some embodiments, the antiviral compound comprises a nucleoside analog, a peptoid, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA-dependent RNA polymerase. In some embodiments, the antiviral compound may include acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, or an interferon. In some embodiments, the interferon is an interferon-a or an interferon-p.

[0410] Also within the scope of this disclosure is use of the pharmaceutical composition in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof of a disease or disorder, such as cancer.

[0411] The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface-active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0412] Preferably, a pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase “parenteral administration,” as used herein, means modes of administration other than enteral and topical administration, usually by injection. It includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the present invention described herein can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically.

[0413] The pharmaceutical compositions may be prepared in many forms, including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, liposomes, and other slow-release formulations, such as shaped polymeric gels. An oral dosage form may be formulated to release the antibody into the intestine after passing through the stomach. Such formulations are described in U.S. Pat. No. 6,306,434 and in the references contained therein.

[0414] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

[0415] An antibody can be formulated for parenteral administration (e.g., by injection, bolus injection or continuous infusion). It may be presented in the unit dosage form in ampules, prefilled syringes, small-volume infusion containers, or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.

[0416] For administration by inhalation, an antibody can be conveniently delivered from an insufflator, nebulizer, a pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoromethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0417] Alternatively, for administration by inhalation or insufflation, an antibody may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch. The powder composition may be presented in a unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator. For intra-nasal administration, an antibody may be administered via a liquid spray, such as via a plastic bottle atomizer.

[0418] Pharmaceutical compositions of the invention may also contain other ingredients such as flavorings, colorings, anti-microbial agents, or preservatives. It will be appreciated that the amount of an antibody required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient. Ultimately, the attendant healthcare provider may determine a proper dosage. In addition, a pharmaceutical composition may be formulated as a single unit dosage form.

[0419] The pharmaceutical composition of the present invention can be in the form of sterile aqueous solutions or dispersions. It can also be formulated in a microemulsion, liposome, or other ordered structure suitable for high drug concentration.

[0420] An antibody of the present invention described herein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of disease symptoms. Thereafter, the patient can be administered a prophylactic regime.

[0421] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition, which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of active ingredient in combination with a pharmaceutically acceptable carrier.

[0422] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. For administration of the antibody, the dosage ranges from about 0.0001 to 800 mg / kg, and more usually 0.01 to 5 mg / kg of the host body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. Preferred dosage regimens for an antibody of this disclosure include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pg / ml, and in some methods, about 25-300 pg / ml. A “therapeutically effective dosage” of an antibody of this disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of cancer in a subject, a “therapeutically effective dosage” may inhibit cell growth by at least about 20%, by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects.

[0423] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0424] Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro-infusion pumps (US 4,487,603); (3) transdermal devices (US 4,486,194); (4) infusion apparatus (US 4,447,233 and 4,447,224); and (5) osmotic devices (US 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference. In some embodiments, the human monoclonal antibodies of the invention described herein can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic compounds of the invention cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g, US 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade (1989) Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman etal. (1995) FEBS Lett. 357: 140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe etal. (1995) Am. Physiol. 1233:134; Schreier etal. (1994). Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346: 123; and Killion and Fidler (1994) Immunomethods 4:273.

[0425] In some embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0426] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see, e.g., Wu etal. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). It may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in a vesicle, in particular, a liposome (see, for example, Langer (1990) Science 249: 1527-1533).

[0427] The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M. el al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995, each incorporated herein in its entirety.

[0428] The pharmaceutical composition can be delivered in a controlled release system in certain situations. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the composition’s target, thus requiring only a fraction of the systemic dose.

[0429] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intracranial, intraperitoneal, intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, sesame oil, soybean oil, etc., may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0430] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0431] Numerous reusable pens and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eh Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to, the SOLOSTAR™ pen (Sanofi- Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name only a few.

[0432] Advantageously, the pharmaceutical compositions for oral or parenteral use described herein are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 300 mg and in about 10 to about 300 mg for the other dosage forms.

[0433] In another aspect, this disclosure provides a kit comprising a pharmaceutically acceptable dose unit of the antibody or antigen-binding fragment thereof or the pharmaceutical composition as described herein. Also within the scope of this disclosure is a kit for the diagnosis, prognosis, or monitoring of cancer treatment in a subject. In some embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described and at least one detection reagent that binds specifically to the antibody or antigen-binding fragment thereof. In some embodiments, the kit includes a container containing the composition and optionally informational material. The informational material can be descriptive, instructional, marketing, or other material related to the methods described herein and / or the use of the agents for therapeutic benefit. In an embodiment, the kit also includes an additional therapeutic agent, as described herein. For example, the kit includes a first container that contains the composition and a second container for the additional therapeutic agent.

[0434] The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the composition, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the composition, e.g., in a suitable dose, dosage form, or mode of administration, to treat a subject in need thereof. In one embodiment, the instructions provide a dosing regimen, dosing schedule, and / or route of administration of the composition or the additional therapeutic agent. The information can be provided in various formats, including printed text, computer-readable material, video recording, audio recording, or information that contains a link or address to substantive material.

[0435] The kit can include one or more containers for the composition. In some embodiments, the kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition can be contained in a bottle or vial, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle or vial that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g. , a dosage form described herein) of the agents.

[0436] The kit optionally includes a device suitable for administration of the composition or other suitable delivery devices. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading. Such a kit may optionally contain a syringe to allow for injection of the antibody contained within the kit into an animal, such as a human.

[0437] Methods of Use

[0438] Methods of Treatment In another aspect, this disclosure further provides a method of treating a disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the heavy chain-only antibody, the fusion protein, the antibody-drug conjugate, or the pharmaceutical composition, as described herein.

[0439] As used herein, the terms “subject” and “patient” are used interchangeably, irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human.

[0440] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or the physician. Thus, the term “treatment” includes preventing a condition from occurring in a patient, particularly when the patient is predisposed to acquiring the condition; reducing and / or inhibiting the condition and / or its development and / or progression; and / or ameliorating and / or reversing the condition. Insofar as some embodiments of the methods of the presently disclosed subject matter are directed to preventing conditions, it is understood that the term “prevent” does not require that the condition be completely thwarted. Instead, as used herein, the term “preventing” refers to the ability of one of ordinary skill in the art to identify a population susceptible to the condition, such that administration of the compositions of the presently disclosed subject matter might occur before the onset of the condition. The term does not imply that the condition must be avoided entirely. The term “inhibiting cell growth” or “inhibiting proliferation of cells” refers to reducing or halting the growth rate of cells. For example, by inhibiting the growth of tumor cells, the rate of increase in size of the tumor may slow. In other embodiments, the tumor may stay the same size or decrease in size, i.e., regress. In particular embodiments, the rate of cell growth or cell proliferation is inhibited by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0441] The term “disease” as used herein is intended to be generally synonymous and is used interchangeably with the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

[0442] In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is characterized by expression of one or more of Nectin-4, Trop2, Her2, EGFR, and CD3.

[0443] As used herein, “cancer,” “tumor,” and “malignancy” all relate equivalently to hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune system, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. The methods described herein can be used to treat lymphatic cells, circulating immune cells, and solid tumors.

[0444] Cancers that can be treated include tumors that are not vascularized or substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (such as hematologic tumors, e.g., leukemias and lymphomas) or solid tumors. The types of cancers to be treated with the disclosed compositions include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemias or malignant lymphoid tumors, benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas. Also included are adult tumor s / cancers and pediatric tumors / cancers.

[0445] Hematologic cancers, including malignancies of the blood, bone marrow, and lymphatic system, are suitable for treatment with the disclosed compositions. Non-limiting examples include leukemias such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), promyelocytic leukemia, erythroleukemia, hairy cell leukemia, and megakaryoblastic leukemia. Lymphomas, including Hodgkin’s lymphoma and various forms of non-Hodgkin’s lymphoma (e.g., B cell, T cell, NK cell, follicular, and mantle cell lymphomas), are also included. Plasma cell malignancies such as multiple myeloma and Waldenstrom’s macroglobulinemia, as well as myeloproliferative neoplasms like polycythemia vera, myelodysplastic syndrome (MDS), and myelofibrosis, are also encompassed.

[0446] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat gland, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma of pheochromocytomas, carcinoma papillary, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma) (such as brainstem glioma and mixed gliomas), glioblastoma (also astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

[0447] Non-limiting examples of tumors that can be treated by the methods described herein include, for example, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. Non-limiting specific examples, include, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, renal cancer, bladder cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancers of all histopathologic types, angiosarcoma, hemangiosarcoma, bone sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, cancers associated with viral infection (such as but not limited to human papilloma virus (HPV) associated tumors (e.g., cancer cervix, vagina, vulva, head and neck, anal, and penile carcinomas)), Ewing’s tumor, leiomyosarcoma, Ewing’s sarcoma, rhabdomyosarcoma, carcinoma of unknown primary (CUP), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom’ s macroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma, bronchogenic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, lung carcinoma, epithelial carcinoma, cervical cancer, testicular tumor, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia, neuroblastoma, small cell lung carcinoma, bladder carcinoma, lymphoma, multiple myeloma, medullary carcinoma, B cell lymphoma, T cell lymphoma, NK cell lymphoma, large granular lymphocytic lymphoma or leukemia, gamma-delta T cell lymphoma or gamma-delta T cell leukemia, mantle cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, hematopoietic neoplasias, thymoma, sarcoma, nonHodgkin’s lymphoma, Hodgkin’s lymphoma, Epstein-Barr virus (EBV) induced malignancies of all types including but not limited to EBV-associated Hodgkin’s and non-Hodgkin’s lymphoma, all forms of post-transplant lymphomas including post-transplant lymphoproliferative disorder (PTLD), uterine cancer, renal cell carcinoma, hepatoma, hepatoblastoma.

[0448] Cancers that may be treated by methods and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.

[0449] In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lympho epithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglio neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythro leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0450] The anti-tumor responses after treatment by the methods disclosed herein may be determined in xenograft tumor models. Tumors may be established using any human cancer cell line expressing the TAAs presented by the viral particles. To establish xenograft tumor models, about 5x 106viable cells may be injected e.g. s.c into nude athymic mice using, for example, Matrigel (Becton Dickinson). The endpoint of the xenograft tumor models can be determined based on the size of the tumors, weight of animals, survival time, and histochemical and histopathological examination of the cancer, using methods known to one skilled in the art.

[0451] In some embodiments, the cancer is epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharynx cancer, cancer of the nose, pancreatic cancer, skin cancer, oral cancer, cancer of the tongue, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0452] Combination Therapies

[0453] Combination therapies may include an antibody described herein and any additional therapeutic agent that may be advantageously combined with an antibody of this disclosure or with a biologically active fragment of an antibody of this disclosure. The antibodies of the present invention may be combined synergistically with one or more drugs or therapies used to treat a disease or disorder (e.g., cancer). In some embodiments, the antibodies of the invention may be combined with a second therapeutic agent to ameliorate one or more symptoms of said disease. In some embodiments, the antibodies of the invention may be combined with a second antibody to provide synergistic activity in ameliorating one or more symptoms of said disease. In some embodiments, the first antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second antibody or antigen-binding fragment thereof.

[0454] For example, the antibody described herein can be used in various detection methods for use in, e.g., monitoring the progression of cancer; monitoring patient response to treatment, etc. The present disclosure provides methods for detecting a neuraminidase polypeptide in a biological sample obtained from an individual. The methods generally involve: a) contacting the biological sample with a subject anti-neuraminidase antibody, and b) detecting binding, if any, of the antibody to an epitope present in the sample. In some instances, the antibody comprises a detectable label. The level of neuraminidase polypeptide detected in the biological sample can indicate the stage, degree, or severity of the disease or disorder. The level of the neuraminidase polypeptide detected in the biological sample can indicate the individual’s response to treatment for the disease or disorder.

[0455] In some embodiments, the second therapeutic agent is another antibody, as described herein. It is contemplated herein to use a combination (“cocktail”) of antibodies as described herein. In some embodiments, non-competing antibodies may be combined and administered to a subject in need thereof. In some embodiments, the antibodies comprising the combination bind to distinct non-overlapping epitopes on the protein. In some embodiments, the second antibody may possess a longer half-life in human serum.

[0456] As used herein, the term “in combination with” means that additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the antibody disclosed herein. The term “in combination with” also includes sequential or concomitant administration of an antibody described herein and a second therapeutic agent.

[0457] The additional therapeutically active component(s) may be administered to a subject prior to administration of an antibody as described herein. For example, a first component may be deemed to be administered “prior to” a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an antibody as described herein of the present invention. For example, a first component may be deemed to be administered “after” a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an antibody as described herein. “Concurrent” administration, for purposes of the present invention, includes, e.g., administration of an antibody as described herein and an additional therapeutically active component to a subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the antibody as described herein and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the antibody as described herein may be administered intravenously, and the additional therapeutically active component may be administered orally). In any event, administering the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered “concurrent administration” for purposes of the present disclosure. For purposes of the present disclosure, administration of an antibody as described herein “prior to,” “concurrent with,” or “after” (as those terms are defined hereinabove) administration of an additional therapeutically active component is considered administration of an antibody as described herein “in combination with” an additional therapeutically active component.

[0458] The present invention includes pharmaceutical compositions in which an antibody as described herein is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

[0459] Administration Regimens

[0460] According to certain embodiments, a single dose of an antibody as described herein (or a pharmaceutical composition comprising a combination of an antibody as described herein and any of the additional therapeutically active agents mentioned herein) may be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of an antibody as described herein body (or a pharmaceutical composition comprising a combination of an antibody as described herein and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an antibody as described herein. As used herein, “sequentially administering” means that each dose of the antibody as described herein is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods that comprise sequentially administering to the patient a single initial dose of an antibody as described herein, followed by one or more secondary doses of the antibody as described herein, and optionally followed by one or more tertiary doses of the antibody as described herein.

[0461] The terms “initial dose,” “secondary doses,” and “tertiary doses,” as used herein, refer to the temporal sequence of administration of the antibody as described herein. Thus, the “initial dose” is the dose, which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses, which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of an antibody as described herein, but generally may differ from one another in terms of frequency of administration. In some embodiments, however, the amount of the antibody as described herein contained in the initial, secondary, and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

[0462] In certain exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered 1 to 48 hours (e.g., 1, 1 %, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11, 11 %, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19, 19%, 20, 20%, 21, 21 %, 22, 22%, 23, 23 %, 24, 24%, 25, 25 %, 26, 26%, or more) after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of an antibody as described herein, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses. In some embodiments, the methods may comprise administering to a patient any number of secondary and / or tertiary doses of an antibody as described herein. For example, in some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in some embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0463] In some embodiments of the invention, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted by a physician during the course of treatment depending on the needs of the individual patient following clinical examination.

[0464] Diagnostic Uses of the Antibodies

[0465] In another aspect, this disclosure additionally provides a method for diagnosing a disease or disorder in a subject or monitoring progression of the disease or disorder in the subject. In some embodiments, the method comprises detecting binding of the heavy chain-only antibody, the fusion protein, or the antibody-drug conjugate, as described herein, to one or more of one or more of Nectin-4, Trop2, Her2, and CD3 in a biological sample of the subject.

[0466] The antibody described herein may be used to detect and / or measure of one or more of Nectin-4, Trop2, Her2, and CD3 in a sample, e.g, for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies as described herein in assays to detect a Her2- associated disease or disorder. Exemplary diagnostic assays may comprise, e.g., contacting a sample obtained from a patient with an antibody described herein, wherein the antibody described herein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate, Her2-, or CD3 -associated cells from patient samples. Alternatively, an unlabeled antibody described herein can be used in diagnostic applications in combination with a secondary antibody, which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as H, C, P, S, or I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, 0- galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure of one or more of Nectin-4, Trop2, Her2, and CD3 in a sample include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0467] In another aspect, this disclosure further provides a method for detecting the presence of of one or more of Nectin-4, Trop2, Her2, and CD3 in a sample comprising the steps of: (i) contacting a sample with the antibody or antigen-binding fragment thereof described herein; and (ii) determining binding of the antibody or antigen-binding fragment to of one or more of Nectin- 4, Trop2, Her2, and CD3 antigens, wherein binding of the antibody to the one or more of , Her2, and / or CD3 antigens is indicative of the presence of one or more of Nectin-4, Trop2, , Her2, and CD3 in the sample.

[0468] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the detection comprises contacting a secondary antibody with the antibody or antigen-binding fragment thereof. In some embodiments, the secondary antibody comprises a label. In some embodiments, the label includes a fluorescent label, a chemiluminescent label, a radiolabel, and an enzyme.

[0469] In some embodiments, the step of detecting comprises detecting fluorescence or chemiluminescence. In some embodiments, the step of detecting comprises a competitive binding assay or ELISA.

[0470] In some embodiments, the method further comprises binding the sample to a solid support. In some embodiments, the solid support includes microparticles, microbeads, magnetic beads, and an affinity purification column.

[0471] Samples used in diagnostic assays may include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of one or more of Nectin-4, Trop2, Her2, and CD3, or fragments thereof, under normal or pathological conditions. Generally, levels of one or more of Nectin-4, Trop2, Her2, and CD3 in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with one or more of Nectin-4, Trop2, Her2, and CD3) will be measured to initially establish a baseline, or standard, level of Her2, and / or CD3. This baseline level of one or more of Nectin-4, Trop2, Her2, and CD3 can then be compared against the levels of one or more of Nectin-4, Trop2, Her2, and CD3 measured in samples obtained from individuals suspected of having a Nectin-4-, Trop2-„ Her2-, and / or CD3 -associated condition or symptoms associated with such condition. The antibodies specific for Nectin-4, Trop2„ Her2, and / or CD3 protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing a N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

[0472] Additional Definitions

[0473] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0474] The term “antibody,” as referred to herein, includes whole antibodies and any antigenbinding fragment or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region comprises CHI, CH2, and CH3 domains. Each light chain comprises 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 hypervariability regions termed complementarity determining regions (CDR), interspersed with more conserved regions, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4. The light chain variable region CDRs and FRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (Clq) of the classical complement system. Camelid / Alpaca VHH antibody (variable domain of the heavy chain of heavy-chain antibodies) is a type of heavy chain-only antibody that preserves both binding and function to antigens similar to conventional antibodies with heavy and light chains. The unique feature of VHH is that it lacks a light chain and has a long CDR3 compared to CDR3 of VH of conventional antibody heavy chain.

[0475] The term “antigen-binding fragment or portion” of an antibody (also referred to herein as “antibody fragment or portion”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It is well established that fragments of full-length antibodies can maintain antigen-binding functionality. Non-limiting examples of such fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked via a disulfide bridge in the hinge region; (iii) a Fab' fragment, which is similar to a Fab but includes part of the hinge region (see, Fundamental Immunology, Paul ed., 3rd ed., 1993); (iv) an Fd fragment, comprising the VH and CHI domains; (v) an Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (as described in Ward et al., Nature 341 : 544-546, 1989), comprising a single VH domain; (vii) an isolated CDR; and (viii) a nanobody, which is a heavy chain variable region comprising a single variable domain and two constant domains.

[0476] Additionally, although the VL and VH domains of an Fv fragment are typically encoded by separate genes, they can be linked using recombinant methods to form a single-chain Fv molecule (scFv), in which the domains are connected by a synthetic peptide linker that permits proper folding and antigen binding (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are included within the scope of the term “antigen-binding fragment or portion” of an antibody. Antibody fragments as described herein may be generated using conventional techniques known in the art and may be screened for utility in the same manner as intact antibodies.

[0477] The term “isolated antibody”, as used herein, refers to an antibody that is substantially free from other antibodies having different antigenic specificities. In some embodiments, the isolated antibody is substantially free from other cellular materials and / or chemical substances. The terms “monoclonal antibody” and “monoclonal antibody composition” refer to a preparation of antibody molecules having a single molecular composition and exhibiting a uniform binding specificity and affinity for a particular epitope.

[0478] The term “human antibody”, as used herein, refers to an antibody in which both the framework and complementarity-determining region (CDR) sequences of the variable region are derived from human germline immunoglobulin gene sequences. If the antibody also comprises a constant region, the constant region is likewise derived from human germline sequences. Human antibodies as described herein may include amino acid substitutions introduced by somatic mutation in vivo or by in vitro mutagenesis techniques and are not limited to sequences naturally occurring in the human repertoire. However, the term “human antibody” expressly excludes antibodies in which CDRs derived from non-human species (e.g., murine) are grafted onto human framework sequences.

[0479] The term “human monoclonal antibody” refers to a monoclonal antibody that displays a single binding specificity and contains variable region sequences (including both framework and CDRs) derived from human germline immunoglobulin genes. In some embodiments, such antibodies may be produced by hybridomas derived from B cells of a transgenic non-human animal, such as a mouse engineered to express human immunoglobulin genes, fused with an immortalized cell line.

[0480] The term “recombinant human antibody”, as used herein, encompasses human antibodies generated, expressed, or isolated through recombinant techniques. This includes: (a) antibodies derived from a transgenic or transchromosomal animal expressing human immunoglobulin genes or hybridomas derived therefrom; (b) antibodies produced by host cells transformed to express human antibodies (e.g., transfectomas); (c) antibodies selected from recombinant combinatorial human antibody libraries; and (d) antibodies produced by any recombinant method involving the manipulation or expression of human immunoglobulin gene sequences. Such recombinant antibodies may possess VH and VL regions derived from or closely related to human germline sequences, including sequences modified by in vitro mutagenesis or in vivo somatic mutation, and may not necessarily be found in the natural human antibody repertoire.

[0481] The term “isotype” refers to the class of an antibody (e.g., IgM, IgGl) as determined by the heavy chain constant region genes. As used herein, the phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably with the phrase “an antibody that binds specifically to an antigen.”

[0482] The term “human antibody derivative” refers to any modified form of a human antibody, including but not limited to conjugates of the antibody with one or more agents (e.g., drugs, toxins, detectable labels) or with another antibody or antibody fragment. Human antibody derivatives may retain antigen-binding specificity while possessing additional functionality conferred by the conjugated moiety.

[0483] The term “humanized antibody” refers to an antibody in which the complementaritydetermining regions (CDRs) are derived from the variable regions of a non-human species, such as a mouse, and are grafted onto human immunoglobulin framework regions. Optional modifications to the human framework regions may be introduced to restore or enhance antigenbinding affinity or to improve stability or expression of the antibody.

[0484] The term “chimeric antibody” refers to an antibody in which the variable region sequences are derived from a first species and the constant region sequences are derived from a different species. For example, a chimeric antibody may include a variable region derived from a murine antibody and a constant region derived from a human antibody. In some embodiments, the variable region or CDR(s) may be derived from one isotype (e.g., IgAl), and the constant region or Fc domain may be derived from another isotype (e.g., IgG, IgA2, IgD, IgE, or IgM).

[0485] The term “chimeric antibody” or “chimeric immunoglobulin” may also refer more broadly to an immunoglobulin molecule comprising amino acid sequences from at least two different immunoglobulin (Ig) loci. For example, a transgenic antibody may include a portion encoded by a human Ig locus and another portion encoded by a non-human (e.g., rat) Ig locus. Chimeric antibodies include molecules having non-human Fc regions or engineered Fc domains, as well as antibodies comprising human idiotypes. Such chimeric immunoglobulins may be produced, for example, using transgenic animals or recombinant expression systems.

[0486] The term “bispecific three-chain antibody-like molecule” or “TCA” refers to an antibodylike molecule comprising, consisting essentially of, or consisting of three polypeptide subunits. Two of these subunits comprise a heavy chain and a light chain from a monoclonal antibody or functional antigen-binding fragments thereof, which together provide binding specificity for a first antigen and comprise at least one CH domain. The third subunit comprises a heavy-chain-only antibody that includes an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CHI domain, and further includes an antigen-binding domain that binds a second antigen or a different epitope of the first antigen. This binding domain may be derived from, or share sequence identity with, the variable region of an antibody heavy or light chain. Variable regions may be encoded by gene segments such as VH, VL, D, JH, or JL and may be represented by rearranged VHDJH, VLDJH, VLJL, or VLJL gene segments. In some embodiments, the third subunit is a heavy chain-only antibody as described herein.

[0487] The term “chimeric antigen receptor” or “CAR” is used herein in its broadest sense to refer to an engineered receptor that combines a desired binding specificity (e.g., derived from a monoclonal antibody or other ligand) with membrane-spanning and intracellular signaling domains. Typically, CARs are expressed on the surface of T cells to redirect specificity toward a selected target antigen. A CAR may comprise, for example, a human VH domain that serves as the extracellular antigen-binding region. Representative CAR designs and therapeutic applications are described in J. Natl. Cancer Inst., 2015, 108(7):dvj439, and Jackson et al., Nat. Rev. Clin. Oncol., 2016, 13:370-383. An exemplary CAR-T construct comprising a human VH binding domain is illustrated in FIG. 6.

[0488] The term “human idiotype” refers to an epitope present in the variable region of a human immunoglobulin heavy or light chain. As used herein, “human idiotype” includes both naturally occurring sequences present in human antibodies and synthetic sequences that are substantially identical to such naturally occurring polypeptides. The term “substantially identical” refers to a degree of amino acid sequence identity of at least about 85% to 95%, and in certain embodiments, greater than 90%, 95%, or 97%.

[0489] The Kabat numbering system is used to number residues in antibody variable regions based on alignment with standard Kabat sequences. Variable regions typically include residues 1-107 of the light chain and 1-113 of the heavy chain. The “EU numbering system” (also referred to as the “EU index”) is commonly used to number residues in the constant region of immunoglobulin heavy chains, and is based on the human IgGl EU antibody as described in Kabat et al., supra. Additional numbering schemes used in the field include the AbM system (Oxford Molecular), the Chothia system (Chothia and Lesk, J. Mol. Biol., 1987, 196:901-17), the Contact system, the IMGT (ImMunoGeneTics) system (see Lafranc et al., Dev. Comp. Immunol., 2003, 27(l):55-77), and the AHo system (Honegger and Pliickthun, J. Mol. Biol., 2001, 309:657-70), all of which are well known to persons of ordinary skill in the art.

[0490] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor, BCR), etc.

[0491] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0492] “Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes mediating ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells preferred. The effector cells may be isolated from a native source, e.g, blood or PBMCs, as described herein.

[0493] “Complement-dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of a complement. The complement activation pathway is initiated by binding the first component of the complement system (Clq) to a molecule (e.g, an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g, described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) may be performed. “Binding affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g, an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1: 1 interaction between members of a binding pair (e.g, antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low- affinity antibodies generally bind antigens slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigens faster and tend to remain bound.

[0494] As used herein, the “Kd” or “Kd value” refers to a dissociation constant determined by BioLayer Interferometry, using an Octet QK384 instrument (Fortebio Inc., Menlo Park, Calif) in kinetics mode. For example, anti-mouse Fc sensors are loaded with mouse-Fc fused antigens and then dipped into antibody-containing wells to measure concentration-dependent association rates (kon). Antibody dissociation rates (koff) are calculated in the final step, where the sensors are dipped into wells containing buffer only. The Kd is the ratio of koff / kon. (For further details, see Concepcion, J et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0495] An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0496] “Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence but are brought together upon folding the protein into its three-dimensional structure.

[0497] “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0498] An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in a number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

[0499] As used herein, an “immune cell” refers to a T cell, B cell, NK cell, NKT cell, or an antigen presenting cell. In some embodiments, an immune cell is a T cell, B cell, NK cell, or NKT cell. In some embodiments, an immune cell is an antigen presenting cell. In some embodiments, an immune cell is not an antigen presenting cell.

[0500] The terms “treatment,” “treating,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effects attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during, or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease.

[0501] A “therapeutically effective amount” is intended for an amount of active agent necessary to impart therapeutic benefit to a subject. For example, a “therapeutically effective amount” is an amount that induces, ameliorates, or otherwise causes an improvement in the pathological symptoms, disease progression, or physiological conditions associated with a disease or which improves resistance to a disorder.

[0502] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In one embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer, individuals with autoimmune diseases, pathogen infections, and the like. Subjects may be human but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc. The invention encompasses isolated or substantially purified nucleic acids, peptides, polypeptides, or proteins. In the context of the present invention, an “isolated” nucleic acid, DNA or RNA molecule or an “isolated” polypeptide is a nucleic acid, DNA molecule, RNA molecule, or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid, DNA molecule, RNA molecule, or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. A “purified” nucleic acid molecule, peptide, polypeptide, or protein, or a fragment thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein, peptide, or polypeptide that is substantially free of cellular material includes preparations of protein, peptide, or polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0503] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0504] A peptide or polypeptide “fragment,” as used herein, refers to a less than full-length peptide, polypeptide, or protein. For example, a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, and at least about 40 amino acids in length, or single unit lengths thereof. For example, fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids in length. In some embodiments, the peptide fragment can elicit an immune response when used to inoculate an animal. A peptide fragment may be used to elicit an immune response by inoculating an animal with a peptide fragment in combination with an adjuvant, a peptide fragment that is coupled to an adjuvant, or a peptide fragment that is coupled to arsanilic acid, sulfanilic acid, an acetyl group, or a picryl group. A peptide fragment can include a non-amide bond and can be a peptidomimetic.

[0505] As used herein, the term “conjugate,” “conjugation,” or “linked,” as used herein, refers to the attachment of two or more entities to form one entity. A conjugate encompasses both peptide- small molecule conjugates and peptide-protein / peptide conjugates.

[0506] The term “recombinant,” as used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated, or obtained by technologies or methods known in the art as recombinant DNA technology, which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0507] A “nucleic acid” or “polynucleotide” refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA) or an RNA molecule (for example, but not limited to, an mRNA), and includes DNA or RNA analogs. A DNA or RNA analog can be synthesized from nucleotide analogs. The DNA or RNA molecules may include portions that are not naturally occurring, such as modified bases, modified backbone, deoxyribonucleotides in an RNA, etc. The nucleic acid molecule can be single-stranded or double-stranded.

[0508] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule with substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide with the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0509] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur- containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet el al. (1992) Science 256: 1443 45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0510] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT,

[0511] I l l which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389- 3402, each of which is incorporated herein by reference.

[0512] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0513] The term “specifically binds,” “binds specifically to,” or the like refers to an antibody that binds to a single epitope, e.g, under physiologic conditions., but does not bind to more than one epitope. Accordingly, an antibody that specifically binds to a polypeptide will bind to an epitope on the polypeptide but not on other polypeptides. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-8 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g, BIACORE™, which bind specifically Her2, and / or CD3.

[0514] In some embodiments, the antibody binds to a spike or S protein with “high affinity,” namely with a KD of 1 X 10-7 M or less, more preferably 5 x 10-8 M or less, more preferably 3 x 10-8 M or less, more preferably 1 x 10-8 M or less, more preferably 5 x 10-9 M or less or even more preferably 1 x 10-9 M or less, as determined by surface plasmon resonance, e.g., BIACORE. The term “does not substantially bind” to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e., binds to the protein or cells with a KD of 1 x 10-6 M or more, more preferably 1 x 10-5 M or more, more preferably 1 x 10-4 M or more, more preferably 1 x 10-3 M or more, even more preferably 1 x 10-2 M or more.

[0515] The term “Kassoc” or “Ka,” as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kdis” or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “KD,” as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a BIACORE system.

[0516] Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known competition experiments. In some embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which antibody is the “blocking antibody” (i.e., the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope, or adjacent epitopes (e.g., as evidenced by steric hindrance). Other competitive binding assays include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland etal., J. Immunol. 137:3614 (1986)); solid phase directly labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer etal., Scand. J. Immunol. 32:77 (1990)).

[0517] The term “epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas of an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, composed of non-linear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, In some embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immune-precipitation assays, wherein overlapping or contiguous peptides from a spike or S protein are tested for reactivity with a given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0518] The term “epitope mapping” refers to the process of identification of the molecular determinants for antibody-antigen recognition.

[0519] The term “binds to an epitope” or “recognizes an epitope” with reference to an antibody or antibody fragment refers to continuous or discontinuous segments of amino acids within an antigen. Those of skill in the art understand that the terms do not necessarily mean that the antibody or antibody fragment is in direct contact with every amino acid within an epitope sequence. The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same, overlapping, or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase “binds to the same epitope” does not necessarily mean that the antibodies bind to or contact the same amino acids. The precise amino acids that the antibodies contact can differ. For example, a first antibody can bind to a segment of amino acids completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to “bind to the same epitope.”

[0520] As used herein, the term “immune response” refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate’s body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T cell, or the inhibition of a Treg cell.

[0521] The term “detectable label” as used herein refers to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens), intercalating dyes and the like. The term “fluorescer” refers to a substance or a portion that can exhibit fluorescence in the detectable range.

[0522] In many embodiments, the terms “subject” and “patient” are used interchangeably, irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In more exemplary aspects, the mammal is a human. As used herein, the expression “a subject in need thereof’ or “a patient in need thereof’ means a human or non-human mammal that exhibits one or more symptoms or indications of disorders (e.g., neuronal disorders, autoimmune diseases, and cardiovascular diseases), and / or who has been diagnosed with inflammatory disorders. In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

[0523] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability or risk of developing a disorder or condition in a subject who does not currently exhibit symptoms of the disorder or condition but is susceptible to or at risk of developing it.

[0524] The terms “decrease,” “reduced,” “reduction,” “inhibit,” and variations thereof, as used herein, generally mean a decrease by a statistically significant amount. Unless otherwise indicated, these terms refer to a reduction of at least 10% relative to a reference level, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or up to and including a 100% decrease (i.e., complete absence), or any amount within the range of 10-100% as compared to a suitable reference.

[0525] As used herein, the term “agent” refers to any chemical compound, a mixture of chemical compounds, a biological macromolecule (e.g., a nucleic acid, antibody, protein, or peptide), or an extract derived from biological materials such as bacteria, plants, fungi, or animal cells or tissues. An agent may exhibit biological, physiological, or pharmacological activity, rendering it suitable as a “therapeutic agent.” A therapeutic agent refers to a substance that, upon administration to a subject, produces a beneficial effect, which may include but is not limited to amelioration of disease symptoms, reduction or prevention of disease onset or recurrence, or enhancement of diagnostic accuracy.

[0526] The terms “therapeutic agent,” “therapeutically active agent,” and “treatment agent” are used interchangeably and encompass any compound or molecule that, when administered to a subject, confers a beneficial effect. Such effects include, but are not limited to, diagnostic enablement, disease amelioration, symptom reduction, disease prevention, or mitigation of a pathological condition. The term “therapeutic effect” refers to a physiological or pharmacological effect in a subject (e.g., a human or other mammal) resulting from administration of a therapeutically active substance, whether systemically or locally.

[0527] The terms “effective amount,” “effective dose,” or “effective dosage” refer to an amount of an agent sufficient to achieve, or at least partially achieve, a desired therapeutic or prophylactic effect. A “therapeutically e...

Claims

CLAIMSWhat is claimed is:

1. An anti-Nectin-4 heavy chain-only antibody that binds to a Nectin-4, comprising a heavy chain variable region that comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

2. The anti-Nectin-4 antibody of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

3. The anti-Nectin-4 antibody of any one of the preceding claims, wherein the anti-Nectin-4 antibody comprises a Fc region comprising human IgGl.

4. The anti-Nectin-4 antibody of any one of the preceding claims, wherein the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

5. The anti-Nectin-4 antibody of any one of the preceding claims, wherein the anti-Nectin-4 antibody is a bispecific or multi-specific antibody.

6. The anti-Nectin-4 antibody of any one of the preceding claims, wherein the anti-Nectin-4 antibody binds to two different epitopes on the same Nectin-4 protein or two different Nectin-4 proteins.

7. A fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2) or (ii) a human epidermal growth factor 2 (Her2), comprising a first polypeptide chain and a second polypeptide chain,wherein the first polypeptide chain comprises a Nectin-4 binding moiety fused to the N- terminus of a first Fc chain that comprises a T336Y substitution (Knob) according to EU numbering or a Y407T substitution (Hole) according to EU numbering; wherein the second polypeptide chain comprises a Trop2 binding moiety or a Her2 binding moiety fused to the N-terminus of a second Fc chain that comprises a Y407T substitution according to EU numbering or a T336Y substitution according to EU numbering; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

8. The fusion protein of claim 7, wherein the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (n) SEQ ID NOs: 9, 10, and 11; (in) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

9. The fusion protein of claim 7, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

10. The fusion protein of any one of claims 7-9, wherein the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

11. The fusion protein of any one of claims 7-9, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

12. The fusion protein of any one of claims 7-11, wherein the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

13. The fusion protein of any one of claims 7-11, wherein the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

14. The fusion protein of any one of claims 7-13, wherein the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

15. The fusion protein of any one of claims 7-13, wherein the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

16. The fusion protein of any one of claims 7-15, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

17. The fusion protein of claim 7-15, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

18. The fusion protein of any one of claims 7-17, wherein the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 38, 40, 66, or 67 or comprises an amino acid sequence of SEQ ID NOs: 38, 40, 66, or 67.

19. A fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2) or (ii) a human epidermal growth factor 2 (Her2), comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a first Trop2 or Her2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the first Trop2 or Her2 binding moiety is fused to the N-terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a second Trop2 or Her2 binding moiety, and the N-terminus of a second Fc chain, and wherein the second Nectin-4 binding moiety or the second Trop2 or Her2 binding moiety is fused to the N- terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

20. The fusion protein of claim 19, wherein the first Nectin-4 binding moiety is fused to the N- terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the N- terminus of the first Nectin-4 binding moiety; and wherein the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

21. The fusion protein of claim 19, wherein the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N- terminus of the first Trop2 or Her2 binding moiety; and wherein the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

22. The fusion protein of claim 19, wherein the first Trop2 or Her2 binding moiety is fused to the N-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C- terminus of the first Fc chain; andwherein the second Trop2 or Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain.

23. The fusion protein of claim 19, wherein the first Nectin-4 binding moiety is fused to the N- terminus of the first Fc chain, and the first Trop2 or Her2 binding moiety is fused to the C- terminus of the first Fc chain; and wherein the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, and the second Trop2 or Her2 binding moiety is fused to the C-terminus of the second Fc chain.

24. The fusion protein of any one of claims 19-23, wherein the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

25. The fusion protein of any one of claims 19-23, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

26. The fusion protein of any one of claims 19-25, wherein the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

27. The fusion protein of any one of claims 19-25, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO:

28. The fusion protein of any one of claims 19-27, wherein the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; (n) SEQ ID NOs: 55, 56, and 57.

29. The fusion protein of any one of claims 19-27, wherein the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

30. The fusion protein of any one of claims 19-29, wherein the Fc region comprises a human IgGl.

31. The fusion protein of any one of claims 19-30, wherein the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

32. The fusion protein of any one of claims 19-31, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

33. The fusion protein of claim 32, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

34. The fusion protein of any one of claims 19-33, wherein the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the first Nectin-4, Trop2 or Her2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Nectin-4, Trop2 or Her2 binding moiety through a linker.

35. The fusion protein of any one of claims 19-33, wherein the first Nectin-4, Trop2, or Her2 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and / or whereinthe second Nectin-4, Trop2, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

36. The fusion protein of any one of claims 19-35, wherein the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58-65, or comprises an amino acid sequence of SEQ ID NOs: 28, 30, 32-37, or 58-65.

37. A fusion protein that binds a Nectin-4, a Trophoblast cell surface antigen-2 (Trop2), and a human epidermal growth factor 2 (Her2), comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a Nectin-4 binding moiety, a Trop2 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the Nectin-4 binding moiety, the Trop2 binding moiety, or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

38. The fusion protein of claim 37, wherein the first Her2 binding moiety is fused to the N- terminus of the first Fc chain, the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Her2 binding moiety; and wherein the second Her2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Her2 binding moiety.

39. The fusion protein of claim 37, wherein the first Nectin-4 binding moiety is fused to the N- terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Trop2 binding moiety is fused to the N-terminus of the first Nectin-4 binding moiety; and wherein the second Nectin-4 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Trop2 binding moiety is fused to the N-terminus of the second Nectin-4 binding moiety.

40. The fusion protein of claim 37, wherein the first Trop2 binding moiety is fused to the N- terminus of the first Fc chain, the first Her2 binding moiety is fused to the C-terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety; and wherein the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

41. The fusion protein of claim 37, wherein the first Trop2 binding moiety is fused to the N- terminus of the first Fc chain, the first Nectin-4 or Her2 binding moiety is fused to the C- terminus of the first Fc chain, and the first Her2 or Nectin-4 binding moiety is fused to the N- terminus of the first Trop2 binding moiety; and wherein the second Trop2 binding moiety is fused to the N-terminus of the second Fc chain, the second Nectin-4 or Her2 binding moiety is fused to the C-terminus of the second Fc chain, and the second Her2 or Nectin-4 binding moiety is fused to the N-terminus of the second Trop2 binding moiety.

42. The fusion protein of any one of claims 37-41, wherein the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

43. The fusion protein of any one of claims 37-41, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

44. The fusion protein of any one of claims 37-43, wherein the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

45. The fusion protein of any one of claims 37-43, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

46. The fusion protein of any one of claims 37-45, wherein the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (h) SEQ ID NOs: 55, 56, and 57.

47. The fusion protein of any one of claims 37-45, wherein the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

48. The fusion protein of any one of claims 37-47, wherein the Fc region comprises a human IgGl.

49. The fusion protein of any one of claims 37-48, wherein the Fc region comprises an amino acid sequence of SEQ ID NO: 7.

50. The fusion protein of any one of claims 37-49, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

51. The fusion protein of claim 50, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

52. The fusion protein of any one of claims 37-51, wherein the first Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the first Her2, Nectin-4, or Trop2 binding moiety through a linker; and / or wherein the second Trop2, Nectin-4, or Her2 binding moiety is fused to the N-terminus of the second Her2, Nectin-4, or Trop2 binding moiety through a linker.

53. The fusion protein of any one of claims 37-51, wherein the first Nectin-4, or Her2 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and / or wherein the second Nectin-4, or Her2 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

54. A fusion protein that binds a Nectin-4, a Trophoblast cell surface antigen-2 (Trop2), and a human epidermal growth factor 2 (Her2), comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4 binding moiety, a Trop2 binding moiety, and a first Fc chain, and wherein the first Nectin-4 binding moiety or the Trop2 binding moiety is fused to the N-terminus of the first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4 binding moiety, a Her2 binding moiety, and a second Fc chain, and wherein the second Nectin-4 binding moiety or the Her2 binding moiety is fused to the N-terminus of the second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

55. The fusion protein of claim 54, wherein the Trop2 binding moiety is fused to the N- terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the N-terminus of the Trop2 binding moiety; and wherein the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the N-terminus of the Her2 binding moiety.

56. The fusion protein of claim 54, wherein the Trop2 binding moiety is fused to the N- terminus of the first Fc chain, and the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain; and wherein the Her2 binding moiety is fused to the N-terminus of the second Fc chain, and the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain.

57. The fusion protein of any one of claims 54-56, wherein the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (iii) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

58. The fusion protein of any one of claims 54-56, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

59. The fusion protein of any one of claims 54-58, wherein the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

60. The fusion protein of any one of claims 54-58, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to anamino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

61. The fusion protein of any one of claims 54-60, wherein the Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (n) SEQ ID NOs: 55, 56, and 57.

62. The fusion protein of any one of claims 54-60, wherein the Her2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 50 or 54, or comprises an amino acid sequence of SEQ ID NO: 50 or 54.

63. The fusion protein of any one of claims 54-62, wherein the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

64. The fusion protein of any one of claims 54-63, wherein the first Fc chain comprises the amino acid sequence of SEQ ID NO: 39 or 41, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 41 or 39.

65. The fusion protein of any one of claims 54-64, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

66. The fusion protein of claim 65, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

67. The fusion protein of any one of claims 54-66, wherein the first Nectin-4 binding moiety is fused to the N-terminus of the first Trop2 binding moiety through a linker; and wherein the second Nectin-4 binding moiety is fused to the N-terminus of the second Her2 binding moiety through a linker.

68. The fusion protein of any one of claims 54-66, wherein the first Nectin-4 binding moiety is fused to the C-terminus of the first Fc chain through a linker; and wherein the second Nectin-4 binding moiety is fused to the C-terminus of the second Fc chain through a linker.

69. The fusion protein of any one of claims 54-68, wherein the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 90-94, or comprises an amino acid sequence of SEQ ID NOs: 90-94.

70. A fusion protein that binds a human CD3 and (i) a Nectin-4, or (ii) a Trophoblast cell surface antigen-2 (Trop2), comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first Nectin-4, or Trop2, a first CD3 binding moiety, and a first Fc chain, and wherein the first Nectin-4, or Trop2, binding moiety is fused to the N-terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C- terminus of a first Fc chain; wherein the second polypeptide chain comprises a second Nectin-4, or Trop2, binding moiety, a second CD3 binding moiety, and a second Fc chain, and wherein the second Nectin-4, Trop2, or Her2 binding moiety is fused to the N-terminus of a second Fc chain, and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain; and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

71. The fusion protein of claim 70, wherein the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (hi) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

72. The fusion protein of claim 70, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

73. The fusion protein of any one of claims 70-72, wherein the first or second Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

74. The fusion protein of any one of claims 70-72, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

75. The fusion protein of any one of claims 70-74, wherein the first or second Her2 binding moiety comprises a Her2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 51, 52, and 53; or (h) SEQ ID NOs: 55, 56, and 57.

76. The fusion protein of any one of claims 70-75, wherein the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98, and wherein the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

77. The fusion protein of any one of claims 70-75, wherein the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chaincomplementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102, and wherein the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

78. The fusion protein of any one of claims 70-75, wherein the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 99, or comprises an amino acid sequence of SEQ ID NO: 99; and / or wherein the CD3 binding light chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95.

79. The fusion protein of any one of claims 70-78, wherein the Fc region comprises a human IgGl.

80. The fusion protein of any one of claims 70-79, wherein the Fc region comprises an amino acid sequence of SEQ ID NOs: 104 and 106.

81. The fusion protein of any one of claims 70-80, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

82. The fusion protein of claim 81, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

83. The fusion protein of any one of claims 70-82, wherein the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminal of the first Fc chain through a linker; and / or wherein the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminal of the second Fc chain through a linker.

84. The fusion protein of any one of claims 70-83, wherein the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

85. A fusion protein that binds a Nectin-4, a Trophoblast cell surface antigen-2 (Trop2), and a human CD3, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a Nectin-4 binding moiety, a first CD3 binding moiety, and a first Fc chain, and wherein the Nectin-4 binding moiety is fused to the N- terminus of a first Fc chain, and the first CD3 binding moiety is fused to the C-terminus of a first Fc chain, wherein the second polypeptide chain comprises a Trop2 binding moiety; a second CD3 binding moiety; and a second Fc chain, and wherein the Trop2 binding moiety is fused to the N- terminus of a second Fc chain; and the second CD3 binding moiety is fused to the C-terminus of a second Fc chain, and wherein the first polypeptide chain and the second polypeptide chain dimerize through the first Fc chain and the second Fc chain that forms a Fc region.

86. The fusion protein of claim 85, wherein the Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (n) SEQ ID NOs: 9, 10, and 11; (m) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

87. The fusion protein of claim 85, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 19 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

88. The fusion protein of any one of claims 85-87, wherein the first CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98, and wherein the second CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102.

89. The fusion protein of any one of claims 85-87, wherein the first CD3 binding moiety comprises a CD3 binding heavy chain variable region comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) having respective amino acid sequences of SEQ ID NOs: 100-102, and wherein the second CD3 binding moiety comprises a CD3 binding light chain variable region comprising three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) having respective amino acid sequences of SEQ ID NOs: 96-98.

90. The fusion protein of any one of claims 85-87, wherein the CD3 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 99, or comprises an amino acid sequence of SEQ ID NO: 99; and / or wherein the CD3 binding light chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 95, or comprises an amino acid sequence of SEQ ID NO: 95.

91. The fusion protein of any one of claims 85-90, wherein the Trop2 binding moiety comprises a Trop2 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of SEQ ID NOs: 25, 26, and 27.

92. The fusion protein of any one of claims 85-91, wherein the Trop2 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to anamino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence of SEQ ID NO: 24.

93. The fusion protein of any one of claims 85-92, wherein the Fc region comprises a human IgGl.

94. The fusion protein of claim 93, wherein the Fc region comprises an amino acid sequence of SEQ ID NOs: 104 and 106.

95. The fusion protein of any one of claims 85-94, wherein the first or second Fc chain comprises a N-terminal hinge region that comprises a C220S substitution according to EU numbering.

96. The fusion protein of claim 95, wherein the N-terminal hinge region comprises the amino acid sequence of SEQ ID NO: 6.

97. The fusion protein of any one of claims 85-96, wherein the first CD3 binding light chain variable region or the first CD3 binding heavy chain variable region is fused to the C-terminus of the first Fc chain through a linker; and / or wherein the second CD3 binding heavy chain variable region or the second CD3 binding light chain variable region is fused to the C-terminus of the second Fc chain through a linker.

98. The fusion protein of any one of claims 85-97, wherein the first or second polypeptide chain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112, or comprises an amino acid sequence of SEQ ID NOs: 103, 105 and 107-112.

99. The fusion protein of any one of claims 85-98, wherein the Fc region comprises a human IgGl.

100. The fusion protein of any one of claims 85-99, wherein the first Fc chain comprises a T336Y substitution according to EU numbering, and the second Fc chain comprises a Y407T substitution according to EU numbering; or wherein the first Fc chain comprises a Y407T substitution according to EU numbering, and the second Fc chain comprises a T336Y substitution according to EU numbering.

101. The fusion protein of any one of claims 85-100, wherein the first and second Fc chains comprise a N297G substitution according to EU numbering.

102. The fusion protein of any one of claims 85-101, wherein the first Fc chain comprises the amino acid sequence of SEQ ID NO: 104 or 106, and the second Fc chain comprises the amino acid sequence of SEQ ID NO: 106 or 104.

103. A fusion protein that binds a Nectin-4 and (i) a Trophoblast cell surface antigen-2 (Trop2), (ii) a human epidermal growth factor 2 (Her2), or (iii) an epidermal growth factor receptor (EGFR), wherein the fusion protein comprises: a first Nectin-4 binding moiety fused to the N- terminus of a first heavy chain variable region of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the N-terminus of a second heavy chain variable region of the anti-Trop2, anti-Her2, or anti-EGFR antibody; or wherein the fusion protein comprises: a first Nectin-4 binding moiety fused to the C- terminus of a first Fc chain of an anti-Trop2, anti-Her2, or anti-EGFR antibody, and a second Nectin-4 binding moiety fused to the C-terminus of a second Fc chain of the anti-Trop2, anti- Her2, or anti-EGFR antibody.

104. The fusion protein of claim 103, wherein the first or second Nectin-4 binding moiety comprises a Nectin-4 binding heavy chain variable region comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) having respective amino acid sequences of (i) SEQ ID NOs: 2, 3, and 4; (ii) SEQ ID NOs: 9, 10, and 11; (hi) SEQ ID NOs: 14, 15, and 16; (iv) SEQ ID NOs: 2, 3, and 19; or (v) SEQ ID NOs: 22, 3, and 19.

105. The fusion protein of claim 104, wherein the Nectin-4 binding heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21, or comprises an amino acid sequence of SEQ ID NO: 1, 8, 13, 18 or 21.

106. The fusion protein of any one of claims 103-105, wherein the anti-Trop2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 43, or comprises an amino acid sequence of SEQ ID NO: 43.

107. The fusion protein of any one of claims 103-105, wherein the anti-Trop2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 45, or comprises an amino acid sequence of SEQ ID NOs: 45.

108. The fusion protein of any one of claims 103-105, wherein the anti-Trop2 antibody is hRS7.

109. The fusion protein of any one of claims 103-108, wherein the anti-Her2 antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 76, or comprises an amino acid sequence of SEQ ID NO: 76.

110. The fusion protein of any one of claims 103-108, wherein the anti-Her2 antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 77, or comprises an amino acid sequence of SEQ ID NO: 77.

111. The fusion protein of any one of claims 103-110, wherein the anti-Her2 antibody is Herceptin.

112. The fusion protein of any one of claims 103-111, wherein the anti-EGFR antibody comprises a heavy chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 85, or comprises an amino acid sequence of SEQ ID NO: 85.

113. The fusion protein of any one of claims 103-111, wherein the anti-EGFR antibody comprises a light chain variable region that comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 86, or comprises an amino acid sequence of SEQ ID NO: 86.

114. The fusion protein of any one of claims 103-111, wherein the anti-EGFR antibody is Cetuximab.

115. An antibody-drug conjugate, comprising the heavy chain-only antibody of any one of claims 1-6 or the fusion protein of any one of claims 7-114 that is connected to a cytotoxic agent through a linker.

116. The antibody-drug conjugate of claim 115, wherein the cytotoxic agent is selected from monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), Trastuzumab emtansine (T-DM1), Ravtansine (DM4), an auristatin, a maytansinoid, a tubulysin, an amberstatin269, an anthracycline, and Exatecan, a DXd, an SN-38, a camptothecin, a pyrrolobenzodiazepine, an indolinobenzodiazepine, a calicheamicin, a duocarmycin, a doxorubicin, an antibiotic, a spliceostatin, a thailanstatin, a derivative thereof, an analog thereof, an isomer thereof, a prodrug thereof, a radioisotope agent, and a pharmaceutically acceptable salt.

117. The antibody-drug conjugate of claim 116, wherein the cytotoxic agent is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

118. The antibody-drug conjugate of any one of claims 115-117, wherein the linker is a non- cleavable linker or a cleavable linker selected from an acid cleavable linker, a disulfide cleavablelinker, a protease cleavable linker, a glycosidase cleavable linker, or a phosphatase cleavable linker.

119. The antibody-drug conjugate of any one of claims 115-118, wherein the linker is cathepsin B, hydrazone, succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), maleimidocaproic acid (me), valine-citrulline (vc), N-hydroxysuccinimidyl 4-(2-pyridyldithio)- 2-sulfobutanoate (sulfo-SPDB), N-hydroxysuccinimidyl 4-(2-pyridydithio)butanoate (SPDB), N- succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), valine-alanine (va), polyethylene glycol 8- valine- citrulline (PEG8-va), mb-vc, CL2A, a cleavable vc-based linker, or a fleximer polymer linker.

120. The antibody-drug conjugate of any one of claims 115-118, wherein a linker-cytotoxic agent pair in the antibody-drug conjugate is mc-vc-PAB-MMAE, vc-MMAE, mc-MMAF, SMCC-DM1, sulfo-SPDB-DM4, SPDB-DM4, SPP-DM1, va-SGD1882, polyethylene glycol 8 (PEG8)-va-SG3199, sulfo- SPDB-DGN462, hydrazone-CMl, vc-seco-DUBA, mb-vc-MGBA, CL2A-SN38, peptide linker with DX-8951 derivative, hydrazone-doxorubicin, cleavable vc- based linker with AurO 101, vc-PF06380101 , fleximer polymer linker with auristatin F, cleavable linker-tubulin inhibitor, or vc-rifalogue.

121. A pharmaceutical composition comprising: the heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, or the antibody-drug conjugate of claims 115-120.

122. A polynucleotide encoding the heavy chain-only antibody of any one of claims 1-6; or the first polypeptide chain and / or the second polypeptide chain of the fusion protein of any one of claims 7-114.

123. A vector comprising the polynucleotide of claim 122.

124. A cell comprising the polynucleotide of 122 or the vector of claim 123.

125. A method of producing the heavy chain-only antibody of any one of claims 1-6 or the fusion protein of any one of claims 7-114, comprising growing the cell of claim 124 under conditions permissive for expression of the heavy chain-only antibody or the fusion protein, and isolating the heavy chain-only antibody or the fusion protein from the cells.

126. A kit comprising of the heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, the antibody-drug conjugate of claims 115-120, or the pharmaceutical composition of claim 121.

127. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, the antibody-drug conjugate of claims 115-120, or the pharmaceutical composition of claim 121.

128. The method of claim 127, wherein the disease or disorder is a cancer.

129. The method of claim 127, wherein the cancer is characterized by expression of one or more of Nectin-4, Trop2, Her2, and CD3.

130. The method of any one of claims 127-129, wherein the cancer is epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharynx cancer, cancer of the nose, pancreatic cancer, skin cancer, oral cancer, cancer of the tongue, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

131. The heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, the antibody-drug conjugate of claims 115-120, or the pharmaceutical composition of claim 121 for use in treatment of cancer.

132. Use of the heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, the antibody-drug conjugate of claims 115-120, or the pharmaceutical composition of claim 121 in the treatment of cancer.

133. A method for diagnosing a disease or disorder in a subject or monitoring progression of the disease or disorder in the subject, comprising detecting binding of the heavy chain-only antibody of any one of claims 1-6, the fusion protein of any one of claims 7-114, or the antibody-drug conjugate of claims 115-120 to one or more of Nectin-4, Trop2, Her2, and CD3 in a biological sample of the subject.

134. An engineered T or NK cell comprising the heavy chain-only antibody of any one of claims 1-6 or the fusion protein of any one of claims 7-114.

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