Multivalent immunoconjugates for targeted radioisotope therapy

Abstract

Described herein is an immunoconjugate comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody comprises a polypeptide comprising a first antigen-binding domain and a second antigen-binding domain.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 373,186, filed August 22, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The specificity of antibodies such as IgG for antigens makes them a highly targeted platform for therapeutics; however, the typical serum half-life of at least 3 weeks for IgG is disadvantageous for the delivery of radioisotopes, including alpha-emitting isotopes such as Ac-225 and beta-emitting isotopes such as Lu-177 and Y-90, particularly due to prolonged exposure and chronic off-target toxicities.

[0003] 225-Ac is the most cytotoxic of the alpha-emitting radioisotopes, and a single decay event can effectively destroy cancer cells by causing double-stranded DNA breaks and subsequent cell death. The potency of alpha-emitting radioisotopes makes them attractive as cell killing agents and can overcome acquired resistance observed in response to other therapies. Summary of the Invention

[0004] Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) that comprise multivalent (e.g., tetravalent) antibodies and are useful for treating cancer. The immunoconjugates described herein are advantageous in that they can achieve high-order avidity interactions with targets while having molecular weights less than conventional antibody molecules (e.g., less than 150,000 daltons) and exhibiting improved safety profiles (e.g., reduced serum half-lives).

[0005] In one aspect, the present specification describes an immunoconjugate comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody comprises a polypeptide comprising (a) a first antigen-binding domain and (b) a second antigen-binding domain. In certain embodiments, the polypeptide further comprises an Fc domain.

[0006] In another aspect, described herein is an immunoconjugate comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody has Formula I: ABC wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, and C comprises an Fc domain.

[0007] Described herein, in another aspect, is an immunoconjugate comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having the structure of Formula II: ACB, wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, and C comprises an Fc domain.

[0008] In another aspect, the present specification describes an immunoconjugate comprising a multivalent antibody, a chelating agent, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula I: ABC, wherein A comprises a first VHH domain, B comprises a second VHH domain, and C comprises an Fc domain, and wherein the first VHH domain binds to FOLR1 or DLL3, and the second VHH domain binds to FOLR1 or DLL3.

[0009] In another aspect, the present specification describes an immunoconjugate comprising a multivalent antibody, a chelating agent, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula II:ACB, wherein A comprises a first VHH domain, B comprises a second VHH domain, and C comprises an Fc domain, and wherein the first VHH domain binds to FOLR1 or DLL3, and the second VHH domain binds to FOLR1 or DLL3.

[0010] In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise an immunoglobulin single-chain variable domain polypeptide. In certain embodiments, the immunoglobulin single-chain variable domain polypeptide comprises a VHH. In certain embodiments, the Fc domain comprises a CH2 domain and a CH3 domain. In certain embodiments, the Fc domain comprises a CH3 domain. In certain embodiments, the Fc domain comprises a CH2 domain. In certain embodiments, the Fc domain comprises a change to one or more amino acid residues that reduces an effector function of the Fc domain. In certain embodiments, the change to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region is a change that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell-phagocytosis (ADCP), or a combination thereof. In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y or 329R (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A, (t) 267G, 267H, 267I or 267K, (u) 268K,(v) 269N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L2 34A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A, L235E and G237A, (aaa) L234A, L235E, G237A and P331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are selected from the list consisting of: (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (II1) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) through (ppp). In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region comprise L234A, L235E, G237A, A330S, and P331S, according to EU numbering. In certain embodiments, the Fc domain comprises:The immunoconjugate may comprise an alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to an amino acid residue selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise I253A, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise H310A, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise H435Q, according to EU numbering. In certain embodiments, the multivalent antibody comprises a homodimer of polypeptides. In certain embodiments, the multivalent antibody comprises a molecular weight of less than about 110,000 daltons. In certain embodiments, the multivalent antibody is a monospecific multivalent antibody. In certain embodiments, the multivalent antibody is a bispecific multivalent antibody. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind to FOLR1, DLL3, or HER2. In certain embodiments,The first antigen-binding domain binds to FOLR1. In certain embodiments, (a) the first antigen-binding domain binds to FOLR1, and (b) the second antigen-binding domain binds to DLL3. In certain embodiments, the first antigen-binding domain comprises a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a complementarity-determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a complementarity-determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the second antigen-binding domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 7, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 113, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 213. a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 307, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 310, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 313, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 407, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 410, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 413, or a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 507, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 510, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 513. In certain embodiments, the first antigen-binding domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 3,the second antigen-binding domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 7; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 113; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 213; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 307, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 310, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 313, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 407, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 410, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 413, or a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 507, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 510, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 513. In certain embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506. In certain embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4.or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, and the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 8, 101-106, 201-206, 301-306, 401-406, or 501-506. is a radioisotope chelator. In certain embodiments, the chelator is an alpha-emitter chelator. In certain embodiments, the chelator is a beta-emitter or a gamma-emitter chelator. In certain embodiments, the chelator is selected from the list consisting of DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, Noneunpa, and combinations thereof. In certain embodiments, the chelator is selected from the list consisting of DOTMA, DOTPA, DO3AM-acetate, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, deferoxamine, DFO*, and combinations thereof. In certain embodiments, the chelator is DOTA. In certain embodiments, the chelator is DOTAGA. In certain embodiments, the chelator is Py4Pa. In certain embodiments, the chelator is directly attached to the antigen-binding region and / or Fc domain. In certain embodiments, the chelator is attached to the antigen-binding region and / or Fc domain via a linker. In certain embodiments, the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa. In certain embodiments, the immunoconjugate further comprises a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a beta emitter.In certain embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0011] A method for killing tumor or cancer cells, the method comprising contacting tumor or cancer cells with an immunoconjugate of the present disclosure, thereby killing the tumor or cancer cells. In certain embodiments, the tumor cells are solid tumor cells. In certain embodiments, the tumor or cancer cells express FOLR1, DLL3, or both.

[0012] A method of treating cancer or tumor in an individual, the method comprising administering to the individual an immunoconjugate, thereby treating the cancer or tumor. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the method further comprises administering to the individual 0.5 μCi to 30.0 μCi per kilogram. In certain embodiments, the method comprises administering to the individual 10 mCi to 75 mCi per square meter of body surface area. In certain embodiments, the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate. In certain embodiments, the immunoconjugate is for use in the method of treating cancer or tumor in an individual.

[0013] Further described herein are methods for delivering a radioisotope to cancer or tumor cells in an individual, the methods comprising administering an immunoconjugate to the individual, thereby delivering the radioisotope to the cancer or tumor cells. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

[0014] Also described herein are methods for imaging a tumor in an individual, the methods comprising administering an immunoconjugate to the individual. In certain embodiments, the individual is a human. In certain embodiments, the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen specifically bound by the immunoconjugate.

[0015] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0016] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Figure 1A] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 1B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs is shown. [Figure 2A] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2B] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 2C] Binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3 is shown. [Figure 3A] 1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 3B]1 shows the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3. [Figure 4] Self-interaction data for anti-HER2 and anti-DLL3 VHH-Fc constructs are shown. [Figure 5] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 6] 1 shows a diagram of the chemical synthesis of the linker molecule. [Figure 7A] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7B] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 7C] The immunoreactive fractions of the different VHH-Fc constructs are shown. [Figure 8] A comparison of imaging with 111In-labeled VHH-Fc compared to the biodistribution of 225Ac-labeled VHH-Fc is shown. [Figure 9A] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9B] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9C] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 9D] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs over time. [Figure 10A] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 10B] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 10C] Tumor:non-tumor tissue ratios are shown for labeled anti-HER2 VHH-Fc constructs. [Figure 11] 1 shows the biodistribution of labeled anti-HER2 VHH-Fc constructs. [Figure 12]Figure 1 shows the systemic clearance of 111In-labeled VHH-Fc (H101) and VHH-Fc variants (H105, H107, and H108). [Figure 13] 1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs over time. [Figure 14] 1 shows the biodistribution of labeled anti-DLL3 VHH-Fc constructs. [Figure 15A] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs. [Figure 15B] Figure 15 shows the biodistribution of 225Ac-labeled anti-HER2 (15A) and anti-DLL3 (15B) VHH-Fc constructs. [Figure 16A] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16B] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 16C] 1 shows the results of a toxicity study performed with 225Ac-labeled anti-HER2 VHH-Fc constructs. [Figure 17] Immunoreactive fractions of different anti-DDL3 VHH-Fc constructs loaded with 177Lu are shown. [Figure 18] 1 shows the chemical structures of certain linker-chelators described herein. [Figure 19A] Schematic diagram of multivalent antibody formats. [Figure 19B] Schematic diagram of multivalent antibody format. [Figure 20] Monospecific and bispecific multivalent formats of FOLR1 and DLL3 are shown. [Figure 21] Binding of multivalent tetramer constructs to cells expressing FOLR1 is shown. Detailed Description of the Invention

[0017] Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) that comprise multivalent (e.g., tetravalent) antibodies and are useful for treating cancer. In certain examples, the immunoconjugates described herein are advantageous in that they achieve higher-order avidity interactions with targets while possessing molecular weights less than conventional antibody molecules (e.g., less than 150,000 daltons). Furthermore, in such examples, the immunoconjugates comprise elements of conventional antibodies, such as an Fc domain or an Fc variant domain, but are able to achieve higher-order avidity interactions at a lower molecular weight. In some embodiments described herein, the immunoconjugates utilize Fc mutations that reduce serum half-life and effector cell function. While reduced serum half-life or effector cell function generally is not associated with improved antibody efficacy, the immunoconjugates described herein can effectively bind to and kill target tumor cells (e.g., within the tumor microenvironment) while achieving improved safety.

[0018] Immunoconjugates Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) comprising multivalent (e.g., tetravalent or greater) polypeptides (e.g., multivalent antibodies or antibody-derived polypeptides). In some embodiments, the multivalent antibodies are tetravalent. In certain embodiments, the multivalent antibodies are monospecific (e.g., bind only to FOLR1 or DLL3). In certain embodiments, the multivalent antibodies are bispecific (e.g., bind both FOLR1 and DLL3). In some embodiments, the multivalent antibodies of the immunoconjugate comprise a molecular weight of less than 150,000 daltons. In certain embodiments, the multivalent antibodies of the immunoconjugate comprise a molecular weight of less than 110,000 daltons.

[0019] In some embodiments, an immunoconjugate (e.g., a radioimmunoconjugate) comprising a multivalent antibody (e.g., having a chelator and a radionuclide) is provided, wherein the multivalent antibody comprises a polypeptide comprising a first antigen-binding domain and a second antigen-binding domain. In certain embodiments, the polypeptide further comprises an Fc domain.

[0020] 19A-B show exemplary multivalent antibody formats described herein. (110) represents a first antigen-binding domain (e.g., an immunoglobulin single chain domain). (120) represents a second antigen-binding domain (e.g., an immunoglobulin single chain domain). (130) represents an Fc domain comprising CH2-CH3 ((132) and (134), respectively). (140) represents an optional linker polypeptide.

[0021] In some embodiments, an immunoconjugate (e.g., a radioimmunoconjugate) is provided comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody has Formula I: ABC wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, and C comprises an Fc domain.

[0022] In some embodiments, an immunoconjugate (e.g., a radioimmunoconjugate) is provided comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody has Formula II: ACB wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, and C comprises an Fc domain.

[0023] In some embodiments, an immunoconjugate (e.g., a radioimmunoconjugate) is provided comprising a multivalent antibody and a chelator, wherein the multivalent antibody has Formula III: A-L1-B-L2-C wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, C comprises an Fc domain, L1 is a polypeptide linker (e.g., a polyGS linker), and L2 is a polypeptide linker (e.g., the same as or different from L1).

[0024] In some embodiments, an immunoconjugate (e.g., a radioimmunoconjugate) is provided comprising a multivalent antibody and a chelator, wherein the multivalent antibody has Formula IV: A-L1-C-L2-B wherein A comprises a first antigen-binding domain, B comprises a second antigen-binding domain, C comprises an Fc domain, L1 is a polypeptide linker (e.g., a polyGS linker), and L2 is a polypeptide linker (e.g., the same as or different from L1).

[0025] In certain embodiments, the multivalent antibody comprises a homodimer of polypeptides (e.g., mediated via Fc domain dimerization). In certain embodiments, the multivalent antibody comprises a molecular weight of less than 150,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 140,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 130,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 120,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of about 50,000 daltons to about 110,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of about 75,000 daltons to about 110,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of about 50,000 daltons to about 105,000 daltons. In certain embodiments, the multivalent antibody comprises a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of about 75,000 daltons to about 105,000 daltons.

[0026] In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise a single-chain variable domain polypeptide selected from the group consisting of scFv, VH, VL, VHH, and VNAR. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise an immunoglobulin single-chain variable domain polypeptide. In certain embodiments, the immunoglobulin single-chain variable domain polypeptide is selected from the group consisting of VH, VL, and VHH. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise a VHH (e.g., a first VHH domain and a second VHH domain).

[0027] In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that modulates (e.g., reduces, inhibits, reduces, prevents, etc.) an effector function of the Fc domain. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that alters (e.g., reduces, inhibits, reduces, prevents, etc.) the serum half-life of the immunoconjugate. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that alters (e.g., reduces, inhibits, reduces, prevents, etc.) binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that modulates (e.g., reduces, inhibits, reduces, prevents, etc.) both (i) an effector function of the Fc domain (e.g., ADCC and / or CDC) and (ii) binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0028] In certain embodiments, the multivalent antibody is monospecific (e.g., binds only to FOLR1 or DLL3). In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind to FOLR1. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind to DLL3.

[0029] In certain embodiments, the multivalent antibody is bispecific (e.g., binds to FOLR1 and DLL3). In certain embodiments, the first antigen-binding domain binds to FOLR1 and the second antigen-binding domain binds to DLL3. In certain embodiments, the first antigen-binding domain binds to DLL3 and the second antigen-binding domain binds to FOLR1.

[0030] In certain embodiments, the immunoconjugate comprises a chelating agent. In certain embodiments, the chelating agent is a radionuclide chelating agent. In certain embodiments, the chelating agent is directly bound to the antigen-binding region and / or the Fc domain. In certain embodiments, the chelating agent is indirectly bound to the antigen-binding region and / or the Fc domain.

[0031] In certain embodiments, the immunoconjugate is a radioimmunoconjugate comprising a radionuclide. In certain embodiments, the radionuclide is an α-emitter. In certain embodiments, the radionuclide is a β-emitter. In certain embodiments, the radionuclide is an α-emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

[0032] An "immunoconjugate" refers to and encompasses a molecular conjugate comprising at least one antigen-binding region (e.g., a variable region or a complementarity-determining region) derived from a multivalent antibody further conjugated to at least one non-antibody-derived molecule, such as a chelator or a cytotoxic agent. The non-antibody-derived molecule may be conjugated, for example, to one or more lysine or cysteine ​​residues of the antigen-binding region or to a constant region bound (by peptide bond or otherwise) to the antigen-binding region. In some embodiments, the immunoconjugate further comprises a chelator (interchangeably, a "chelator"). In some embodiments, the immunoconjugate comprises an antibody construct of the present invention linked, directly or indirectly, to a cytotoxic agent or a radioisotope.

[0033] Antigen-binding Domains The term "variable region" or "variable domain" refers to and encompasses the domain of an antibody heavy or light chain or immunoglobulin single-chain variable domain (e.g., VHH) antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies, or VHHs, generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co. (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. A single VHH is sufficient to confer antigen-binding specificity.

[0034] An "immunoglobulin single-chain variable domain," or "immunoglobulin single-chain variable domain antibody," or "immunoglobulin single variable domain," or "single chain antibody," or "VHH," as used interchangeably herein, refers to and encompasses an immunoglobulin molecule in which the antigen-binding site is present on, and formed by, a single immunoglobulin domain (e.g., a variable domain).

[0035] Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site, i.e., provide a total of six CDRs for antigen-binding site formation. In this case, the complementarity-determining regions (CDRs) of both the VH and VL can contribute to the antigen. The antigen-binding domain of a conventional antibody (such as an IgG, IgM, IgA, IgD, or IgE molecule with cognate VL and VH), Fab fragment, F(ab')2 fragment, Fv fragment such as a disulfide-linked Fv, scFv fragment, or diabody derived from such a conventional four-chain antibody, is different from a single-chain variable domain antibody.

[0036] The VH, VL, or VHH regions can be subdivided into regions of hypervariability called "complementarity determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FR or FW).

[0037] The extent of framework regions and CDRs can be defined in several ways (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; AbM definitions used in Oxford Molecular's AbM antibody modeling software). Generally, see, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia", numbering scheme). Methods encompassed and included herein are the Chothia, AbM, Kabat, Contact, and / or IMGT.

[0038] "Complementarity determining region" or "CDR" refers to and encompasses the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). Immunoglobulin single variable domain antibodies contain three CDRs (CDR1, CDR2, and CDR3).

[0039] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), which are interrupted by three complementarity-determining regions ("CDRs"), referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively. Thus, a single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit that essentially consists of a single variable domain, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). The immunoglobulin single-chain variable domain can be, for example, a heavy chain ISVD, such as a VH, a VHH, including a camelized VH, or a humanized VHH. Preferably, it is a VHH, including a camelized VH or a humanized VHH.

[0040] "VHH domains," also known as VHHs, VHH antibody fragments, and VHH antibodies, were originally described as antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies devoid of light chains"; Hamers-Casterman et al. Nature 363:446-448, 1993). The terms "VHH domain" and "immunoglobulin single-chain variable domain" are used to distinguish these variable domains from the heavy-chain variable domains present in conventional four-chain antibodies (referred to herein as "VH domains") and the light-chain variable domains present in conventional four-chain antibodies (referred to herein as "VL domains"). For a further description of VHHs, see the review article by Muyldermans (reviewed in Molecular Biotechnology 74:277-302, 2001).

[0041] "Humanized VHHs" include amino acid sequences that correspond to the amino acid sequence of a naturally occurring VHH domain, but that have been "humanized," i.e., by substituting one or more amino acid residues in the amino acid sequence (particularly the framework sequence) of said naturally occurring VHH sequence with one or more amino acid residues present at the corresponding positions in a VH domain derived from a conventional four-chain antibody of human origin (e.g., as shown above). This can be performed in a manner known per se, which will be clear to those skilled in the art, for example, based on the further explanations herein and the prior art (e.g., WO2008 / 020079). Again, it should be noted that such humanized VHHSs can be obtained in any suitable manner known per se, and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material.

[0042] Affinity encompasses and / or refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, binding affinity encompasses and refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described throughout.

[0043] An affinity matured antibody includes and / or refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, such as alterations that result in an improvement in the affinity of the antibody for an antigen.

[0044] Binding, and determination of binding, can be readily determined by methods known in the art (e.g., ELISA, surface plasmon resonance, bio-layer interferometry, isothermal calorimetry, etc.). In some embodiments, binding is determined by ELISA. In some aspects, binding comprises a KD of, e.g., less than 10^-5 M (10 μM) as measured by surface plasmon resonance, bio-layer interferometry, or isothermal calorimetry. In some aspects, binding comprises a KD of, e.g., less than 10^-6 M (1 μM) by surface plasmon resonance, bio-layer interferometry, or isothermal calorimetry. In some embodiments, binding comprises a KD of, e.g., less than 10^-7 M (100 nM) by surface plasmon resonance, bio-layer interferometry, or isothermal calorimetry.

[0045] In certain embodiments, the antibody comprises one or more naturally occurring amino acids. In certain embodiments, the antibody consists of naturally occurring amino acids. As used herein, naturally occurring amino acids include and / or refer to amino acids that are found in nature and have not been engineered by humans. In particular examples, naturally occurring amino acids include and / or are further referred to as the 20 conventional amino acids: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0046] In some embodiments, the antibody comprises a variant sequence of the antibody. In certain instances, amino acid substitutions can be made in the sequence of any of the antibodies described herein without necessarily reducing or eliminating its activity (e.g., as measured by binding or functional assays described herein). Thus, in some embodiments, the variant sequence comprises one or more amino acid substitutions (e.g., within the variable region or one or more CDRs). In some embodiments, the variant sequence comprises one or more substitutions in one or more CDRs. In certain embodiments, the variant sequence comprises one amino acid substitution. In certain embodiments, the variant sequence comprises two amino acid substitutions. In certain embodiments, the variant sequence comprises three amino acid substitutions. In certain instances, the substitutions comprise conservative substitutions (e.g., substitution with an amino acid having equivalent chemical characteristics). In certain cases, a nonpolar amino acid may be substituted and replaced with another nonpolar amino acid, including alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In certain cases, neutrally charged polar amino acids can be substituted with other neutrally charged polar amino acids, and neutrally charged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine.In certain cases, positively charged amino acids can be substituted with other positively charged amino acids, and positively charged amino acids include arginine, lysine, and histidine.In certain cases, negatively charged amino acids can be substituted with other negatively charged amino acids, and negatively charged amino acids include aspartic acid and glutamic acid.Examples of amino acid substitution also include substituting L-amino acids with their corresponding D-amino acids, substituting cysteine ​​with homocysteine ​​or other unnatural amino acids.

[0047] In certain embodiments, the antibody comprises one or more unnatural amino acids. In certain embodiments, the antibody is composed of unnatural amino acids. As used herein, unnatural amino acids and / or unnatural amino acids include and / or refer to amino acid structures that cannot be biosynthetically produced in any organism using unmodified or modified genes from any organism. For example, these include, but are not limited to, modified amino acids and / or amino acid analogs that are not one of the 20 naturally occurring amino acids (e.g., unnatural side chain variant amino acids), D-amino acids, homoamino acids, β-homoamino acids, N-methyl amino acids, and α-methyl amino acids. As further examples, unnatural amino acids also include 4-benzoylphenylalanine (Bpa), aminobenzoic acid (Abz), aminobutyric acid (Abu), aminohexanoic acid (Ahx), aminoisobutyric acid (Aib), citrulline (Cit), diaminobutyric acid (Dab), diaminopropanoic acid (Dap), diaminopropionic acid (Dap), gamma-carboxyglutamic acid (Gla), homoalanine (Hala), homoarginine (Harg), homoasparagine (Hasn), homoaspartic acid (Hasp), homocysteine ​​(Hcys), homoglutamic acid (Hglu), homoglutamine (Hgln), homoisoleucine (Hile ... Non-naturally occurring amino acid residues include moroicine (Hleu), homomethionine (Hmet), homophenylalanine (Hphe), homoserine (Hser), homotyrosine (Htyr), homovaline (Hval), hydroxyproline (Hp), isonipestic acid (Inp), naphthylalanine (Nal), nipecotic acid (Nip), norleucine (Nle), norvaline (Nva), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), phenylglycine (Phg), pyroglutamic acid (Pyr), sarcosine (Sar), t-butylglycine (Tle), and tetrahydro-isoquinoline-3-carboxylic acid (Tic). Such non-naturally occurring amino acid residues can be introduced by substitution of naturally occurring amino acids and / or by insertion of the non-naturally occurring amino acid into a naturally occurring antibody sequence.Non-natural amino acid residues can also be incorporated to confer desired functionality onto the apelin molecule, for example, the ability to attach a functional moiety (eg, PEG).

[0048] A stable formulation refers to and / or encompasses a formulation in which a protein (e.g., an antibody) therein essentially retains its physical and / or chemical stability and / or biological activity upon storage at the intended storage temperature, e.g., 2-8°C. In some embodiments, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period can be selected based on the intended shelf life of the formulation. Furthermore, the formulation is stable after freezing (e.g., to -20°C) and thawing the formulation, e.g., after one or more cycles of freezing and thawing. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and / or quantitatively in a variety of different ways, including assessing aggregate formation (e.g., by measuring turbidity and / or by visual inspection using size exclusion chromatography), assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis, SDS-PAGE analysis to compare reduced and intact antibodies, assessing antibody biological activity or antigen-binding function, and methods described herein. Instability can include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, etc.

[0049] A pharmaceutically acceptable carrier encompasses and / or refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0050] Polypeptide or protein are used interchangeably and include and / or refer to polymers of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, a polypeptide can contain modifications to the native or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as by site-directed mutagenesis, or can be accidental, such as by mutations of hosts producing the protein or by errors resulting from PCR amplification.

[0051] The determination of percent identity or percent similarity between two sequences can be achieved using a mathematical algorithm.A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877.Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410.Alternatively, PSI-Blast can be used to perform an iterative search to detect distant relationships between molecules. When using BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Alternatively, sequence alignments can be performed using the CLUSTAL algorithm (eg, as provided in the program CLUSTAL-omega), as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0052] As used herein, the terms individual, patient, or subject include and / or refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease, disorder, or condition that the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0053] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genomic integrated vector or "integrated vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. Vectors capable of inducing the expression of an operably linked gene are referred to herein as "expression vectors." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements, such as promoters, enhancers, and polyadenylation signals used to control transcription, can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene for facilitating recognition of transformants may further be incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors can be linearized for integration into the genomic region. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus.

[0054] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence homology can be determined using the latest version of BLAST as of the filing date of this application.

[0055] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise transgenic suitable cells, thus enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for the production of antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct;2(5):466-477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, mammalian cells that are cell lines useful for antibody production are Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In certain embodiments, the nucleic acids encoding the antibodies are integrated into the genomic locus of cells useful for producing the antibodies. In certain embodiments, methods of producing antibodies are described herein, the methods comprising culturing in vitro cells containing nucleic acid encoding the antibody under conditions sufficient to allow production and secretion of the antibody.

[0056] In certain embodiments, described herein is a master cell bank, the master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises (a) a CHO cell line comprising a nucleic acid encoding an antibody of the present disclosure, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0057] Also described herein are methods for producing the antibodies described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the antibodies in cell culture medium under conditions sufficient to allow expression and secretion of the antibodies, and further harvesting the antibodies from the cell culture medium. Harvesting can further include one or more purification steps to remove viable cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the further purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, and / or ion exchange chromatography.

[0058] "Treat," "treatment," or "treating," as used herein, refers to the deliberate intervention in a physiological disease state that results in, for example, a reduction in the severity of a disease or condition, a shortening of the duration of a disease condition course, an improvement or elimination of one or more symptoms associated with a disease or condition, or the provision of a beneficial effect to a subject with a disease or condition. Treatment does not require a cure of the underlying disease or condition.

[0059] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, any amount of drug, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0060] As used herein, "pharmaceutically acceptable" with respect to a "carrier," "excipient," or "diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0061] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like, as well as those derived from nontoxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0062] As used herein, treatment or treating includes and / or refers to pharmaceutical or other intervention regimens used to obtain beneficial or desired results in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit may refer to the eradication or amelioration of the condition or underlying disease being treated. Therapeutic benefit can also be achieved by the eradication or amelioration of one or more physiological symptoms associated with an underlying disorder, such that an improvement is observed in a subject, even though the subject may still be afflicted with the underlying disorder. Prophylactic benefit includes delaying, preventing, or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, subjects at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may receive treatment even if the disease has not been diagnosed. Those skilled in the art will recognize that when considering a population of potential individuals for treatment, not all will respond to treatment, or will not respond equally. Such individuals are considered to be treated.

[0063] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening naive or synthetic libraries, such as phage display. The generation of immunoglobulin sequences, such as VHHs and immunoglobulin single-chain variable domains, is described in various publications, including WO94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The resulting nanobody repertoire is then further screened for nanobodies that bind to the target antigen. In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0064] The multivalent antibodies described herein comprise antigen-binding domains. In certain examples, any independent antigen-binding domains having a molecular weight of less than about 25,000 daltons (e.g., a VHH having a molecular weight of about 15,000 daltons) may be used herein, with the multivalent antibodies of the immunoconjugate having a total molecular weight of less than 150,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 25,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 20,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 19,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 18,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 17,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 16,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of less than about 15,000 daltons.

[0065] In certain embodiments, the antigen-binding domain comprises a molecular weight of about 12,000 daltons to about 25,000 daltons. In certain embodiments, the antigen-binding domain comprises a molecular weight of about 12,000 daltons to about 13,000 daltons, about 12,000 daltons to about 14,000 daltons, about 12,000 daltons to about 15,000 daltons, about 12,000 daltons to about 16,000 daltons, about 12,000 daltons to about 17,000 daltons, about 12,000 daltons to about 18,000 daltons, about 12,000 daltons to about 19,000 daltons, about 12,000 daltons to about 20,000 daltons, about 12,000 daltons to about 25,000 daltons, or about 13,000 Daltons to about 14,000 Daltons, about 13,000 Daltons to about 15,000 Daltons, about 13,000 Daltons to about 16,000 Daltons, about 13,000 Daltons to about 17,000 Daltons, about 13,000 Daltons to about 18,000 Daltons, about 13,000 Daltons to about 19,000 Daltons, about 13,000 Daltons to about 20,000 Daltons, about 13,000 Daltons to about 25,000 Daltons, about 14,000 Daltons to about 15,000 Daltons, about 14,000 Daltons to about 16,000 Daltons about 14,000 Daltons to about 17,000 Daltons, about 14,000 Daltons to about 18,000 Daltons, about 14,000 Daltons to about 19,000 Daltons, about 14,000 Daltons to about 20,000 Daltons, about 14,000 Daltons to about 25,000 Daltons, about 15,000 Daltons to about 16,000 Daltons, about 15,000 Daltons to about 17,000 Daltons, about 15,000 Daltons to about 18,000 Daltons, about 15,000 Daltons to about 19,000 Daltons, about 15,000 Daltons to about 20,000 Daltons Daltons, about 15,000 Daltons to about 25,000 Daltons, about 16,000 Daltons to about 17,000 Daltons, about 16,000 Daltons to about 18,000 Daltons, about 16,000 Daltons to about 19,000 Daltons, about 16,000 Daltons to about 20,000 Daltons, about 16,000 Daltons to about 25,000 Daltons, about 17,000 Daltons to about 18,000 Daltons, about 17,000 Daltons to about 19,000 Daltons, about 17,000 Daltons to about 20,000 Daltons, about 17,000 Daltons to about 25,In certain embodiments, the antigen-binding domain comprises a molecular weight of about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 16,000 daltons, about 17,000 daltons, about 18,000 daltons, about 19,000 daltons, about 20,000 daltons, or about 25,000 daltons. ,

[0066] In certain embodiments, the antigen-binding domain comprises a single-chain variable domain polypeptide selected from the group consisting of scFv, VH, VL, VHH, and VNAR. In certain embodiments, the antigen-binding domain comprises an immunoglobulin single-chain variable domain polypeptide. In certain embodiments, the immunoglobulin single-chain variable domain polypeptide is selected from the group consisting of VH, VL, and VHH. In certain embodiments, the antigen-binding domain comprises a VHH (e.g., a first VHH domain and a second VHH domain).

[0067] For example, in certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise a single-chain variable domain polypeptide selected from the group consisting of scFv, VH, VL, VHH, and VNAR. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise an immunoglobulin single-chain variable domain polypeptide. In certain embodiments, the immunoglobulin single-chain variable domain polypeptide is selected from the group consisting of VH, VL, and VHH. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise a VHH (e.g., a first VHH domain and a second VHH domain).

[0068] Provided herein are antigen-binding domains that bind to FOLR1. "FOLR1" or "Folate receptor alpha" or "MOv18" or "Folate Receptor 1" refers to and encompasses the protein encoded by the FOLR1 gene (see NC_000011.10 (72189709..72196323), NCBI Gene 2348, or UniProt ID P15328).

[0069] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, where CDRs 1-3 are defined using the Kabat definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, where CDRs 1-3 are defined using the Chothia definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, where CDRs 1-3 are defined using the AbM definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, where CDRs 1-3 are defined using the Contact definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, where CDRs 1-3 are defined using the IMGT definition.

[0070] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a complementarity-determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a complementarity-determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 3, wherein the immunoglobulin single-chain domain binds to FOLR1.

[0071] In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0072] Provided herein are antigen-binding domains that bind to HER2. "HER2" or "ERBB2" or "erb-b2 receptor tyrosine kinase 2" refers to and encompasses the protein encoded by the HER2 gene (see NC_000017.11 (39688094..39728658), NCBI Gene 2064, or UniProt ID Q9UK79).

[0073] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 24, where CDRs 1-3 are defined using the Kabat definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 24, where CDRs 1-3 are defined using the Chothia definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 24, where CDRs 1-3 are defined using the AbM definition. In some embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising complementarity determining region (CDR) 1, complementarity determining region (CDR) 2, and complementarity determining region (CDR) 3 of SEQ ID NO: 24, where CDRs 1-3 are defined using the Contact definition. In some embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising complementarity determining region (CDR) 1, complementarity determining region (CDR) 2, and complementarity determining region (CDR) 3 of SEQ ID NO: 24, where CDRs 1-3 are defined using the IMGT definition.

[0074] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a complementarity-determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a complementarity-determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the immunoglobulin single-chain domain binds to HER2.

[0075] In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0076] Provided herein are antigen-binding domains that bind to DLL3. "DLL3," or "delta-like canonical Notch ligand 3," or "SCDO1," refers to and includes the protein encoded by the DLL3 gene (see NC_000019.10 (39498947..39508469), NCBI Gene 10683, or UniProt ID Q9NYJ7).

[0077] In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4, and the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506.

[0078] In some embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, where CDRs 1-3 are defined using the Kabat definition. In some embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, where CDRs 1-3 are defined using the Chothia definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, where CDRs 1-3 are defined using the AbM definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain comprising complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, where CDRs 1-3 are defined using the Contact definition. In some embodiments, the antigen-binding domain is an immunoglobulin single chain domain comprising complementarity determining region (CDR) 1, complementarity determining region (CDR) 2, and complementarity determining region (CDR) 3 of SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406, or 501-506, where CDRs 1-3 are defined using the IMGT definition.

[0079] In some embodiments, the antigen-binding domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 6, a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 7, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 110, a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 113, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 210, a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 213, or a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 220. a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 307, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 310, a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 313, a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 407, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 410, a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 413, or a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 507, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 510, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 513.

[0080] In some embodiments, the multivalent peptide is monospecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to the same target (e.g., antigen). In some embodiments, the multivalent peptide is monospecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to FOLR1. In some embodiments, the multivalent peptide is monospecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to DLL3. In some embodiments, the multivalent peptide is monospecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to HER2.

[0081] In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to different targets (e.g., antigens). In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain that binds to FOLR1 and a second antigen-binding domain that binds to DLL3.

[0082] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 407, (2) a CDR2 comprising SEQ ID NO: 410, and (3) a CDR3 comprising SEQ ID NO: 413. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 408, (2) a CDR2 comprising SEQ ID NO: 411, and (3) a CDR3 comprising SEQ ID NO: 414. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 409, (2) a CDR2 comprising SEQ ID NO: 412, and (3) a CDR3 comprising SEQ ID NO: 415.

[0083] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 307, (2) a CDR2 comprising SEQ ID NO: 310, and (3) a CDR3 comprising SEQ ID NO: 313. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 308, (2) a CDR2 comprising SEQ ID NO: 311, and (3) a CDR3 comprising SEQ ID NO: 314. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 309, (2) a CDR2 comprising SEQ ID NO: 312, and (3) a CDR3 comprising SEQ ID NO: 315.

[0084] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 207, (2) a CDR2 comprising SEQ ID NO: 210, and (3) a CDR3 comprising SEQ ID NO: 213. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 208, (2) a CDR2 comprising SEQ ID NO: 211, and (3) a CDR3 comprising SEQ ID NO: 214. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 209, (2) a CDR2 comprising SEQ ID NO: 212, and (3) a CDR3 comprising SEQ ID NO: 215.

[0085] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 507, (2) a CDR2 comprising SEQ ID NO: 510, and (3) a CDR3 comprising SEQ ID NO: 513. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 508, (2) a CDR2 comprising SEQ ID NO: 511, and (3) a CDR3 comprising SEQ ID NO: 514. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 509, (2) a CDR2 comprising SEQ ID NO: 512, and (3) a CDR3 comprising SEQ ID NO: 515.

[0086] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 107, (2) a CDR2 comprising SEQ ID NO: 110, and (3) a CDR3 comprising SEQ ID NO: 113. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 108, (2) a CDR2 comprising SEQ ID NO: 111, and (3) a CDR3 comprising SEQ ID NO: 114. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 109, (2) a CDR2 comprising SEQ ID NO: 112, and (3) a CDR3 comprising SEQ ID NO: 115.

[0087] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 5, (2) a CDR2 comprising SEQ ID NO: 6, and (3) a CDR3 comprising SEQ ID NO: 7.

[0088] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 403. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 303. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 304. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 305. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 503. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 103. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO:4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO:8.

[0089] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 403. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 303. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 304. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 305. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 503. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO: 103. In a specific embodiment, the antigen binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO:4, and the antigen binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO:8.

[0090] In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to different targets (e.g., antigens). In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain that binds to FOLR1 and a second antigen-binding domain that binds to HER2.

[0091] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises (1) a CDR1 comprising SEQ ID NO: 1, (2) a CDR2 comprising SEQ ID NO: 2, and (3) a CDR3 comprising SEQ ID NO: 3, and the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 4, and the antigen-binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH comprising SEQ ID NO: 4, and the antigen-binding domain that binds to HER2 comprises a VHH comprising SEQ ID NO: 24.

[0092] In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain and a second antigen-binding domain that bind to different targets (e.g., antigens). In some embodiments, the multivalent peptide is bispecific and comprises a first antigen-binding domain that binds to HER2 and a second antigen-binding domain that binds to DLL3.

[0093] In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 407, (2) a CDR2 comprising SEQ ID NO: 410, and (3) a CDR3 comprising SEQ ID NO: 413. In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 408, (2) a CDR2 comprising SEQ ID NO: 411, and (3) a CDR3 comprising SEQ ID NO: 414. In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 409, (2) a CDR2 comprising SEQ ID NO: 412, and (3) a CDR3 comprising SEQ ID NO: 415.

[0094] In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 307, (2) a CDR2 comprising SEQ ID NO: 310, and (3) a CDR3 comprising SEQ ID NO: 313. In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 308, (2) a CDR2 comprising SEQ ID NO: 311, and (3) a CDR3 comprising SEQ ID NO: 314. In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 309, (2) a CDR2 comprising SEQ ID NO: 312, and (3) a CDR3 comprising SEQ ID NO: 315.

[0095] In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 207, (2) a CDR2 comprising SEQ ID NO: 210, and (3) a CDR3 comprising SEQ ID NO: 213. In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 208, (2) a CDR2 comprising SEQ ID NO: 211, and (3) a CDR3 comprising SEQ ID NO: 214. In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 209, (2) a CDR2 comprising SEQ ID NO: 212, and (3) a CDR3 comprising SEQ ID NO: 215.

[0096] In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 507, (2) a CDR2 comprising SEQ ID NO: 510, and (3) a CDR3 comprising SEQ ID NO: 513. In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 508, (2) a CDR2 comprising SEQ ID NO: 511, and (3) a CDR3 comprising SEQ ID NO: 514. In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 509, (2) a CDR2 comprising SEQ ID NO: 512, and (3) a CDR3 comprising SEQ ID NO: 515.

[0097] In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 107, (2) a CDR2 comprising SEQ ID NO: 110, and (3) a CDR3 comprising SEQ ID NO: 113. In certain embodiments, the antigen binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 108, (2) a CDR2 comprising SEQ ID NO: 111, and (3) a CDR3 comprising SEQ ID NO: 114. In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 109, (2) a CDR2 comprising SEQ ID NO: 112, and (3) a CDR3 comprising SEQ ID NO: 115.

[0098] In certain embodiments, the antigen-binding domain that binds to HER2 comprises (1) a CDR1 comprising SEQ ID NO: 21, (2) a CDR2 comprising SEQ ID NO: 22, and (3) a CDR3 comprising SEQ ID NO: 23, and the antigen-binding domain that binds to DLL3 comprises (1) a CDR1 comprising SEQ ID NO: 5, (2) a CDR2 comprising SEQ ID NO: 6, and (3) a CDR3 comprising SEQ ID NO: 7.

[0099] In certain embodiments, the antigen binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 403. In certain embodiments, the antigen binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 303. In certain embodiments, the antigen binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 304. In certain embodiments, the antigen-binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 305. In certain embodiments, the antigen-binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 503. In certain embodiments, the antigen-binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 103. In certain embodiments, the antigen-binding domain that binds to HER2 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 24, and the antigen-binding domain that binds to DLL3 comprises a VHH comprising a sequence having at least 90% sequence identity to SEQ ID NO: 8.

[0100] In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 403. In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 303. In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 304. In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 305. In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 503. In certain embodiments, the antigen binding domain that binds HER2 comprises a VHH comprising SEQ ID NO: 24, and the antigen binding domain that binds DLL3 comprises a VHH comprising SEQ ID NO: 103. In a specific embodiment, the antigen binding domain that binds to HER2 comprises a VHH comprising SEQ ID NO:24, and the antigen binding domain that binds to DLL3 comprises a VHH comprising SEQ ID NO:8.

[0101] Fc domain "Fc region" (fragment crystallizable region), or "Fc domain," or "Fc" refers to and encompasses the C-terminal, non-antigen-binding region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (Clq) of the classical complement system. An antibody constant region typically comprises the CL region (e.g., in the case of a light chain) or the CH1-CH2-CH3 region. Generally, the Fc domain generally refers to and encompasses the CH2-CH3 region of the heavy chain constant region. For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cy2 and Cy3), and optionally all or a portion of the hinge region between CH1 (Cyl) and CH2 (Cy2). In some embodiments, the Fc domain comprises, from the N-terminus to the C-terminus, CH2-CH3 and hinge-CH2-CH3. In some embodiments, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4, comprising the hinge-CH2-CH3 domain / region. Furthermore, in certain embodiments, the Fc domain is a human IgG1 Fc domain, and the hinge comprises a C220S amino acid substitution. Additionally, in some embodiments where the Fc domain is a human IgG4 Fc domain, the hinge contains a S228P amino acid substitution. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues E216, C226, or A231 at its carboxyl terminus, numbered according to EU. In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγRs or FcRn, as described more fully below. The Fc domain can be a native sequence Fc, including any allotypic variants, or a variant Fc (containing one or more mutations that reduce effector cell function and / or FcRN).

[0102] "Hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to and encompasses the flexible polypeptide comprising the amino acids between the first (CH1) and second (CH2) heavy chain constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. For IgG, the antibody hinge comprises positions 216 (E216 in IgG1) to 230 (P230 in IgG1), numbering according to the EU index in Kabat. In some embodiments, for example, in the context of the Fc region, the hinge (full length or a fragment of the hinge) is included, generally referring to positions 216-230.

[0103] "Isotype" refers to and encompasses antibody classes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant domain genes. The full-length amino acid sequences of each wild-type human IgG constant region (including all domains, i.e., CH1, hinge, CH2, and CH3 domains) are cataloged in the UniProt database available online, e.g., as P01857 (IgG1), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or their different allotypes. When a domain of a heavy chain constant region comprises the amino acid sequence of a corresponding domain of each isotype, or a variant thereof (which has higher homology to the corresponding domain of each isotype than to the domain of another isotype), the domain is of the "IgG1 isotype," "IgG2 isotype," "IgG3 isotype," or "IgG4 isotype." An "allotype" refers to a naturally occurring variant within a particular isotype group, which variant differs by several amino acids (see, for example, Jefferies et al. (2009) mAbs 1:1). In certain embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is of the IgG4 isotype.

[0104] In some embodiments, the Fc domain comprises a variant Fc domain comprising one or more mutations that modulate (e.g., reduce, inhibit, decrease, prevent, etc.) an effector function associated with the heavy chain constant region, FcRn binding, or both.

[0105] The immunoglobulin heavy chain constant region may be a variant constant region comprising one or more changes to amino acid residues that confer additional utility and advantageous properties to the immunoconjugates described herein. In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or alter binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region or reduce binding of the immunoconjugate to neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0106] In some embodiments, the Fc domain comprises an alteration to the heavy chain constant region that reduces an effector function associated with the heavy chain constant region, such as the ability to fix complement, promote phagocytosis, or recruit other immune effector cells (e.g., NK cells) to the heavy chain constant region. In certain embodiments, the alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof. In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f ) 234A, 234V or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y or 329R, (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 25 4P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A, (t) 267G, 267H, 267I or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D,(mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235 E, (vv) L234A and L235A, (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A, L235E and G237A, (aaa) L234A, L235E, G237A and P331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (II1) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a)-(ooo). In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region include, according to EU numbering, L234A, L235E, G237A, A330S, and P331S.

[0107] In some embodiments, the Fc domain comprises an alteration to the heavy chain constant region that reduces the serum half-life of the immunoconjugate. In certain embodiments, the amino acid alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of 253, 254, 310, 435, 436, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253A, H310A, H435Q, and combinations thereof, according to EU numbering. In certain embodiments, the change that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: H310A, H435Q, and combinations thereof, according to EU numbering.

[0108] In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of an immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q, or 327T, (j) 329A, 329V, or 329V, 9G, 329Y or 329R (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A, (t) 267G, 267H, 267I or 267K, (u ) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (l l) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S,(zz) L234A, L235E and G237A, (aaa)L234A, L235E, G237A and P331S, (bbb)L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc)L234A, L235A and P329A, (ddd)G236R and L328R, (eee)G237A, (fff)F241A, (ggg)V264A, (hhh)D265A, ( iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (II1) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (ppp).

[0109] In certain embodiments, the changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region comprise, according to EU numbering, L234A, L235E, G237A, A330S, and P331S. In certain embodiments, the Fc domain comprises a change to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the change to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of, according to EU numbering, I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to an amino acid residue selected from the list consisting of I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are to an amino acid residue selected from the list consisting of I253A, H310A, H435Q, and combinations thereof, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise I253A, according to EU numbering. In certain embodiments, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise H310A, according to EU numbering. In a specific embodiment, the changes to one or more amino acid residues that alter binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprise H435Q according to EU numbering.

[0110] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 11, wherein the heavy chain constant region comprises a I253A substitution according to EU numbering.

[0111] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 12. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 12. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 12, wherein the heavy chain constant region comprises a S254A substitution according to EU numbering.

[0112] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 13. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 13. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 13, wherein the heavy chain constant region comprises an H310A substitution according to EU numbering.

[0113] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 14. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 14, wherein the heavy chain constant region comprises an H435Q substitution according to EU numbering.

[0114] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 15. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 15, and the heavy chain constant region comprises a Y436A substitution according to EU numbering.

[0115] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 16. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 16, wherein the heavy chain constant region comprises H310A / H435Q substitutions according to EU numbering.

[0116] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 17. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 17, wherein the heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S substitutions according to EU numbering.

[0117] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 18. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 18, wherein the heavy chain constant region comprises L234A, L235E, G237A, H310A, A330S, and P331S substitutions according to EU numbering.

[0118] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 19. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 19. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 19, wherein the heavy chain constant region comprises L234A, L235E, G237A, H435Q, A330S, and P331S substitutions according to EU numbering.

[0119] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 20. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence that is identical to SEQ ID NO: 20 according to EU numbering.

[0120] Changes that affect FcRn binding can decrease the serum half-life of the immunoconjugate, thus allowing one of skill in the art to select a half-life appropriate for a particular imaging or therapeutic goal. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours to about 120 hours. In certain embodiments, the immunoconjugate is administered for about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 108 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 108 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, about 36 hours to about The serum half-life is 108 hours, about 36 hours to about 120 hours, about 48 hours to about 60 hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours, about 48 hours to about 96 hours, about 48 hours to about 108 hours, about 48 hours to about 120 hours, about 60 hours to about 72 hours, about 60 hours to about 84 hours, about 60 hours to about 96 hours, about 60 hours to about 108 hours, about 60 hours to about 120 hours, about 72 hours to about 84 hours, about 72 hours to about 96 hours, about 72 hours to about 108 hours, about 72 hours to about 120 hours, about 84 hours to about 96 hours, about 84 hours to about 108 hours, about 84 hours to about 120 hours, about 96 hours to about 108 hours, about 96 hours to about 120 hours, or about 108 hours to about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 108 hours.In certain embodiments, the immunoconjugate has a serum half-life of at most about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours.

[0121] In certain embodiments, the immunoconjugate has a serum half-life of about 1 to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 to about 2 days, about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 1 to about 6 days, about 1 to about 7 days, about 1 to about 8 days, about 1 to about 9 days, about 1 to about 10 days, about 2 to about 3 days, about 2 to about 4 days, about 2 to about 5 days, about 2 to about 6 days, about 2 to about 7 days, about 2 to about 8 days, about 2 to about 9 days, about 2 to about 10 days, about 3 to about 4 days, about 3 to about 5 days, about 3 to about 6 days, about 3 to about 7 days, about 3 to about 8 days, about 3 to about 8 days, about 3 to about 3 days. The serum half-life is from about 3 to about 9 days, from about 3 to about 10 days, from about 4 to about 5 days, from about 4 to about 6 days, from about 4 to about 7 days, from about 4 to about 8 days, from about 4 to about 9 days, from about 4 to about 10 days, from about 5 to about 6 days, from about 5 to about 7 days, from about 5 to about 8 days, from about 5 to about 9 days, from about 5 to about 10 days, from about 6 to about 7 days, from about 6 to about 8 days, from about 6 to about 9 days, from about 6 to about 10 days, from about 7 to about 8 days, from about 7 to about 9 days, from about 7 to about 10 days, from about 8 to about 9 days, from about 8 to about 10 days, or from about 9 to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days. In certain embodiments, the immunoconjugate has a serum half-life of at most about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0122] In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, or about 20 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0123] chelating agents As described herein, a chelating agent can be attached to an immunoconjugate, an antigen-binding region / immunoglobulin heavy chain constant region molecule, a VHH antigen-binding region / immunoglobulin heavy chain constant region molecule (wild-type or mutant), or a VHH antigen-binding region / immunoglobulin Fc molecule (wild-type or mutant). The chelating agent allows the immunoconjugate to be loaded with a suitable radioisotope, such as a beta-emitter or an alpha-emitter. The chelating agent can be attached to the immunoconjugate via the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such attachment can suitably be via a covalent bond to one or more amino acids of the immunoconjugate, the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof.

[0124] In some embodiments, the chelator of the immunoconjugate is covalently bound to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In some embodiments, the chelator is covalently bound directly (e.g., without a spacer, stretcher, or linker) to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In some embodiments, the chelator is covalently bound to the antigen-binding arm via a linker that is covalently bound to the chelator and to the antigen-binding arm. In some embodiments, the linker is hydrophilic (e.g., a PEG chain). In some embodiments, the linker is hydrophobic (e.g., an alkyl or alkene chain). The chelator can be linked or attached to the immunoconjugate as described in Sadiki, A. et al. "Site-specific conjugation of native antibody." Antibody Therapeutics 2020, 3, 271-284.

[0125] In some embodiments, immunoconjugates are formed by site-specific conjugation of chelator-linkers directed to specific amino acid or glycan residues. In some embodiments, site-specific conjugation involves directed functionalization of specific lysine residues in framework regions with chelator-linkers. In other embodiments, the residues can be functionalized with a different reactive functional group, which then reacts with the chelator-linker in a second step to provide the immunoconjugate. In some embodiments, the reactive functional group is thiopropionate.

[0126] In some embodiments, non-naturally occurring cysteine ​​residues are engineered into the antibody framework as sites for thiol-directed conjugation to provide immunoconjugates, hi some embodiments, other non-naturally occurring amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for chelator-linkers or secondary reactive groups to which chelator-linkers are conjugated to provide immunoconjugates.

[0127] In some embodiments, a non-natural amino acid containing a bridging group is engineered into the framework for chelator-linker attachment. In some embodiments, the non-natural amino acid contains an azide.

[0128] In some embodiments, the chelator-linker is attached to the glutamine residue via the action of a transglutaminase enzyme, while in other embodiments, a secondary reactive group is attached by transglutaminase, onto which the chelator-linker is added to provide the immunoconjugate.

[0129] In some embodiments, the chelator-linker is attached by modifying one or more N-glycans with reactive functional groups by the action of glycosidase, followed by conjugation of the chelator-linker to that site. In some embodiments, the glycan is modified by the action of β-galactosidase. In some embodiments, the glycan is modified with an azide-containing glycoside for attachment of an appropriately functionalized chelator-linker.

[0130] In some embodiments, the immunoconjugate comprises more than one chelating agent, which may be the same or different.

[0131] In some embodiments, an immunoconjugate having more than one chelator has more than one chelator attached to the same antigen-binding arm.

[0132] In some embodiments, an immunoconjugate having more than 1 chelator and less than 11 chelators has more than 2 chelators, more than 3 chelators, more than 4 chelators, more than 5 chelators, more than 6 chelators, more than 7 chelators, more than 8 chelators, or more than 9 chelators. In some embodiments, the chelators are the same. In some embodiments, each antigen-binding arm is linked, directly or indirectly, to more than one chelator.

[0133] In some embodiments, the chelator comprises a radioisotope chelating component and a functional group that allows for covalent attachment to the antigen-binding arm. In some embodiments, the functional group is directly attached to the radioisotope chelating component. In some embodiments, the chelator further comprises a linker between the functional group and the radioisotope chelating component.

[0134] In some embodiments, the radioisotope chelating moiety comprises DOTA or a DOTA derivative. In some embodiments, the radioisotope chelating moiety comprises DOTAGA. In some embodiments, the radioisotope chelating moiety comprises Macropa or a Macropa derivative. In some embodiments, the radioisotope chelating moiety comprises Py4Pa or a Py4Pa derivative.

[0135] In a preferred embodiment, the chelator of the immunoconjugate is not attached to an antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0136] In some embodiments, the chelator of the immunoconjugate is non-covalently associated with the antigen-binding arm, hi preferred embodiments, the chelator is not associated with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0137] In some embodiments, the chelator comprises DOTA or a DOTA derivative. In some embodiments, the chelator comprises DOTAGA. In some embodiments, the chelator comprises Macropa or a Macropa derivative. In some embodiments, the chelator comprises Py4Pa or a Py4Pa derivative. In some embodiments, the chelator comprises siderocalin or a siderocalin derivative.

[0138] In certain embodiments, described herein is an immunoconjugate conjugated to a chelating agent. In certain embodiments, the chelating agent is a radioisotope chelating agent. In certain embodiments, the radioisotope chelating agent is selected from the list consisting of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), α-(2-carboxyethyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa). In certain embodiments, the radioisotope chelating agent is DOTA. In certain embodiments, the radioisotope chelating agent is DOTAGA. In certain embodiments, the radioisotope chelating agent is Py4Pa. In certain embodiments, the radioisotope chelating agent is a radioisotope directly attached to an antigen-binding region and / or an immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelator is attached to the antigen binding region or the immunoglobulin heavy chain constant region by a linker. In certain embodiments, the linker is selected from those derived from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino-benzyl-oxycarbonyl (PAB), and conjugates with the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercapto-pentanoic acid (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), polyethylene glycol (PEG), polyethylene glycol polymer (PEGn), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is selected from polyethylene glycol (PEG), polyethylene glycol polymer (PEG), and S-2-(4-isothiocyanatobenzyl) (SCN). In certain embodiments, the linker is PEG5. In certain embodiments, the linker is SCN.In certain embodiments, the radioisotope chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0139] The chelating agent may be conjugated to the protein or antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In certain embodiments, the radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 6:1. In certain embodiments, the radioisotope chelating agent is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0140] In some embodiments, immunoconjugates of the invention comprise a linker, for example, for linking the antigen-binding arm to a chelator (interchangeably "chelator"), or to a radioisotope, or to a cargo (e.g., a cytotoxin). The linker may comprise one or more linker moieties. In some embodiments, immunoconjugates of the invention are engineered to have a terminal lysine available for conjugation with a chelator or linker.

[0141] For example, bifunctional chelators are used to conjugate radioisotopes to the radioisotope delivery platforms of the present invention to produce immunoconjugates of the present invention. (See, e.g., Scheinberg D, McDevitt M, Curr Radiopharm 4:306-20 (2011)). Examples of bifunctional chelators known in the art include DOTA, DTPA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX'A"-DTPA, p-SCN-Bn-TCMC, macropa-NCS, crown, p-SCN-Ph-Et-Py4Pa, 3,2-HOPO, and TCMC.

[0142] Examples of bifunctional chelating agents are 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogs of the foregoing. Such chelating agents are suitable for coordinating metal ions, such as α- and β-emitting radionuclides.

[0143] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the invention is a bifunctional chelator, such as DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-TCMC, Macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), Crown (Yang H, et al. Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. al. Int. J. Rad. One. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelators, see, e.g., Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166 (2008)).

[0144] In some embodiments, the chelator of the immunoconjugate or radioimmunoconjugate of the invention is a bifunctional chelator, such as DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A"-DTPA, p-SCN-Bn-TCMC, Macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56:1 (2017)), Crown (Yang H, et al. Chem. Eur. J. 26:11435 (2020)), P-SCN-Ph-Et-Py4Pa (Li L, et al. Bioconjugate Chem. ASAP (2020)), 3,2-HOPO (Wickstroem K, et al. al. Int. J. Rad. One. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelators, see, e.g., Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW QJ Nucl. Med. Mol. Imaging 52:166 (2008)).

[0145] For 225-Ac immunoconjugates, there are a variety of acyclic and cyclic ligands known in the art as suitable chelators (see, e.g., Davis I, et al., Nucl Med Biol 26:581 (1999); Chappell L, et al., Bioconjug Chem 11:510 (2000); Chappell, L, et al., Nucl Med Biol 30:581 (2003); McDevitt M, et al., Appl Radiat Isot 57:841 (2002); Gouin S, et al., Org Biomol Chem 3:453 (2005); Thiele N, et al., Angew Chem Int Ed Engl 56:14712 (2017)).

[0146] In certain embodiments, the chelating agent is a chelating agent suitable for alpha emitter chelation. Some suitable chelators for alpha emitters are described in Yang et al., “Harnessing alpha-Emitting Radionuclides for Therapy: Radiolabeling Method Review.” J Nucl Med. 2022 Jan;63(1):5-13.

[0147] In certain embodiments, chelators suitable for alpha emitter chelation include DOTA 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid, DO3A 1,4,7-tris(carboxy-methyl) 1,4,7,10-tetra-azacyclododecane, DOTAGA α-(2-carboxyethyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAGA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl) 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, Py4Pa 6,6',6'',6''''-(((pyridine-2,6-diylbis(methylene))bis(azatriyl))tetrakis(methylene))tetrapicolinic acid, Py4Pa-NCS is 6,6'-((((4-isothiocyanatopyridine-2,6-diyl)bismethylene))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Crown 2,2',2'',2'''-(1,10-dioxa-4,7,13,16-tetra-azacyclooctadecane-4,7,13,16-tetrayl)tetra-acetic acid, Macropa 6,6'-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid, Macropa-NCS 6-((16-((6-carboxypyridin-2-yl)methyl) 1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl) 4-isothiocyanatopicolinic acid, HEHA 1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid, CHX octapa 6,6'-[(1R,2R) 1,2-cyclohexanediylbis[[(carboxy-methyl)imino]methylene]]bis[2-pyridinecarboxylic acid], Bispa 3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylic acid, 7-[(6-carboxy-2-pyridinyl)methyl]-9-hydroxy-3-methyl-2,4-di-2-pyridinyl-,1,5-dimethyl ester, Noneunpa, 6,6'-(((oxybis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, and combinations thereof.

[0148] In certain embodiments, the chelating agent is suitable for beta-emitter chelation or gamma-emitter chelation. In certain embodiments, chelating agents suitable for beta-emitter chelation or gamma-emitter chelation include DOTMA (1R,4R,7R,10R) a,a',a",a"'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl) 1,4,7,10-tetraazacyclododecane), DOTPA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid, DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl) 1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTP 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid), DOTMP 1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTP 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid, TETA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, PEPA 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N'”,N""-pentaacetic acid, H4Octapa N,N'-bis(6-carboxy-2-pyridylmethyl) ethylenediamine-N,N'-diacetic acid, H2Dedpa 1,2-[[6-(carboxy) Pyridin-2-yl]-methylamino]ethane, H6phospa N,N'-(methylene-phosphonate) N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane, TTHA triethylenetetramine-N,N,N',N'',N'”,N”'-hexaacetic acid, DO2P tetraazacyclododecanedimethanephosphonic acid, HP-DO3A hydroxy-propyltetraazacyclododecanetriacetic acid, EDTA ethylenediaminetetraacetic acid, DTPA diethylenetriaminepentaacetic acid, DTPA-BMA Diethylenetriamine-pentaacetic acid-bismethylamide, HOPO octadentate hydroxypyridinone, 3,2,3-LI(HOPO)N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydropyridine-).

[0149] 2-Carboxamido)propyl) 6-oxo-1,6-dihydropyridine-2-carboxamido), 3,2-HOPO N,N'

[0150] -(((2-(4-aminobenzyl) 3-((2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydro-pyridine-4-carboxamido)ethyl)(2-(3-hydroxy-2-oxo-1,2-dihydropyridine-4-carbox-amido)ethyl)amino)propyl)azanediyl)bis(ethane-2,1-diyl))bis(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide), Neunpa 6,6'-(((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Neunpa-NCS = 6,6'-(((((4-isothiocyanatophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid, Octapa 6,6'-((ethane-1,2-diylbis((carboxymethyl)(azanediyl))bis(methylene))dipicolinic acid, Octox 2,2'-(ethane-1,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)(azanediyl))diacetic acid, PyPa 6,6'-(((pyridine-2,6-diylbis(methylene))bis((carboxymethyl)(azadiyl))bis(methyl-ene))dipicolinic acid, porphyrin 21,22,23,24-tetraazapentacyclo[16.2.1.13,6.18,11.113,16]tetracosa-1,3,5,7,9,11(23),12,14,16,18(21),19-undecene, deferoxamine 30-amino-3,14,25-trihydroxy-3,9,14,20,25-penta-azatriacontane-2,10,13,21,24-pentaone, DFO*N1-[5-(acetyl-hydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetra-azahexacosanediamide, and combinations thereof.

[0151] Alternatively, or in addition, an isothiocyanate linker, such as p-SCN-Bn-DOTA, may be used which comprises a lysine residue in the immunoconjugates of the invention.

[0152] Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and the linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate forming linker moiety 4-((2,5-dioxopyrrolidin-1-yl)methyl)cyclohexanecarboxylic acid ("SMCC", also referred to herein as "MCC"), 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate forming Linker moieties include those derived from the conjugation of 4-mercaptobutanoic acid ("SPDB"), N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), and ethyleneoxy-CHCHO- as one or more repeating units ("EO," "PEO," or "PEG"). Additional linker components are known in the art, some of which are described herein. A variety of linker components are known in the art, some of which are described below.

[0153] In certain embodiments, the linker is SCN. In certain embodiments, the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa. In certain embodiments, the chelator is TFP-Ad-PEG5-DOTAGA. In certain embodiments, the chelator is p-SCN-Bn-DOTA. In certain embodiments, the chelator is p-SCN-Ph-Et-Py4Pa. In certain embodiments, the chelator is TFP-Ad-PEG5-Ac-Py4Pa. Such linkers are shown in Figure 18.

[0154] The linker may be a "cleavable linker" that facilitates release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-31 (1992); U.S. Patent No. 5,208,020) may be used.

[0155] In certain embodiments, the linker has the formula:

[0156] [ka] As shown in the figure.

[0157] wherein A is a Stretcher unit, a is an integer from 0 to 1, W is an amino acid unit, w is an integer from 0 to 12, Y is a Spacer unit, y is 0, 1 or 2, and Ab, D, and p are as defined above. Exemplary embodiments of such linkers are described in US20050238649.

[0158] In some embodiments, a linker component may comprise a "stretcher unit" that links the immunoconjugate to another linker component or a drug moiety. Exemplary stretcher units include:

[0159] [ka] (where the wavy line indicates the site of covalent attachment to the immunoconjugate).

[0160] In some embodiments, linkers can be conjugated to antibodies via cysteine ​​bridging functionalities such as ThioBridge® or DBM (dibromomaleimide). These linkers can act to restabilize intrachain disulfides after reduction and conjugation (Bird M, et al., Antibody-Drug Conjugates pp. 113-129 (2019), and Behrens CR, et al. Mol. Pharmaceutics 12:3986 (2015)). Exemplary rebridging stretcher elements are listed below.

[0161] [ka] (where the wavy line indicates the site of covalent attachment to the immunoconjugate).

[0162] In some embodiments, the linker component may comprise an amino acid unit. In one such embodiment, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to an intracellular protease, such as a lysosomal enzyme (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21:778-4). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid units can include naturally occurring amino acid residues, as well as minor amino acids and unnatural amino acid analogs, such as citrulline. The amino acid units can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0163] In some embodiments, the linker component may include a "spacer" unit that links the immunoconjugate to the drug moiety directly or via a stretcher unit and / or an amino acid unit. The spacer unit may be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety upon enzymatic (e.g., proteolytic) cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Other combinations of peptide spacers susceptible to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease would result in release of the glycine-glycine-drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0164] A "self-immolative" spacer unit allows for release of the drug moiety without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the benzyl alcohol and the cytotoxic agent (see, e.g., Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-103). In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene portion of the p-aminobenzyl unit is substituted with Qm, where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. Examples of self-immolative spacer units further include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, e.g., US2005 / 0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., 1972, 94:5815), and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., 1990, 55:5867). Elimination of amine-containing drugs substituted at the alpha position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27:1447) is also an example of a self-immolative spacer useful in ADCs.

[0165] In some embodiments, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, shown below, which can be used to incorporate and release multiple drugs.

[0166] [ka]

[0167] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; n is 0 or 1; and p is ranging from 1 to about 20.

[0168] In some embodiments, the immunoconjugate comprises a linker, such as a dendritic linker, for covalently attaching more than one drug moiety to an antibody via, for example, a branched polyfunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-5; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-8). Dendritic linkers can increase the drug-to-antibody molar ratio, i.e., loading, which is related to the potency of the ADC. Thus, cysteine-engineered antibodies have only one reactive cysteine ​​thiol group, and multiple drug moieties can be attached via dendritic linkers.

[0169] Examples of linker moieties and combinations thereof are shown below, which are also suitable for use in the above formula.

[0170] [ka]

[0171] Further non-limiting examples of linkers include those described in WO 2015095953.

[0172] Linker components, including stretcher, spacer, and amino acid units, can be synthesized by methods known in the art, such as those described in US20050238649.

[0173] In some embodiments, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,189, filed August 22, 2022, or a U.S. non-provisional or international application claiming priority thereto, which are incorporated herein by reference with respect to such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, and 1-34 described in U.S. Application No. 63 / 373,189, filed August 22, 2022, which is incorporated herein by reference for such compounds.

[0174] In some aspects, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,183, filed August 22, 2022, or U.S. non-provisional or international applications claiming priority thereto, which are incorporated by reference herein with respect to such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, and 2-13, described in U.S. Application No. 63 / 373,183, filed August 22, 2022, which are incorporated by reference herein with respect to such compounds.

[0175] In some embodiments, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,190, filed August 22, 2022, or a U.S. non-provisional application claiming priority thereto, which are incorporated by reference herein with respect to such compounds. In some embodiments, the chelator comprises a linker and is selected from compounds 3-1, 3-2, 3-3, 3-4, 3-5, 3-13, 3-16, described in U.S. Application No. 63 / 373,190, filed August 22, 2022, which are incorporated by reference herein in their entirety with respect to such compounds.

[0176] Radio-immunoconjugates A "radionuclide" or "radioisotope" refers to an alpha-emitting isotope (interchangeably, α-emitting isotope), a beta-emitting isotope (interchangeably, β-emitting isotope), or a β-emitting isotope (interchangeably, β-emitting isotope), such as, for example, any one of 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 225-Ac, 213-Bi, 213-Po, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd. "γ-emitting isotope" refers to and includes a γ-emitting isotope, and / or a gamma-emitting isotope (gamma-emitting isotope) (interchangeably, a γ-emitting isotope).

[0177] A "radio-immunoconjugate" (also used interchangeably with "immunoconjugate" when used in the context of treatment) refers to and encompasses a molecular complex comprising: (1) an immunoconjugate according to the present invention; and (2) a radioisotope. In some embodiments, the radioisotope is an α-emitting isotope. In some embodiments, the radioisotope is a β-emitting isotope. In some embodiments, the radioisotope is a γ-emitting isotope. In some embodiments, the present invention provides a radioimmunoconjugate comprising an α-emitting isotope and a β-emitting isotope. The term "radioconjugate" is also used interchangeably herein with the term "radioimmunoconjugate." In some embodiments, the radioisotope is attached to a chelator of the radioimmunoconjugate. In some embodiments, the radioisotope is directly linked to the immunoconjugate.

[0178] In some embodiments, the present invention provides immunoconjugates. In some embodiments, the immunoconjugates, when so labeled, linked, or loaded with an α-emitter, are capable of delivering an α-emitter in vivo. In some embodiments, the immunoconjugates, when so labeled, linked, or loaded, are also capable of delivering other radioisotopes (β-emitters and / or γ-emitters) and / or other atoms in vivo. In some embodiments, the immunoconjugates, when so labeled, linked, or loaded, are capable of delivering an imaging metal (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga, or 134-Ce) in vivo.

[0179] The immunoconjugates of the present disclosure may be loaded with a radioisotope for therapeutic or diagnostic effects. In certain embodiments, the chelating agent may further comprise a radioisotope. In certain embodiments, the radioisotope is an alpha emitter. In certain embodiments, the radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a beta emitter. In certain embodiments, the radioisotope is a beta emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0180] Also described herein are methods for producing radioimmunoconjugates, which include loading or conjugating the immunoconjugates of the present disclosure to a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In certain embodiments, the radioisotope is 225-Ac. In certain embodiments, the radioisotope is a β-emitter. In certain embodiments, the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

[0181] In some aspects, the present disclosure provides a radioimmunoconjugate comprising an immunoconjugate of the present disclosure and an α-emitting isotope. In some embodiments, the α-emitting isotope of the radioimmunoconjugate is selected from the group including 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the α-emitting isotope of the radioimmunoconjugate is selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the α-emitting isotope of the radioimmunoconjugate is 225-Ac. In some embodiments, the α-emitting isotope of the radioimmunoconjugate is 223-Ra. In some embodiments, the α-emitting isotope of the radioimmunoconjugate is 224-Ra. In some embodiments, the alpha-emitting isotope of the radioimmunoconjugate is 227-Th. In some embodiments, the alpha-emitting isotope of the radioimmunoconjugate is 212-Pb. In some embodiments, the alpha-emitting isotope of the radioimmunoconjugate is 212-Bi. In some embodiments, the alpha-emitting isotope of the radioimmunoconjugate is 213-Bi.

[0182] In some embodiments, the immunoconjugates of the present invention are combined with a radioisotope to provide radioimmunoconjugates of the present invention. In some embodiments, the radioisotope is 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, or 103-Pd. In some embodiments, the radioisotope is an alpha emitter, such as, for example, 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the radioisotope is a beta particle emitter, such as, for example, 177-Lu, 90-Y, 67-Cu, 153-Sm, etc. In some embodiments, the radioisotope is both an alpha particle emitter and a beta and / or gamma particle emitter. In some embodiments, the radioisotope is both a beta particle emitter and a gamma particle and / or photon emitter. In some embodiments, the radioimmunoconjugate is labeled, linked, or loaded with, or thus comprises, both an alpha emitter and a beta emitter. In some embodiments, the radioisotope is selected for use in radioimaging from, for example, 68-Ga, 64-Cu, 89-Zr, 111-In, 134-Ce.

[0183] The immunoconjugates and radioimmunoconjugates of the invention can contain other cargoes or payloads, in addition to radioisotopes, including, for example, various cytotoxic agents such as a small molecule chemotherapeutic agent, a cytotoxic antibiotic, an alkylating agent, antimetabolite, a topoisomerase inhibitor, and / or a tubulin inhibitor. For example, the immunoconjugates of the invention can be used to deliver a non-radioisotope cytotoxin to a target cell. Non-limiting examples of cytotoxic agents include aziridine, cisplatin, tetrazine, procarbazine, hexamethyl-lmelamine, vinca alkaloids, taxanes, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin, dolastatins, maytansine, docetaxel, adriamycin, calicheamicin, auristatins, pyrrolo-benzodiazepines, carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, 1,3-bis(2-chloroethyl) These include 1-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, and gemcitabine.

[0184] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from the group including 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 211-At, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd.

[0185] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from the group consisting of 225-Ac, 86-Y, 90-Y, 177-Lu, 186-Re, 188-Re, 89-Sr, 153-Sm, 213-Bi, 213-Po, 211-At, 212-Bi, 223-Ra, 224-Ra, 227-Th, 149-Tb, 68-Ga, 64-Cu, 67-Cu, 89-Zr, 137-Cs, 212-Pb, and 103-Pd.

[0186] In some embodiments, the radioisotope is an alpha-particle-emitting radioisotope, including 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, or 213-Bi.

[0187] In some embodiments, the radioisotope is an alpha particle-emitting radioisotope selected from the group consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

[0188] Immunoconjugate Derivatives and Other Modifications Covalent modification of the immunoconjugates of the present invention is included within the scope of the present invention. One type of covalent modification involves reacting targeted amino acid residues of the immunoconjugates of the present disclosure with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues of the immunoconjugate. Derivatization with bifunctional agents is useful, for example, for crosslinking the immunoconjugates of the present disclosure to a water-insoluble support matrix or surface for use in methods for purifying the immunoconjugates of the present disclosure, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl) 2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., with 4-azidosalicylic acid, 3,3′-dithiobis(succinimidyl propionate), homobifunctional imidoesters including disuccinimidyl esters, bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0189] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, respectively, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0190] In some embodiments, the immunoconjugates provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of immunoconjugates include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone). Polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the immunoconjugate can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the immunoconjugate to be improved, whether the immunoconjugate derivative will be used therapeutically under defined conditions, etc.

[0191] PEG-derivatized immunoconjugates of the present invention may contain one or more -CH2CHO- containing linkers, which can be used to alter the biodistribution and pharmacokinetics of the immunoconjugate. PEG can be prepared in polymeric form or as individual oligomers. Bifunctionalized versions of these polymers can link the immunoconjugate with chelators and / or provide additional size and / or solubility to the overall molecule. In some embodiments, PEG-derivatized immunoconjugates exhibit reduced immunogenicity compared to their underivatized parent molecules.

[0192] Methods for producing immunoconjugates The present invention provides an immunoconjugate according to any of the above embodiments, or a composition comprising one or more of the immunoconjugates described herein. In another embodiment, the present invention provides an isolated nucleic acid encoding the radioisotope delivery platform described herein. Also provided herein are nucleic acids encoding protein components of the immunoconjugates of the present invention, expression vectors comprising the nucleic acids, and host cells comprising the expression vectors.

[0193] In another embodiment, the present invention provides a host cell comprising the nucleic acid and / or vector provided herein. In some embodiments, the host cell of the present invention is isolated or purified. In some embodiments, the host cell of the present invention is in cell culture medium. The nucleic acids, expression vectors, and host cells of the present disclosure can be used to generate compositions comprising one or more of the immunoconjugates of the present disclosure. In some embodiments, the host cell is eukaryotic. In some embodiments, the host cell is mammalian. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is prokaryotic. In some embodiments, the host cell is E. coli.

[0194] Exemplary techniques for producing immunoconjugates and radioimmunoconjugates of the invention for use according to the methods of the invention are described below. In some embodiments, the invention provides a process for making an immunoconjugate of the present disclosure, the method comprising culturing a host cell provided herein under conditions suitable for an expression vector encoding a radioisotope delivery platform, and recovering or purifying the radioisotope delivery platform. In some embodiments, the method further comprises radiolabeling the radioisotope delivery platform with a suitable isotope, e.g., an α- or β-particle emitter.

[0195] Generation and Identification of Antigen-Binding Domains, Immunoconjugates, and Nucleic Acids

[0196] The antigen-binding domain useful as the antigen-binding region herein can be identified in either monoclonal and / or polyclonal antibodies. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional procedures. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells (see, for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol Revs. 130:151-188 (1992)).

[0197] In some embodiments, the antigen-binding domain of an immunoconjugate of the present invention, or a fragment thereof, is isolated by screening a phage library containing phage displaying various fragments of antibody variable regions (Fv, scFv, or VHH) fused to a phage coat protein. Such a phage library is screened for binding to a desired target antigen or epitope. Clones expressing Fv fragments, scFv, or VHH that can bind to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0198] In some embodiments, antibodies or antibody fragments thereof are isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc Acids Res. 21:2265-2266 (1993)). Variable domains can be functionally displayed on phage, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994), in which VH and VL are covalently linked via a short, flexible peptide, as single-chain Fv (scFv) fragments, or as Fab fragments, in which they are each fused to a constant domain and interact noncovalently.

[0199] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding clones, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Naive libraries for screening can be constructed from non-immunized sources to provide high-affinity antibodies to the antigen (see, e.g., Griffiths et al., EMBO J. 12:725-734 (1993)). Another example is a naive library synthetically constructed by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0200] Screening of libraries can be accomplished by a variety of techniques known in the art. For example, target antigens can be used to coat the wells of adsorption plates, expressed on host cells immobilized on adsorption plates, used in cell sorting, conjugated to biotin for capture by streptavidin-coated beads, or used in any other method for panning display libraries. Selection of antibodies with slow dissociation rates (and strong binding affinity) can be facilitated by the use of long washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO1992 / 09690, and low antigen coating density, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0201] Techniques for screening cDNA libraries are well known in the art. Libraries can be screened using probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening of a cDNA or genomic library with a selected probe can be performed using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means for isolating the gene encoding the immunoconjugate of the present invention is to use PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0202] DNA encoding the immunoconjugates of the invention can be obtained from a cDNA library prepared from tissue believed to harbor the immunoconjugate mRNA of the invention and express it at detectable levels. Thus, human immunoconjugate DNA of the invention can be conveniently obtained from a cDNA library prepared from human tissue. Immunoconjugate invention-encoding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis). In some embodiments, the desired polynucleotide sequence encoding an antibody can be isolated and sequenced from antibody-producing cells, such as hybridoma cells.

[0203] The sequences identified in such library screening methods can be compared and aligned with other known sequences deposited and available in public databases, such as GenBank or other private sequence databases. Sequence identity (either at the amino acid level or nucleotide level) within a defined region of a molecule or over the entire sequence can be determined using methods known in the art and described herein. Any of the antibody CDRs or heavy chain variable fragments of the present invention can be obtained by designing an appropriate antigen screening procedure to select a phage clone of interest, and then constructing an antibody clone using the variable domain and / or CDR sequences from the phage clone of interest and an appropriate constant region (Fc) sequence as described in Kabat et al., 1991, supra.

[0204] Production of immunoconjugates; host cells and expression vectors of the invention The following description primarily relates to producing the antibody constructs of the present invention by culturing cells transformed or transfected with a vector-containing immunoconjugate of a nucleic acid encoding the antibody construct. It is, of course, contemplated that alternative methods, well known in the art, may be used to prepare the antibody constructs of the present invention. For example, the appropriate amino acid sequence, or portions thereof, may be generated by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J., Am. Chem. Soc., 85:2149-54 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. The various portions of the immunoconjugates of the present invention can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugate of the present invention.

[0205] Antibody constructs can be produced using recombinant methods and compositions such as those described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids encoding the antibodies described herein are provided. Such nucleic acids can encode an amino acid sequence comprising the VH and / or VL amino acid sequence (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In some embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody (e.g., has been transformed with the vector). In some other embodiments, the host cell comprises (1) a vector comprising a nucleic acid encoding 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 encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In some embodiments, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20 cell). In some embodiments, a method of making an immunoconjugate of the invention is provided, the method comprising culturing a host cell comprising nucleic acid encoding an antibody provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0206] For recombinant production of the immunoconjugates of the present invention, nucleic acids encoding, for example, antibody constructs as described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and / or light chains of the antibody). Nucleic acid molecules encoding the amino acid sequences (including sequence variants) of the immunoconjugates of the present invention can be prepared by a variety of methods known to those skilled in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared mutant or non-mutant versions of the antibody construct.

[0207] Engineering host cells for immunoconjugate production For the production of the immunoconjugates of the present invention, host cells are transfected or transformed with the expression or cloning vectors described herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Culture conditions, such as medium, temperature, and pH, can be selected by those skilled in the art without undue experimentation. Generally, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991), and Sambrook et al., supra.

[0208] Suitable host cells for cloning or expressing the nucleic acid and vector encoding the immunoconjugate include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, US 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describe the expression of antibody fragments in E. coli. After expression, the immunoconjugate can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0209] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for immunoconjugate-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0210] Suitable host cells for the expression of glycosylated immunoconjugates are also derived from multicellular organisms (e.g., invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains suitable for use with insect cells, particularly for transfection of Spodoptera frugiperda cells, have been identified. Plant cell cultures can also be used as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429).

[0211] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 line, human embryonic kidney line (e.g., 293 or 293 cells described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV 1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MOCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep 02), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC5 cells, and FS4 cells, described in NYAcad. Sci. 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)), and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for immunoconjugate production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0212] Methods for eukaryotic cell transfection and prokaryotic cell transformation, which involve introducing DNA into a host so that it is replicable, either extrachromosomally or as a chromosomal integrant, are well known to those skilled in the art and include, for example, CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described by Sambrook et al. (supra), or electroporation is commonly used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859, published June 29, 1989. For mammalian cells lacking such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be used. A general aspect of mammalian cell host system transfection is described in U.S. Pat. No. 4,399,216. Transformation into yeast is typically performed according to the methods of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc Natl Acad Sci USA 76:3829 (1979). However, other methods for introducing DNA into cells, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations such as polybrene and polyornithine, can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0213] Prokaryotic Host Cells Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as K12 strains MM294 (ATCC 31,446), X1776 (ATCC 31,537), W3110 (ATCC 27,325), and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Enterobacteriaceae, such as Bacilli, such as B. subtilis and B. licheniformis (e.g., B. licheniformis, as disclosed in DD 266,710 published April 12, 1989). 41P), P. aeruginosa, Rhizobia, Pseudomonas such as Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative rather than limiting. E. coli strain W3110 is one advantageous host or parent host because it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) can be altered to introduce genetic mutations into proteins encoding genes endogenous to the host; examples of such hosts include E. coli W3110 strain 1A2 with the complete tonA genotype, E. coli W3110 strain 9E4 with the complete tonA ptr3 genotype, E. coli W3110 strain 27C7 (ATCC 55,244) with the complete tonA ptr3 phoA E15(argF-lac)169degP ompT kanr genotype, and E. coli W3110 strain 27C7 with the complete tonA ptr3 phoA E15(argF-lac)169degP ompT rbs7 ilvG genotype. Suitable strains include E. coli W3110 strain 37D6 with kanr, E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation, E. coli W3110 strain 33D3 with the genotype W3110 ΔfhuA(ΔtonA) ptr3 lac lacL8 ΔompT Δ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635), and E. coli strains with mutant periplasmic proteases as disclosed in U.S. Pat. No. 4,946,783, issued August 7, 1990. Other strains and their derivatives are also suitable, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608). These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, the appropriate bacterium must be selected taking into account the replicability of the replicon in the bacterial cell.For example, Escherichia coli, Serratia, or Salmonella species may be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated into the cell culture. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0214] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially if glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. After expression, the immunoconjugate can be isolated from the E. coli cell paste in the soluble fraction and purified, for example, through a protein A or G column depending on the isotype. Final purification can be performed similarly to the process for purifying antibodies expressed in, for example, CHO cells.

[0215] Eukaryotic Host Cells In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for immunoconjugation of invention-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other examples include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383 published May 2, 1985), Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9:968-75 (1991)), such as K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), K. thermotolerans and K. marxianus, yarrowia (EP402,226), Pichia pastoris (EP183,070; Sreekrishna et al., J. Basic Microbiol.,28:265-278(1988)), Candida, Trichoderma reesia(EP244,234), Neurospora crassa(Case et al., Proc Natl Acad Sci USA 76:5259-5263 (1979)), Schwanniomyces, e.g., Schwanniomyces occidentalis (EP 394,538 published October 31, 1990), as well as filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991), Aspergillus hosts, e.g., A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc Natl Acad Sci USA 81:1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotrophic yeasts, as appropriate herein, include, but are not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0216] Suitable host cells for the expression of glycosylated immunoconjugates of the present invention are derived from multicellular organisms.Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures.A large number of baculovirus strains and mutants have been identified, as well as corresponding permissible insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses according to the present invention and as described herein, particularly for transfection of Spodoptera frugiperda cells.

[0217] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatoma line (Hep G2).

[0218] Host cells are transformed with the above-described expression or cloning vectors for producing the immunoconjugates of the invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0219] Selection and use of replicable vectors

[0220] For recombinant production of the radioisotope delivery platform of the present invention, the nucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the immunoconjugate is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of an antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are either of prokaryotic or eukaryotic (generally mammalian) origin.

[0221] Vectors can be, for example, in the form of plasmids, cosmids, virus particles, or phage.Appropriate nucleic acid sequences can be inserted into vectors by various procedures.Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0222] The immunoconjugates of the present invention can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, which can be signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence can be a component of the vector, or it can be a part of the DNA encoding the immunoconjugates of the present invention that is inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence can be, for example, the yeast invertase leader, the alpha-factor leader (including the Saccharomyces and Kluyveromyces alpha-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990), or the signals described in WO 90 / 13646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct protein secretion, for example, signal sequences from secreted polypeptides of homologous or related species, as well as viral secretory leaders.

[0223] Cultivation of Host Cells Producing Radioisotope Delivery Platforms The host cells used to produce the immunoconjugates of the present invention may be cultured in a variety of media and culture conditions.

[0224] Prokaryotic Host Cell Cultures Prokaryotic cells used to produce the polypeptides of the present invention are grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria Broth (LB) and necessary nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing an ampicillin resistance gene.

[0225] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or in mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol.

[0226] Prokaryotic host cells are cultured at an appropriate temperature. For example, for E. coli growth, preferred temperatures range from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably about 30°C. The pH of the medium can be any pH in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0227] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for the activation of the promoter.In some embodiments of the present invention, the PhoA promoter is used to control the transcription of polypeptide.Therefore, transformed host cells are cultured in a phosphate-limited medium for induction.In some embodiments, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-47).As known in the art, various other inducers can be used depending on the vector construct used.

[0228] In some embodiments, the expressed polypeptides of the present invention are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganisms, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the produced protein. The expressed polypeptides can be further isolated and identified using well-known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0229] In some embodiments of the present invention, immunoconjugates are produced in large quantities by a fermentation process. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably between about 1,000 and 100,000 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, particularly glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volumetric capacity of about 100 liters or less, and can range from about 1 liter to about 100 liters.

[0230] In fermentation processes, induction of protein expression typically begins after cells have been grown under appropriate conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in early stationary phase. As known in the art and described above, various inducers can be used depending on the vector construct used. Cells can be grown for a shorter period before induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction times can be used.

[0231] Various fermentation conditions can be changed to improve the production yield and quality of the polypeptide of the present invention. For example, to improve the proper assembly and folding of the secreted immunoconjugate polypeptide, an additional vector overexpressing a chaperone protein, such as Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis, trans-isomerase with chaperone activity), can be used to co-transform the host prokaryotic cell. Chaperone proteins have been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-5; U.S. Patent No. 6,083,715, U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-5; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-13; Arie et al. (2001) Mol. Microbiol. 39:199-210.

[0232] To minimize proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified by introducing genetic mutations into genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available, see, for example, Joly et al. (1998), U.S. Patent No. 5,264,365, U.S. Patent No. 5,508,192, supra, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0233] In some embodiments, E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins are used as host cells in the expression systems of the present invention.

[0234] Eukaryotic Host Cell Cultures Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing the host cells. Additionally, see Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. No. 4,767,704; Any of the media described in US Pat. Nos. 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Patent Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (trace elements), and the like. The culture medium may be supplemented with nutrients (defined as inorganic compounds, usually present at final concentrations in the micromolar range), and glucose, or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions such as temperature, pH, and the like will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0235] Purification of immunoglobulin-derived structures of the invention The immunoconjugates of the present invention can be recovered from culture medium or host cell lysates. If membrane-bound, they can be released from the membrane using an appropriate detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used to express the immunoconjugates of the present invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0236] It may be desirable to purify the immunoconjugates of the present invention from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures, including fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, a protein A Sepharose column to remove contaminants such as IgG, and a metal chelate column to bind epitope-tagged forms of the immunoconjugates of the present invention. Various methods of protein purification can be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification steps selected will depend, for example, on the nature of the production process used and the particular immunoconjugate of the present invention being produced.

[0237] When using recombinant technology, immunoconjugates can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the immunoconjugate is produced intracellularly, the first step is to remove particulate debris, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the immunoconjugate is secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0238] Immunoconjugate compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the immunoconjugate. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the immunoconjugate contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSE™ chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the immunoconjugate to be recovered.

[0239] After any preliminary purification steps, the mixture containing the immunoconjugate of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography, using an elution buffer with a pH between about 2.5 and 4.5, and generally a low salt concentration (e.g., about 0 to 0.25 M salt).

[0240] Immunoconjugates (including antibody drug conjugates (ADCs)) In a further embodiment of the invention, an immunoconjugate of the invention according to any of the above embodiments or described herein is conjugated to a heterologous moiety or agent, including, for example, any of the additional exogenous materials described below and herein.

[0241] In some embodiments, the present invention provides immunoconjugates comprising a multivalent antibody construct of the present invention conjugated to one or more therapeutic agents or radioisotopes.

[0242] In some embodiments, the immunoconjugate comprises a multivalent antibody construct described herein conjugated to a radioactive atom to form a radioconjugate. As described herein, a variety of radioisotopes are available for the generation of the radioconjugates of the present invention.

[0243] Immunoconjugates or antibody construct conjugates can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate H), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(pdiazoniumbenzoyl)-ethylenediamine)), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see, e.g., WO 1994 / 11026). The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker can be used (see, e.g., Chari et al., Cancer Res. 52:127-131 (1992); US Pat. No. 5,208,020).

[0244] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared using crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as commercially available SVSB (succinimidyl-(4-vinylsulfone)benzoate) (available, for example, from Pierce Biotechnology, Inc., Rockford, IL, US).

[0245] As will be appreciated by those skilled in the art, the particular methods described above are also useful for preparing radioimmunoconjugates and targeted imaging conjugates (notwithstanding the text's reference to only immunoconjugates or antibody constructs), and such preparation methods are also encompassed by the present invention.

[0246] Immunoconjugation using chelators and / or linkers Methods for attaching radioisotopes to immunoconjugates or antibody constructs (i.e., "labeling" multivalent antibodies with radioisotopes) are well known to those skilled in the art. Some of these methods are described, for example, in WO2017 / 155937.

[0247] For example, bifunctional chelators such as DOTA, DTPA, and related analogs are suitable for coordinating metal ions such as α- and β-radionuclides. For example, these chelating molecules can be linked to targeting molecules by forming a new amide bond between an amine (e.g., a functional group of a lysine residue) on the antibody construct and a carboxylate on DOTA / DTPA. In the case of peptide synthesis, characterization and purification of the linker addition can be part of the overall synthesis of a multivalent antibody platform or immunoconjugate for radioisotope conjugates.

[0248] In some embodiments, the method of producing the immunoconjugate comprises a click chemistry step as described by Poty, S et al., Chem Commun. (Camb) 54:2599 (2018).

[0249] In some embodiments, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors, including, for example, fluorine-19, instead of hydrogen. In some embodiments, a radiolabel can be incorporated into the peptide. In some embodiments, a radiolabel can be linked to the peptide. To incorporate iodine-123, the IODOGEN method (Fraker et al. (1978) Biochem Biophys Res Commun. 80:49-57) can be used. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0250] Pharmaceutical Composition Provided herein are compositions comprising the immunoconjugates or radioimmunoconjugates described herein. The present invention further provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the present invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the present invention and (2) a pharmaceutically acceptable carrier.

[0251] The immunoconjugate or radioimmunoconjugate may be formulated in any form suitable for delivery to target cells / tissues. Pharmaceutical formulations of the immunoconjugates of the present invention are prepared by mixing such immunoconjugates having the desired purity with one or more pharmaceutically acceptable carriers, diluents, and / or excipients in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed and include, for example, sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (fewer than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; A chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as polyethylene glycol (PEG).

[0252] Pharmaceutical formulations to be used for in vivo administration are generally sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0253] Examples of lyophilized antibody formulations are described in US 6,267, 958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0254] Pharmaceutically acceptable carriers herein further include an interstitial drug dispersing agent, such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0255] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, such active ingredients being present in suitable combinations in amounts that are effective for the purpose intended.

[0256] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0257] In some embodiments, the immunoconjugate may be formulated as an immunoliposome. A "liposome" is a vesicle composed of various types of lipids, phospholipids, and / or surfactants that is useful for delivering drugs to mammals. The components of a liposome are usually arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing immunoconjugates are prepared by methods known in the art, such as those described in Epstein et al., Proc Natl Acad Sci USA 82:3688 (1985); Hwang et al., Proc Natl Acad Sci USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 1997 / 38731, published October 23, 1997. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with defined pore sizes to obtain liposomes with desired diameters. Chemotherapeutic agents are optionally contained within the liposomes (see Gabizon et al., J. National Cancer Inst. 81:1484 (1989)). Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0258] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0259] How to use The immunoconjugates (e.g., radioimmunoconjugates) described herein are useful for treating a disease, disorder, or condition (e.g., tumor or cancer) in a patient in need thereof, the method comprising administering an immunoconjugate or radioimmunoconjugate or composition described herein. The immunoconjugates are also useful for killing tumor or cancer cells (e.g., solid tumor cells that express FOLR1 or DLL3). Furthermore, the immunoconjugates described herein can be used in methods for labeling or detecting tumor or cancer cells.

[0260] In some embodiments, provided herein are methods for killing tumor or cancer cells, the methods comprising contacting tumor or cancer cells with a radioimmunoconjugate (e.g., an immunoconjugate comprising a radionuclide) described herein, thereby killing the tumor or cancer cells. In some embodiments, the tumor cells are solid tumor cells. In certain embodiments, the tumor cell cancer and / or cancer cells express FOLR1 or DLL3. In certain embodiments, the tumor cells and / or cancer cells are in an individual.

[0261] In some embodiments, provided herein are methods of treating cancer or tumor in an individual, the methods comprising administering to the individual an immunoconjugate (e.g., a radioimmunoconjugate) described herein, thereby treating the cancer or tumor. In certain embodiments, the individual is a human. In certain embodiments, the tumor is a solid tumor and / or the cancer comprises a solid tumor.

[0262] The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the patient's disease and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0263] In some embodiments, the immunoconjugates or radioimmunoconjugates or compositions of the present invention can be used in a method for binding a target antigen in an individual suffering from a disorder associated with increased target antigen expression and / or activity, the method comprising administering the immunoconjugate or radioimmunoconjugate or composition to the individual such that the target antigen in the individual is bound. In some embodiments, the target antigen is a human target antigen, and the individual is a human individual. The immunoconjugates or radioimmunoconjugates or compositions of the present invention can be administered to humans for therapeutic purposes. Furthermore, the immunoconjugates or radioimmunoconjugates or compositions of the present invention can be administered to non-human mammals (e.g., primates, pigs, rats, or mice) expressing a target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts for veterinary purposes or as animal models of human disease. Regarding the latter, such animal models can be useful for evaluating the therapeutic efficacy of the immunoconjugates or radioimmunoconjugates or compositions of the present invention (e.g., testing dosages and time courses of administration).

[0264] The immunoconjugates or radioimmunoconjugates or compositions of the invention (and any additional therapeutic agents or adjuvants) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, as well as, if local treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, antibodies (e.g., multivalent antibodies) are suitably administered by pulse infusion, particularly with declining doses of antibody (e.g., multivalent antibodies). Dosing can be by any suitable route, e.g., injections, such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic.

[0265] The immunoconjugates or radioimmunoconjugates or compositions of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The immunoconjugates of the present invention are administered to human patients according to known methods, for example, intravenous administration as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, intravenous or subcutaneous administration of the immunoconjugates or radioimmunoconjugates or compositions of the present disclosure is preferred.

[0266] For disease prevention or treatment, the dosage and mode of administration will be selected by a physician according to known criteria. The appropriate dosage of the immunoconjugate or radioimmunoconjugate or composition of the present disclosure depends on the type of disease being treated, as defined above, the severity and course of the disease, whether the immunoconjugate or radioimmunoconjugate or composition of the present disclosure is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the immunoconjugate or radioimmunoconjugate or composition, and the discretion of the attending physician. The immunoconjugate or radioimmunoconjugate or composition of the present disclosure is appropriately administered to the patient at one time or over a series of treatments. Preferably, the immunoconjugate or radioimmunoconjugate or composition is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, about 1 μg / kg to about 50 mg / kg body weight (e.g., about 0.1 to 15 mg / kg / dose) of the immunoconjugate or radioimmunoconjugate or composition may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. A dosing regimen may involve administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of the invention. However, other dosing regimens may also be useful. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the disease, treatment is continued until a desired suppression of disease symptoms occurs. The progress of this therapy can be easily monitored by conventional methods and assays and based on criteria known to the physician or other skilled artisan.

[0267] The dosage and administration schedule can be selected and adjusted based on the level of disease or tolerance in the subject, and this can be monitored during the course of treatment.The conjugate of the present invention can be administered once a day, once a week, multiple times a week but less than once a day, multiple times a month but less than once a day, multiple times a month but less than once a week, once a month, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, or intermittently to alleviate or relieve the symptoms of disease.Administration can be continued at any of the intervals disclosed until the symptoms of the tumor or cancer being treated are alleviated.Administration can be continued after symptom alleviation or relief is achieved, and such alleviation or relief is prolonged by such continued administration.

[0268] In some embodiments, an effective amount of the immunoconjugate or radioimmunoconjugate or composition may be provided as a single dose.

[0269] The immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with conventional and / or novel treatment or therapy methods, or separately as monotherapy. In some embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used with one or more radiation sensitizer agents. Such agents include any agent that can increase the sensitivity of cancer cells to radiation therapy. In other embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with novel and / or conventional agents that can enhance the biological effects of radiation therapy. Irradiation of tumors can induce various biological outcomes that can be exploited by combining the immunoconjugates and radioimmunoconjugates of the present invention with agents that target relevant pathways. In some embodiments, such agents can reduce tumor angiogenesis, inhibit local invasion and metastasis, prevent regrowth, enhance the immune response, deregulate cellular energy, reduce cell population, alter tumor metabolism, increase tumor damage, or reduce DNA repair. In certain embodiments, agents for use in combination with the immunoconjugates and radioimmunoconjugates of the present invention may include DDR inhibitors, e.g., PARP, ATR, Chk1, or DNA-PK, or survival signaling inhibitors, e.g., mTOR, PI3k, NF-kB, or anti-hypoxia agents, e.g., HIF-1α, CAP, or UPR, or metabolic inhibitors, e.g., MCT1, MCT4 inhibitors, or immunotherapeutics, e.g., anti-CTLA4, anti-PD-1, or inhibitors of growth factor signaling, e.g., EGFR or MAPK inhibitors, or anti-invasives, e.g., kinase inhibitors, chemokine inhibitors, or integrin inhibitors, or anti-angiogenic agents, e.g., VEGF inhibitors.

[0270] The immunoconjugates and radioimmunoconjugates of the present invention may (i) inhibit the growth or proliferation of cells to which they bind, (ii) induce the death of cells to which they bind, (iii) inhibit delamination of cells to which they bind, (iv) inhibit metastasis of cells to which they bind, or (v) inhibit the angiogenesis of tumors containing cells to which they bind. In this context, "inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0271] By way of example, an immunoconjugate that inhibits tumor cell proliferation is one that results in measurable growth inhibition of tumor cells (e.g., cancer cells). In some embodiments, the immunoconjugates or radioimmunoconjugates of the invention are capable of inhibiting the growth of cancer cells that present the antigen bound by the immunoconjugate or radioimmunoconjugate. Preferred growth-inhibitory immunoconjugates or radioimmunoconjugates inhibit the growth of antigen-expressing tumor cells by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%), relative to a suitable control, which is typically tumor cells that have not been treated with the immunoconjugate or radioimmunoconjugate being tested.

[0272] In some embodiments, the majority of the immunoconjugate or radioimmunoconjugate or composition administered to a subject typically consists of unlabeled immunoconjugate, with a minority being labeled radioimmunoconjugate. The ratio of labeled to unlabeled immunoconjugate can be adjusted using known methods. Thus, according to certain aspects of the invention, the immunoconjugate or radioimmunoconjugate may be provided in a total protein amount of up to 100 mg, for example less than 60 mg, or between 5 mg and 45 mg, or between 0.1 μg / kg and 1 mg / kg patient body weight, for example between 1 μg / kg and 1 mg / kg patient body weight, or between 10 μg / kg and 1 mg / kg patient body weight, or between 100 μg / kg and 1 mg / kg patient body weight, or between 0.1 μg / kg and 100 μg / kg and 50 μg / kg patient body weight, or between 0.1 μg / kg and 10 μg / kg patient body weight, or between 0.1 μg / kg and 40 μg / kg patient body weight, or between 1 μg / kg and 40 μg / kg patient body weight, or between 0.1 mg / kg and 1.0 mg / kg patient body weight, for example between 0.2 mg / kg and 0.6 mg / kg patient body weight.

[0273] In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at about 0.5 mg / kg to about 30 mg / kg.In certain embodiments, the immunoconjugate / radioimmunoconjugate is administered at a concentration of about 0.5 mg / kg to about 1 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 3 mg / kg, about 0.5 mg / kg to about 4 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 1 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 3 mg / kg, about 1 mg / kg to about 4 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 30 mg / kg, about 2 m g / kg~about 5mg / kg, about 2mg / kg~about 10mg / kg, about 2mg / kg~about 3mg / kg, about 2mg / kg~about 4mg / kg, about 2mg / kg~about 5mg / kg, about 2mg / kg~about 10mg / kg, about 2mg / kg~about 20mg / kg, about 2mg / kg~about 30 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 3 mg / kg, approximately 5 mg / kg to approximately 4 mg / kg, approximately 5 mg / kg to approximately 5 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 20 mg / kg, approximately 5 mg / kg to approximately 30 mg / kg, approximately 10mg / kg~about 3mg / kg, about 10mg / kg~about 4mg / kg, about 10mg / kg~about 5mg / kg, about 10mg / kg~about 10mg / kg, about 10mg / kg~about 20mg / kg, about 10mg / kg~about 30mg / kg, about 3mg / kg~about 4mg / kg, about 3m It may be administered at a dose of about 10 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3 mg / kg to about 20 mg / kg, about 3 mg / kg to about 30 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4 mg / kg to about 20 mg / kg, about 4 mg / kg to about 30 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at at least about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, or about 20 mg / kg. In certain embodiments, the immunoconjugate / radioimmunoconjugate may be administered at up to about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, or about 30 mg / kg.

[0274] In some embodiments, the method comprises administering an effective amount of a radioimmunoconjugate comprising 225-Ac that is between 0.01 and 0.1 mCi, or between 0.1 mCi and 1.0 mCi, or between 1.0 mCi and 2.0 mCi, or between 2.0 mCi and 4.0 mCi.

[0275] In some embodiments, the method comprises administering an effective amount of a radioimmunoconjugate comprising 225-Ac at 0.1 μCi / kg to 2.0 μCi / kg of the subject's body weight, or 0.1 μCi / kg to 1.0 μCi / kg of the subject's body weight, or 1.0 μCi / kg to 3.0 μCi / kg of the subject's body weight, or 3.0 μCi / kg to 10.0 μCi / kg of the subject's body weight, or 10.0 μCi / kg to 20.0 μCi / kg of the subject's body weight, or 10.0 μCi / kg to 30.0 μCi / kg of the subject's body weight.

[0276] In certain embodiments, the effective amount of 225-Ac is about 0.1 microcuries to about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcuries to about 0.2 microcuries, about 0.1 microcuries to about 0.5 microcuries, about 0.1 microcuries to about 1 microcurie, about 0.1 microcuries to about 2 microcuries, about 0.1 microcuries to about 3 microcuries, about 0.1 microcuries to about 4 microcuries, about 0.1 microcuries to about 5 microcuries, or about 0.1 microcuries to about 10 microcuries. Approximately 0.1 microcuries to approximately 20 microcuries, approximately 0.2 microcuries to approximately 0.5 microcuries, approximately 0.2 microcuries to approximately 1 microcurie, approximately 0.2 microcuries to approximately 2 microcuries, approximately 0.2 microcuries to approximately 3 microcuries, approximately 0.2 microcuries to approximately 4 microcuries, approximately 0.2 microcuries to approximately 5 microcuries, approximately 0.2 microcuries to approximately 10 microcuries, approximately 0.2 microcuries to approximately 20 microcuries, approximately 0.5 microcuries to approximately 1 microcurie microcuries, about 0.5 microcuries to about 2 microcuries, about 0.5 microcuries to about 3 microcuries, about 0.5 microcuries to about 4 microcuries, about 0.5 microcuries to about 5 microcuries, about 0.5 microcuries to about 10 microcuries, about 0.5 microcuries to about 20 microcuries, about 1 microcurie to about 2 microcuries, about 1 microcurie to about 3 microcuries, about 1 microcurie to about 4 microcuries, about 1 microcurie to about 5 microcuries. About 1 microcurie to about 10 microcuries, about 1 microcurie to about 20 microcuries, about 2 microcuries to about 3 microcuries, about 2 microcuries to about 4 microcuries, about 2 microcuries to about 5 microcuries, about 2 microcuries to about 10 microcuries, about 2 microcuries to about 20 microcuries, about 3 microcuries to about 4 microcuries, about 3 microcuries to about 5 microcuries, about 3 microcuries to about 10 microcuries.The effective dose is about 3 microcuries to about 20 microcuries, about 4 microcuries to about 5 microcuries, about 4 microcuries to about 10 microcuries, about 4 microcuries to about 20 microcuries, about 5 microcuries to about 10 microcuries, about 5 microcuries to about 20 microcuries, or about 10 microcuries to about 20 microcuries. In certain embodiments, the effective dose of 225-Ac is about 0.1 microcuries, about 0.2 microcuries, about 0.5 microcuries, about 1 microcurie, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, about 10 microcuries, or about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is at least about 0.1 microcuries, about 0.2 microcuries, about 0.5 microcuries, about 1 microcuries, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, or about 10 microcuries. In certain embodiments, the effective amount of 225-Ac is at most about 0.2 microcuries, about 0.5 microcuries, about 1 microcuries, about 2 microcuries, about 3 microcuries, about 4 microcuries, about 5 microcuries, about 10 microcuries, or about 20 microcuries. According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, the effective amount is, for example, less than 15.0 mCi (i.e., when the amount of 111-In administered to the subject delivers a total body radiation dose of less than 15.0 mCi).

[0277] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, an effective amount is less than 15.0 mCi, less than 14.0 mCi, less than 13.0 mCi, less than 12.0 mCi, less than 11.0 mCi, less than 10.0 mCi, less than 9.0 mCi, less than 8.0 mCi, less than 7.0 mCi, less than 6.0 mCi, less than 5.0 mCi, less than 4.0 mCi, less than 3.5 mCi, less than 3.0 mCi, less than 2.5 mCi, less than 2.0 mCi, less than 1.5 mCi, less than 1.0 mCi, less than 0.5 mCi, less than 0.4 mCi, less than 0.3 mCi, less than 0.2 mCi, or less than 0.1 mCi.

[0278] According to embodiments where the radioactive isotope of the radioimmunoconjugate is 111-In, an effective amount is between 0.1 mCi and 1.0 mCi, between 0.1 mCi and 2.0 mCi, between 1.0 mCi and 2.0 mCi, between 1.0 mCi and 3.0 mCi, between 1.0 mCi and 4.0 mCi, between 1.0 mCi and 5.0 mCi, between 1.0 mCi and 10.0 mCi, between 1.0 mCi and 15.0 mCi, between 1.0 mCi and 20.0 mCi. i, 2.0mCi~3.0mCi, 3.0mCi~4.0mCi, 4.0mCi~5.0mCi, 5.0mCi~10.0mCi, 5.0mCi~15.0mCi, 5.0mCi~20.0 mCi, 6.0mCi~14.0mCi, 7.0mCi~13.0mCi, 8.0mCi~12.0mCi, 9.0mCi~11.0mCi, or 10.0mCi~15.0mCi.

[0279] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 111-In, an effective amount is 15.0 mCi, 14.0 mCi, 13.0 mCi, 12.0 mCi, 11.0 mCi, 9.0 mCi, 8.0 mCi, 7.0 mCi, 6.0 mCi, 5.0 mCi, 4.0 mCi, 3.5 mCi, 3.0 mCi, 2.5 mCi, 2.0 mCi, 1.5 mCi, 1.0 mCi, 0.5 mCi, 0.4 mCi, 0.3 mCi, 0.2 mCi, or 0.1 mCi.

[0280] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is, for example, less than 30.0 μCi / kg (i.e., when the amount of 225-Ac administered to a subject delivers a radiation dose of less than 30.0 μCi per kilogram of the subject's body weight).

[0281] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, effective amounts are 30 μCi / kg, 25 μCi / kg, 20 μCi / kg, 17.5 μCi / kg, 15.0 μCi / kg, 12.5 μCi / kg, 10.0 μCi / kg, 9 μCi / kg, 8 μCi / kg, 7 μCi / kg, 6 μCi / kg, 5 μCi / kg, 4.5 μCi / kg, 4.0 μCi / kg. kg, 3.5μCi / kg, 3.0μCi / kg, 2.5μCi / kg, 2.0μCi / kg, 1.5μCi / kg, 1.0μCi / kg, 0.9μCi / kg, 0.8μCi / kg, 0.7 less than μCi / kg, 0.6 μCi / kg, 0.5 μCi / kg, 0.4 μCi / kg, 0.3 μCi / kg, 0.2 μCi / kg, 0.1 μCi / kg, or 0.05 μCi / kg.

[0282] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is from 0.05 μCi / kg to 0.1 μCi / kg, 0.1 μCi / kg to 0.2 μCi / kg, 0.2 μCi / kg to 0.3 μCi / kg, 0.3 μCi / kg to 0.4 μCi / kg, 0.4 μCi / kg to 0.5 μCi / kg, 0.5 μCi / kg to 0.6 μCi / kg, 0.6 μCi / kg to 0.7 μCi / kg, 0.7 μCi / kg to 0.8 μCi / kg, 0.8 μCi / kg to 0.9 μCi / kg, 0.9 μCi / kg to 0.10 μCi / kg, 0.9 μCi / kg to 0.11 μCi / kg, 0.9 μCi / kg to 0.12 μCi / kg, 0.9 μCi / kg to 0.13 μCi / kg, 0.9 μCi / kg to 0.14 μCi / kg, 0.9 μCi / kg to 0.15 μCi / kg, 0.9 μCi / kg to 0.16 μCi / kg, 0.9 μCi / kg to 0.17 μCi / kg, 0.9 μCi / kg to 0.18 μCi / kg, 0.9 μCi / kg to 0.19 μCi / kg, 0.10 μCi / kg to 0.20 μCi / kg, 0.10 μCi / kg to 0.21 μCi / kg, 0.10 μCi / kg to 0.225 μCi / kg, 0.10 μCi / kg to 0.23 μCi / kg .8μCi / kg~0.9μCi / kg, 0.9μCi / kg~1.0μCi / kg, 1.0μCi / kg~1.5μCi / kg, 1.5μCi / kg~2.0μCi / kg, 2.0μCi / kg~2.5μCi / kg, 2.5 μCi / kg~3.0μCi / kg, 3.0μCi / kg~3.5μCi / kg, 3.5μCi / kg~4.0μCi / kg, 4.0μCi / kg~4.5μCi / kg, or 4.5μCi / kg~5.0μCi / kg.

[0283] According to embodiments in which the radioactive isotope of the radioimmunoconjugate is 225-Ac, an effective amount is 0.05 μCi / kg, 0.1 μCi / kg, 0.2 μCi / kg, 0.3 μCi / kg, 0.4 μCi / kg, 0.5 μCi / kg, 0.6 μCi / kg, 0.7 μCi / kg, 0.8 μCi / kg, 0.9 μCi / kg, 1.0 μCi / kg, 1.5 μCi / kg, 2.0 μCi / kg, 2.5 μCi / kg, 3.0 μCi / kg, 3.5 μCi / kg, 4.0 μCi / kg, 4.5 μCi / kg, 5.0 μCi / kg, 6.0 μCi / kg, 6.5 μCi / kg, 7.0 μCi / kg, 7.5 μCi / kg, 8.0 μCi / kg, 8.5 μCi / kg, 9.0 μCi / kg, 9.0 μCi / kg, 10 ... Ci / kg, 3.0μCi / kg, 3.5μCi / kg, 4.0μCi / kg, or 4.5μCi / kg, 5.0μCi / kg, 6.0μCi / kg, 7.0μCi / kg, 8.0μCi / kg, 9.0μCi / kg, 10.0μCi / kg, 12.5μCi / kg, 15.0μCi / kg, 17.5μCi / kg, 20.0μCi / kg, 25μCi / kg, or 30μCi / kg.

[0284] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu, the effective amount is between 0.1 uCi and 100 mCi per square meter of body surface area.

[0285] In certain embodiments, where the radioisotope of the radioimmunoconjugate is 177-Lu, an effective amount is between 1 mCi and 100 mCi per square meter of body surface area. In certain embodiments, an effective amount is between about 1 mCi per square meter and about 100 mCi per square meter. In certain embodiments, an effective amount is between about 1 mCi per square meter and about 5 mCi per square meter, between about 1 mCi per square meter and about 10 mCi per square meter, between about 1 mCi per square meter and about 15 mCi per square meter, between about 1 mCi per square meter and about 20 mCi per square meter, between about 1 mCi per square meter and about 25 mCi per square meter, between about 1 mCi per square meter and about 75 mCi per square meter, between about 1 mCi per square meter and about 100 mCi per square meter. Approximately 100, approximately 5 per square meter to approximately 10 per square meter, approximately 5 per square meter to approximately 15 per square meter, approximately 5 per square meter to approximately 20 per square meter, approximately 5 per square meter to approximately 25 per square meter, approximately 5 per square meter to approximately 75 per square meter, approximately 5 per square meter to approximately 100 per square meter, approximately 10 per square meter to approximately 15 per square meter, approximately Approximately 10 per square meter to approximately 20 per square meter, approximately 10 per square meter to approximately 25 per square meter, approximately 10 per square meter to approximately 75 per square meter, approximately 10 per square meter to approximately 100 per square meter, approximately 15 per square meter to approximately 20 per square meter, approximately 15 per square meter to approximately 25 per square meter, approximately 15 per square meter to approximately 75 per square meter, approximately about 15 per square meter to about 100 per square meter, about 20 per square meter to about 25 per square meter, about 20 per square meter to about 75 per square meter, about 20 per square meter to about 100 per square meter, about 25 per square meter to about 75 per square meter, about 25 per square meter to about 100 per square meter, or about 75 per square meter to about 100 per square meter.In certain embodiments, the effective amount is about per square meter, about per 5 square meters, about per 10 square meters, about per 15 square meters, about per 20 square meters, about per 25 square meters, about per 75 square meters, or about per 100 square meters. In certain embodiments, the effective amount is at least about 1 per square meter, about 5 per square meter, about 10 per square meter, about 15 per square meter, about 20 per square meter, about 25 per square meter, or about 75 per square meter. In certain embodiments, the effective amount is at most about 5 per square meter, about 10 per square meter, about 15 per square meter, about 20 per square meter, about 25 per square meter, about 75 per square meter, or about 100 per square meter.

[0286] According to certain embodiments of the present invention, a preparation of a radioimmunoconjugate of the present disclosure or a composition thereof (e.g., a pharmaceutical composition) may comprise a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), and the labeled:unlabeled ratio may be about 1:1000 to 1:1.

[0287] Additionally, pharmaceutical compositions can be provided as single-dose compositions tailored to a particular patient, i.e., as patient-specific therapeutic compositions, where the amounts of labeled and unlabeled immunoconjugates (for clarity, labeled immunoconjugates are the same as radioactive immunoconjugates herein) in the composition can depend, at least, on the patient's weight, height, body surface area, age, sex, and / or disease or health condition. Thus, the total volume of the patient-specific therapeutic composition can be provided in a vial configured to be administered entirely to a patient in a single treatment session, such that little or no composition remains in the vial after administration.

[0288] Currently, depending on the stage of the cancer, cancer treatment includes one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Treatment using the radioimmunoconjugates of the present invention (interchangeably, "radiolabeled immunoconjugates") may be particularly desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well, and in metastatic disease where radiation therapy has limited usefulness. In some embodiments, treatment using the radiolabeled immunoconjugates of the present invention is useful for alleviating target antigen-expressing cancers at the time of initial diagnosis of disease or during recurrence.

[0289] In some embodiments, determining whether a cancer is suitable for treatment with the methods disclosed herein involves detecting the presence of a target antigen in a subject or a sample derived from the subject. Various detection assays are available for determining target antigen expression in cancer. In some embodiments, target antigen overexpression is analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies are subjected to IHC assays and subjected to target antigen staining intensity criteria. Alternatively, or additionally, FISH assays such as INFORM® (sold by Ventana, AZ, USA) or PATHVISION® (Vysis, IL, USA) can be performed on formalin-fixed, paraffin-embedded tumor tissue to determine the degree (if any) of target antigen overexpression in the tumor.

[0290] Overexpression or amplification of a target antigen can be assessed using an in vivo detection assay, for example, by administering a molecule (such as an antibody construct or immunoconjugate of the invention) that binds to the molecule to be detected and is tagged with a detectable label (e.g., a radioisotope or fluorescent label) and externally scanning the patient for label localization.

[0291] 2. Use of Immunoconjugates and Radioimmunoconjugates of the Invention to Kill Cells

[0292] The immunoconjugates or radioimmunoconjugates of the present invention can be used, for example, in vitro, ex vivo, and in vivo methods. In some embodiments, the present invention provides methods for inhibiting cell growth or proliferation either in vivo or in vitro, comprising exposing cells to the immunoconjugates or radioimmunoconjugates of the present disclosure under conditions that allow binding of the immunoconjugates or radioimmunoconjugates to target antigens. The immunoconjugates or radioimmunoconjugates of the present invention can also (i) inhibit the growth or proliferation of cells to which they bind, (ii) induce the death of cells to which they bind, (iii) inhibit the delamination of cells to which they bind, (iv) inhibit the metastasis of cells to which they bind, or (v) inhibit the angiogenesis of tumors containing cells to which they bind.

[0293] In some embodiments, the present invention provides a method for killing antigen-expressing cells, the method comprising contacting cells with an immunoconjugate or radioimmunoconjugate of the present invention (or a composition thereof). The method can be used, for example, to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells. The method can be used, for example, to kill, deplete, or eliminate target antigen-expressing cells from a population of mixed cells as a step in the purification of other cells. The method can be performed in vitro or in vivo, including ex vivo, on primary patient cell or tissue compositions to prepare such compositions for transplantation.

[0294] In some embodiments, immunoconjugates or radioimmunoconjugates of the invention are used to treat or prevent cell proliferative disorders. In certain embodiments, the cell proliferative disorder includes solid tumor cancer. Solid tumor cancers are cancers involving an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder includes liquid tumor cancers or hematological cancers, e.g., leukemias and lymphomas, used interchangeably, where such cancers are present in bodily fluids. In certain embodiments, the cell proliferative disorder is associated with increased expression and / or activity of a target antigen. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression of a target antigen on the surface of a cell. In certain embodiments, the cell proliferative disorder is a tumor or cancer. In certain embodiments, the cell proliferative disorder includes solid tumor cancer. Solid tumor cancers include cancers involving an abnormal mass of tissue, e.g., carcinomas and sarcomas. In certain other embodiments, the cell proliferative disorder includes liquid tumor cancers or hematological cancers, used interchangeably, where such cancers are present in bodily fluids.

[0295] In some embodiments, the present invention provides a method for treating a cell proliferative disorder, the method comprising administering to an individual an effective amount of an immunoconjugate or radioimmunoconjugate of the present disclosure.

[0296] In addition to direct cell killing of target cells expressing the cell surface antigen specifically bound by the immunoconjugates or radioimmunoconjugates of the present invention, the immunoconjugates or radioimmunoconjugates of the present invention can optionally be used to deliver additional cargo to the vicinity of or inside the target cells. Delivery of additional exogenous substances can be used, for example, for cytotoxicity, cytostatic, interrogation, and / or diagnostic functions. Non-cytotoxic, or optionally toxic, variants of the immunoconjugates or radioimmunoconjugates of the present invention can be used to deliver cargo to and / or label the interior of cells expressing the target antigen. Non-limiting examples of cargo include cytotoxic agents, detection-enhancing agents, and small molecule chemotherapeutic agents.

[0297] As described herein, in some embodiments, the antibody (e.g., multivalent antibody) constructs, immunoconjugates, radioimmunoconjugates, and targeted imaging conjugates of the present invention have a variety of non-therapeutic applications. In some embodiments, the compositions of the present invention can be used to identify patient populations predicted to benefit from a particular therapeutic approach or treatment modality, such as treatment with an immunoconjugate or radioimmunoconjugate of the present disclosure. In some embodiments, the compositions of the present invention can be useful for staging (e.g., by radioimaging) target antigen-expressing cancers or as prognostic indicators of disease progression. In some embodiments, the compositions are also useful for in vitro detection and quantitation of target epitopes, for example, in ELISA or Western blots, and for purification or immunoprecipitation of target antigens from cell or tissue samples.

[0298] In some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention are used in methods for detecting the presence or level of antigens, for example, in biological samples in vitro or using imaging techniques in vivo.Detection of immunoconjugates and radioimmunoconjugates can be achieved through different techniques known to those skilled in the art and described herein, such as IHC and PET imaging.When the immunoconjugates or radiolabeled immunoconjugates of the present invention are used for detection, they can contain radioactive atoms, such as 99m-Tc or 111-In, for scintigraphy studies.

[0299] The labeled immunoconjugates of the present invention are useful as imaging biomarkers and probes for various biomedical and molecular imaging methods and techniques, such as (i) MRI (magnetic resonance imaging), (ii) MicroCT (computed tomography), (iii) SPECT (single-photon emission computed tomography), (iv) PET (positron emission tomography) Chen et al. Bioconjugate Chem. 15:41-9 (2004), (v) bioluminescence, (vi) fluorescence, and (vii) ultrasound. Immunoscintigraphy is an imaging procedure in which a radiolabeled antibody is administered to an animal or human patient, and photographs are taken of the site in the body where the antibody (e.g., a polyvalent antibody) localizes (US6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

[0300] Another embodiment of the present invention is a method for determining the presence of a target antigen in a sample suspected of containing the target antigen, the method comprising exposing the sample to an immunoconjugate that binds to the target antigen and determining binding of the immunoconjugate to the target antigen in the sample, the presence of such binding indicating the presence of the target antigen in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the target antigen. The immunoconjugate used in the method may optionally be detectably labeled, bound to a solid support, etc.

[0301] Another embodiment of the present invention provides a method for diagnosing the presence of a tumor in a subject, the method comprising: (a) contacting a test sample comprising tissue cells obtained from a mammal with an immunoconjugate that binds to a target antigen; and (b) detecting the formation of a conjugate between the immunoconjugate and the target antigen in the test sample, wherein the formation of the conjugate indicates the presence of a tumor in the mammal. Optionally, the immunoconjugate is detectably labeled, attached to a solid support, or the like, and / or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0302] In some embodiments, the immunoconjugates of the invention, including compositions including those described above and / or provided herein, are useful, for example, for detecting the presence of a target antigen in vivo or in a biological sample. The immunoconjugates of the invention can be used in a variety of different assays, including, but not limited to, ELISA, bead-based immunoassays, and mass spectrometry.

[0303] In some embodiments, the immunoconjugates of the present invention are useful for quantifying the amount of a target antigen in a sample. In some embodiments, the biological sample is a biological fluid such as whole blood or whole blood components, including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, urine, and other components of the body that may contain the target antigen of interest. In various embodiments, the sample is a body sample from any animal. In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is biopsy material. In some embodiments, the biological sample is biopsy material from a clinical patient. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is primary cell culture material. In some embodiments, the biological sample is primary cell culture material from a clinical patient. In some embodiments, the biological sample is derived from a clinical patient or a patient treated with a therapeutic antibody or an antibody that binds to the same target antigen.

[0304] In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject, for example, when measuring antigen expression in a clinical sample. In some embodiments, the biological sample is from a clinical patient or a patient treated with a therapy / therapeutic (e.g., an antibody therapy targeting the same target antigen). In some embodiments, the biological sample is serum or plasma. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is biopsy material. In some embodiments, the biological sample is biopsy material from a clinical patient. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is primary cell culture material. In some embodiments, the biological sample is primary cell culture material from a clinical patient.

[0305] In some embodiments, compositions comprising "labeled" immunoconjugates are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are indirectly detected, e.g., via an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, J3-galactosidase, glucoamylase, lysozyme, sugar oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, dye precursors such as HRP, lactoperoxidase, or microperoxidase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that use hydrogen peroxide to oxidize biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0306] Conventional methods can be used to covalently bind these labels to protein or polypeptide.For example, by using coupling agents such as dialdehyde, carbodiimide, dimaleimide, bis-imidate, bis-diazotized benzidine, etc., the immunoconjugate or antibody construct of the present invention can be tagged with the above-mentioned fluorescent label, chemiluminescent label, and enzyme label (see, for example, US 3,645,090 (enzyme), US 3,940,475 (fluorometry), Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J.Immunol.Methods, 40:219-230 (1981); Nygren, J.Histochem and Cytochem, 30:407-412 (1982)).

[0307] Conjugation of such labels, including enzymes, to immunoconjugates or antibody constructs is a standard procedure for those skilled in the art of immunoassay technology (see, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166). Suitable commercially available labeled antibodies can also be used.

[0308] Finally, after adding the labeled immunoconjugate, the amount of bound immunoconjugate is determined by washing away excess unbound labeled immunoconjugate, then measuring the amount of bound label using a detection method appropriate for the label, and correlating the measured amount with the amount of the immunoconjugate of interest in the biological sample. For example, in the case of an enzyme, the amount of color developed and measured is a direct measurement of the amount of the immunoconjugate of interest present. Specifically, when HRP is the label, color can be detected using the substrate TMD, using a reading wavelength of 450 nm and a reference wavelength of 620 or 630 nm.

[0309] In one example, after the enzyme-labeled secondary antibody against the unlabeled immunoconjugate is washed from the stationary phase, color or chemiluminescence is developed and measured by incubating the immobilized capture reagent with a substrate for the enzyme. The concentration of the antibody of interest (e.g., a multivalent antibody) is then calculated by comparing the color or chemiluminescence produced by a parallel run of the immunoconjugate of interest.

[0310] In some embodiments, the method involves bead-based immunoassays, ELISA assays, or mass spectrometry techniques. The mass analyzer of such a mass spectrometer includes, but is not limited to, a quadrupole (Q), time-of-flight (TOF), ion trap, magnetic sector, or Fourier transform ion cyclotron resonance (FT-ICR), or a combination thereof. The ion source of the mass spectrometer must primarily generate sample molecular ions or pseudo-molecular ions and specific, characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are the two most commonly used methods for ionizing proteins for mass analysis of small molecules, such as by liquid chromatography-mass spectrometry (LC / MS) (Lee, M., LC / MS Applications in Drug Development (2002) J. Wiley & Sons, New York). Another example is surface-enhanced laser desorption / ionization (SELDI). SELDI is a surface-based ionization technique that enables high-throughput mass spectrometry. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI uses chemically reactive surfaces, such as "protein chips," to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with and immobilize the analytes thereon. Thus, analytes of the present invention can be partially purified on the chip and then rapidly analyzed in a mass spectrometer. Throughput can be increased by providing multiple reactive moieties at different sites on the substrate surface.

[0311] In another embodiment, the present invention provides a method for detecting an antigen in a biological sample, the method comprising: (a) contacting the biological sample with an immunoconjugate described herein to allow formation of the immunoconjugate; and (b) detecting or measuring the level of immunoconjugate bound to the sample. In some embodiments, the immunoconjugate is immobilized on a solid support. In some embodiments, the immobilized immunoconjugate is conjugated to biotin and bound to a streptavidin-coated microtiter plate. [Example]

[0312] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0313] Example 1. Preparation of VHH-Fc VHH-Fc plasmids were generated by cloning VHH sequences containing the hinge and Fc regions (human IgG1 CH2-CH3) into a mammalian expression vector. In some cases, mutations were introduced into the Fc region. To produce recombinant VHH-Fc and its variants, the plasmids were transfected into HEK293.SUS cells (ATUM, or similar). After 3–5 days of secretion, the cells were removed from the antibody-containing supernatant by centrifugation and sterile filtration. The antibodies were purified using a Mab Select SuRe PCC column (GE, Cat#: 11003495) and buffer exchange into PBS, pH 7.0. Protein was quantified using A280 or BCA. Antibody purity was tested by SDS-PAGE, capillary electrophoresis, HPLC-SEC, and LC-MS using standard protocols. Regarding VHH polypeptides, see, for example, McMahon et al., Nature Structural & Molecular Biology|VOL 25|MARCH 2018|289-296 Yeast surface display platform for rapid discovery of conformationally selective nanobodies; Moutel et al., eLife 2016;5:e16228 NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. De Genst E, Saerens D, Muyldermans S, Conrath K. Antibody repertoire development in camelids. Dev Comp Immunol. 2006;30(1-2):187-98. doi:10.1016 / j.dci.2005.06.010. PMID:16051357.Vincke C,Gutierrez C,Wernery U,Devoogdt N,Hassanzadeh-Ghassabeh G,Muyldermans S.Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.Methods Mol Biol.2012;907:145-76.doi:10.1007 / 978-1-61779-974-7_8.PMID:22907350.Arbabi Ghahroudi M,Desmyter A,Wyns L,Hamers R,Muyldermans S.Selection and identification of single domain antibody fragments from camel heavy-chain antibodies.FEBS Lett.1997 Sep 15;414(3):521-6.doi:10.1016 / s0014-5793(97)01062-4.PMID:9323027.

[0314] For VHH humanization, see, e.g., Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi:10.1074 / jbc.M806889200. Epub 2008 Nov 14. PMID:19010777.

[0315] For VHH stability, see e.g. Kunz P,Flock T,Soler N,Zaiss M,Vincke C,Sterckx Y,Kastelic D,Muyldermans S,Hoheisel JD.Exploiting sequence and stability information for directing nanobody stability engineering.Biochim Biophys Acta Gen Subj.2017 Sep;1861(9):2196-2205.doi:10.1016 / j.bbagen.2017.06.014.Epub 2017 Jun 20.PMID:28642127;PMCID:PMC5548252;Kunz P, Zinner K, Mucke N, Bartoschik T, Muyldermans S, Hoheisel JD.The structural basis of nanobody unfolding reversibility and thermoresistance.Sci Rep.2018 May 21;8(1):7934. doi:10.1038 / s41598-018-26338-z. PMID:29784954; PMCID:PMC5962586.

[0316] Several VHH-Fc prototypes and variants were engineered using VHH sequences such as the anti-HER2 clone 2RS15d VHH (see, e.g., WO2016 / 016021) (SEQ ID NO: 20) and the anti-DLL3 clone hz10D9v7.251 VHH sequence (see, e.g., WO2020 / 07967) (SEQ ID NO: 30), and unless otherwise stated herein, the data collected and presented was obtained using the VHH antigen-binding regions of these clones.

[0317] [Table 1]

[0318] Example 2. Antibody Binding Properties: Target Protein and Target Cell Assays

[0319] VHH-Fc was assessed by ELISA for binding to target soluble proteins—human, mouse, and cynomolgus monkey orthologs, as appropriate—according to standard protocols. Antigens were either commercially available or produced by cloning known antigen sequences (Uniprot) into mammalian expression vectors with HIS, FLAG, or equivalent tags for purification and detection purposes. A commercially available control anti-target IgG was included. Plates (96-well maxisorp, Corning 3368) were coated with 50–100 μL of each target protein of interest at a concentration optimized for coating. Purified VHH-Fc and hIgG1 isotype control (Sigma, Cat# II5154) were prepared at starting concentrations of 200–400 nM and titrated 1:4. The primary antibody was incubated for 1 hour at room temperature (RT) and washed, after which 0.2 μg / ml of secondary HRP-labeled antibody was added and incubated for 1 hour at RT (goat anti-human IgG-Fc-HRP Jackson, Cat#109-035-098). The reaction was detected using 50 μL / well of TMB (Neogen, Cat#308177). Color development was stopped with 1 M HCl (50 μL). Optical density (OD) was measured at 450 nm using a Spectromax plate reader, and the data were processed using SoftMaxPro. The data show that the anti-target VHH-Fc binds to human, mouse, and cynomolgus monkey target proteins. The recombinant DLL3 proteins used were human DLL3.FLAG (Adipogen #AG-40B-0151, amino acids 27-466), human DLL3.HIS (Abcam #ab255797, amino acids 27-492), mouse DLL3.HIS (IPA Custom, amino acids 25-477), and cynomolgus monkey DLL3.HIS (Acrobiosystems #, amino acids 27-490). The control antibody for DLL3 binding was rovalpituzumab (Creative Biolabs #TAB-216CL). The recombinant HER2 proteins used were Her2.HIS (Sinobiologics, #10004-H08H) and mouse HER2.HIS (Sinobiologics #50714-M08H).The control antibody for HER2 binding was trastuzumab (DIN: 02240692, ROCHE). Figures 1A and 1B show anti-Her2 and anti-DLL3 VHH-Fcs that specifically bind to soluble target antigen in ELISA. Additional VHH-Fcs containing Fc region mutations that reduce effector function and / or FcRn binding were tested but did not significantly affect binding to the target antigen.

[0320] VHH-Fc were screened for binding to various target-positive cancer cell lines by flow cytometry. All cell lines were obtained from ATCC unless otherwise noted and were cultured according to the manufacturer's instructions and recommended media. HER2-positive cell lines used were SKBR3 (ATCC #HTB-30), BT474 (ATCC #HTB-20), and HEK293-6E (NRC) cells. DLL3-positive cell lines tested included SHP-77 (ATCC CRI-2195), NCI-H82 (ATCC HTB-175), NCI-H69 (ATCC HTB-119), and HEK-DLL3 (Creative Biogene #CSC-RO0531). HER2-negative cell lines tested included SHP-77. DLL3-negative cell lines tested included HCT-116 (CCL-247), BT-474, and SKBR3. Primary antibodies, diluted in the same manner as in ELISA, were added to the cells and incubated on ice for 1 hour. The cells were washed twice with 1% FBS in PBS, centrifuged at 450×G for 4 minutes, and incubated with 2 μg / mL AlexaFluor 647-conjugated anti-human IgG (Jackson, Cat. #109-605-098) or AlexaFluor 647-conjugated anti-mouse IgG (Jackson, Cat. #115-605-164) containing 1:1000 DAPI (Biolegend, Cat. #422801) for 30 minutes on ice. After two additional washes, the cells were resuspended and analyzed by flow cytometry on the iQue screening platform (Intellicyt). Data were processed in Forecyt according to standard protocols. Figures 2A, 2B, and 2C show binding to target-positive cell lines, demonstrating that binding was specific to target-positive cells (i.e., through comparison with binding to negative control cells). Additional experiments showed that Fc mutations to reduce effector function and / or FcRn binding did not affect binding to cancer cells compared to wild-type Fc.

[0321] Example 3. Internalization assay VHH-Fc was tested for internalization by target-expressing cells using a secondary antibody conjugated to a pH-sensitive dye. A goat anti-huIgG-Fc secondary antibody was amine-conjugated to a pH-sensitive pHAb dye (Promega Cat# G9845) according to the manufacturer's instructions. pHAb dyes have low or no fluorescence at pH >7 but become fluorescent in acidic environments upon antibody internalization. Target-positive and target-negative cells were seeded at 1.0 × 106 / mL in a 96-well V-bottom plate. VHH-Fc and hIgG1 isotype controls were diluted to 75 nM in medium. Cells were centrifuged, the supernatant removed, and the cells were resuspended in the prepared primary antibody and incubated on ice for 1 hour. Excess primary antibody was washed from the cells, followed by incubation with pHAb-labeled secondary antibody on ice for 30 minutes. Excess secondary antibodies were then washed away, and the cells were resuspended in medium. One set of samples was placed in a 37°C incubator for internalization, while the other set was placed on ice (0°C) as a binding-only control. Cells were sampled at different time points ranging from 0 to 24 hours. Cells were stained with DAPI and analyzed by flow cytometry in the 572 / 28 channel using the iQue screening platform. VHH-Fc exhibited higher fluorescence on target-positive cells than negative controls (isotype, buffer). Figures 3A and 3B show that H101 and D102 were internalized by SHP-77 and HEK-DLL3 cells.

[0322] Example 4. Antibody Thermal Stability Determination The denaturation temperature (Tm) of VHH-Fc was determined by differential scanning fluorimetry (DSF) using the Protein Thermo Shift Dye Kit™ (ThermoFisher, Cat#: 4461146). Briefly, a total of 1 μg of antibody was used for each reaction. Antibody melting curves were generated using an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System with the recommended settings described in the kit manual. The Tm of the antibodies in Table 1 was then determined using ThermoFisher Protein Thermal Shift software (v.1.3). The Tm1 of VHH-Fc was determined by DSF. Both H101 and D102 showed good thermal stability of 67.5 ± 0.1 °C. Furthermore, VHH-Fcs containing mutations in the Fc region to reduce effector function and / or FcRn binding were tested for thermal stability, resulting in slightly lower thermal stability (1–2°C), but still within the acceptable range.

[0323] Example 5. Receptor Density Determination To test the efficacy of immunoconjugate binding relative to target density, target receptor density was measured on target-positive cell lines. Target density was measured using an ABC (Antibody Binding Capacity) assay. Cancer cells expressing the target of interest, as well as negative control cell lines, were harvested with cell dissociation buffer and seeded at approximately 5 × 10 cells per well in a 96-well V-bottom plate (Sarstedt 82.1583.001). Cells were tested for receptor expression using QuantiBRITE PE beads (BD Cat# 340495) and PE-conjugated anti-hu IgG (Biolegend clone HP6017) according to the manufacturer's instructions. Briefly, VHH-Fc and isotype control antibodies were prepared at appropriate saturating concentrations based on previous experiments. Antibody sample dilutions were incubated with the cell line panel for 1 hour on ice. Cells were washed twice with 1% FBS in 1x PBS (FACS buffer) and centrifuged at 400g for 4 minutes. They were then incubated with 4µg / mL mouse PE-conjugated anti-hu and DAPI (1:1000) on ice for 30 minutes. The cells were washed twice with FACS buffer, centrifuged at 400g for 4 minutes, and resuspended in FACS buffer. The fluorescence intensity on the PE channel was measured using the iQue Screener platform, and the data was processed using ForeCyt software. The amount of PE signal generated from different primary antibodies was then fitted to a standard curve based on known PE molecule / Quantibrite bead samples to determine the number of antibody binding sites per cell. The relative antibody binding sites correlate with the number of antigens or receptors on the cell surface. Table 2 shows the receptor density of anti-DLL3 and anti-HER2 VHH-Fc binding to a panel of cancer cell lines, which was in a range similar to that reported in the literature.

[0324] [Table 2]

[0325] Example 6. Affinity of antibodies to target proteins Antibody affinity was assessed using Octet Red96e (ForteBio). The association rate constant (k), dissociation rate constant (K), and affinity constant (K) were measured by biolayer interferometry using an anti-hIgG Fc (AHC) capture biosensor (ForteBio cat# 18-5063). Each cycle was performed at an orbital shake speed of 1,000 rpm. The antigen was titrated 1:2 from the appropriate starting concentration in kinetics buffer (ForteBio, cat# 18-1105). The AHC biosensor set was immersed in kinetics buffer for a 60-second baseline step. Anti-target VHH-Fc (5 μg / mL in kinetics buffer) was loaded onto the biosensor for 240 seconds, followed by a second 30-second baseline step. To compensate for the natural dissociation of the captured IgG, the IgG capture sensor was immersed in a buffer for a single reference subtraction. Each biosensor was then immersed in the corresponding concentration of target protein (human, mouse, or cynomolgus monkey monomeric protein) for 600 seconds, followed by dissociation for 1800 seconds in kinetics buffer or under optimized conditions. A new set of AHC biosensors was used for every VHH-Fc. Data were analyzed using a global fit 1:1 model for the association and dissociation steps (Octet software version v11.0). Table 3 shows the binding affinity data.

[0326] [Table 3]

[0327] Example 7. Determination of FcRn and Fc effector mutant affinities The FcRn affinity of a VHH-Fc can generally be used to predict the half-life of antibody serum clearance. (See, for example, Datta-Mannan A et al. "FcRn affinity-pharmacokinetic relationship of five human IgG4 antibodies engineered for improved in vitro FcRn binding properties in cynomolgus monkeys," Drug Metab Dispos. 2012 Aug;40(8):1545-55.) Briefly, 10 nM biotinylated hFcRn (Sino Biological, Cat#:CT071-H27H-B) was captured on an SA biosensor using an Octet RED96e (Fortebio). The hFcRN-coated biosensor was immersed in a sample solution in sodium phosphate buffer (100 mM NaHPO, 150 mM NaCl w / 0.05% Tween-20, pH 6.0) containing serial concentrations of test antibody, and association was measured. Dissociation was measured by immersing the biosensor in sodium phosphate buffer without antibody. KD values ​​were determined using Octet Data Analysis HT 11.0 software. A 2:1 (heterologous ligand) binding model was used for the analysis. Table 4 shows the effect of specific mutations in the Fc on FcRn affinity for wild-type VHH-Fc and for the mutants. The changes in FcRn affinity were consistent between targets. Constructs with only Fc effector mutations do not affect FcRn affinity. Addition of Fc effector mutations to FcRn mutant constructs does not affect FcRn affinity. Table 4a shows the affinity of VHH-Fc and Fc mutants for FcRn.

[0328] [Table 4]

[0329] VHH-Fc was also tested for affinity to FcγR by biolayer interferometry using the Octet Red96e platform. Each cycle was performed at an orbital shaking speed of 1,000 rpm. Streptavidin (SA) biosensors (Sartorius 18-5019) were rehydrated for 10 minutes using kinetics buffer (PBS + 0.1% BSA + 0.02% Tween-20). Biotinylated FcγR (Acro Biosystems) was then loaded onto the SA biosensor at concentrations ranging from 1 to 5 μg / mL in PBS for 40 to 100 seconds. VHH-Fc was serially diluted 1:2 in sample buffer (PBS + 0.02% Tween-20) to starting concentrations ranging from 5000 nM to 37.5 nM. The loaded biosensor was then allowed to bind to VHH-Fc for 60 to 120 seconds. VHH-Fc dissociation was measured in sample buffer for 30 to 900 seconds. Bound VHH-Fc was then removed using three cycles of 5 seconds of regeneration buffer (150 mM NaCl, 300 mM sodium citrate) and 5 seconds of sample buffer. Data were analyzed using either a global fit 1:1 Langmuir binding model (FcγRI) or steady-state analysis (Octet software version HT v11.1).

[0330] The analysis shows reduced binding to FcγR (represented by a higher KD) for constructs with incorporated mutations, as shown in Table 4b.

[0331] [Table 5]

[0332] Example 8. Study of self-bonding using AC-SINS The self-association tendency of VHH-Fc was determined by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using gold nanoparticles (Au-NPs) (Ted Pella, Cat#: 15705) (PMID: 24492294, 30395473). Briefly, Au-NPs were coated with goat IgG and goat anti-human Fc IgG (1:4 molar ratio). The conjugated Au-NPs were mixed with 5 μg of each VHH-Fc in quadruplicates in a 96-well plate. Wavelength scans were measured using a Synergy Neo2 plate reader. The maximum absorbance difference (Δλmax) was calculated by subtracting the λmax of each reaction from the λmax of PBS buffer. Data were analyzed using the Linest function in Excel using second-order polynomial fitting. A control antibody with a known high ACSINS score (above the literature established cutoff of 11 for IgG) was included in the assay. Figure 4 shows the ACSINS scores for the test articles and controls.

[0333] Example 9. Polyreactivity studies The polyreactivity of VHH-Fc against negatively charged biomolecules was determined by ELISA (e.g., Avery et al., "Establishing in vitro and in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics." MAbs. 2018 Feb / Mar;10(2):244-255). Briefly, ELISA plates were coated overnight with 5 μg / mL human insulin (SigmaAlrich, Cat#: I9278) and 10 μg / mL double-stranded DNA (SigmaAlrich, Cat#: D1626-250MG). The plates were blocked with ELISA buffer (PBS, 1 mM EDTA, 0.05% Tween-20, pH 7.4). 10 μg / mL of test VHH-Fc was loaded onto the plates in quadruplicate and incubated for 2 hours. HRP-conjugated goat anti-human Fc (0.01 μg / ml) was then added, and the plate was incubated for 1 hour. Signals were developed with TMB, and A450 absorbance was measured on a Synergy Neo2 plate reader. Signals were normalized to the signal of uncoated wells for each antibody tested. Table 5 shows the polyreactivity scores compared to the control antibody.

[0334] [Table 6]

[0335] Example 10. Fc variants effectively reduce VHH-Fc half-life In certain instances, shortening the drug half-life of α-emitters is important for safety and to avoid unwanted toxicity associated with treatment.However, antibodies usually have a half-life of 14 days or more.Therefore, the half-life of VHH-Fc variants was tested to observe and measure any reduction in half-life.

[0336] Twenty-eight 8-week-old male B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ (Tg32 hom, JAX stock #014565) mice were distributed into seven groups with four mice per group, as outlined in the table. Tg32 mice contain a humanized FcRn and are generally considered surrogate for human antibody pharmacokinetics when compared to non-human primates. (See, e.g., Avery LB et al. "Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies," MAbs. 2016 Aug-Sep;8(6):1064-78.) On day 0, mice were weighed and the test article was administered IV at 3 mg / kg and 5 ml / kg. 25 μL blood samples were collected from each mouse at timed intervals. Blood samples were collected in 1 μL of K3EDTA, processed to plasma, diluted 1 / 10 with 50% glycerol in PBS, transferred to specialized 96-well storage plates, and stored at −20° C. All plasma samples were evaluated by hIgG ELISA, selected for its high sensitivity to all seven test articles.

[0337] [Table 7]

[0338] As can be seen in Table 6, the introduction of mutations within FcRn generally reduced the half-life of anti-HER2 VHH-Fc. Interestingly, in contrast to published results in the field, not all Fc variants when included in the tested immunoconjugates exhibited a reduced half-life consistent with previously published results found in the literature (see, e.g., Burvenich IJ et al., "Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal the importance of S254 and Y436 in binding human neonatal Fc receptor," MAbs. 2016 May-Jun;8(4):775-86).

[0339] [Table 8]

[0340] As can be seen in Table 7, the introduction of mutations within FcRn was generally able to shorten the half-life of anti-DLL3 VHH-Fc. Similar to the HER2-binding immunoconjugates, and contrary to published results, not all Fc mutants showed a decrease in half-life consistent with previously published results found in the literature.

[0341] Example 11.VHH-Fc Intact Mass Analysis The conjugates were deglycosylated and then analyzed using in-house Endo-S enzyme (final concentration 10 μg / mL) at 37°C for 1 h.

[0342] For intact mass analysis, 8 μL of sample was injected into a Waters Acquity UPLC-Q-TOF equipped with a UPLC BEH200SEC 1.7 μM 4.6 × 150 mm column. The samples were eluted with a mobile phase of water / ACN (70 / 30, v / v) containing 0.1% TFA and 0.1% FA (formic acid) for 11 min at a flow rate of 0.25 mL / min.

[0343] Example 12. Procurement of bifunctional chelating agents Several chelators pre-functionalized for antibody conjugation are known to those skilled in the art. p-SCN-Bn-DOTA (1) is available from Macrocyclics (Plano, TX). Other linker variants of DOTA can be generated from the advanced intermediate DOTAGA-tetra(t-Bu ester) (2) (Macrocyclics, Plano, TX) according to the following general procedure.

[0344] Other reagents used in these procedures are available from Millipore Sigma, CombiBlocks, Chem-Impex, and Broadpharm. All solvents were obtained from VWR and used as received without anhydrous handling conditions unless otherwise indicated. Mass spectra were acquired using an Agilent HPLC-MS or Waters HPLC-MS with a C18 reverse-phase column and an acetonitrile / water (+0.1% formic acid) gradient. Flash chromatography was performed using a Biotage IsoleraOne instrument equipped with an appropriately sized normal-phase silica gel cartridge, with fractions collected at 254 nm. The final compound was purified by Agilent preparative-scale HPLC using an acetonitrile / water (+0.1% TFA) gradient. NMR spectra were acquired on a Bruker 400 MHz NMR instrument and processed with MestReNova v.14. Detailed NMR data were compiled using the multiplet analysis function in manual mode.

[0345] Figure 5 shows the synthesis of PEG5-DOTA, including compounds numbered (2) to (5) below: Compound 3 was prepared by HATU coupling followed by TFA deprotection. It is available without chromatographic purification.

[0346] Synthesis of compound (3) 4-({2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamoyl)-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid: Tetrakis(trifluoroacetic acid): Compound 2 (100 mg, 0.143 mmol) was dissolved in DMF (2 mL) and HATU (65.1 mg, 0.171 mmol) was added, followed by DIPEA (0.099 mL, 73.8 mg, 0.57 mmol). After 3 minutes, a solution of Boc-NH-PEG5-amine (65.1 mg, 0.17 mmol) was added to the reaction. After stirring for 10 minutes, HPLC indicated the reaction was complete. After 1 h, the reaction was quenched with approximately 5 mL of NaHCO (sat.), then 5 mL of water was added, and the mixture was extracted four times with 30 mL of Et O. The combined organics were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude protected intermediate in good purity. m / z found=1063.6 (M+H).

[0347] The above intermediate was directly dissolved in DCM (5 mL) and TFA (5 mL) was added. The reaction was stirred for 24 h until HPLC showed complete removal of the Boc and tBu esters. The reaction solution was concentrated in vacuo and coevaporated twice with 25 mL of DCM. The residue was precipitated from DCM containing EtO, and the remaining solid was then thoroughly triturated with sonication (15–30 min) to afford the title compound (128 mg, 86% two-step) as an off-white powder in good purity. 1H NMR (400 MHz, deuterium oxide) δ 4.15-3.68(m,7H),3.62(d,J=4.7 Hz,2H),3.59-3.49(m,20H),3.47(t,J=5.5 Hz,2H),3.35-2.78(m,16H),2.52-2.37(m,2H),1.97-1.79(m,2H).m / z found=739.5(M+H).

[0348] Synthesis of compound (4) bis(2,3,5,6-tetrafluorophenyl)hexanedioate: Adipic acid (1.00 g, 6.84 mmol) and EDC (3.28 g, 17.1 mmol) were dissolved in 20 mL of DCM and cooled to 0 °C in an ice bath, followed by the addition of a solution of 2,3,5,6-tetrafluorophenol in 20 mL of DCM. Conversion to the product was monitored by TLC (Rf = 0.5; 75% DCM / Hexanes). The reaction mixture was concentrated in vacuo and purified by flash chromatography (0 to 100% DCM / Hexanes) to give the title compound (2.48 g, 82%) as a crystalline white powder. 1H NMR (400 MHz, chloroform-d) δ 7.03 (tt, J = 9.9, 7.0 Hz, 2H), 3.00-2.63 (m, 4H), 1.95 (t, J = 3.3 Hz, 4H). This compound has poor signals by LCMS.

[0349] Compound (5) -{[2-(2-{2-[6-oxo-6-(2,3,5,6 tetrafluorophenoxy)hexanamido]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10 tetraazacyclododecan-1-yl]butanoic acid: To a solution of compound 3 (22.1 mg, 0.017 mmol) in DMF (1.5 mL) was added bis(2,3,5,6-tetrafluorophenyl)hexanedioate (4) (45.2 mg, 0.102 mmol) and triethylamine (0.0086 mL, 6.2 mg, 0.061 mmol). Complete conversion to the product was confirmed by HPLC. After stirring for 2 h, the reaction was diluted with DMSO (1.5 mL) and purified by direct injection onto preparative HPLC (Agilent, Hanover, CT) with a gradient of 15–50% MeCN / water + 0.1% TFA to afford the title compound (10.6 mg, 50%) as a white powder (2× TFA salt). 1H NMR (400 MHz, deuterium oxide) δ7.20(tt,J=10.4,7.2 Hz,1H),3.97-3.65(m,5H),3.58-3.51(m,20H),3.49(q,J=5.1 Hz,2H),3.43-3.32(m,6H),3.26(t,J=5.3 Hz,2H),3.20-2.82(m,12H),2.69(t,J=6.8 Hz,2H),2.52-2.34(m,2H),2.19(t,J=6.8 Hz,2H),1.99-1.82(m,2H),1.75-1.46(m,4H).m / z found =1015.3(M+H).

[0350] FIG. 6 shows the synthesis of PEG5-Py4Pa, including compounds numbered (6) to (10) below.

[0351] Synthesis of Compound (6) tert-butyl 6-[({[4-(benzyloxy)-6-{[bis({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino]methyl}pyridin-2-yl]methyl}({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino)methyl]pyridine-2-carboxylate: To a stirred solution of 1-[6-(aminomethyl)-4-(benzyloxy)pyridin-2-yl]methanamine (0.65 g, 2.67 mmol) (available from N. Delsuc, et al. Angew Chem. Int. Ed. 2007, 46, 214-217) in acetonitrile (50 mL) was added DIPEA (1.40 mL, 1.04 mg, 8.01 mmol) and tert-butyl 6-(Bromomethyl)pyridine-2-carboxylate (4.36 g, 16.0 mmol) (available from P. Coomba, et al. Inorg. Chem. 2016, 55, 12531-12543) was added, and the solution was heated to reflux. After 16 hours, the reaction was cooled, and the solvent was removed in vacuo. The crude material was dissolved in 200 mL of DCM and washed with 2 × 75 mL of NaHCO3 (sat.) and 2 × 75 mL of saturated brine. The DCM layer was then dried over sodium sulfate, filtered, and concentrated in vacuo to give a crude brown oil (950 mg), which could be used in the next step without further purification. The intermediate from above was dissolved in EtOH, ammonium formate (297 mg, 4.71 mmol) was added, and the flask was purged with N2. 10% Pd / C (250 mg, 0.23 mmol) was added, followed by another purging with N, and then 30% Pd / C (50 mg, 0.14 mmol). After another purging with N, the reaction was heated to 50 °C and stirred for 6 h, at which point the reaction was complete by LCMS. The reaction mixture was filtered through Celite, washed with 3 × 50 mL of MeOH, and then concentrated in vacuo to give a pale yellow oil. The crude material was purified by flash chromatography using a Biotage Sfar amino D cartridge and a gradient of 40-100% EtOAc / hexanes, followed by 0-20% MeOH / DCM to give the title compound as a yellow solid (278 mg, 11%).1H NMR (400 MHz, methanol-d4) δ7.88(dd,J=7.7,1.3 Hz,4H),7.82(t,J=7.7 Hz,4H),7.73(dd,J=7.7,1.2 Hz,4H),6.41(s,2H),4.00(s,8H),3.94(s,4H),1.61(s,36H).m / z found=918.4(M+H).

[0352] Synthesis of compound (7) tert-butyl N-[17-(2-bromoacetamido)-3,6,9,12,15-pentaoxaheptadecan-1-yl]carbamate: A solution of tert-butyl N-(17-amino-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamate (200 mg, 0.53 mmol) and DIPEA (0.146 mL, 109 mg, 0.84 mmol) in 5 mL of DCM was cooled to 0 °C. A solution of 2-bromoacetyl bromide (0.069 mL, 159 mg, 0.79 mmol) in 5 mL of DCM cooled to 0 °C was added dropwise over 2 minutes. The reaction was allowed to warm to room temperature, and after 90 minutes, HPLC showed full conversion to the product. The reaction was concentrated and partitioned between EtO and water, NaHCO (sat.) was added, and the mixture was then extracted 3 x 25 mL with EtO. The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was co-evaporated once with acetonitrile to remove water. The title compound was recovered as a brownish oil (261 mg, 99%). 1H NMR (400 MHz, chloroform-d) δ3.90(s,2H),3.75-3.64(m,18H),3.61(d,J=4.5 Hz,2H),3.56(t,J=5.1 Hz,2H),3.52(t,J=5.2 Hz,2H),3.37-3.30(m,2H),1.46(s,9H).m / z found=523.2(M+Na).

[0353] Synthesis of compound (8) tert-butyl 6-({[(6-{[bis({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino]methyl}-4-{[(17-{[(tert-butoxy)carbonyl]amino}-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamoyl]methoxy}pyridin-2-yl)methyl]({6-[(tert-butoxy)carbonyl]pyridin-2-yl}methyl)amino}methyl)pyridine-2-carboxylate: Compound 6 (100 mg, 0.11 mmol) and compound 7 (81.9 mg, 0.163 mmol) were dissolved in acetonitrile (5 mL), then potassium carbonate (30.1 mg, 0.218 mmol) was added and the reaction was stirred at 60° C. After 24 h, no starting material remained by HPLC. The reaction was concentrated and purified by flash chromatography (Biotage amino D cartridge, gradient 0.2-15% MeOH / DCM) to give the title compound as a yellow film (106 mg, 73%). 1H NMR (400 MHz, methanol-d4) δ7.89(d,J=7.8 Hz,4H),7.83(t,J=7.7 Hz,4H),7.66(d,J=7.6 Hz,4H),6.95(s,2H),4.66(s,2H),4.04(s,8H),3.92(s,4H),3.75-3.55(m, 20H),3.53-3.43(m,2H),3.30-3.13(m,2H),1.52(s,36H),1.43(s,9H).m / z found=670.0(M+2H / 2).

[0354] Synthesis of compound (9) 6-({[(4-{[(17-amino-3,6,9,12,15-pentaoxaheptadecan-1-yl)carbamoyl]methoxy}-6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)pyridin-2-yl)methyl][(6-carboxypyridin-2-yl)methyl]amino}methyl)pyridine-2-carboxylic acid: Compound 8 (125 mg, 0.093 mmol) was dissolved in DCM (5 mL) and TFA (5 mL) was added. After 18 h, HPLC showed no starting material or t-butyl intermediate remained. The reaction was concentrated in vacuo and co-evaporated once with DCM. The crude oil was triturated twice with Et2O with sonication and collected by filtration to give 100 mg (64%, as 5x TFA salt) of the title compound as a brown solid. 1H NMR (400 MHz, methanol-d4) δ 8.04(d,J=7.7 Hz,4H),7.96(t,J=7.8 Hz,4H),7.66(t,J=8.4 Hz,4H),7.45(s,2H),4.84(s,2H),4.74-4.49(m,12H),3.74(t,J=5.0 Hz,2H),3.71-3.63(m,14H),3.60(t,J=5.3Hz,2H),3.48(t,J=5.6 Hz,2H),3.20-3.12(m,2H).m / z found=1014.3(M+H).

[0355] Synthesis of compound (10) 6-[({[6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)-4-[({17-[6-oxo-6-(2,3,5,6-tetrafluoro-phenoxy)hexanamido]-3,6,9,12,15-pentaoxaheptadecan-1-yl}carbamoyl)methoxy]pyridin-2-yl]methyl}[(6-carboxypyridin-2-yl)methyl]amino)methyl]pyridine-2-carboxylic acid: To a solution of compound 9 (80 mg, 0.079 mmol) in DMF (2.5 mL) was added bis(2,3,5,6-tetrafluorophenyl)hexanedioate (4) (140 mg, 0.32 mmol) and triethylamine (0.027 mL, 20 mg, 0.197 mmol). Complete conversion to the product was confirmed by HPLC. After stirring for 4 h, the reaction was diluted with DMSO (1.5 mL) and purified by direct injection onto preparative HPLC (Agilent, Hanover, CT) with a gradient of 25-60% MeCN / water + 0.1% TFA to afford the title compound (57.5 mg, 56%) as a white powder (3x TFA salt). 1H NMR (400 MHz, deuterium oxide) δ 7.85(t,J=7.8 Hz,4H),7.78(dd,J=7.8,1.2 Hz,4H),7.50(dd,J=7.8,1.2 Hz,4H),7.11(tt,J=10.4,7.2 Hz,1H),6.99(s,2H),4.59(s,2H),4.49(s,8H),4.45(s,4H),3.60-3.45(m,18H),3.46(t,J=5.3 Hz,2H),3.36(t,J=5.3 Hz,2H),3.22(t,J=5.3 Hz,2H),2.59(t,J=6.7 Hz,2H),2.14(t,J=6.7 Hz,2H),1.61-1.46(m,4H).m / z found=1290.3(M+H).

[0356] Synthesis of compound (11) 6-[({[6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)-4-{2-[4-(cyanosulfanyl)phenyl]ethoxy}pyridin-2-yl]methyl}[(6-carboxypyridin-2-yl)methyl]amino)methyl]pyridine-2-carboxylic acid; bis(trifluoroacetic acid): The title compound was prepared according to the conditions in L Li et al. Bioconjugate Chem. 2021, 32, 1348-1363. Spectral and LCMS data matched the reported values.

[0357] Example 13. Conjugation of VHH-Fc proteins using chelator-linkers Conjugation can be carried out using many of the methods available for the preparation of IgG radioconjugates and IgG antibody-drug conjugates. For information on the range of applicable methodologies, see PW Howard, Antibody-Drug Conjugates (ADCs), Protein Therapeutics, First Edition, chapter 9, pp. 278-279 (2017).

[0358] For a typical lysine-based conjugate, VHH-Fc was buffer-exchanged into 0.1 M NaHCO3, pH 8.5–9.5, using either a Microsep Advance Centrifugal Device (Pall 10K MWCO, Cat#: MCP010C41) or a Zeba column (ThermoFisher, Cat#: 87768), followed by sterilization in a Costar Spin-X Centrifuge Tube, 0.22 μm (Corning, Cat#: 8160). Buffer-exchanged antibody was quantified by BCA assay. An appropriate molar excess (5–20 equivalents) of chelator-linker (50 mM in DMSO) was added to VHH-Fc (2 mg / mL final concentration), and the reaction was incubated at 25°C for 2 hours or overnight in a Thermomixer. After the reaction was complete, the sample was passed through a Zeba column (ThermoFisher, Cat#: 87770) to remove unused chelator-linker and buffer exchanged into PBS (pH 7.4) (LifeTechnologies, Cat#: 10010-023) according to the manufacturer's protocol. The VHH-Fc-chelator conjugate (VFCC) was stored at 4°C until analysis and purification.

[0359] Example 14. Purification of VHH-Fc-chelator conjugates (VFCC) by SEC To remove high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS), VHH-Fc was purified by SEC using an AKTA Pure FPLC system equipped with a Cytiva HiLoad 16 / 600 Superdex 200 pg column. TBS buffer (50 mM Tris, 150 mM NaCl, OmniTrace Ultra water [VWR, Cat#: CAWX0003-2]) at pH 7.6 was used as the SEC buffer. Fractions containing intact VHH-Fc were pooled together and concentrated using a Microsep Advance Centrifugal Device (Pall 10k MWCO, Cat#: MCP010C41). The concentrated sample was transferred to an Ultrafree-MC GV centrifugal filter, 0.22 μm, 0.5 mL (Millipore, Cat#: UFC30GV0S) and centrifuged at 3,000 × g for 3 minutes.

[0360] Example 15. Protein quantification VHH-Fc protein content was quantified with cetuximab (LIST / E:094822, DIN 02271249, 2 mg / mL) using a standardized Pierce BCA protein assay kit (Thermo, Cat#:23225).

[0361] Example 16. Chelating Agent (CAR) Analysis for VHH-Fc Ratio The chelator loading ratio, herein referred to as CAR, can be analyzed by methods applicable to those skilled in the art of antibody conjugates. For a review of these methods in the context of ADCs, see A Wakankar et al., mAbs 3:161 (2011). The CAR of each conjugate was analyzed by DG-SEC-MS.

[0362] The conjugates were analyzed by deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this case, the mass distribution is obtained after spectral deconvolution, which allows the calculation of the average CAR of the preparation.

[0363] The conjugates were analyzed by deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this case, the distribution of masses is obtained after spectral deconvolution, which allows the calculation of the average CAR of the preparation.

[0364] Example 17. Binding of VHH-Fc conjugates to cells expressing target proteins In some cases, conjugation can negatively affect the binding of VHH-Fc to target proteins. Therefore, the binding of VHH-Fc conjugates was tested as described above. Table 8 shows the cell binding data of VHH-Fc chelator conjugates.

[0365] [Table 9]

[0366] As seen in Table 8, binding was observed for both long and short DOTA linkers. As shown in Table 8, binding was also observed over increasing chelator VHH-Fc ratios (CAR).

[0367] Example 18. Percent Intact Analysis The percentage of intact i...

Claims

1. 1. An immunoconjugate comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody comprises a polypeptide comprising (a) a first antigen-binding domain, and (b) a second antigen-binding domain.

2. The immunoconjugate of claim 1 , wherein the polypeptide further comprises an Fc domain.

3. 1. An immunoconjugate comprising a multivalent antibody and a chelating agent, wherein said multivalent antibody has Formula I: A-B-C and a polypeptide having the structure During the ceremony, A comprises a first antigen-binding domain; B comprises a second antigen-binding domain, and C comprises an Fc domain.

4. An immunoconjugate comprising a multivalent antibody and a chelating agent, wherein the multivalent antibody has the formula II: A-C-B and a polypeptide having the structure During the ceremony, A comprises a first antigen-binding domain; B comprises a second antigen-binding domain, and C comprises an Fc domain.

5. The immunoconjugate of any one of claims 1 to 4, wherein the first antigen-binding domain and the second antigen-binding domain each comprise an immunoglobulin single chain variable domain polypeptide.

6. The immunoconjugate of any one of claims 1 to 5, wherein the immunoglobulin single chain variable domain polypeptide comprises a VHH.

7. The immunoconjugate of any one of claims 2 to 6, wherein the Fc domain comprises a CH2 domain and a CH3 domain.

8. The immunoconjugate of any one of claims 2 to 7, wherein the Fc domain comprises a CH3 domain.

9. The immunoconjugate of any one of claims 2 to 7, wherein the Fc domain comprises a CH2 domain.

10. 10. The immunoconjugate of any one of claims 2 to 9, wherein the Fc domain comprises an alteration to one or more amino acid residues that reduces an effector function of said Fc domain.

11. The immunoconjugate of claim 10, wherein the changes to one or more amino acid residues that reduce an effector function of the immunoglobulin heavy chain constant region are changes that reduce complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or a combination thereof.

12. The changes to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region are, according to EU numbering, (a) 297A, 297Q, 297G or 297D, (b) 279F, 279K or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R or 235S, (e) 237A, 237E, 237K, 237N or 237R, (f) 234A, 234V or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y or is 329R (k) 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T or 254V, (p) 255N, (q) 256H, 256K, 256R or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y or 265A, (t) 267G, 267H, 267I or 267K, (u) 268K, (v) 2 69N or 269Q, (w) 270A, 270G, 270M or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm ) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A,L235E and G237A, (aaa) L234A, L235E, G237A and P331S, (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D26 12. The immunoconjugate of any one of claims 1 to 11, wherein the amino acid sequence is selected from the list consisting of: (a) L234A, (b) G237A, (c) P331A or P331S, (d) L233P, (e) L234A, (f) L235E, (g) G237A, (h) P331A or P331S, (i) L234A, (i) L235E, (j) D265A and N297G, (k) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (ppp).

13. 12. The immunoconjugate of any one of claims 2 to 11, wherein the changes to one or more amino acid residues in the immunoglobulin heavy chain constant region that reduce effector function comprise L234A, L235E, G237A, A330S, and P331S, according to EU numbering.

14. 14. The immunoconjugate of any one of claims 2 to 13, wherein the Fc domain comprises an alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

15. 15. The immunoconjugate of any one of claims 2 to 14, wherein the change to one or more amino acid residues that alters binding of the immunoconjugate to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: 1253A, 1253D, 1253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof, according to EU numbering.

16. 15. The immunoconjugate of any one of claims 2 to 14, wherein the change to one or more amino acid residues that alters binding of the immunoconjugate to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof, according to EU numbering.

17. 15. The immunoconjugate of any one of claims 2 to 14, wherein the change to one or more amino acid residues that alters binding of the immunoconjugate to neonatal Fc receptor (FcRn) is a change to an amino acid residue selected from the list consisting of: I253A, H310A, H435Q, and combinations thereof, according to EU numbering.

18. 15. The immunoconjugate of any one of claims 2 to 14, wherein the change to one or more amino acid residues that alters binding of the immunoconjugate to neonatal Fc receptor (FcRn) comprises I253A, according to EU numbering.

19. 15. The immunoconjugate of any one of claims 2 to 14, wherein the changes to one or more amino acid residues that alter binding of the immunoconjugate to neonatal Fc receptor (FcRn) comprise H310A, according to EU numbering.

20. 15. The immunoconjugate of any one of claims 2 to 14, wherein the changes to one or more amino acid residues that alter binding of the immunoconjugate to neonatal Fc receptor (FcRn) comprise H435Q, according to EU numbering.

21. The immunoconjugate of any one of claims 1 to 20, wherein the multivalent antibody comprises a homodimer of the polypeptide.

22. The immunoconjugate of any one of claims 1 to 21, wherein the multivalent antibody comprises a molecular weight of less than about 110,000 daltons.

23. The immunoconjugate of any one of claims 1 to 22, wherein the multivalent antibody is a monospecific multivalent antibody.

24. The immunoconjugate of any one of claims 1 to 23, wherein the multivalent antibody is a bispecific multivalent antibody.

25. 25. The immunoconjugate of any one of claims 1 to 24, wherein the first antigen-binding domain and the second antigen-binding domain bind to FOLR1, DLL3, or HER2.

26. The immunoconjugate of any one of claims 1 to 25, wherein the first antigen-binding domain binds to FOLR1.

27. 27. The immunoconjugate of any one of claims 1 to 26, wherein (a) the first antigen-binding domain binds to FOLR1, and (b) the second antigen-binding domain binds to DLL3.

28. the first antigen-binding domain binds to FOLR1; and Complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO:

3.

28. The immunoconjugate of any one of claims 1 to 27, comprising:

29. the second antigen-binding domain binds to DLL3; and a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 7; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 113; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 213; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 307, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 310, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 313; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 407, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 410, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 413; or Complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 507, complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 510, and complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 513 28. The immunoconjugate of any one of claims 1 to 27, comprising:

30. the first antigen-binding domain binds to FOLR1; and Complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 1, complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO:

3. wherein the second antigen-binding domain binds to DLL3; and a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 7; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 113; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 207, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 213; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 307, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 310, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 313; a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 407, a complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 410, and a complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 413; or Complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 507, complementarity determining region (CDR) 2 comprising the amino acid sequence set forth in SEQ ID NO: 510, and complementarity determining region (CDR) 3 comprising the amino acid sequence set forth in SEQ ID NO: 513 27. The immunoconjugate of any one of claims 1 to 26, comprising:

31. 31. The immunoconjugate of any one of claims 1 to 30, wherein the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:

4.

32. 32. The immunoconjugate of any one of claims 1 to 31, wherein the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 8, 101-106, 201-206, 301-306, 401-406, or 501-506.

33. 33. The immunoconjugate of any one of claims 1 to 32, wherein the first antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:4, and the second antigen-binding domain comprises an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8, 101-106, 201-206, 301-306, 401-406, or 501-506.

34. The immunoconjugate of any one of claims 1 to 33, wherein the chelating agent is a radioisotope chelating agent.

35. The immunoconjugate of any one of claims 1 to 33, wherein the chelating agent is an alpha-emitter chelating agent.

36. 34. The immunoconjugate of any one of claims 1 to 33, wherein the chelating agent is a beta-emitter chelating agent or a gamma-emitter chelating agent.

37. 37. The immunoconjugate of any one of claims 1 to 36, wherein the chelating agent is selected from the list consisting of DOTA, DO3A, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, Noneunpa, and combinations thereof.

38. The chelating agent is DOTMA, DOTPA, DO3AM-acetic acid, DOTP, DOTMP, DOTA-4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H4Octapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI (HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, porphyrin, deferoxamine, DFO ＊ 38. The immunoconjugate of any one of claims 1 to 37, selected from the list consisting of:

39. The immunoconjugate of any one of claims 1 to 38, wherein the chelating agent is DOTA.

40. The immunoconjugate of any one of claims 1 to 38, wherein the chelating agent is DOTAGA.

41. The immunoconjugate of any one of claims 1 to 38, wherein the chelating agent is Py4Pa.

42. 38. The immunoconjugate of any one of claims 1 to 37, wherein the chelating agent is directly attached to the antigen binding region and / or the Fc domain.

43. 38. The immunoconjugate of any one of claims 1 to 37, wherein the chelator is attached to the antigen binding region and / or the Fc domain by a linker.

44. 38. The immunoconjugate of any one of claims 1 to 37, wherein the chelator is a linker-chelator selected from the list consisting of TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

45. The immunoconjugate of any one of claims 1 to 44, further comprising a radioisotope.

46. An immunoconjugate comprising a multivalent antibody, a chelating agent, and a radioisotope, wherein the multivalent antibody has the formula I: A-B-C and a homodimer of a polypeptide having the structure During the ceremony, A comprises a first VHH domain, B comprises a second VHH domain, and C comprises an Fc domain; wherein said first VHH domain binds to FOLR1 or DLL3, and wherein said second VHH domain binds to FOLR1 or DLL3.

47. An immunoconjugate comprising a multivalent antibody, a chelating agent, and a radioisotope, wherein the multivalent antibody has the formula II: A-C-B and a homodimer of a polypeptide having the structure During the ceremony, A comprises a first VHH domain, B comprises a second VHH domain, and C comprises an Fc domain; wherein said first VHH domain binds to FOLR1 or DLL3, and wherein said second VHH domain binds to FOLR1 or DLL3.

48. 46. ​​The immunoconjugate of claim 45, wherein the radioisotope is an alpha emitter.

49. 49. The immunoconjugate of claim 48, wherein said radioisotope is an alpha emitter selected from the list consisting of 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

50. 50. The immunoconjugate of claim 48 or 49, wherein the radioisotope is 225-Ac.

51. 49. The immunoconjugate of claim 48, wherein the radioisotope is a beta emitter.

52. 52. The immunoconjugate of claim 51, wherein the radioisotope is a β-emitter selected from 177-Lu, 90-Y, 67-Cu, and 153-Sm.

53. 53. A method of killing a tumor or cancer cell, said method comprising contacting said tumor or cancer cell with the immunoconjugate of any one of claims 1-52, thereby killing said tumor or cancer cell.

54. 54. The method of claim 53, wherein the tumor cells are solid tumor cells.

55. 55. The method of any one of claims 53 or 54, wherein the tumor or cancer cells express FOLR1, DLL3, or both.

56. 53. A method of treating cancer or tumor in an individual, said method comprising administering to said individual an immunoconjugate of any one of claims 1-52, thereby treating said cancer or tumor.

57. 57. The method of claim 56, wherein the individual is a human individual.

58. 58. The method of any one of claims 56 or 57, wherein the cancer or tumor is a solid cancer or tumor.

59. 58. The method of any one of claims 56 or 57, wherein the cancer or tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

60. 60. The method of any one of claims 56 to 59, comprising administering to the individual between 0.5 μCi and 30.0 μCi per kilogram.

61. 61. The method of any one of claims 56 to 60, comprising administering to the individual between 10 mCi and 75 mCi per square metre of body surface area.

62. 62. The method of any one of claims 56-61, wherein the cancer or tumor expresses an antigen that is specifically bound by the immunoconjugate.

63. 53. The immunoconjugate of any one of claims 1 to 52 for use in a method of treating cancer or a tumor in an individual.

64. 100. A method of delivering a radioisotope to cancer or tumor cells in an individual, said method comprising administering to said individual an immunoconjugate of any one of claims 1-52, thereby delivering said radioisotope to said cancer or tumor cells.

65. 65. The method of claim 64, wherein the individual is a human individual.

66. 66. The method of claim 64 or 65, wherein the cancer or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

67. 67. The method of any one of claims 64 to 66, wherein the cancer or tumor cells express an antigen that is specifically bound by the immunoconjugate.

68. 53. A method of imaging a tumor in an individual, said method comprising administering to said individual an immunoconjugate of any one of claims 1 to 52.

69. 69. The method of claim 68, wherein the individual is a human individual.

70. 70. The method of any one of claims 68 or 69, wherein the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer.

71. 71. The method of any one of claims 68 to 70, wherein the tumor expresses an antigen that is specifically bound by the immunoconjugate.

72. A nucleic acid encoding a polypeptide according to any one of claims 1 to 47.

73. 73. An expression vector comprising the nucleic acid of claim 72.

74. 74. A cell comprising the nucleic acid of claim 72 or the expression vector of claim 73.

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