Multivalent immunoconjugates for targeted radioisotope therapy

Abstract

Disclosed herein are immunoconjugates 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

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 373,186, filed on August 22, 2022, which is incorporated herein by reference in its entirety. Background of the Invention

[0003] The specificity of antibodies (such as IgG) for their antigens makes antibodies a prime targeting platform for therapeutic agents; however, the typical serum half-life of IgG is at least three weeks, which is disadvantageous for the delivery of radioisotopes (including α-emitting isotopes such as Ac-225 and β-emitting isotopes such as Lu-177 and Y-90), especially due to prolonged exposure and chronic off-target toxicity.

[0004] 225-Ac is one of the most cytotoxic radioisotopes among α-emitting radioisotopes, and a single decay event can effectively destroy cancer cells by causing double-strand DNA breaks and subsequent cell death. The potency of α-emitting radioisotopes makes them attractive as cell-killing agents, capable of overcoming acquired resistance observed in response to other therapies. Summary of the Invention

[0005] Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) that include a multivalent (e.g., tetravalent) antibody and can be used to treat cancer. The immunoconjugates described herein are advantageous because the immunoconjugates are capable of achieving higher-order affinity interactions with a target, while having a molecular weight less than that of a conventional antibody molecule (e.g., less than 150,000 daltons), and while displaying improved safety characteristics (e.g., reduced serum half-life).

[0006] In one aspect, described herein is an immunoconjugate that includes a multivalent antibody and a chelator, wherein the multivalent antibody includes a polypeptide that includes: (a) a first antigen-binding domain; and (b) a second antigen-binding domain. In certain embodiments, the polypeptide further includes an Fc domain.

[0007] In another aspect, described herein is an immunoconjugate that includes a multivalent antibody and a chelator, wherein the multivalent antibody includes a polypeptide having a structure of Formula I:

[0008] A - B - C

[0009] wherein: A includes a first antigen-binding domain; B includes a second antigen-binding domain; and C includes an Fc domain.

[0010] In another aspect, the present disclosure describes an immunoconjugate comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having the structure of Formula II: A-C-B; wherein: A comprises a first antigen-binding domain; B comprises a second antigen-binding domain; and C comprises an Fc domain.

[0011] In another aspect, the present disclosure describes an immunoconjugate comprising a multivalent antibody, a chelator, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula I: A-B-C; wherein: A comprises a first VHH domain; B comprises a second VHH domain; and C comprises an Fc domain, wherein the first VHH domain binds to FOLR1 or DLL3; and wherein the second VHH domain binds to FOLR1 or DLL3.

[0012] In another aspect, the present disclosure describes an immunoconjugate comprising a multivalent antibody, a chelator, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula II: A-C-B; wherein: A comprises a first VHH domain; B comprises a second VHH domain; and C comprises an Fc domain, wherein the first VHH domain binds to FOLR1 or DLL3; and wherein the second VHH domain binds to FOLR1 or DLL3.

[0013] In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain comprises 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 an alteration to one or more amino acid residues that reduces the effector function of the Fc domain. In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell phagocytosis ADCP, or a combination thereof. In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected from, 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)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; (lll)A330L; (mmm)P331A or P331S; or (nnn)E233P; (ooo)L234A, L235E, G237A, A330S and P331S;or any combination of (ppp)(a)-(ppp). In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S according to EU numbering. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the following according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the following according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to amino acid residues selected from the following according to EU numbering: I253A, H310A, H435Q, and combinations thereof. In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprises I253A according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprises H310A according to EU numbering. In certain embodiments, the alteration to one or more amino acid residues that modifies the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) comprises H435Q according to EU numbering. In certain embodiments, the multivalent antibody comprises a homodimer of a polypeptide. In certain embodiments, the multivalent antibody has 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 FOLR1, DLL3, or HER2. In certain embodiments, the first antigen-binding domain binds FOLR1. In certain embodiments, (a) the first antigen-binding domain binds FOLR1; and (b) the second antigen-binding domain binds DLL3. In certain embodiments, the first antigen-binding domain comprises: complementarity-determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:1; complementarity-determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:2;and a complementarity determining region (CDR) 3 comprising the amino acid sequence as 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 as set forth in SEQ ID NO: 5; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 6; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 7; a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 107; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 110; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 113; a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 207; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 210; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 213; a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 307; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 310; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 313; a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 407; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 410; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 413 or a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 507; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 510; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as 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 as set forth in SEQ ID NO: 1; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 2; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 3, and wherein the second antigen-binding domain comprises: a complementarity determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 5; a complementarity determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 6; and a complementarity determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 7;Complementary determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 107; Complementary determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 110; and Complementary determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 113; Complementary determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 207; Complementary determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 210; and Complementary determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 213; Complementary determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 307; Complementary determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 310; and Complementary determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 313; Complementary determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 407; Complementary determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 410; and Complementary determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO: 413 or Complementary determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO: 507; Complementary determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO: 510; and Complementary determining region (CDR) 3 comprising the amino acid sequence as 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 having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence as 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 having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequences as 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 having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence as set forth in SEQ ID NO: 4;And the second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity 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 chelator is a radioisotope chelator. In certain embodiments, the chelator is an α-emitter chelator. In certain embodiments, the chelator is a β-emitter chelator or a γ-emitter chelator. In certain embodiments, the chelator is selected from: 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: 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, desferrioxamine, 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 conjugated to the antigen-binding region and / or the Fc domain. In certain embodiments, the chelator is conjugated to the antigen-binding region and / or the Fc domain via a linker. In certain embodiments, the chelator is a linker-chelating agent selected from: 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 α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from: 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.;

[0014] A method of killing tumor cells or cancer cells, the method comprising: contacting the tumor cells or cancer cells with an immunoconjugate of the present disclosure so as to kill the tumor cells or cancer cells. In certain embodiments, the tumor cells are solid tumor cells. In certain embodiments, the tumor cells or cancer cells express FOLR1, DLL3, or both.

[0015] A method of treating cancer or a tumor in an individual, the method comprising administering to the individual an immunoconjugate herein so as to treat 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 comprises administering to the individual from 0.5 μCi to 30.0 μCi per kilogram. In certain embodiments, the method comprises administering to the individual from 10 mCi to 75 mCi per square meter of body surface area. In certain embodiments, the cancer or tumor expresses an antigen to which the immunoconjugate specifically binds. In certain embodiments, the use of an immunoconjugate for the method of treating cancer or a tumor in an individual.

[0016] Also described herein is a method of delivering a radioisotope to cancer cells or tumor cells in an individual, the method comprising administering to the individual an immunoconjugate so as to deliver the radioisotope to the cancer cells or tumor cells. In certain embodiments, the individual is a human individual. In certain embodiments, the cancer cells or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells. In certain embodiments, the cancer cells or tumor cells express an antigen to which the immunoconjugate specifically binds.

[0017] Also described herein is a method of imaging a tumor in an individual, the method comprising administering to the individual an immunoconjugate. In certain embodiments, the individual is a human individual. In certain embodiments, the tumor comprises lung cancer, breast cancer, ovarian cancer, or neuroendocrine cancer. In certain embodiments, the tumor expresses an antigen to which the immunoconjugate specifically binds.

[0018] Incorporated by reference

[0019] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 and the accompanying drawings that illustrate exemplary embodiments in which the principles of the present disclosure are utilized:

[0021] Figure 1A and 1B show the binding of anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0022] Figure 2A 、 2B and 2C show the binding of anti-HER2 and anti-DLL3 VHH-Fc constructs to cells expressing HER2 and / or DLL3.

[0023] Figure 3A and 3B show the internalization of anti-HER2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3.

[0024] Figure 4 show the self-interaction data of anti-HER2 and anti-DLL3 VHH-Fc constructs.

[0025] Figure 5 show a schematic diagram of the chemical synthesis of the linker molecule.

[0026] Figure 6 show a schematic diagram of the chemical synthesis of the linker molecule.

[0027] Figure 7A 、 7B and 7C show the immunoreactivity scores of different VHH-Fc constructs.

[0028] Figure 8 show the comparison of imaging with 111In-labeled VHH-Fc and biodistribution of 225Ac-labeled VHH-Fc.

[0029] Figure 9A 、 9B 、9C and 9D show the biodistribution of the labeled anti-HER2 VHH-Fc construct over time.

[0030] Figure 10A 、 10B and 10C show the tumor:non-tumor tissue ratio of the labeled anti-HER2 VHH-Fc construct.

[0031] Figure 11 show the biodistribution of the labeled anti-HER2 VHH-Fc construct.

[0032] Figure 12 show the whole-body clearance of 111In-labeled VHH-Fc(H101) and VHH-Fc variants (H105, H107, and H108).

[0033] Figure 13Show biodistribution over time of radiolabeled anti-DLL3 VHH-Fc constructs.

[0034] Figure 14 Show biodistribution of radiolabeled anti-DLL3 VHH-Fc constructs.

[0035] Figure 15A and 15B Show biodistribution of 225Ac-labeled anti-HER2(15A) and anti-DLL3(15B) VHH-Fc constructs.

[0036] Figure 16A 、 16B and 16C show results of toxicity studies with 225Ac-labeled anti-HER2 VHH-Fc constructs.

[0037] Figure 17 Show immunoreactivity fractions of different anti-DDL3 VHH-Fc constructs loaded with 177Lu.

[0038] Figure 18 Show chemical structures of certain linker chelator agents described herein.

[0039] Figure 19A and 19B Show schematic of multivalent antibody formats.

[0040] Figure 20 Show monospecific and bispecific multivalent formats of FOLR1 and DLL3.

[0041] Figure 21 Show binding of multivalent tetramer constructs to cells expressing FOLR1. DETAILED DESCRIPTION

[0042] Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) that include multivalent (e.g., tetravalent) antibodies and can be used to treat cancer. In some cases, the immunoconjugates described herein are advantageous because they achieve higher-order avidity interactions with targets while having a molecular weight less than that of conventional antibody molecules (e.g., less than 150,000 daltons). Additionally, in such cases, immunoconjugates that include elements of conventional antibodies (such as an Fc domain or an Fc variant domain) can also 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. Although reducing serum half-life or effector cell function is generally not associated with improved antibody efficacy, the immunoconjugates described herein can achieve improved safety while being able to effectively bind and kill target tumor cells (e.g., within the tumor microenvironment).

[0043] Immunoconjugate

[0044] Provided herein are immunoconjugates (e.g., radiolabeled immunoconjugates) comprising a multivalent (e.g., tetravalent or higher) polypeptide (e.g., a multivalent antibody or a polypeptide derived from an antibody). In some embodiments, the multivalent antibody is tetravalent. In certain embodiments, the multivalent antibody is monospecific (e.g., binds only FOLR1 or DLL3). In certain embodiments, the multivalent antibody is bispecific (e.g., binds both FOLR1 and DLL3). In some embodiments, the multivalent antibody of the immunoconjugate has a molecular weight of less than 150,000 daltons. In certain embodiments, the multivalent antibody of the immunoconjugate has a molecular weight of less than 110,000 daltons.

[0045] In some embodiments, provided are immunoconjugates (e.g., radioimmunoconjugates) comprising a multivalent antibody (e.g., having a chelator and a radionuclide), 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.

[0046] Figure 19A -B shows exemplary multivalent antibody forms described herein. 110 depicts a first antigen-binding domain (e.g., an immunoglobulin single-chain domain). 120 depicts a second antigen-binding domain (e.g., an immunoglobulin single-chain domain). 130 depicts an Fc domain comprising CH2-CH3 (132 and 134, respectively). 140 depicts an optional linker polypeptide.

[0047] In some embodiments, provided are immunoconjugates (e.g., radioimmunoconjugates) comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula I:

[0048] A - B - C

[0049] wherein: A comprises a first antigen-binding domain; B comprises a second antigen-binding domain; and C comprises an Fc domain.

[0050] In some embodiments, provided are immunoconjugates (e.g., radioimmunoconjugates) comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula II:

[0051] A - C - B

[0052] wherein: A comprises a first antigen-binding domain; B comprises a second antigen-binding domain; and C comprises an Fc domain.

[0053] In some embodiments, provided are immunoconjugates (e.g., radioimmunoconjugates) comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula III:

[0054] A-L1-B-L2-C

[0055] 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).

[0056] In some embodiments, provided are immunoconjugates (e.g., radioimmunoconjugates) comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula IV:

[0057] A-L1-C-L2-B

[0058] 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).

[0059] In certain embodiments, the multivalent antibody comprises a homodimer of a polypeptide (e.g., mediated by Fc domain dimerization). In certain embodiments, the multivalent antibody has a molecular weight of less than 150,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 140,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 130,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 120,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain has a molecular weight of from about 50,000 daltons to about 110,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain has a molecular weight of from about 75,000 daltons to about 110,000 daltons. In certain embodiments, the antigen-binding domain has a molecular weight of from about 50,000 daltons to about 105,000 daltons. In certain embodiments, the multivalent antibody has a molecular weight of less than 110,000 daltons. In certain embodiments, the antigen-binding domain has a molecular weight of from about 75,000 daltons to about 105,000 daltons.

[0060] In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain comprises a single-chain variable domain polypeptide selected from: scFv, VH, VL, VHH, and VNAR. In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain comprises an immunoglobulin single-chain variable domain polypeptide. In certain embodiments, the immunoglobulin single-chain variable domain polypeptide is selected from: VH, VL, and VHH. In certain embodiments, each of the first antigen-binding domain and the second antigen-binding domain comprises a VHH (e.g., a first VHH domain and a second VHH domain).

[0061] In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that modulates (e.g., reduces, inhibits, decreases, prevents, etc.) the effector function of the Fc domain. In certain embodiments, the Fc domain comprises an alteration to one or more amino acid residues that modifies (e.g., reduces, inhibits, decreases, 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 modifies (e.g., reduces, inhibits, decreases, prevents, etc.) the 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 both (i) the effector function of the Fc domain (e.g., ADCC and / or CDC) and (ii) the binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0062] In certain embodiments, the multivalent antibody is monospecific (e.g., binds only FOLR1 or DLL3). In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind FOLR1. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain bind DLL3.

[0063] In certain embodiments, the multivalent antibody is bispecific (e.g., binds FOLR1 and DLL3). In certain embodiments, the first antigen-binding domain binds FOLR1; and the second antigen-binding domain binds DLL3. In certain embodiments, the first antigen-binding domain binds DLL3; and the second antigen-binding domain binds FOLR1.

[0064] In certain embodiments, the immunoconjugate comprises a chelator. In certain embodiments, the chelator is a radionuclide chelator. In certain embodiments, the chelator is directly conjugated to the antigen-binding region and / or the Fc domain. In certain embodiments, the chelator is indirectly conjugated to the antigen-binding region and / or the Fc domain.

[0065] In certain embodiments, the immunoconjugate is a radioimmunoconjugate and comprises 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: 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

[0066] "Immunoconjugate" refers to and encompasses a molecular complex that comprises at least one antigen-binding region (e.g., variable region or complementarity-determining region) derived from a multivalent antibody, which antigen-binding region is further conjugated to at least one non-antibody-derived molecule, such as a chelating agent or a cytotoxic agent. The non-antibody-derived molecule can be conjugated, for example, to one or more lysine or cysteine residues of the antigen-binding region, or to a constant region conjugated (by a peptide bond or otherwise) to the antigen-binding region. In some embodiments, the immunoconjugate further comprises a chelator (interchangeably, "chelating agent"). In some embodiments, the immunoconjugate comprises an antibody construct of the invention directly or indirectly linked to a cytotoxic agent or a radioisotope.

[0067] Antigen-binding domain

[0068] "Variable region" or "variable domain" refers to and encompasses the domain of an antibody heavy or light chain or an immunoglobulin single-chain variable domain (e.g., VHH) antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) or VHH of a native antibody generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. A single VHH is sufficient to confer antigen-binding specificity.

[0069] "Immunoglobulin single-chain variable domain" or "immunoglobulin single-chain variable domain antibody" or "immunoglobulin single variable domain" or "single-chain antibody" or "VHH", which are used interchangeably herein, refers to and encompasses an immunoglobulin molecule in which the antigen-binding site resides on and is formed by a single immunoglobulin domain (e.g., variable domain).

[0070] Typically, in conventional immunoglobulins, the variable heavy domain (VH) and the variable light domain (VL) interact to form an antigen-binding site, providing a total of six CDRs for antigen-binding site formation. In this context, the complementarity-determining regions (CDRs) of VH and VL can contribute to the antigen. The antigen-binding domains of conventional antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules with homologous VL and VH), Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv), scFv fragments, or diabodies derived from such conventional four-chain antibodies, differ from single-chain variable domain antibodies.

[0071] The VH, VL, or VHH regions can be subdivided into hypervariable regions, called "complementarity-determining regions" (CDRs), interspersed with more conserved regions, called "framework regions" (FR or FW).

[0072] The framework regions and the CDRs can be defined by a variety of methods (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by the AbM antibody modeling software of Oxford Molecular). See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (eds. 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 in the following: 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). The methods herein encompass and include Chothia, AbM, Kabat, Contact, and / or IMGT.

[0073] “Complementary determining region” or “CDR” refers to and encompasses the sequences of amino acids within the variable region of an antibody that confer 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). An immunoglobulin single-chain variable domain antibody contains 3 CDRs (CDR1, CDR2, and CDR3).

[0074] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions (“FR”), which are 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”); these framework regions are interrupted by three complementarity-determining regions (“CDR”), which are referred to in the art and herein as “complementarity-determining region 1” (“CDR1”); “complementarity-determining region 2” (“CDR2”); and “complementarity-determining region 3” (“CDR3”). Thus, the 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; provided that it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially 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 VH, VHH, including camelized VH or humanized VHH. Preferably, it is a VHH, including camelized VH or humanized VHH.

[0075] The “VHH domain”, also known as VHH, VHH antibody fragment and VHH antibody, was originally described as the antigen-binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., “antibodies lacking a light chain”; 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 4-chain antibodies (which are referred to herein as “VH domains”) and the light chain variable domains present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For further description of VHH, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0076] "Humanized VHH" includes an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain, but the amino acid sequence has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and particularly in the framework sequence) with one or more amino acid residues that occur at the corresponding positions in the VH domain of a conventional four-chain antibody from humans (e.g., as indicated above). This can be done in a manner essentially known, which will be obvious to those skilled in the art, for example based on the further description herein and the prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized VHHS can be obtained in any suitable manner essentially known, and thus are not strictly limited to polypeptides obtained using polypeptides containing a naturally occurring VHH domain as the starting material.

[0077] 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 that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can be represented by the 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 the text.

[0078] Affinity matured antibody encompasses and / or refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs), which result in an increase in the affinity of the antibody for an antigen as compared to the parental antibody that does not have such alterations.

[0079] Assays for binding and binding can be readily determined by methods known in the art (e.g., ELISA, surface plasmon resonance, biolayer interferometry, isothermal calorimetry, etc.). In some embodiments, binding is determined by ELISA. In some embodiments, binding, as measured by surface plasmon resonance, biolayer interferometry or isothermal calorimetry, comprises a KD of less than, for example, 10^-5 M (10 uM). In some embodiments, binding, as measured by surface plasmon resonance, biolayer interferometry or isothermal calorimetry, comprises a KD of less than, for example, 10^-6 M (1 uM). In some embodiments, binding, as measured by surface plasmon resonance, biolayer interferometry or isothermal calorimetry, comprises a KD of less than, for example, 10^-7 M (100 nM).

[0080] In certain embodiments, an antibody comprises one or more naturally occurring amino acids. In certain embodiments, an 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 are not subject to human manipulation. In certain instances, naturally occurring includes and / or further refers to 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).

[0081] In some embodiments, an antibody comprises a variant sequence of the antibody. In certain instances, amino acid substitutions can be made in the sequence of any antibody described herein without necessarily reducing or eliminating its activity (as measured by, for example, the 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 within 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 include conservative substitutions (e.g., substituting an amino acid with another amino acid having comparable chemical characteristics). In certain instances, a nonpolar amino acid can be substituted and replaced with another nonpolar amino acid, where nonpolar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan, and methionine. In certain instances, a polar amino acid with a neutral charge can be substituted and replaced with another polar amino acid with a neutral charge, where polar amino acids with a neutral charge include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In certain instances, a positively charged amino acid can be substituted and replaced with another positively charged amino acid, where positively charged amino acids include arginine, lysine, and histidine. In certain instances, a negatively charged amino acid can be substituted and replaced with another negatively charged amino acid, where negatively charged amino acids include aspartic acid and glutamic acid. Examples of amino acid substitutions also include substituting its corresponding D-amino acid with an L-amino acid, substituting homocysteine with cysteine, or other non-natural amino acids.

[0082] In certain embodiments, the antibody comprises one or more non-natural amino acids. In certain embodiments, the antibody consists of non-natural amino acids. As used herein, non-natural amino acids and / or unnatural amino acids include and / or refer to amino acid structures that cannot be biosynthetically generated 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 sequence amino acids), D-amino acids, homoamino acids, β-homoamino acids, N-methyl amino acids, α-methyl amino acids, or. By way of further example, non-natural amino acids also include 4-benzoyl-L-phenylalanine (Bpa), anthranilic acid (Abz), aminobutyric acid (Abu), aminohexanoic acid (Ahx), aminoisobutyric acid (Aib), citrulline (Cit), diaminobutyric acid (Dab), diaminopropanoic acid (Dap), diaminopropionic acid (Dap), γ-carboxyglutamic acid (Gla), homoalanine (Hala), homoarginine (Harg), homoasparagine (Hasn), homoaspartic acid (Hasp), homocysteine (Hcys), homoglutamic acid (Hglu), homoglutamine (Hgln), homoisoleucine (Hile), homoleucine (Hleu), homomethionine (Hmet), homophenylalanine (Hphe), homoserine (Hser), homotyrosine (Htyr), homovaline (Hval), hydroxyproline (Hyp), 4-piperidinecarboxylic acid (Isonipecotic Acid, Inp), naphthylalanine (Nal), 3-piperidinecarboxylic acid (Nipecotic Acid, Nip), norleucine (Nle), norvaline (Nva), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), phenylglycine (Phg), pyroglutamic acid (Pyr), sarcosine (Sar), tert-butylglycine (Tle), and tetrahydroisoquinoline-3-carboxylic acid (Tic). Such non-natural amino acid residues can be introduced by substituting naturally occurring amino acids and / or by inserting non-natural amino acids into naturally occurring antibody sequences. Non-natural amino acid residues can also be incorporated to confer the desired functionality on the apelin molecule, e.g., the ability to link functional moieties (e.g., PEG).

[0083] A stable formulation is a formulation in which and / or that encompasses a protein (e.g., an antibody) that substantially retains its physical stability and / or chemical stability and / or biological activity when stored at the intended storage temperature (e.g., 2 - 8 °C). In some embodiments, the formulation substantially retains its physical and chemical stability as well as its biological activity upon storage. The storage period can be selected based on the intended shelf life of the formulation. In addition, the formulation is stable upon freezing (e.g., to -20 °C) and thawing of the formulation, e.g., after 1 or more freeze - thaw cycles. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247 - 301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29 - 90 (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively evaluated in a variety of different ways, including evaluating aggregate formation (e.g., using size - exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing charge heterogeneity by using cation - exchange chromatography or capillary zone electrophoresis; performing SDS - PAGE analysis to compare reduced and intact antibodies; evaluating the biological activity or antigen - binding function of the antibody; and the methods described herein. Instability can involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / fragmentation (e.g., hinge - region fragmentation), succinimide formation, unpaired cysteines, etc.

[0084] A pharmaceutically acceptable carrier encompasses and / or refers to the components of a pharmaceutical formulation other than the active ingredient that are non - toxic to the subject. Pharmaceutically acceptable carriers encompass, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0085] Polypeptide and protein can be used interchangeably and encompass and / or refer to a polymer 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 binding peptides) can include amino acid residues, including natural and / or non - natural amino acid residues. The term also includes post - expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, the polypeptide can contain modifications relative to the native or natural sequence, so long as the protein retains the desired activity. These modifications can be intentional, such as by site - directed mutagenesis, or can be accidental, such as by mutations in the host that produces the protein or due to errors in PCR amplification.

[0086] Mathematical algorithms can be used to determine the percent identity or percent similarity between two sequences. Non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are 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 that detects distant relationships between molecules. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are 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 alignment can be performed using the CLUSTAL algorithm (e.g., as provided in the program Clustal-omega), as described in Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0087] As used herein, the terms individual, patient, or subject include and / or refer to an individual who has been diagnosed with at least one disease, disorder, or condition, is suspected of having at least one disease, disorder, or condition, or is at risk of developing at least one disease, disorder, or condition, and the described compositions and methods can be used to treat the at least one disease, disorder, or condition. 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.

[0088] The antibodies described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains 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 has been linked. One type of vector is a genomic integration vector or "integrating vector", which can integrate into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, for example, a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a gene operably linked thereto is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In an expression vector, regulatory elements for controlling transcription such as promoters, enhancers, polyadenylation signals 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 selectable gene that facilitates the identification of transformants can also be introduced additionally. Vectors derived from viruses can be employed, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. A plasmid vector can be linearized for integration into a genomic region. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, adenovirus or adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus.

[0089] As used herein, when used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the terms "homologous", "homology" or "percent homology" can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of a sequence can be determined using the most recent version of BLAST as of the filing date of this application.

[0090] The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise genetically engineer suitable cells with the nucleic acids, such that the cells are capable of producing the antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. September–October 2010; 2(5):466–477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines that can be used to produce antibodies, are Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells, or cells. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell that can be used to produce the antibody. In certain embodiments, methods for preparing antibodies are described herein, which include culturing cells comprising a nucleic acid encoding the antibody under in vitro conditions sufficient to permit production and secretion of the antibody.

[0091] In certain embodiments, master cell banks are described herein that comprise: (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 capable of withstanding liquid nitrogen freezing.

[0092] Methods for preparing the antibodies described herein are also described. Such methods include incubating cells or cell lines comprising a nucleic acid encoding the antibody in a cell culture medium under conditions sufficient to permit expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium. Harvesting can also include one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture components. In certain embodiments, the additional purification steps include centrifugation, ultracentrifugation, Protein A purification, Protein G purification, Protein A / G purification, or Protein L purification and / or ion exchange chromatography.

[0093] As used herein, "Treat", "treatment" or "treating" refers to, for example, an intentional intervention in a physiological disease state that results in a reduction in the severity of the disease or disorder; a shortening of the duration of the disorder process; an improvement or elimination of one or more symptoms associated with the disease or disorder; or the provision of a beneficial effect to a subject having the disease or disorder. Treatment does not require a cure of the underlying disease or disorder.

[0094] A "therapeutically effective amount", "effective dose", "effective dosage" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease (manifested as a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the disease symptom-free period, or the prevention of damage or disability caused by the affliction of the disease). The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by measuring the activity of the agent in in vitro assays.

[0095] 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, etc. 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., the antibody) can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0096] The pharmaceutical compounds described herein can 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 produce any undesired toxicological effects (see, for example, Berge, S.M. et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids (such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.) and those derived from non-toxic organic acids (such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include those derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.) and those derived from non-toxic organic amines (such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0097] As used herein, treatment or treating includes and / or refers to a pharmaceutical or other intervention regimen used to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic and / or prophylactic benefits. A therapeutic benefit may refer to the eradication or amelioration of a symptom or underlying condition being treated. Additionally, a therapeutic benefit can be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying condition. Prophylactic effects include 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 and condition, or any combination thereof. For prophylactic benefits, a subject at risk of developing a particular disease or reporting one or more physiological symptoms of a disease may undergo treatment even though a diagnosis of the disease may not have been made. One of skill in the art will recognize that, given the population of potential individuals to be treated, not all individuals will respond to treatment or respond equally. Such individuals are considered to have been treated.

[0098] Generally, generating immunoglobulins involves immunizing experimental animals, fusing immunoglobulin-producing cells to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries, e.g., by phage display. The generation of immunoglobulin sequences such as VHHs and immunoglobulin single-chain variable domains has been described in various publications, of which WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001) may be illustrative. In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. Nanobodies that bind the target antigen are further screened from the nanobody library obtained from the immunization. In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from natural sources or during recombinant production.

[0099] The multivalent antibodies described herein comprise antigen-binding domains. In some cases, any individual antigen-binding domain having a molecular weight of less than about 25,000 daltons (e.g., a VHH having a molecular weight of about 15,000 daltons) can be used herein, wherein the total molecular weight of the multivalent antibody of the immunoconjugate is less than 150,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 25,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 20,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 19,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 18,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 17,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 16,000 daltons. In some embodiments, the antigen-binding domain has a molecular weight of less than about 15,000 daltons.

[0100] In certain embodiments, the antigen-binding domain has a molecular weight of from about 12,000 Daltons to about 25,000 Daltons. In certain embodiments, the antigen-binding domain has a molecular weight of from about 12,000 Daltons to about 13,000 Daltons, from about 12,000 Daltons to about 14,000 Daltons, from about 12,000 Daltons to about 15,000 Daltons, from about 12,000 Daltons to about 16,000 Daltons, from about 12,000 Daltons to about 17,000 Daltons, from about 12,000 Daltons to about 18,000 Daltons, from about 12,000 Daltons to about 19,000 Daltons, from about 12,000 Daltons to about 20,000 Daltons, from about 12,000 Daltons to about 25,000 Daltons, from about 13,000 Daltons to about 14,000 Daltons, from about 13,000 Daltons to about 15,000 Daltons, from about 13,000 Daltons to about 16,000 Daltons, from about 13,000 Daltons to about 17,000 Daltons, from about 13,000 Daltons to about 18,000 Daltons, from about 13,000 Daltons to about 19,000 Daltons, from about 13,000 Daltons to about 20,000 Daltons, from about 13,000 Daltons to about 25,000 Daltons, from about 14,000 Daltons to about 15,000 Daltons, from about 14,000 Daltons to about 16,000 Daltons, from about 14,000 Daltons to about 17,000 Daltons, from about 14,000 Daltons to about 18,000 Daltons, from about 14,000 Daltons to about 19,000 Daltons, from about 14,000 Daltons to about 20,000 Daltons, from about 14,000 Daltons to about 25,000 Daltons, from about 15,000 Daltons to about 16,000 Daltons, from about 15,000 Daltons to about 17,000 Daltons, from about 15,000 Daltons to about 18,000 Daltons, from about 15,000 Daltons to about 19,000 Daltons, from about 15,000 Daltons to about 20,000 Daltons, from about 15,000 Daltons to about 25,000 Daltons, from about 16,000 Daltons to about 17,000 Daltons, from about 16,000 Daltons to about 18,000 Daltons, from about 16,000 Daltons to about 19,000 Daltons, from about 16,000 Daltons to about 20,000 Daltons, from about 16,000 Daltons to about 25,000 Daltons, from about 17,000 Daltons to about 18,000 Daltons, from about 17,000 Daltons to about 19,000 Daltons, from about 17,000 Daltons to about 20,000 Daltons, from about 17,000 Daltons to about 25,000 Daltons, from about 18,000 Daltons to about 19,000 Daltons, from about 18,000 Daltons to about 20,000 Daltons, from about 18,000 Daltons to about 25,000 Daltons, from about 19,000 Daltons to about 20,a molecular weight of 000 Daltons, about 19,000 to about 25,000 Daltons or about 20,000 to about 25,000 Daltons. In certain embodiments, the antigen-binding domain has 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.,

[0101] 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: VH, VL, and VHH. In certain embodiments, the antigen-binding domain comprises VHH (e.g., a first VHH domain and a second VHH domain).

[0102] 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 VH, VL, and VHH. In certain embodiments, the first antigen-binding domain and the second antigen-binding domain each comprise VHH (e.g., a first VHH domain and a second VHH domain).

[0103] Antigen-binding domains that bind to FOLR1 are provided herein. "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 UniProtID P15328).

[0104] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, wherein CDR1-3 are defined using the Kabat definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, wherein CDR1-3 are defined using the Chothia definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, wherein CDR1-3 are defined using the AbM definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, wherein CDR1-3 are defined using the Contact definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3 of SEQ ID NO: 4, wherein CDR1-3 are defined using the IMGT definition.

[0105] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO: 1; complementarity-determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO: 2; and complementarity-determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO: 3, wherein the immunoglobulin single-chain domain binds FOLR1.

[0106] In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:4. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises the amino acid sequence as set forth in SEQ ID NO:4.

[0107] Provided herein are antigen-binding domains that bind 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).

[0108] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 of SEQ ID NO:24, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3, wherein CDR1-3 are defined using the Kabat definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 of SEQ ID NO:24, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3, wherein CDR1-3 are defined using the Chothia definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 of SEQ ID NO:24, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3, wherein CDR1-3 are defined using the AbM definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 of SEQ ID NO:24, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3, wherein CDR1-3 are defined using the Contact definition. In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 of SEQ ID NO:24, complementarity-determining region (CDR) 2, and complementarity-determining region (CDR) 3, wherein CDR1-3 are defined using the IMGT definition.

[0109] In some embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises: complementarity-determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:21; complementarity-determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:22; and complementarity-determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:23, wherein the immunoglobulin single-chain domain binds HER2.

[0110] In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:24. In certain embodiments, the antigen-binding domain is an immunoglobulin single-chain domain that comprises the amino acid sequence as set forth in SEQ ID NO:24.

[0111] Provided herein are antigen-binding domains that bind DLL3. “DLL3” or “delta-like canonical Notch ligand 3” or “SCDO1” refers to and encompasses the protein encoded by the DLL3 gene (see NC_000019.10(39498947..39508469); NCBI Gene 10683 or UniProt ID Q9NYJ7).

[0112] In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NOs: 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 having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 4; and the second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NOs: 8, 101-106, 201-206, 301-306, 401-406 or 501-506.

[0113] 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, wherein CDR1-3 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, wherein CDR1-3 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, wherein CDR1-3 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, wherein CDR1-3 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, wherein CDR1-3 are defined using the IMGT definition.

[0114] In certain embodiments, the antigen-binding domain comprises: complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:5; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:6; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:7; complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:107; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:110; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:113; complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:207; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:210; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:213; complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:307; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:310; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:313; complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:407; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:410; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:413; or complementarity-determining region (CDR) 1 comprising the amino acid sequence as set forth in SEQ ID NO:507; complementarity-determining region (CDR) 2 comprising the amino acid sequence as set forth in SEQ ID NO:510; and complementarity-determining region (CDR) 3 comprising the amino acid sequence as set forth in SEQ ID NO:513.

[0115] 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., an 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.

[0116] 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 FOLR1 and a second antigen-binding domain that binds to DLL3.

[0117] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0118] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:403. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:303. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:304. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:305. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:503. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:103. In certain embodiments, the antigen-binding domain that binds FOLR1 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:4; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:8.

[0124] In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:403. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:303. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:304. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:305. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:503. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:103. In certain embodiments, the antigen-binding domain that binds to FOLR1 comprises a VHH that comprises SEQ ID NO:4; and the antigen-binding domain that binds to DLL3 comprises a VHH that comprises SEQ ID NO:8.

[0125] 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.

[0126] 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 that comprises 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 that comprises 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 that comprises SEQ ID NO:4; and the antigen-binding domain that binds to HER2 comprises a VHH that comprises SEQ ID NO:24.

[0127] 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.

[0128] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0129] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0130] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0131] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0132] In certain embodiments, the antigen-binding domain that binds 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 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 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 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 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 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.

[0133] In certain embodiments, the antigen-binding domain that binds 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 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.

[0134] In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:403. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:303. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:304. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:305. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:503. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:103. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises a sequence having at least 90% sequence identity to SEQ ID NO:8.

[0135] In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:403. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:303. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:304. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:305. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:503. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ IDNO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:103. In certain embodiments, the antigen-binding domain that binds HER2 comprises a VHH that comprises SEQ ID NO:24; and the antigen-binding domain that binds DLL3 comprises a VHH that comprises SEQ ID NO:8.

[0136] Fc domain

[0137] "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, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or the first component of the classical complement system (Clq). The antibody constant region generally includes the CL region (e.g., for the light chain) or the CH1-CH2-CH3 regions. Generally, the Fc domain generally refers to and encompasses the CH2-CH3 regions of the heavy chain constant region. For IgG, the Fc domain contains the immunoglobulin domains CH2 and CH3 (Cy2 and Cy3), and optionally all or part of the hinge region between CHI (Cyl) and CH2 (Cy2). In some embodiments, the Fc domain includes CH2-CH3 and hinge-CH2-CH3 from the N-terminus to the C-terminus. In some embodiments, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4, which contains the hinge-CH2-CH3 domain / region. Additionally, in certain embodiments, where the Fc domain is a human IgG1 Fc domain, the hinge includes the C220S amino acid substitution. Further, in some embodiments, where the Fc domain is a human IgG4 Fc domain, the hinge includes the S228P amino acid substitution. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as including the carboxy-terminal residues E216, C226, or A231, where numbering is according to EU. In some embodiments, as described more fully below, amino acid modifications are made to the Fc region, e.g., to alter binding to one or more FcyRs or to FcRn. The Fc domain can be a native sequence Fc, which includes any allotypic variant or variant Fc (containing one or more mutations that reduce effector cell function and / or FcRN).

[0138] "Hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to and encompasses a flexible polypeptide containing 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 includes positions 216 (E216 in IgG1) to 230 (P230 in IgG1), where numbering is according to the EU index as in Kabat. In some embodiments, e.g., in the context of the Fc region, including the hinge (the full length or a fragment of the hinge) generally refers to positions 216-230.

[0139] "Isotype" refers to and encompasses antibody classes encoded by heavy chain constant domain genes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies). The full-length amino acid sequences of each wild-type human IgG constant region (including all domains, i.e., CH1 domain, hinge, CH2 domain, and CH3 domain) are cataloged in the publicly available UniProt database, such as P01857 (IgG1), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or their various isotypes. If the domain of the heavy chain constant region contains the amino acid sequence of the corresponding domain of the respective isotype or its variant (with higher homology to the corresponding domain of the respective isotype than to the corresponding domain of other isotypes), then that domain belongs to the "IgG1 isotype", "IgG2 isotype", "IgG3 isotype", or "IgG4 isotype". "Allotype" refers to naturally occurring variants within a particular isotype group that differ in a few amino acids (see 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 an IgA, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG1 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype.

[0140] In some embodiments, the Fc domain comprises a variant Fc domain that contains one or more mutations that modulate (e.g., reduce, inhibit, decrease, prevent, etc.) effector function, FcRn binding, or both, associated with the heavy chain constant region.

[0141] The immunoglobulin heavy chain constant region can be a variant constant region that contains one or more amino acid residue alterations that confer additional utility and advantageous properties to the immunoconjugates described herein. In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region or decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region and decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region contains alterations to one or more amino acid residues that decrease the binding of the immunoconjugate to the neonatal Fc receptor (FcRn).

[0142] In some embodiments, the Fc domain comprises an alteration to the heavy chain constant region that reduces effector functions 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 the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement-dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cell phagocytosis ADCP, or a combination thereof. In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected, according to EU numbering, from: (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) 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; (lll) 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 alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S and P331S according to EU numbering.;

[0143] In some embodiments, the Fc domain comprises alterations to the heavy chain constant region that shorten the serum half-life of the immunoconjugate. In certain embodiments, amino acid alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) shorten the serum half-life of the immunoconjugate. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following according to EU numbering: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following according to EU numbering: 253, 254, 310, 435, 436, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following according to EU numbering: I253A, H310A, H435Q, and combinations thereof. In certain embodiments, the alterations that alter or reduce the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to amino acid residues selected from the following: H310A, H435Q, and combinations thereof (according to EU numbering).

[0144] In certain embodiments, the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected from, 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) 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; (lll) A330L; (mmm) P331A or P331S; or (nnn) E233P; (ooo) L234A, L235E, G237A, A330S, and P331S; or (ppp) any combination of (a)-(ppp).;

[0145] In certain embodiments, alterations to one or more amino acid residues that reduce the effector function of the immunoglobulin heavy chain constant region include L234A, L235E, G237A, A330S, and P331S according to EU numbering. In certain embodiments, the Fc domain includes alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to the following amino acid residues selected according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to the following amino acid residues selected according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof. In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) are directed to the following amino acid residues selected according to EU numbering: I253A, H310A, H435Q, and combinations thereof. In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) include I253A according to EU numbering. In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) include H310A according to EU numbering. In certain embodiments, the alterations to one or more amino acid residues that alter the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) include H435Q according to EU numbering.

[0146] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:11. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:11. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:11, wherein the heavy chain constant region comprises an I253A substitution (according to EU numbering).

[0147] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:12. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:12. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:12, wherein the heavy chain constant region comprises an S254A substitution (according to EU numbering).

[0148] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:13. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:13. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:13, wherein the heavy chain constant region comprises an H310A substitution (according to EU numbering).

[0149] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:14. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:14. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:14, wherein the heavy chain constant region comprises an H435Q substitution (according to EU numbering).

[0150] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:15. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:15. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:15, wherein the heavy chain constant region comprises a Y436A substitution (according to EU numbering).

[0151] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:16. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:16. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:16, wherein the heavy chain constant region comprises H310A / H435Q substitutions (according to EU numbering).

[0152] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:17. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:17. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity 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).

[0153] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO:18. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:18, wherein the heavy chain constant region comprises L234A, L235E, G237A, H310A, A330S and P331S substitutions (according to EU numbering).

[0154] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:19. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:19. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:19, wherein the heavy chain constant region comprises L234A, L235E, G237A, H435Q, A330S and P331S substitutions (according to EU numbering).

[0155] In certain embodiments, the heavy chain constant region of the immunoconjugate comprises a sequence having at least 90%, 95%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:20. In certain embodiments, the heavy chain constant region of the immunoconjugate comprises the same sequence as SEQ ID NO:20 (according to EU numbering).

[0156] Alterations that affect FcRn binding can shorten the serum half-life of the immunoconjugate, thus allowing the skilled person to select a half-life suitable for a particular imaging or therapeutic goal. In certain embodiments, the serum half-life of the immunoconjugate is from about 12 hours to about 120 hours. In certain embodiments, the serum half-life of the immunoconjugate is from about 12 hours to about 24 hours, from about 12 hours to about 36 hours, from about 12 hours to about 48 hours, from about 12 hours to about 60 hours, from about 12 hours to about 72 hours, from about 12 hours to about 84 hours, from about 12 hours to about 96 hours, from about 12 hours to about 108 hours, from about 12 hours to about 120 hours, from about 24 hours to about 36 hours, from about 24 hours to about 48 hours, from about 24 hours to about 60 hours, from about 24 hours to about 72 hours, from about 24 hours to about 84 hours, from about 24 hours to about 96 hours, from about 24 hours to about 108 hours, from about 24 hours to about 120 hours, from about 36 hours to about 48 hours, from about 36 hours to about 60 hours, from about 36 hours to about 72 hours, from about 36 hours to about 84 hours, from about 36 hours to about 96 hours, from about 36 hours to about 108 hours, from about 36 hours to about 120 hours, from about 48 hours to about 60 hours, from about 48 hours to about 72 hours, from about 48 hours to about 84 hours, from about 48 hours to about 96 hours, from about 48 hours to about 108 hours, from about 48 hours to about 120 hours, from about 60 hours to about 72 hours, from about 60 hours to about 84 hours, from about 60 hours to about 96 hours, from about 60 hours to about 108 hours, from about 60 hours to about 120 hours, from about 72 hours to about 84 hours, from about 72 hours to about 96 hours, from about 72 hours to about 108 hours, from about 72 hours to about 120 hours, from about 84 hours to about 96 hours, from about 84 hours to about 108 hours, from about 84 hours to about 120 hours, from about 96 hours to about 108 hours, from about 96 hours to about 120 hours, or from about 108 hours to about 120 hours. In certain embodiments, the serum half-life of the immunoconjugate is 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 serum half-life of the immunoconjugate is 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 serum half-life of the immunoconjugate is 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.

[0157] In certain embodiments, the serum half-life of the immunoconjugate is from about 1 day to about 10 days. In certain embodiments, the serum half-life of the immunoconjugate is from about 1 day to about 2 days, from about 1 day to about 3 days, from about 1 day to about 4 days, from about 1 day to about 5 days, from about 1 day to about 6 days, from about 1 day to about 7 days, from about 1 day to about 8 days, from about 1 day to about 9 days, from about 1 day to about 10 days, from about 2 days to about 3 days, from about 2 days to about 4 days, from about 2 days to about 5 days, from about 2 days to about 6 days, from about 2 days to about 7 days, from about 2 days to about 8 days, from about 2 days to about 9 days, from about 2 days to about 10 days, from about 3 days to about 4 days, from about 3 days to about 5 days, from about 3 days to about 6 days, from about 3 days to about 7 days, from about 3 days to about 8 days, from about 3 days to about 9 days, from about 3 days to about 10 days, from about 4 days to about 5 days, from about 4 days to about 6 days, from about 4 days to about 7 days, from about 4 days to about 8 days, from about 4 days to about 9 days, from about 4 days to about 10 days, from about 5 days to about 6 days, from about 5 days to about 7 days, from about 5 days to about 8 days, from about 5 days to about 9 days, from about 5 days to about 10 days, from about 6 days to about 7 days, from about 6 days to about 8 days, from about 6 days to about 9 days, from about 6 days to about 10 days, from about 7 days to about 8 days, from about 7 days to about 9 days, from about 7 days to about 10 days, from about 8 days to about 9 days, from about 8 days to about 10 days, or from about 9 days to about 10 days. In certain embodiments, the serum half-life of the immunoconjugate is 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 serum half-life of the immunoconjugate is 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 serum half-life of the immunoconjugate is 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.

[0158] In certain embodiments, the molecular weight of the heavy chain constant region is from about 10 kDa to about 25 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is from about 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 25 kDa, from about 15 kDa to about 20 kDa, from about 15 kDa to about 25 kDa, or from about 20 kDa to about 25 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the molecular weight of the heavy chain constant region is at most about 15 kDa, about 20 kDa, or about 25 kDa.

[0159] chelating agent

[0160] As described herein, chelators can be conjugated to immunoconjugates, antigen-binding region / immunoglobulin heavy chain constant region molecules, VHH antigen-binding region / immunoglobulin heavy chain constant region molecules (wild-type or variant), VHH antigen-binding region / immunoglobulin Fc molecules (wild-type or variant). The chelator allows the immunoconjugate to be loaded with a suitable radioisotope, such as a β-emitter or an α-emitter. The chelating factor can be conjugated to the immunoconjugate via the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. Such conjugation can be effected, for example, by covalent attachment to one or more amino acids of the immunoconjugate, antigen-binding region, heavy chain constant region, immunoglobulin Fc region, or any combination thereof.

[0161] In some embodiments, the chelator of the immunoconjugate is covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof. In some embodiments, the chelator is directly covalently linked to the antigen-binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof (e.g., without using a spacer, stretcher, or linker). In some embodiments, the chelator is covalently linked to the antigen-binding arm via a linker that is covalently linked to the chelator and covalently linked 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 olefin chain). The chelating factor can be linked or conjugated to the immunoconjugate as described in Sadiki, A. et al., “Site-specific conjugation of native antibody.” Antibody Therapeutics 2020, 3, 271-284.

[0162] In some embodiments, the immunoconjugate is formed by attaching a chelator-linker in a site-specific manner (directed to a specific amino acid or glycan residue). In some embodiments, site-specific conjugation involves the directed functionalization of a specific lysine residue in the pair of framework regions with a chelator-linker. In other embodiments, this residue can be functionalized with a different reactive functional group and then reacted with the chelator-linker in a second step to obtain the immunoconjugate. In some embodiments, this reactive functional group is a thiopropionate group.

[0163] In some embodiments, a non-natural cysteine residue is engineered into the framework of the antibody as a site for thiol-directed conjugation to obtain the immunoconjugate. In some embodiments, other non-natural amino acids or amino acid sequences are engineered into the framework to serve as attachment sites for the chelator-linker or a second reactive group, to which the chelator-linker will conjugate to obtain the immunoconjugate.

[0164] In some embodiments, non-natural amino acids containing crosslinking groups are engineered into the framework for attaching chelator-linkers. In some embodiments, such non-natural amino acids contain azides.

[0165] In some embodiments, the chelator-linker is attached to a glutamine residue through the action of transglutaminase. In other embodiments, a second reactive group is attached through transglutaminase to add the chelator-linker to the transglutaminase to obtain an immunoconjugate.

[0166] In some embodiments, one or more N-glycans are modified with a reactive functional group through the action of a glycosidase, and then the chelator-linker is conjugated to the site to attach the chelator-linker. In some embodiments, the glycan is modified through the action of β-galactosidase. In some embodiments, the glycan is modified with a glycoside containing an azide for attaching a suitably functionalized chelator-linker.

[0167] In some embodiments, the immunoconjugate comprises more than one chelator, which are the same or different.

[0168] In some embodiments, an immunoconjugate having more than one chelator has more than one chelator attached to the same antigen-binding arm.

[0169] In some embodiments, an immunoconjugate having more than one chelator and fewer than eleven chelators has more than two chelators, more than three chelators, more than four chelators, more than five chelators, more than six chelators, more than seven chelators, more than eight chelators, or more than nine chelators. In some embodiments, the chelators are the same. In some embodiments, each antigen-binding arm is directly or indirectly linked to more than one chelator.

[0170] In some embodiments, the chelator comprises a radioactive isotope chelating component and a functional group that allows covalent linkage to the antigen-binding arm. In some embodiments, the functional group is directly attached to the radioactive isotope chelating component. In some embodiments, the chelator further comprises a linker between the functional group and the radioactive isotope chelating component.

[0171] In some embodiments, the radioactive isotope chelating component comprises DOTA or a DOTA derivative. In some embodiments, the radioactive isotope chelating component comprises DOTAGA. In some embodiments, the radioactive isotope chelating component comprises macropa or a macropa derivative. In some embodiments, the radioactive isotope chelating component comprises Py4Pa or a Py4Pa derivative.

[0172] In a preferred embodiment, the chelator of the immunoconjugate is not attached to the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0173] In some embodiments, the chelator of the immunoconjugate is non-covalently associated with the antigen-binding arm. In a preferred embodiment, the chelating moiety is not associated with the antigen-binding region in the antigen-binding arm of the immunoconjugate.

[0174] 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.

[0175] In certain embodiments, an immunoconjugate is described herein that is conjugated to a chelator. In certain embodiments, the chelator is a radioisotope chelator. In certain embodiments, the radioisotope chelator is selected from: 1,4,7,10-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 chelator is DOTA. In certain embodiments, the radioisotope chelator is DOTAGA. In certain embodiments, the radioisotope chelator is Py4Pa. In certain embodiments, the radioisotope, wherein the radioisotope chelator is directly conjugated to the antigen-binding region and / or the immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region or the immunoglobulin heavy chain constant region via a linker. In certain embodiments, the linker is selected from: 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzoxycarbonyl (PAB), and those obtained by conjugation with the following linker reagents: 4-(2-pyridylthio)valeric acid N-succinimidyl ester (SPP) that forms the linker moiety 4-mercaptovaleric acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), 4-(2-pyridyldithio)butyric acid N-succinimidyl ester (SPDB), (4-iodo-acetyl)aminobenzoic acid N-succinimidyl ester (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-chelate factor selected from: TFP-Ad-PEG5-DOTAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.

[0176] Chelating factors can be conjugated to the protein or antigen-binding region and / or immunoglobulin heavy chain constant region at a certain ratio. In certain embodiments, the radioisotope chelator is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 1:1 to 8:1. In certain embodiments, the radioisotope chelator 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 chelator is conjugated to the antigen-binding region and / or immunoglobulin heavy chain constant region at a ratio of 2:1 to 6:1.

[0177] In some embodiments, the immunoconjugates of the present invention comprise a linker to, for example, conjugate the antigen-binding arm to a chelator (interchangeably, "chelating factor") or a radioisotope or a cargo (e.g., a cytotoxin). The linker can comprise one or more linker components. In some embodiments, the immunoconjugates of the present invention are engineered to have a terminal lysine available for conjugation to a chelator or a linker.

[0178] For example, bifunctional chelating factors are used to conjugate radioisotopes to the radioisotope delivery platform of the present invention, resulting in the immunoconjugates of the present invention. (See, for example, Scheinberg D, McDevitt M, Curr Radiopharm 4:306-20 (2011)). Examples of bifunctional chelating factors 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.

[0179] Examples of bifunctional chelating factors are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), and related analogs thereof. Such chelating factors are suitable for coordinating metal ions, such as α- and β-emitting radionuclides.

[0180] In some embodiments, the chelating agent of the immunoconjugate or radioimmunoconjugate of the present invention is selected from bifunctional chelating agents, 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., Int. J. Rad. Oncol. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelating agents, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0181] In some embodiments, the chelating agent of the immunoconjugate or radioimmunoconjugate of the present invention is selected from: bifunctional chelating agents, 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., Int. J. Rad. Oncol. Biol. Phys. 105:410 (2019)) (for reviews of these and other bifunctional chelating agents, see, for example, Price EW and Orvig C Chem. Soc. Rev., 2014, 43:260 (2014) and Brechbiel MW Q. J. Nucl. Med. Mol. Imaging 52:166 (2008)).

[0182] For 225-Ac immunoconjugates, there are a variety of acyclic and cyclic ligands known in the art as suitable chelating factors (see, for example, 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)).

[0183] In certain embodiments, the chelating factor is a chelating factor suitable for chelating α-emitters. Some chelating factors for α-emitting species are described in Yang et al., “Harnessing α-Emitting Radionuclides for Therapy: Radiolabeling Method Review.” J Nucl Med. 2022 Jan;63(1):5-13.

[0184] In certain embodiments, chelating factors suitable for α-emitter chelation are selected from: DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DO3A, 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane; 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(aza-3-yl))tetrakis(methylene))tetrapicolinic acid; Py4Pa-NCS, 6,6'-((((4-isothiocyanatopyridine-2,6-diyl)bis(methylene))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; Crown, 2,2',2”,2”'-(1,10-dioxo-4,7,13,16-tetraazacyclooctadecane-4,7,13,16-tetrayl)tetraacetic acid; Macropa, 6,6'-((1,4,10,13-tetraoxo-7,16-diaza-cyclooctadecane-7,16-diyl)bis(methylene))dipicolinic acid; Macropa-NCS, 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxo-7,16-diazacyclooctadecane-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[[(carboxymethyl)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; Nonunpa, 6,6'-(((oxy-bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid; and combinations thereof.

[0185] In certain embodiments, the chelating agent is a chelating agent suitable for chelating β or γ emitters. In certain embodiments, the chelating agent suitable for chelating β or γ emitters is selected from: 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-tetra(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-tetra(acetamidomethylenephosphonic 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'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane; TTHA, triethylenetetramine-N,N,N',N”,N”',N”'-hexaacetic acid; DO2P, tetraazacyclododecane dimethanephosphonic acid; HP-DO3A, hydroxypropyltetraazacyclododecane triacetic acid; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; DTPA-BMA, diethylenetriaminepentaacetic acid-bisdimethylamide; HOPO, octadentate hydroxypyridone; 3,2,3-LI(HOPO), N,N'-(butane-1,4-diyl)bis(1-hydroxy-N-(3-(1-hydroxy-6-oxo-1,6-dihydropyridin-

[0186] (2-formamidopropyl)-6-oxo-1,6-dihydropyridine-2-carboxamide); 3,2-HOPO, N,N'

[0187] -(((2-(4-aminobenzyl)-3-((2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)(2-(3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)propyl)aza-diyl)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)azanediyl))bis(methylene))dipicolinic acid; porphyrin, 21,22,23,24-tetraazapentacyclo[16.2.1.13,6.1.8,11.113,16]tetracosa-1,3,5,7,9,11(23),12,14,16,18(21),19-undecene; desferrioxamine, 30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaazatriacontane-2,10,13,21,24-pentone; DFO*, N1-[5-(acetylhydroxyamino)pentyl]-N26-(5-aminopentyl)-N26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetraazadecane-1,2-diamide; and combinations thereof.

[0188] Alternatively or additionally, an isothiocyanate linker such as sp-SCN-Bn-DOTA can be used, which contains lysine residues within the immunoconjugates of the present invention.

[0189] Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), and those obtained by conjugation with the following linker reagents: 4-(2-pyridylthio)valeric acid N-succinimidyl ester (“SPP”) which forms the linker moiety 4-mercaptovaleric acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-succinimidyl ester (“SMCC”, also referred to herein as “MCC”) which forms the linker moiety 4-((2,5-dioxopyrrolidin-1-yl)methyl)cyclohexanecarboxylic acid, 4-(pyridin-2-yldithio)butyric acid 2,5-dioxopyrrolidin-1-ester (“SPDB”) which forms the linker moiety 4-mercaptobutyric acid, (4-iodo-acetyl)aminobenzoic acid N-succinimidyl ester (“SIAB”), ethyleneoxy-CH2CH2O- (“EO”, “PEO” or “PEG”) in one or more repeating units. Additional linker components are known in the art and some are described herein. Various linker components are known in the art, some of which are described below.

[0190] In certain embodiments, the linker is SCN. In certain embodiments, the chelator is a linker-chelate factor selected from: 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 .

[0191] The linker can be a “cleavable linker” which facilitates the release of the drug in the cell. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used ((Chari et al., Cancer Research 52:127-31 (1992); U.S. Patent No. 5,208,020).

[0192] In certain embodiments, the linker is as shown in the following formula:

[0193] -A a -W w -Y y -

[0194] Wherein A is an extension sequence unit, and a is an integer from 0 to 1; W is an amino acid unit, and w is an integer from 0 to 12; Y is a spacer sequence unit, and y is 0, 1, or 2; and Ab, D, and p are as defined above. Exemplary embodiments of such linkers are described in US20050238649.

[0195] In some embodiments, the linker component may include an "extension sequence unit" that attaches the immunoconjugate to another linker component or drug moiety. Exemplary extension sequence units are shown below (where the wavy line indicates the site of covalent attachment to the immunoconjugate):

[0196]

[0197] In some embodiments, the linker may be conjugated to the antibody through a cysteine bridging functional group such as or DBM (dibromomaleimide). These linkers can re-stabilize the intra-chain disulfide bonds 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 re-bridging extension sequence elements are shown below (where the wavy line indicates the site of covalent attachment to the immunoconjugate):

[0198]

[0199] In some embodiments, the linker component can comprise amino acid units. In one such embodiment, the amino acid units allow the linker to be cleaved by a protease, thereby facilitating the release of the drug from the immunoconjugate upon exposure to intracellular proteases such as lysosomal enzymes (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 comprise naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. The amino acid units can be designed and optimized for selective enzymatic cleavage by a specific enzyme (e.g., a tumor-associated protease, cathepsin B, C, and D, or a plasmin protease).

[0200] In some embodiments, the linker component can comprise a "spacer sequence" unit that directly or through an extension sequence unit and / or an amino acid unit links the immunoconjugate to the drug moiety. The spacer sequence unit can be "self-immolative" or "non-self-immolative". A "non-self-immolative" spacer sequence unit is a spacer sequence unit in which some or all of the spacer sequence unit remains bound to the drug moiety upon enzymatic (e.g., proteolytic) cleavage of the ADC. Examples of non-self-immolative spacer sequence units include, but are not limited to, glycine spacer sequence units and glycine-glycine spacer sequence units. Other combinations of peptide spacers sensitive to sequence-specific enzymatic cleavage are also contemplated. For example, enzymatic cleavage of an ADC containing a glycine-glycine spacer sequence unit by a tumor cell-associated protease will result in the release of glycine-glycine-drug moiety from the remainder of the ADC. In one such embodiment, the glycine-glycine-drug moiety then undergoes a separate hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer sequence unit from the drug moiety.

[0201] "Self-immolative" spacer units allow 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 formed 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 moiety 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 0-4. Examples of self-immolative spacer units also include, but are not limited to, aromatic compounds electronically similar to p-aminobenzyl alcohol (see, e.g., US2005 / 0256030A1), 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-aminobutyramide (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-aminophenylpropionamide (Amsberry et al., J. Org. Chem., 1990, 55:5867). Elimination of amine-containing drugs substituted at the a-position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27:1447) is also an example of a self-immolative spacer that can be used for ADCs.

[0202] In some embodiments, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as depicted below, which can be used to incorporate and release a variety of drugs.

[0203]

[0204] where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p ranges from 1 to about 20.

[0205] In some embodiments, the immunoconjugate comprises a linker, such as a dendritic linker for covalently linking more than one drug moiety to an antibody via a branched, multi-functional linker moiety (Sun et al., (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-5; Sun et al., (2003) Bioorganic & Medicinal Chemistry 11:1761-8). The dendritic linker can increase the molar ratio of drug to antibody, i.e., the payload, which is related to the potency of the ADC. Thus, in the case where a cysteine-engineered antibody bears only one reactive cysteine thiol group, multiple drug moieties can be attached via the dendritic linker.

[0206] Examples of linker components and their combinations are shown below, which are also suitable for use in the above formula:

[0207] Val-Cit or VC

[0208]

[0209] Additional non-limiting examples of linkers include those described in WO 2015095953.

[0210] Linker components, including the extension sequence, spacer sequence, and amino acid units, can be synthesized by methods known in the art, such as those described in US20050238649.

[0211] 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 application or international application claiming priority thereto, which applications are hereby incorporated by reference for 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 of U.S. Application No. 63 / 373,189, filed August 22, 2022, which application is hereby incorporated by reference for such compounds.

[0212] In some embodiments, the chelator comprises a linker and is selected from one of the compounds described in U.S. Application No. 63 / 373,183, filed Aug. 22, 2022, or a U.S. non-provisional application or international application claiming priority therefrom, which applications are hereby incorporated by reference for 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 of U.S. Application No. 63 / 373,183, filed Aug. 22, 2022, which application is hereby incorporated by reference for such compounds.

[0213] 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 Aug. 22, 2022, or a U.S. non-provisional application claiming priority therefrom, which applications are hereby incorporated by reference for 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 of U.S. Application No. 63 / 373,190, filed Aug. 22, 2022, which application is hereby incorporated by reference in its entirety for such compounds.

[0214] Radioimmunoconjugate

[0215] "Radionuclide" or "radioisotope" means and encompasses an alpha-emitting isotope (interchangeably, α-emitting isotope), a beta-emitting isotope (interchangeably, β-emitting isotope), and / or a gamma-emitting isotope (interchangeably, γ-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.

[0216] "Radioimmunoconjugate" (also interchangeably used with "immunoconjugate" when used for 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 radioisotope. In some embodiments, the radioisotope is a β-emitting radioisotope. In some embodiments, the radioisotope is a γ-emitting isotope. In some embodiments, the present invention provides a radioimmunoconjugate comprising an α-emitting and a β-emitting radioisotope. The term "radioactive conjugate" is also interchangeably used herein with the term "radioimmunoconjugate". In some embodiments, the radioisotope is associated with a chelator of the radioimmunoconjugate. In some embodiments, the radioisotope is directly linked to the immunoconjugate.

[0217] In some embodiments, the present invention provides an immunoconjugate. In some embodiments, when so labeled, linked or loaded with an α-emitter, the immunoconjugate is capable of delivering the α-emitter in vivo. In some embodiments, when so labeled, linked or loaded, the immunoconjugate is also capable of delivering other radioisotopes (β-emitters and / or γ-emitters) and / or other atoms in vivo. In some embodiments, when so labeled, linked or loaded, the immunoconjugate is capable of delivering imaging metals (e.g., 111-In, 89-Zr, 64-Cu, 68-Ga or 134-Ce) in vivo.

[0218] The immunoconjugates of the present disclosure can be loaded with radioisotopes to achieve therapeutic or diagnostic effects. In certain embodiments, the chelating moiety can also comprise a radioisotope. In certain embodiments, the radioisotope is an α-emitter. In certain embodiments, the radioisotope is an α-emitter selected from: 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.

[0219] The present disclosure also describes a method for preparing a radioimmunoconjugate, the method comprising loading or conjugating an immunoconjugate 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: 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.

[0220] In some embodiments, the present invention provides a radioimmunoconjugate comprising an immunoconjugate of the present invention and an α-emitting radioisotope. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is selected from: 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is selected from: 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 225-Ac. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 223-Ra. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 224-Ra. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 227-Th. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 212-Pb. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 212-Bi. In some embodiments, the α-emitting radioisotope of the radioimmunoconjugate is 213-Bi.

[0221] In some embodiments, the immunoconjugates of the invention are combined with a radioisotope to provide the radioimmunoconjugates of the 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 α-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 β-particle emitter, such as, for example, 177-Lu, 90-Y, 67-Cu, 153-Sm. In some embodiments, the radioisotope is both an α-particle emitter and a β- and / or γ-particle emitter. In some embodiments, the radioisotope is both a β-particle emitter and a γ-particle and / or photon emitter. In some embodiments, the radioimmunoconjugate is labeled, linked or loaded with an α-emitter and a β-emitter and thus contains them. In some embodiments, for example, the radioisotope is selected from among 68-Ga, 64-Cu, 89-Zr, 111-In, 134-Ce for use in radioimaging.

[0222] The immunoconjugates and radioimmunoconjugates of the present invention may comprise other cargo or payloads in addition to the radioisotope, including various cytotoxic agents such as, for example, small molecule chemotherapeutic agents, cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, and / or tubulin inhibitors. For example, the immunoconjugates of the present invention can be used to deliver non-radioisotope cytotoxins to target cells. Non-limiting examples of cytotoxic agents include aziridines, cisplatins, tetrazines, procarbazine, hexamethyl-lmelamine, vinca alkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin, dolastatins, maytansines, docetaxel, adriamycin, calicheamicin, auristatin, pyrrolo-benzodiazepine, carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin, cisplatin, methotrexate, and gemcitabine.

[0223] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from: 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.

[0224] In some embodiments, the radioimmunoconjugates of the present invention comprise a radioisotope selected from: 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.

[0225] In some embodiments, the radioisotope is an alpha-emitting radioisotope, which comprises 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, or 213-Bi.

[0226] In some embodiments, the radioisotope is an alpha-emitting radioisotope selected from: 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

[0227] Immunoconjugate derivatives and other modifications

[0228] Covalent modifications of the immunoconjugates of the present invention are included within the scope of the present invention. One type of covalent modification involves reacting a target amino acid residue of the immunoconjugates of the present invention with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of the immunoconjugate. Derivatization using bifunctional agents can be used, for example, to crosslink the immunoconjugates of the present invention to a water-insoluble support matrix or surface for use in methods for purifying the immunoconjugates of the present invention, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane; glutaraldehyde; N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid; homobifunctional imidates, including disuccinimide esters, such as 3,3'-dithiobis(succinimidyl propionate); bismaleimides, such as bis-N-maleimido-1,8-octane; and agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate.

[0229] Other modifications include: deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino groups of the side chains of lysine, arginine, and histidine (T.E. Creighton, 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.

[0230] In some embodiments, the immunoconjugates provided herein can be further modified to contain additional non-proteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of immunoconjugates include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacture due to its stability in water. The polymer 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. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the immunoconjugate to be enhanced, whether the immunoconjugate derivative will be used in therapy under defined conditions, etc.

[0231] The PEG-derivatized immunoconjugates of the invention can contain a linker comprising one or more -CH 2 CH 2 O- and can be used to alter the biodistribution and pharmacokinetics of the immunoconjugate. PEG can be prepared in polymeric form or as discrete oligomers. The bifunctionalized forms of these polymers can link the immunoconjugate to a chelator and / or provide additional size and / or solubility to the overall molecule. In some embodiments, the PEG-derivatized immunoconjugates exhibit reduced immunogenicity compared to their underivatized parent molecules.

[0232] Methods of producing immunoconjugates

[0233] The present invention provides a composition comprising one or more of the immunoconjugates as in any of the above embodiments or as described herein. In another embodiment, the present invention provides an isolated nucleic acid encoding a radioisotope delivery platform as described herein. Also provided herein are nucleic acids encoding the protein components of the immunoconjugates of the present invention, expression vectors comprising the foregoing nucleic acids, and host cells comprising the foregoing expression vectors.

[0234] In another embodiment, the present invention provides a host cell comprising the nucleic acid and / or vector as provided herein. In some embodiments, the host cells of the present invention are isolated or purified. In some embodiments, the host cells of the present invention are in cell culture medium. The nucleic acids, expression vectors, and host cells of the present invention can be used to produce a composition comprising one or more of the immunoconjugates of the present invention. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is Escherichia coli.

[0235] Illustrative techniques for generating the immunoconjugates and radioimmunoconjugates of the present invention for use in the methods according to the present invention are described below. In some embodiments, the present invention provides a method for preparing an immunoconjugate of the present invention, the method comprising culturing a host cell as 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 (such as, for example, an α or β particle emitter).

[0236] Generation and identification of antigen-binding domains, immunoconjugates, and nucleic acids

[0237] Antigen-binding domains that can be used herein as antigen-binding regions can be identified in antibodies, which are monoclonal antibodies and / or polyclonal antibodies. The DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures. Once isolated, the DNA can be placed into an expression vector and then transfected into a host cell (such as Escherichia 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 the monoclonal antibody in the recombinant host cell (see, for example, Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol Revs. 130:151-188 (1992)).

[0238] In some embodiments, the antigen-binding domain or a fragment thereof of the immunoconjugates of the present invention is isolated by screening a phage library containing phages displaying various antibody variable region fragments (Fv, scFv or VHH) fused to a phage coat protein. Such phage libraries are screened for binding to the desired target antigen or epitope. Clones expressing an Fv fragment, scFv or VHH capable of binding the desired antigen are adsorbed to the antigen and are thus separated from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0239] In some embodiments, the antibody or antibody fragment thereof is isolated from an antibody phage library 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 using phage libraries, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc Acids Res. 21:2265-2266 (1993)). The variable domains can be functionally displayed on the phage as single-chain Fv (scFv) fragments (in which VH and VL are covalently linked by a short flexible peptide) or as Fab fragments (in which they are each fused to a constant domain and interact non-covalently), as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994).

[0240] VH and VL gene libraries can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then their antigen-binding clones can be searched as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The initial library for screening can be constructed from non-immunized sources to provide high-affinity antibodies against antigens (see, for example, Griffiths et al., EMBO J, 12:725-734 (1993)). Another example is an initial library constructed synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions and performing rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0241] Screening of the library can be accomplished by various techniques known in the art. For example, the target antigen can be used to coat the wells of an adsorption plate, expressed on host cells attached to the adsorption plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning a display library. Selection of antibodies with slow dissociation kinetics (and strong binding affinity) can be facilitated by using long washes and monovalent phage display (as described by Bass et al., Proteins, 8:309-314 (1990) and WO 1992 / 09690) and low antigen coating density (as described by Marks et al., Biotechnol., 10:779-783 (1992)).

[0242] Techniques for screening cDNA libraries are well known in the art. The library can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify genes or proteins encoded thereby. Standard procedures (such as those described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) can be used to screen cDNA or genomic libraries with the selected probes. An alternative means of isolating genes encoding the immunoconjugates of the present invention is to use PCR methods (Sambrook et al. (as above); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0243] DNA encoding the immunoconjugates of the present invention can be obtained from a cDNA library prepared from tissues that are believed to have the mRNA for the immunoconjugates of the present invention and express it at detectable levels. Thus, the human immunoconjugate DNA of the present invention can be conveniently obtained from a cDNA library prepared from human tissues. The gene encoding the immunoconjugates of the present invention can also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). For some embodiments, the desired polynucleotide sequences encoding the antibodies can be isolated from antibody-producing cells such as hybridoma cells and sequenced.

[0244] 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 (at the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein. Any of the antibody CDRs or heavy chain variable fragments of the present invention can be obtained by designing antigen screening procedures suitable for selecting phage clones of interest and then constructing antibody clones using the variable domain and / or CDR sequences from the phage clones of interest and the appropriate constant region (Fc) sequences as described in Kabat et al., 1991 (as above).

[0245] Production of immunoconjugates; host cells and expression vectors of the present invention

[0246] The following description mainly relates to the production of the antibody constructs of the present invention by culturing cells transformed or transfected with a vector containing nucleic acid encoding the immunoconjugates of the present invention. Of course, alternative methods well known in the art are envisioned for preparing the antibody constructs of the present invention. For example, appropriate amino acid sequences or portions thereof can be produced 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 can be performed using manual techniques or by automation. For example, an Applied Biosystems peptide synthesizer (Foster City, CA) can be used to complete automated synthesis using the manufacturer's instructions. The individual parts of the immunoconjugates of the present invention can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired immunoconjugates of the present invention.

[0247] Antibody constructs can be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. In some embodiments, an isolated nucleic acid encoding an antibody as described herein is provided. Such nucleic acids can encode an amino acid sequence comprising an antibody VH and / or an amino acid sequence comprising a VL (e.g., an antibody light chain and / or heavy chain). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In some embodiments, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In some other embodiments, the host cell comprises: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a nucleic acid encoding an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In some embodiments, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20 cells). In some embodiments, a method for preparing an immunoconjugate of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression and optionally recovering the antibody from the host cell (or the host cell culture medium).

[0248] For recombinant production of the immunoconjugates of the invention, a nucleic acid encoding, e.g., an antibody construct as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the antibody heavy and / or light chains). Nucleic acid molecules encoding the amino acid sequences (including sequence variants) of the immunoconjugates of the invention can be prepared by a variety of methods known to those of skill 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 earlier prepared variants or non-variant forms of the antibody constructs.

[0249] Engineering host cells for immunoconjugate production

[0250] The host cells are transfected or transformed with the expression or cloning vectors described herein to effect the production of the conjugates of the invention and are cultured in a conventional nutrient medium that has been appropriately modified to induce the promoter, select for transformants, or amplify the gene encoding the desired sequences. The culture conditions, such as the medium, temperature, pH, etc., can be selected by the skilled person without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing the productive capacity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al. (as above).

[0251] Host cells suitable for the cloning or expression of nucleic acids and vectors encoding immunoconjugates include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For antibody fragment and polypeptide expression in bacteria, see, e.g., US 5,648,237; US 5,789,199; US 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, 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 the form of a soluble fraction and can be further purified.

[0252] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding immunoconjugates, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to result in the production of antibodies with a partial or fully human glycosylation pattern (see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006)).

[0253] Host cells suitable 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. A number of baculovirus strains have been identified that are suitable for use in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells. 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).

[0254] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV 1 line transformed by SV40 (COS-7); human embryonic kidney line, 293 or 293 cells, as described, e.g., by Graham et al., J Gen Viral. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells, TM4 cells, as described, e.g., by 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 hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep02); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include 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 certain mammalian host cell lines suitable for immunoconjugate production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0255] Methods for eukaryotic cell transfection and prokaryotic cell transformation are known to those skilled in the art, which refer to introducing DNA into a host in an extrachromosomal form or through chromosomal integrants such that the DNA is replicable, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques suitable for such cells. Calcium treatment or electroporation using calcium chloride as described by Sambrook et al. (as above) is commonly used for prokaryotes. Agrobacterium tumefaciens infection is used to transform certain plant cells as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859 published on June 29, 1989. For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of transfection of mammalian cell host systems have been described in U.S. Patent No. 4,399,216. Transformation into yeast is typically carried out 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 can also be used, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations, such as polybrene, polyornithine. 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).

[0256] Prokaryotic host cell

[0257] Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae, such as Escherichia coli. Various strains of E. coli are publicly available, such as strain K12 MM294 (ATCC 31,446); X1776 (ATCC 31,537); W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens) and Shigella, as well as Bacillus (such as B. subtilis and B. licheniformis) (e.g., B. licheniformis 41P as disclosed in DD 266,710 published on April 12, 1989), Pseudomonas (such as P. aeruginosa), Rhizobia, Vitreoscilla, Paracoccus and Streptomyces. These examples are illustrative and not restrictive. E. coli strain W3110 is a preferred host or parental host because it is a common host strain for the fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes.For example, the strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) can be modified to effect a gene mutation of a gene encoding a protein endogenous to a host, examples of such hosts including: Escherichia coli strain W3110 1A2, which has the complete genotype tonA; Escherichia coli strain W3110 9E4, which has the complete genotype tonA ptr3; Escherichia coli strain W3110 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kanr; Escherichia coli strain W3110 37D6, which has the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT rbs7 ilvG kanr; Escherichia coli strain W3110 40B4, which is strain 37D6 having a non-kanamycin resistant degP deletion mutation; Escherichia coli strain W3110 33D3, which has the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Patent No. 5,639,635); and an Escherichia coli strain having a mutant periplasmic protease as disclosed in U.S. Patent No. 4,946,783 issued August 7, 1990. Other strains and their derivatives, such as Escherichia coli 294 (ATCC 31,446), Escherichia coli B, Escherichia coli λ1776 (ATCC 31,537), and Escherichia coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not restrictive. Methods for constructing derivatives of any of the above-described bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to consider the replicability of the replicon in the bacterial cell when selecting an appropriate bacterium. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to provide the replicon, Escherichia coli, Serratia, or Salmonella species can be suitable as hosts. Generally, the host cell should secrete a minimal amount of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0258] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, particularly when 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 the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. 5,648,237, U.S. 5,789,199, and U.S. 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, depending on the isotype, by, for example, protein A or G columns. Final purification can be carried out similarly to the process for purifying antibodies expressed, for example, in CHO cells.

[0259] Eukaryotic host cells

[0260] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for the vectors encoding the immunoconjugates of the present invention. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include: Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981); EP 139,383 published on May 2, 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology, 9:968-75 (1991)), such as, for example, Kluyveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc Natl Acad Sci USA 76:5259-5263 (1979)); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538 published on October 31, 1990); and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published on January 10, 1991) and Aspergillus hosts, such as Aspergillus 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 Aspergillus niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotrophic yeast is suitable herein and includes, but is not limited to, yeast capable of growing on methanol, which is selected from Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. A list of specific species that are examples of such yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0261] Suitable host cells for expressing the 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 cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. A number of baculovirus strains and variants, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0262] However, there is the greatest interest in vertebrate cells, and the proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); 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 CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

[0263] The host cells are transformed with the expression or cloning vectors described above to effect production of the immunoconjugates of the invention and are cultured in a conventional nutrient medium modified as appropriate to induce the promoter, select for transformants or amplify the gene encoding the desired sequences.

[0264] Selection and Use of Replicable Vectors

[0265] For recombinant production of the radioisotope delivery platform of the invention, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. The DNA encoding the immunoconjugate can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody). There are many vectors available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of prokaryotic or eukaryotic (usually mammalian) origin.

[0266] The vector can be in the form of, for example, a plasmid, cosmid, viral particle, or phage. Appropriate nucleic acid sequences can be inserted into the vector by a number of procedures. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0267] The immunoconjugates of the present invention can be produced not only by direct recombination but also as a fusion polypeptide with a heterologous polypeptide, which can be a signal sequence or other polypeptide having a specific cleavage site 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 part of the DNA encoding the immunoconjugate of the present invention inserted into the vector. The signal sequence can be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or the heat-stable enterotoxin II leader sequence. For yeast secretion, the signal sequence can be, for example, the yeast invertase leader sequence, the α-factor leader sequence (including Saccharomyces and Kluyveromyces α-factor leader sequences, the latter being described in U.S. Patent No. 5,010,182), or the acid phosphatase leader sequence, the Candida albicans glucoamylase leader sequence (EP 362,179 published on April 4, 1990), or the signal described in WO 90 / 13646 published on November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct the secretion of proteins, such as signal sequences of secreted polypeptides from the same or related species and viral secretion leader sequences.

[0268] Culturing host cells for producing the radioisotope delivery platform

[0269] Host cells for producing the immunoconjugates of the present invention can be cultured in a variety of media and culture conditions.

[0270] Prokaryotic host cell cultures

[0271] Prokaryotic cells for producing 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) plus necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing the ampicillin resistance gene.

[0272] In addition to a carbon, nitrogen, and inorganic phosphate source, any necessary supplements can be included at appropriate concentrations, introduced either singly or as a mixture with another supplement or a medium such as a complex nitrogen source. Optionally, the medium can contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cysteamine, thioglycolate, dithiothreitol, and dithioerythritol.

[0273] Prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, the preferred temperature range is 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 from about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.

[0274] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter. In some embodiments of the present invention, the PhoA promoter is used to control transcription of the polypeptide. Thus, the transformed host cells are cultured in a phosphate-limited medium for induction. In some embodiments, the phosphate-limited medium is C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-47). Depending on the vector construct employed, a variety of other inducers can be used, as are known in the art.

[0275] In some embodiments, the polypeptides expressed in the present invention are secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves disruption of the microorganism, typically 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 medium and isolated therein. The cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of the resulting protein. Commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays can be used to further separate and identify the expressed polypeptides.

[0276] In some embodiments of the present invention, immunoconjugates are produced in large quantities by a fermentation method. Different large-scale fed-batch fermentation procedures can be used for the production of recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, preferably a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in a fermenter with a volume capacity of no more than about 100 liters, and the range can be from about 1 liter to about 100 liters.

[0277] In the fermentation process, induction of protein expression is typically initiated after the cells have grown to the desired density under suitable conditions (e.g., an OD550 of about 180 - 220) (at this stage the cells are in the early stationary phase). Depending on the vector construct employed, a variety of inducers can be used, as are known in the art and described above. The cells can be allowed to grow for a short period before induction. The cells are typically induced for about 12 - 50 hours, but longer or shorter induction times can be used.

[0278] To increase the yield and quality of the polypeptides of the present invention, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of the secreted immunoconjugate polypeptides, host prokaryotic cells can be co-transformed with additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl-prolyl cis,trans isomerase with chaperone activity). It has been demonstrated that chaperone proteins facilitate the correct 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.

[0279] To minimize proteolysis of heterologous proteins expressed (especially those sensitive to proteolysis), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to achieve one or more gene mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some Escherichia coli protease-deficient strains are available and described, for example, in Joly et al., (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0280] In some embodiments, an Escherichia coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as the host cell in the expression system of the present invention.

[0281] Eukaryotic host cell cultures

[0282] 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 host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent No. 30,985 can be used as the medium for host cells. Any of these media can be supplemented, as needed, with 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 GENTAMYCINTM drug), trace elements (defined as inorganic compounds that are usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements at appropriate concentrations known to those skilled in the art can also be included. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression and are obvious to those of ordinary skill in the art.

[0283] Purification of Immunoglobulin-Derived Structures of the Invention

[0284] The immunoconjugates of the invention can be recovered from the culture medium or from host cell lysates. If membrane-bound, they can be detached from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used for expressing the immunoconjugates of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0285] It may be necessary to purify the immunoconjugates of the invention from recombinant cell proteins or polypeptides. The following procedures are examples of suitable purification procedures: fractionation on an ion-exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on silica or cation-exchange resins such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A agarose columns to remove contaminants such as IgG; and metal chelate columns to bind the immunoconjugates of the invention in epitope-tagged form. A variety of protein purification methods can be employed, and such methods are known in the art and 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 method used and the particular immunoconjugate of the invention produced.

[0286] When recombinant techniques are used, the immunoconjugates can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the immunoconjugates are produced intracellularly, as a first step, particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describe procedures 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 approximately 30 min. Cell debris can be removed by centrifugation. When the immunoconjugates are secreted into the culture medium, the supernatant from such expression systems is usually first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors (such as PMSF) can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0287] Immunoconjugate compositions prepared from cells can be purified using, for example, hydroxyapatite 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 type 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 murine isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl) benzene can achieve faster flow rates and shorter processing times compared to what is achieved using agarose. When the immunoconjugate contains a CH3 domain, Bakerbond ABXTM resin (J.T. Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques can also be used depending on the immunoconjugate to be recovered, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSETM chromatography, anion or cation exchange resin (such as polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0288] After any preliminary purification step, low pH hydrophobic interaction chromatography can be performed on a mixture containing the immunoconjugate of interest and contaminants using an elution buffer with a pH between about 2.5 - 4.5 and typically at a low salt concentration (e.g., about 0 - 0.25 M salt).

[0289] Immunoconjugates (including antibody-drug conjugates (ADCs))

[0290] In another embodiment of the invention, an immunoconjugate according to any one of the above embodiments or of the invention as described herein is conjugated to a heterologous moiety or agent, such as, for example, those described below and including any additional exogenous materials as described herein.

[0291] In some embodiments, the invention provides an immunoconjugate comprising a multivalent antibody construct of the invention conjugated to one or more therapeutic agents or radioisotopes.

[0292] In some embodiments, the immunoconjugate comprises a multivalent antibody construct as described herein conjugated to a radioactive atom to form a radio-conjugate. As described herein, a variety of radioisotopes can be used to produce the radio-conjugates of the invention.

[0293] Conjugates of immunoconjugates or antibody constructs can be prepared 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 hexamethylenediamine dimethylester H), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazotized compounds (such as bis(p-azidobenzoyl)hexanediamine), diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies (see, for example, WO 1994 / 11026). The linker can be a "cleavable linker" that facilitates the release of cytotoxic drugs in cells. For example, acid-labile linkers, protease-sensitive linkers, photo-labile linkers, dimethyl linkers or disulfide-containing linkers can be used (see, for example, Chari et al., Cancer Res. 52:127-131 (1992); US 5,208,020).

[0294] The immunoconjugates or ADCs herein specifically contemplate but are not limited to such conjugates prepared with crosslinking agents, 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 SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., available from Pierce Biotechnology, Inc., Rockford, IL., U.S.).

[0295] As will be appreciated by those of ordinary skill in the art, some of the methods above can also be used to prepare radiolabeled immunoconjugates and targeted imaging complexes (although the text only mentions immunoconjugates or antibody constructs), and such preparation methods are also encompassed within the present invention.

[0296] Immunoconjugation using chelating factors and / or linkers

[0297] 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 WO 2017 / 155937.

[0298] Bifunctional chelating agents such as, for example, DOTA, DTPA, and related analogs are suitable for coordinating metal ions such as α and β-emitting radionuclides. For example, these chelating molecules can be linked to the targeting molecule by forming a new amide bond between an amine on the antibody construct (e.g., the functional group of a lysine residue) and a carboxylate ester on DOTA / DTPA. In the case of peptide synthesis, the characterization and purification of linker addition can be part of the overall synthesis of the multivalent antibody platform or immunoconjugate for radioisotope conjugation.

[0299] For some embodiments, the method of producing an immunoconjugate involves a click chemistry step as described by Poty, S et al., Chem Commun. (Camb) 54:2599 (2018).

[0300] For some embodiments, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors (involving, for example, replacing hydrogen with fluorine 19). In some embodiments, a radioactive label can be incorporated into the peptide. In some embodiments, a radioactive label can be linked to the peptide. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al., (1978) Biochem Biophys Res Commun. 80:49-57). Other methods are described in detail in “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989).

[0301] Pharmaceutical Compositions

[0302] Compositions are provided herein that comprise an immunoconjugate or radioimmunoconjugate as described herein. The invention also provides pharmaceutical compositions and formulations that comprise at least one immunoconjugate of the invention and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the invention and (2) a pharmaceutically acceptable carrier.

[0303] An immunoconjugate or radioimmunoconjugate is formulated in any suitable form for delivery to a target cell / tissue. Pharmaceutical formulations of the immunoconjugates of the invention are prepared in the form of lyophilized preparations or aqueous solutions by mixing such immunoconjugates having the desired purity with one or more optional pharmaceutically acceptable carriers, diluents, and / or excipients (Remington's Pharmaceutical Sciences, 16th edition, ed. A. Osol (1980)). Pharmaceutically acceptable carriers, diluents, and excipients are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to: sterile water; buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as cetrimonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0304] Drug formulations to be used for in vivo administration are usually sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0305] 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 of which contain a histidine-acetate buffer.

[0306] The pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20( Baxter International, Inc.). In some embodiments, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0307] The formulations of the present invention may also contain, if desired, more than one active ingredient for the particular indication being treated, preferably those having complementary activities which do not adversely affect each other. Such active ingredients are present in a suitable combination in an amount effective for the intended purpose.

[0308] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, Ed. (1980).

[0309] In some embodiments, the immunoconjugate can be formulated as an immunoliposome. An "immunoliposome" is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants that can be used to deliver a drug to a mammal. The components of a liposome are generally arranged in a bilayer, similar to the lipid arrangement of a biological membrane. Immunoliposomes containing an immunoconjugate 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. Patent Nos. 4,485,045 and 4,544,545; and WO 1997 / 38731 published on October 23, 1997. Particularly suitable immunoliposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising lecithin, cholesterol, and a PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters having a defined pore size to produce liposomes of the desired diameter. The chemotherapeutic agent is optionally included within the liposome (see Gabizon et al., J. National Cancer Inst. 81:1484 (1989)). Liposomes with increased circulation times are disclosed in U.S. Patent No. 5,013,556.

[0310] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, membranes or microcapsules.

[0311] Methods of Use

[0312] The immunoconjugates (e.g., radioimmunoconjugates) described herein can be used to treat a disease, disorder or condition (e.g., a tumor or cancer) in a patient in need thereof, the method comprising administering an immunoconjugate or radioimmunoconjugate or composition described herein. The immunoconjugates can also be used to kill tumor or cancer cells (e.g., solid tumor cells expressing FOLR1 or DLL3). In addition, the immunoconjugates described herein can be used in methods for labeling or detecting tumor cells or cancer cells.

[0313] In some embodiments, provided herein are methods of killing tumor cells or cancer cells, the method comprising: contacting the tumor cells or cancer cells with a radioimmunoconjugate described herein (e.g., an immunoconjugate comprising a radionuclide), thereby killing the tumor cells or cancer cells. In some embodiments, the tumor cells are solid tumor cells. In certain embodiments, the tumor cells and / or cancer cells express FOLR1 or DLL3. In certain embodiments, the tumor cells and / or cancer cells are in an individual.

[0314] In some embodiments, provided herein are methods of treating cancer or a tumor in an individual, the method comprising administering to the individual an immunoconjugate described herein (e.g., a radioimmunoconjugate), 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.

[0315] The pharmaceutical compositions of the invention can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0316] In some embodiments, the immunoconjugates, or radioimmunoconjugates, or compositions of the invention can be used in methods for binding to a target antigen in an individual suffering from a condition associated with increased expression and / or activity of the target antigen, the method comprising administering to the individual an immunoconjugate, or radioimmunoconjugate, or composition 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 invention can be administered to a human for therapeutic purposes. In addition, the immunoconjugates, or radioimmunoconjugates, or compositions of the invention can be administered to a non-human mammal (e.g., a primate, pig, rat or mouse) expressing a target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts, for veterinary purposes or as an animal model of a human disease. With respect to the latter, such animal models can be used to evaluate the therapeutic efficacy of the immunoconjugates or radioimmunoconjugates or compositions of the invention (e.g., testing the dosage and duration of administration).

[0317] The immunoconjugates or radioimmunoconjugates or compositions of the present invention (and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenterally, subcutaneously, intraperitoneally, intraluminally, and intranasally, and, in cases where local treatment is required, intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, antibodies (e.g., multivalent antibodies) are suitable for administration by pulse infusion, particularly in cases where the antibody (e.g., multivalent antibody) dose is tapered. Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term.

[0318] The immunoconjugates or radioimmunoconjugates or compositions of the present invention will be formulated, dosed, and administered in a manner that is in keeping with good medical practice. Factors to be considered 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 to which the agent is to be delivered, the method of administration, the scheduling of administration, and other factors known to the practicing physician. The immunoconjugates of the present invention are administered to human patients according to known methods, such as intravenous administration, for example as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intrathecal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. For some embodiments, intravenous or subcutaneous administration of the immunoconjugates or radioimmunoconjugates or compositions of the present invention is preferred.

[0319] For the prevention or treatment of a disease, the dosage and mode of administration will be selected by the physician according to known criteria. The appropriate dosage of the immunoconjugate or radioimmunoconjugate or composition of the present invention will depend on the type of disease to be treated (as defined above), the severity and course of the disease, whether the immunoconjugate or radioimmunoconjugate or composition of the present invention is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the immunoconjugate or radioimmunoconjugate or composition, and the judgment of the attending physician. The immunoconjugate or radioimmunoconjugate or composition of the present invention is suitable for administration to a patient either as a single dose or in a series of treatments. Preferably, the immunoconjugate or radioimmunoconjugate or composition is administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, an initial candidate dose of from about 1 μg / kg body weight to about 50 mg / kg body weight (e.g., about 0.1 - 15 mg / kg / dose) of the immunoconjugate or radioimmunoconjugate or composition may be administered to the patient, whether, for example, by one or more separate administrations or by continuous infusion. The dosing regimen may include an initial loading dose of about 4 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of the present invention, followed by a maintenance dose of about 2 mg / kg of the immunoconjugate or radioimmunoconjugate or composition of the present invention administered weekly. However, other dosing regimens may be useful. The typical daily dose range may be 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 condition, the treatment is continued until suppression of the desired disease symptoms occurs. The progress of such therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other skilled persons in the art.

[0320] The dosage and administration schedule can be selected and adjusted based on the disease level or tolerance of the subject, which can be monitored during the course of treatment. The conjugate of the present invention can be administered once daily, once weekly, more than once a week but less than once daily, more than once a month but less than once daily, more than once a month but less than once a week, once a month, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or intermittently to alleviate or mitigate the symptoms of the disease. Administration can be continued at any of the disclosed intervals until remission of the tumor or the symptoms of the cancer being treated. In cases where remission or alleviation is prolonged by continued administration, administration can be continued after remission or alleviation of the symptoms has been achieved.

[0321] For some embodiments, an effective amount of the immunoconjugate or radioimmunoconjugate or composition can be provided as a single dose.

[0322] The immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with conventional and / or novel treatments or therapies or administered alone as monotherapy. In some embodiments, the immunoconjugates and radioimmunoconjugates of the present invention can be used in combination with one or more radiosensitizers. Such agents include any agent that can increase the sensitivity of cancer cells to radiotherapy. 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 radiotherapy. Irradiation of tumors can cause a variety of biological consequences, which 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, or inhibit local invasion and metastasis, or prevent repopulation, or enhance the immune response, or deregulate cellular energy, or reduce the population, or alter tumor metabolism, or increase tumor damage, or reduce DNA repair. In certain embodiments, the agents used in combination with the immunoconjugates and radioimmunoconjugates of the present invention can include: DDR inhibitors, such as PARP, ATR, Chk1 or DNA-PK; or survival signaling inhibitors, such as mTOR, PI3k, NF-kB; or anti-hypoxia agents, such as HIF-1-α, CAP or UPR; or metabolic inhibitors, such as MCT1, MCT4 inhibitors; or immunotherapeutic agents, such as anti-CTLA4, anti-PD-1; or growth factor signal transduction inhibitors, such as EGFR or MAPK inhibitors; or anti-invasion agents, such as kinase inhibitors, chemokine inhibitors or integrin inhibitors; or anti-angiogenic agents, such as VEGF inhibitors.

[0323] The immunoconjugates and radioimmunoconjugates of the present invention can (i) inhibit the growth or proliferation of the cells to which they bind; (ii) induce the death of the cells to which they bind; (iii) inhibit the stratification of the cells to which they bind; (iv) inhibit the metastasis of the cells to which they bind; or (v) inhibit the angiogenesis of tumors containing the cells to which they bind. In this context, "inhibit cell growth or proliferation" means reducing the growth or proliferation of cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and includes inducing cell death.

[0324] By way of example, an immunoconjugate that inhibits the growth of tumor cells is an immunoconjugate that results in a 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 display the antigen to which the immunoconjugate or radioimmunoconjugate binds. Compared to an appropriate control, a preferred growth-inhibiting immunoconjugate or radioimmunoconjugate inhibits the growth of tumor cells expressing the antigen by greater than 20%, preferably about 20% to about 50%, and even more preferably greater than 50% (e.g., about 50% to about 100%), where the control is typically tumor cells not treated with the test immunoconjugate or radioimmunoconjugate.

[0325] For some embodiments, most of the immunoconjugates or radioimmunoconjugates or compositions administered to a subject typically consist of unlabeled immunoconjugates, with a minority consisting of labeled radioimmunoconjugates. The ratio of labeled radioimmunoconjugate to unlabeled immunoconjugate can be adjusted using known methods. Thus, in certain aspects of the invention, an immunoconjugate or radioimmunoconjugate can be provided having a total protein mass of up to 100 mg, such as less than 60 mg, or 5 mg to 45 mg, or having a total protein mass between 0.1 μg / kg and 1 mg / kg of patient body weight, such as 1 μg / kg to 1 mg / kg of patient body weight, or 10 μg / kg to 1 mg / kg of patient body weight, or 100 μg / kg to 1 mg / kg of patient body weight, or 0.1 μg / kg to 100 μg / kg of patient body weight, or 0.1 μg / kg to 50 μg / kg of patient body weight, or 0.1 μg / kg to 10 μg / kg of patient body weight, or 0.1 μg / kg to 40 μg / kg of patient body weight, or 1 μg / kg to 40 μg / kg of patient body weight, or 0.1 mg / kg to 1.0 mg / kg of patient body weight, such as 0.2 mg / kg of patient body weight to 0.6 mg / kg of patient body weight.

[0326] In certain embodiments, an immunoconjugate / radioimmunoconjugate can be administered at about 0.5 mg / kg to about 30 mg / kg.In certain embodiments, an immunoconjugate / radioimmunoconjugate may be administered at 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 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 3 mg / kg, about 2 mg / kg to about 4 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 20 mg / kg, about 2 mg / kg to about 30 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 3 mg / kg, about 5 mg / kg to about 4 mg / kg, about 5 mg / kg to about 5 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 3 mg / kg, about 10 mg / kg to about 4 mg / kg, about 10 mg / kg to about 5 mg / kg, about 10 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, about 3 mg / kg to about 4 mg / kg, about 3 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, an 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, an 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, an immunoconjugate / radioimmunoconjugate may be administered at at most 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.

[0327] In some embodiments, the method includes administering an effective amount of a radioimmunoconjugate that comprises 0.01 to 0.1 mCi, or 0.1 mCi to 1.0 mCi, or 1.0 mCi to 2.0 mCi, or 2.0 mCi to 4.0 mCi of 225-Ac.

[0328] In some embodiments, the method includes administering an effective amount of a radioimmunoconjugate that comprises 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 of 225-Ac.

[0329] In certain embodiments, the effective amount of 225-Ac is from about 0.1 microcurie to about 20 microcuries. In certain embodiments, the effective amount of 225-Ac is from about 0.1 microcurie to about 0.2 microcurie, from about 0.1 microcurie to about 0.5 microcurie, from about 0.1 microcurie to about 1 microcurie, from about 0.1 microcurie to about 2 microcurie, from about 0.1 microcurie to about 3 microcurie, from about 0.1 microcurie to about 4 microcurie, from about 0.1 microcurie to about 5 microcurie, from about 0.1 microcurie to about 10 microcurie, from about 0.1 microcurie to about 20 microcurie, from about 0.2 microcurie to about 0.5 microcurie, from about 0.2 microcurie to about 1 microcurie, from about 0.2 microcurie to about 2 microcurie, from about 0.2 microcurie to about 3 microcurie, from about 0.2 microcurie to about 4 microcurie, from about 0.2 microcurie to about 5 microcurie, from about 0.2 microcurie to about 10 microcurie, from about 0.2 microcurie to about 20 microcurie, from about 0.5 microcurie to about 1 microcurie, from about 0.5 microcurie to about 2 microcurie, from about 0.5 microcurie to about 3 microcurie, from about 0.5 microcurie to about 4 microcurie, from about 0.5 microcurie to about 5 microcurie, from about 0.5 microcurie to about 10 microcurie, from about 0.5 microcurie to about 20 microcurie, from about 1 microcurie to about 2 microcurie, from about 1 microcurie to about 3 microcurie, from about 1 microcurie to about 4 microcurie, from about 1 microcurie to about 5 microcurie, from about 1 microcurie to about 10 microcurie, from about 1 microcurie to about 20 microcurie, from about 2 microcurie to about 3 microcurie, from about 2 microcurie to about 4 microcurie, from about 2 microcurie to about 5 microcurie, from about 2 microcurie to about 10 microcurie, from about 2 microcurie to about 20 microcurie, from about 3 microcurie to about 4 microcurie, from about 3 microcurie to about 5 microcurie, from about 3 microcurie to about 10 microcurie, from about 3 microcurie to about 20 microcurie, from about 4 microcurie to about 5 microcurie, from about 4 microcurie to about 10 microcurie, from about 4 microcurie to about 20 microcurie, from about 5 microcurie to about 10 microcurie, from about 5 microcurie to about 20 microcurie, or from about 10 microcurie to about 20 microcurie. In certain embodiments, the effective amount of 225-Ac is about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20 microcurie. In certain embodiments, the effective amount of 225-Ac is at least about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, or about 10 microcurie. In certain embodiments, the effective amount of 225-Ac is at most about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20 microcurie. According to embodiments in which the radioisotope of the radioimmunoconjugate is 111-In, the effective amount is less than, for example, 15.0 mCi (i.e., the amount of 111-In administered to the subject delivers a whole body radiation dose of less than 15.0 mCi).

[0330] In embodiments where the radioisotope of the radioimmunoconjugate is 111-In, the 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.

[0331] In embodiments where the radioisotope of the radioimmunoconjugate is 111-In, the effective amount is from 0.1 mCi to 1.0 mCi, 0.1 mCi to 2.0 mCi, 1.0 mCi to 2.0 mCi, 1.0 mCi to 3.0 mCi, 1.0 mCi to 4.0 mCi, 1.0 mCi to 5.0 mCi, 1.0 mCi to 10.0 mCi, 1.0 mCi to 15.0 mCi, 1.0 mCi to 20.0 mCi, 2.0 mCi to 3.0 mCi, 3.0 mCi to 4.0 mCi, 4.0 mCi to 5.0 mCi, 5.0 mCi to 10.0 mCi, 5.0 mCi to 15.0 mCi, 5.0 mCi to 20.0 mCi, 6.0 mCi to 14.0 mCi, 7.0 mCi to 13.0 mCi, 8.0 mCi to 12.0 mCi, 9.0 mCi to 11.0 mCi or 10.0 mCi to 15.0 mCi.

[0332] In embodiments where the radioisotope of the radioimmunoconjugate is 111-In, the effective amount is 15.0 mCi, 14.0 mCi, 13.0 mCi, 12.0 mCi, 11.0 mCi, 10.0 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.

[0333] In embodiments where the radioisotope of the radioimmunoconjugate is 225-Ac, the effective amount is less than, for example, 30.0 μCi / kg (i.e., where the amount of 225-Ac administered to the subject delivers a radiation dose of less than 30.0 μCi / kg of subject body weight).

[0334] In embodiments where the radioisotope of the radioimmunoconjugate is 225-Ac, the effective amount is less than 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, 3.5 μCi / kg, 3.0 μCi / kg, 2.5 μCi / kg, 2.0 μCi / kg, 1.5 μCi / kg, 1.0 μCi / kg, 0.9 μCi / kg, 0.8 μCi / kg, 0.7 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.

[0335] In embodiments where the radioisotope of the radioimmunoconjugate is 225-Ac, the 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 1.0 μCi / kg, 1.0 μCi / kg to 1.5 μCi / kg, 1.5 μCi / kg to 2.0 μCi / kg, 2.0 μCi / kg to 2.5 μCi / kg, 2.5 μCi / kg to 3.0 μCi / kg, 3.0 μCi / kg to 3.5 μCi / kg, 3.5 μCi / kg to 4.0 μCi / kg, 4.0 μCi / kg to 4.5 μCi / kg or 4.5 μCi / kg to 5.0 μCi / kg.

[0336] In an embodiment where the radioisotope of the radioimmunoconjugate is 225-Ac, the 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, or 4.5 μCi / kg, 5.0 μCi / kg, 6.0 μCi / kg, 7.0 μCi / kg, 8.0 μCi / kg, 9.0 μCi / kg, 10.0 μCi / kg, 12.5 μCi / kg, 15.0 μCi / kg, 17.5 μCi / kg, 20.0 μCi / kg, 25 μCi / kg or 30 μCi / kg.

[0337] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu, the effective amount is from 0.1 uCi to 100 mCi per square meter of body surface area.

[0338] In certain embodiments where the radioisotope of the radioimmunoconjugate is 177-Lu, the effective amount is from 1 mCi to 100 mCi per square meter of body surface area. In certain embodiments, the effective amount is from about 1 / m² to about 100 / m². In certain embodiments, the effective amount is from about 1 / m² to about 5 / m², from about 1 / m² to about 10 / m², from about 1 / m² to about 15 / m², from about 1 / m² to about 20 / m², from about 1 / m² to about 25 / m², from about 1 / m² to about 75 / m², from about 1 / m² to about 100 / m², from about 5 / m² to about 10 / m², from about 5 / m² to about 15 / m², from about 5 / m² to about 20 / m², from about 5 / m² to about 25 / m², from about 5 / m² to about 75 / m², from about 5 / m² to about 100 / m², from about 10 / m² to about 15 / m², from about 10 / m² to about 20 / m², from about 10 / m² to about 25 / m², from about 10 / m² to about 75 / m², from about 10 / m² to about 100 / m², from about 15 / m² to about 20 / m², from about 15 / m² to about 25 / m², from about 15 / m² to about 75 / m², from about 15 / m² to about 100 / m², from about 20 / m² to about 25 / m², from about 20 / m² to about 75 / m², from about 20 / m² to about 100 / m², from about 25 / m² to about 75 / m², from about 25 / m² to about 100 / m², or from about 75 / m² to about 100 / m². In certain embodiments, the effective amount is about 1 / m², about 5 / m², about 10 / m², about 15 / m², about 20 / m², about 25 / m², about 75 / m², or about 100 / m². In certain embodiments, the effective amount is at least about 1 / m², about 5 / m², about 10 / m², about 15 / m², about 20 / m², about 25 / m², or about 75 / m². In certain embodiments, the effective amount is at most about 5 / m², about 10 / m², about 15 / m², about 20 / m², about 25 / m², about 75 / m², or about 100 / m².

[0339] In certain embodiments of the invention, a formulation or composition thereof (e.g., a pharmaceutical composition) of the radioimmunoconjugate of the invention may comprise a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), wherein the ratio of the labeled fraction:unlabeled fraction may be from about 1:1000 to 1:1.

[0340] In addition, the pharmaceutical composition may be provided as a single-dose composition customized for a particular patient, i.e., as a patient-specific therapeutic composition, wherein the amounts of the labeled and unlabeled immunoconjugates (the labeled immunoconjugate, for clarity, being the same as the radioimmunoconjugate herein) in the composition may depend at least on the patient's weight, height, body surface area, age, gender, and / or disease state or health status. Accordingly, the patient-specific therapeutic composition in a total volume may be provided in a vial configured to be administered entirely to the patient in one treatment such that little of the composition remains in the vial after administration.

[0341] Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy, and chemotherapy. Therapy using the radioimmunoconjugates (interchangeably, "radiolabeled immunoconjugates") of the present invention may be particularly desirable in elderly patients who cannot well tolerate the toxicity and side effects of chemotherapy and in metastatic diseases where the effectiveness of radiation therapy is limited. For some embodiments, therapy using the radiolabeled immunoconjugates of the present invention can be used to mitigate cancers expressing a target antigen after initial diagnosis of the disease or during recurrence.

[0342] In some embodiments, determining whether a cancer is suitable for treatment by the methods disclosed herein involves detecting the presence of a target antigen in a subject or in a sample from the subject. To determine target antigen expression in cancer, various detection assays can be used. In some embodiments, target antigen overexpression is analyzed by immunohistochemistry (IHC). The IHC assay is performed on paraffin-embedded tissue sections from a tumor biopsy and made to conform to a target antigen staining intensity criterion. Alternatively or additionally, a FISH assay can be performed on formalin-fixed, paraffin-embedded tumor tissue, such as (sold by Ventana, AZ, U.S.A.) or (Vysis, IL, U.S.A.) to determine the extent (if any) of target antigen overexpression in the tumor.

[0343] Target antigen overexpression or amplification can be evaluated using in vivo detection assays, for example, by administering a molecule (such as an antibody construct or immunoconjugate of the present invention) that binds to the molecule to be detected and is tagged with a detectable label (e.g., a radioisotope or a fluorescent label), and externally scanning the patient for the location of the label.

[0344] 2. Use of the immunoconjugates and radioimmunoconjugates of the present invention to kill cells

[0345] The immunoconjugates or radioimmunoconjugates of the present invention can be used in, for example, in vitro, ex vivo, and in vivo methods. In some embodiments, the present invention provides methods for inhibiting cell growth or proliferation in vivo or in vitro, the methods comprising exposing cells to the immunoconjugates or radioimmunoconjugates of the present invention under conditions that permit binding of the immunoconjugate or radioimmunoconjugate to the target antigen. The immunoconjugates or radioimmunoconjugates of the present invention can also (i) inhibit the growth or proliferation of the cells to which they bind; (ii) induce cell death of the cells to which they bind; (iii) inhibit the stratification of the cells to which they bind; (iv) inhibit the metastasis of the cells to which they bind; or (v) inhibit angiogenesis of tumors comprising the cells to which they bind.

[0346] In some embodiments, the present invention provides a method of killing cells expressing an antigen, the method comprising contacting the cells with the immunoconjugates or radioimmunoconjugates (or a composition thereof) of the present invention. This method can be used, for example, to kill, deplete, or eliminate cells expressing a target antigen from a mixed cell population. This method can be used, for example, to kill, deplete, or eliminate cells expressing a target antigen from a mixed cell population as a step in purifying other cells. This method can be performed in vitro or in vivo, including ex vivo on primary patient cells or tissue compositions to prepare such compositions for use in transplantation.

[0347] In some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention are used for treating or preventing cell proliferative disorders. In certain embodiments, cell proliferative disorders include solid tumor cancers. Solid tumor cancers are cancers that comprise abnormal tissue masses, such as carcinomas and sarcomas. In certain other embodiments, cell proliferative disorders include liquid tumor cancers or hematological cancers, used interchangeably, such cancers being present in body fluids, such as leukemias and lymphomas. In certain embodiments, cell proliferative disorders are associated with increased expression and / or activity of a target antigen. For example, in certain embodiments, cell proliferative disorders are associated with increased expression of a target antigen on the cell surface. In certain embodiments, cell proliferative disorders are tumors or cancers. In certain embodiments, cell proliferative disorders include solid tumor cancers. Solid tumor cancers are cancers that comprise abnormal tissue masses, such as carcinomas and sarcomas. In certain other embodiments, cell proliferative disorders include liquid tumor cancers or hematological cancers, used interchangeably, such cancers being present in body fluids, such as leukemias and lymphomas.

[0348] In some embodiments, the present invention provides methods for treating cell proliferative disorders, the methods comprising administering to an individual an effective amount of the immunoconjugates or radioimmunoconjugates of the present invention.

[0349] In addition to directly killing target cells that express a cell surface antigen to which the immunoconjugates or radioimmunoconjugates of the present invention specifically bind, the immunoconjugates or radioimmunoconjugates of the present invention may optionally be used to deliver additional cargo to the vicinity or interior of the target cells. Delivery of additional exogenous substances may be used, for example, for cytotoxic, cytostatic, information gathering, and / or diagnostic functions. Non-cytotoxic variants of the immunoconjugates or radioimmunoconjugates of the present invention, or optionally cytotoxic variants, may be used to deliver cargo to the interior of cells that express the target antigen and / or label their interior. Non-limiting examples of cargo include cytotoxic agents, detection enhancers, and small molecule chemotherapeutics.

[0350] As described herein, in some embodiments, the antibody (e.g., multivalent antibody) constructs, immunoconjugates, radioimmunoconjugates, and targeted imaging complexes of the present invention have various non-therapeutic applications. In some embodiments, the compositions of the present invention may be used to identify patient populations that are predicted to benefit from a particular treatment method or modality, such as, for example, treatment using the immunoconjugates or radioimmunoconjugates of the present invention. In some embodiments, the compositions of the present invention may be used to stage cancers that express the target antigen (e.g., by radioimaging) or serve as a prognostic indicator of disease progression. In some embodiments, the compositions may also be used to detect and quantify target epitopes in vitro, such as in ELISA or Western blotting, and to purify or immunoprecipitate the target antigen from cell or tissue samples.

[0351] For some embodiments, the immunoconjugates or radioimmunoconjugates of the present invention are used in methods for detecting the presence or level of an antigen, such as, for example, in vitro in a biological sample or in vivo using imaging techniques. Detection by immunoconjugates and radioimmunoconjugates can be accomplished via different techniques known to those skilled in the art and as described herein, such as IHC and PET imaging. When the immunoconjugates or radiolabeled immunoconjugates of the present invention are used for detection, it may comprise a radioactive atom for scintigraphic studies, such as 99m-Tc or 111-In.

[0352] The labeled immunoconjugates of the present invention can be used as imaging biomarkers and probes by various methods and techniques of biomedical and molecular imaging 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 an antibody labeled with a radioactive substance is administered to an animal or human patient, and a photograph is taken of the site in the body where the antibody (e.g., a multivalent antibody) is located (US 6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

[0353] 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, wherein the method comprises exposing the sample to an immunoconjugate that binds to the target antigen, and determining the binding of the immunoconjugate to the target antigen in the sample, wherein the presence of such binding indicates the presence of the target antigen in the sample. Optionally, the sample can contain cells (which can be cancer cells) suspected of expressing the target antigen. The immunoconjugate employed in the method can optionally be detectably labeled, attached to a solid support, etc.

[0354] Another embodiment of the present invention relates to a method for diagnosing the presence of a tumor in a subject, wherein the method comprises (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 complex between the immunoconjugate and the target antigen in the test sample, wherein the formation of the complex indicates the presence of a tumor in the mammal. Optionally, the immunoconjugate is detectably labeled, attached to a solid support, etc., and / or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0355] In some embodiments, the immunoconjugates of the present invention, including compositions comprising the foregoing and / or the immunoconjugates provided herein, can be used to detect the presence of a target antigen, for example, in vivo or in a biological sample. The immunoconjugates of the present invention can be used in a variety of different assays, including but not limited to ELISA, bead-based immunoassays, and mass spectrometry.

[0356] In some embodiments, the immunoconjugates of the present invention can be used to quantify the amount of 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, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, saliva, sputum, tears, sweat, mucus, cerebrospinal fluid, urine, and other body components 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 from a clinical patient or a patient treated with one or more therapeutic antibodies that bind the same target antigen.

[0357] In some embodiments, the sample is from a mammal. In some embodiments, the sample is from a human subject, e.g., 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 agent (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.

[0358] In some embodiments, a composition comprising a 'labeled' immunoconjugate is provided. Labels include, but are not limited to, labels or moieties for direct detection (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radiolabels), and moieties for indirect detection, such as enzymes or ligands, for example, by enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, fluorophores (such as rare earth chelates or fluorescein and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, J3-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), conjugates with enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, phage labels, stable free radicals, etc.

[0359] These labels can be covalently attached to a protein or polypeptide using conventional methods. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, diimidates, di-azotized benzidines, etc. can be used to label the immunoconjugates or antibody constructs of the present invention with the fluorescent labels, chemiluminescent labels, and enzyme labels described above (see, for example, US 3,645,090 (enzymes); 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)).

[0360] The conjugation of such labels (including enzymes) to immunoconjugates or antibody constructs is standard operating procedure for those of ordinary skill in the art of immunoassay techniques (see, e.g., O'Sullivan et al., “Methods for the Preparation of Enzyme - antibody Conjugates for Use in Enzyme Immunoassay,” Methods in Enzymology, J. Langone and H. Van Vunakis eds., Vol. 73 (Academic Press, New York, New York, 1981), pp. 147 - 166). Suitable commercially available labeled antibodies may also be used.

[0361] After addition of the final labeled immunoconjugate, the amount of bound immunoconjugate is determined by washing to remove excess unbound labeled immunoconjugate and then measuring the amount of attached label using a detection method appropriate for the label, and correlating the measured amount to the amount of immunoconjugate of interest in the biological sample. For example, in the case of an enzyme, the development of color and the amount of color measured will be a direct measure of the amount of immunoconjugate of interest present. Specifically, if HRP is the label, substrate TMD may be used and the color detected using a reading wavelength of 450 nm and a reference wavelength of 620 or 630 nm.

[0362] In one example, after washing the enzyme - labeled second antibody directed against the unlabeled immunoconjugate from the solid phase, color or chemiluminescence is developed and measured by incubating the immobilized capture reagent with an enzyme substrate. The concentration of the antibody of interest (e.g., a multivalent antibody) is then calculated by comparison to the color or chemiluminescence generated by the immunoconjugate of interest run in parallel.

[0363] In some embodiments, the method involves bead-based immunoassays, ELISA assays, or mass spectrometry techniques. The mass analyzers of such mass spectrometers include, but are not limited to, quadrupole (Q) analyzers, time-of-flight (TOF) analyzers, ion trap analyzers, sector magnetic field analyzers, or Fourier transform ion cyclotron resonance (FT-ICR) analyzers, or combinations thereof. The ion source of the mass spectrometer should primarily generate sample molecular ions or quasi-molecular ions and certain characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), as well as matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are the two most commonly employed methods for ionizing proteins for mass spectrometry analysis of small molecules (such as, for example, 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. Generally, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface (such as a "protein chip") to interact with analytes (e.g., proteins) in solution. Such surfaces selectively interact with the analytes and immobilize them thereon. Thus, the analytes of the present invention can be partially purified on the chip and then rapidly analyzed in a mass spectrometer. By providing multiple reactive moieties at different sites on the substrate surface, the throughput can be increased.

[0364] 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 as described herein to allow the formation of an immune complex; (b) detecting or measuring the level of the 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 binds to a streptavidin-coated microtiter plate.

[0365] Examples

[0366] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0367] Example 1. Preparation of VHH-Fc

[0368] The VHH-Fc plasmid was generated by cloning the VHH sequence with a hinge and Fc portion (human IgG1 CH2-CH3) into a mammalian expression vector. In some cases, mutations were introduced into the Fc portion. To produce recombinant VHH-Fc and its variants, the plasmid was transfected into HEK293.SUS cells (ATUM or similar cells). After 3-5 days of secretion, the cells were removed from the supernatant containing the antibody by centrifugation and sterile filtration. The antibody was purified using a Mab Select SuRe PCC column (GE, Cat#: 11003495) and buffer exchanged into PBS at pH 7.0. The protein was quantified using A280 or BCA. The purity of the antibody was tested by SDS-PAGE, capillary electrophoresis, HPLC-SEC, and LC-MS using standard protocols.For 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, Gutiérrez 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。

[0369] For VHH humanization, see, for example, 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.

[0370] For VHH stability, see, for example, 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, Mücke 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.

[0371] Many VHH-Fc prototypes and variants were engineered using VHH sequences such as the anti-HER2 clone 2RS15d VHH (see, e.g., W02016 / 016021) (SEQ ID NO:20) and the anti-DLL3 clone hz10D9v7.251 VHH sequence (see, e.g., W02020 / 07967) (SEQ ID NO:30), and unless otherwise stated herein, the data collected and presented were obtained using the VHH antigen-binding regions of these clones.

[0372]

[0373] Example 2. Antibody Binding Properties: Assays of Target Proteins and Target Cells

[0374] According to standard protocols, the binding of VHH-Fc to the target soluble protein (human, murine, and cynomolgus macaque orthologs, as appropriate) was evaluated by ELISA. Antigens were either commercially sourced or produced by cloning the known antigen sequence (Uniprot) into a mammalian expression vector with HIS, FLAG, or equivalent tags for purification and detection purposes. Commercially available control anti-target IgG was included. Plates (96-well maxisorp, Corning 3368) were coated with 50 μL to 100 μL of each target protein of interest at an optimized concentration for coating. Purified VHH-Fc and hIgG1 isotype control (Sigma, Cat#I5154) were prepared at an initial concentration of 200 nM to 400 nM and titrated down 1:4. After incubating the primary antibody for 1 hour at room temperature (RT) and washing, 0.2 μg / ml HRP-conjugated secondary antibody was added and incubated for 1 h 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 1M HCl (50 μl). Optical density (OD) was measured at 450 nm using a Spectromax plate reader, and data were processed using SoftMaxPro. Data showed that anti-target VHH-Fc bound to human, murine, and cynomolgus macaque target antigens. The recombinant DLL3 proteins used were human DLL3.FLAG (Adipogen#AG-40B-0151, amino acids 27-466), or human DLL3.HIS (abcam#ab255797, amino acids 27-492), or murine DLL3.HIS (IPA custom, amino acids 25-477) or cynomolgus macaque DLL3.HIS (Acrobiosystems#, amino acids 27-490). The control antibody for DLL3 binding was lovatuzumab (Creative Biolabs#TAB-216CL). The recombinant HER2 proteins used were human Her2.HIS (Sinobiologics, #10004-H08H) and murine HER2.HIS (Sinobiologics#50714-M08H). The control antibody for HER2 binding was trastuzumab (DIN: 02240692, ROCHE). Figure 1A and 1B Anti-Her2 and anti-DLL3 VHH-Fc that specifically bind soluble target antigens in ELISA were tested for additional VHH-Fc with mutations in the Fc region that reduce effector function and / or FcRn binding, but do not significantly affect binding to the target antigen.

[0375] The VHH-Fc was screened for binding to a panel of target-positive cancer cell lines by flow cytometry. Unless otherwise stated, all cell lines were obtained from ATCC and were cultured according to the manufacturer's instructions and recommended media. The HER2-positive cell lines used were SKBR3 (ATCC#HTB-30), BT474 (ATCC#HTB-20), and HEK293-6E (NRC) cells. The DLL3-positive cell lines tested included SHP-77 (ATCC CRl-2195), NCI-H82 (ATCC HTB-175), NCI-H69 (ATCC HTB-119), HEK-DLL3 (Creative Biogene#CSC-RO0531). The HER2-negative cell lines tested included SHP-77. The DLL3-negative cell lines tested included HCT-116 (CCL-247), BT-474, and SKBR3. The primary antibody diluted in the same manner as in ELISA was added to the cells and incubated on ice for 1 hour. The cells were washed twice with PBS containing 1% FBS, centrifuged at 450G for 4 minutes, and incubated on ice for 30 minutes 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) together with 1:1000 DAPI (Biolegend, Cat#422801). After two additional washes according to standard protocols, the cells were resuspended and analyzed by flow cytometry on an iQue screener platform (Intellicyt), and the data were processed using Forecyt. Figure 2A , 2B and 2C showed binding to the target-positive cell lines and showed that the binding was specific to the target-positive cells (i.e., by comparison with binding to negative control cells). Additional experiments indicated that Fc mutations that reduce effector function and / or FcRn binding compared to wild-type Fc did not affect binding to cancer cells.

[0376] Example 3. Internalization Assay

[0377] The internalization of VHH-Fc by cells expressing the target was tested using a secondary antibody conjugated to a pH-sensitive dye. According to the manufacturer's instructions, a goat anti-huIgG-Fc secondary antibody was amine-conjugated to the pH-sensitive pHAb dye (Promega Cat#G9845). The pHAb dye has low or no fluorescence at pH > 7 but fluoresces in an acidic environment after antibody internalization. The target-positive cells and target-negative cells were seeded at 1.0x10 6Plate at / mL in 96-well V-bottom plates. Dilute VHH-Fc and hIgG1 isotype control to 75 nM in the medium. Centrifuge the cells to remove the supernatant, resuspend them with the prepared primary antibody and incubate on ice for 1 hour. Wash the excess primary antibody from the cells and then incubate them with the pHAb-labeled secondary antibody on ice for 30 minutes. Then wash the excess secondary antibody and resuspend the cells in the medium. Place one sample set in an incubator at 37 °C to allow internalization, and place another set on ice (0 °C) as a binding-only control. Sample the cells at different time points from 0 to 24 hours. Stain the cells with DAPI and read them by flow cytometry on the 572 / 28 channel using the iQue screener platform. VHH-Fc showed higher fluorescence on target-positive cells than the negative controls (isotype, buffer). Figure 3A and 3B Showed that H101 and D102 were internalized by SHP-77 and HEK-DLL3 cells.

[0378] Example 4. Antibody Thermal Stability Determination

[0379] The denaturation temperature (Tm) of VHH-Fc was determined using the Protein Thermo Shift Dye KitTM (ThermoFisher, Cat#: 4461146) according to differential scanning fluorimetry (DSF). Briefly, a total of 1 μg of antibody was used in each reaction. Using the Applied Biosystems QuantStudio 7Flex real-time PCR system, melting curves of the antibody were generated using the recommended settings described in the kit manual. Then the Tm of the antibody in Table 1 was determined using the ThermoFisher Protein ThermalShift software (v.1.3). The Tm1 of VHH-Fc was determined by DSF. Both H101 and D102 showed good thermal stability at 67.5 ± 0.1 °C. Additionally, the thermal stability of VHH-Fc containing mutations in the Fc region that reduce effector function and / or FcRn binding was tested, and it resulted in slightly lower thermal stability (1 to 2 °C), but still within an acceptable range.

[0380] Example 5. Receptor Density Determination

[0381] To test the efficacy of immunoconjugate binding relative to target density, the receptor density on a target-positive cell line was measured. The target density was measured using the ABC (antibody binding capacity) assay. Harvest the cancer cells expressing the target of interest and the negative control cell line with cell dissociation buffer and plate them at approximately 5x 10 4Cells were seeded at

[0382]

[0383] Example 6. Affinity of antibody for target protein

[0384] The antibody affinity was evaluated using Octet Red96e (ForteBio). The biosensor (Fortebio cat#18-5063) was captured with anti-hIgG Fc (AHC), and the association rate constant (ka), dissociation rate constant (kd), and affinity constant (KD) were measured by biolayer interferometry. Each cycle was performed at an orbital shaking speed of 1,000 rpm. The antigen was titrated 1:2 from a suitable starting concentration in kinetic buffer (Fortebio, Cat#18-1105). The set of AHC biosensors was immersed in the kinetic buffer for a 60 s baseline step. The anti-target VHH-Fc (5 μg / mL, in kinetic buffer) was loaded onto the biosensors for 240 s, followed by a 30 s second baseline step. The IgG-captured sensors were immersed in buffer for a single reference subtraction to compensate for the natural dissociation of the captured IgG. Then each biosensor was immersed in the corresponding concentration of target protein (human, mouse, or cynomolgus monomeric protein) for 600 s, followed by a dissociation time of 1,800 s in kinetic buffer or under optimized conditions. A new set of AHC biosensors was used for each VHH-Fc. The data for the association and dissociation steps were analyzed by global fitting to a 1:1 model (Octet software version v11.0).

[0385] Table 3 shows the binding affinity data.

[0386]

[0387] Example 7. Determination of FcRn and Fc effector mutant affinities

[0388] The FcRn affinity of VHH-Fc can generally be used to predict the half-life of antibody serum clearance. (See, e.g., 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, using Octet RED96e (Fortebio), 10 nM biotinylated hFcRn (Sino Biological, Cat#: CT071-H27H-B) was captured with an SA biosensor. The hFcRN-coated biosensor was immersed in a sample solution in sodium phosphate buffer (100 mM Na2HPO4, 150 mM NaCl w / 0.05% Tween-20, pH 6.0), and a series of concentrations of the test antibody and association were measured. Dissociation was measured by immersing the biosensor in sodium phosphate buffer without antibody. The KD value was determined using Octet DataAnalysis HT 11.0 software. A 2:1 (Heterogeneous Ligand) binding model was used in the analysis. Table 4 shows the affinity of FCRN for wild-type VHH-Fc and the effect of specific mutations in Fc on the affinity for mutants. The change in FcRn affinity was consistent across targets. Constructs with only Fc effector mutations had no effect on FcRn affinity. Adding Fc effector mutations to FcRn mutant constructs did not affect FcRn affinity. Table 4a shows the affinity of VHH-Fc and Fc variants for FcRn.

[0389]

[0390] The Octet Red96e platform was also used to test the affinity of VHH-Fc for FcγR by biolayer interferometry. Each cycle was carried out at an orbital shaking speed of 1,000 rpm. The streptavidin (SA) biosensor (Sartorius 18-5019) was rehydrated for 10 min using kinetic buffer (PBS + 0.1% BSA + 0.02% Tween-20). Then biotinylated FcγR (Acro Biosystems) was loaded onto the SA biosensor at a concentration ranging from 1 - 5 μg / mL (diluted in PBS) for 40 - 100 s. VHH-Fc was serially diluted 1:2 in sample buffer (PBS + 0.02% Tween-20) at an initial concentration ranging from 5000 nM to 37.5 nM. Then the loaded biosensor was associated with VHH-Fc for 60 - 120 s. The dissociation of VHH-Fc was measured in sample buffer for 30 - 900 s. Then the bound VHH-Fc was removed using three 5 s cycles of regeneration buffer (150 mM NaCl, 300 mM sodium citrate) and 5 s of sample buffer. Data were analyzed using a globally fit 1:1 Langmuir binding model (FcγRI) or steady state analysis (Octet software version HT v11.1).

[0391] Analysis showed that for constructs with those mutations incorporated as shown in Table 4b, the binding to FcγR (represented by a higher KD) was reduced.

[0392]

[0393] Example 8. Self-association studies using AC-SINS

[0394] The tendency of self-association of VHH-Fc was determined using gold nanoparticles (Au-NP) (Ted Pella, Cat#: 15705) according to affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). (PMID: 24492294, 30395473) Briefly, Au-NP were coated with goat IgG and goat anti-human Fc IgG (1:4 molar ratio). The conjugated Au-NP were mixed with 5 μg of each VHH-Fc in a 96-well plate in quadruplicate. Wavelength scans were measured using a Synergy Neo2 plate reader. The difference in maximum absorbance (Δλmax) was calculated by subtracting the λmax of each reaction from that of the PBS buffer. Data were analyzed using the Linest function in Excel with a second-order polynomial fit. Controls with known high ACSINS scores (above the IgG cut-off of 11 determined in the literature) were included in the assay. Figure 4Show the ACSINS scores of the test article and the control.

[0395] Example 9. Polyreactivity study

[0396] The polyreactivity of VHH-Fc towards negatively charged biomolecules was determined by ELISA (as in Avery et al., “Establishing in vitro 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 (Sigma Alrich, Cat#: I9278) and 10 μg / mL double-stranded DNA (Sigma Alrich, 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 the test VHH-Fc was loaded onto the plates in quadruplicate and incubated for 2 hours. Then, goat anti-human Fc conjugated to HRP (0.01 ug / ml) was added and the plates were incubated for 1 hour. Signal was generated using TMB and the A450 absorbance was measured using a Synergy Neo2 plate reader. For each tested antibody, the signal was normalized with the signal from the uncoated wells. Table 5 shows the polyreactivity scores compared to the control antibody.

[0397]

[0398]

[0399] Example 10. Fc variants effectively shorten the half-life of VHH-Fc

[0400] In some cases, shortening the drug half-life of α-emitters is important for safety and avoiding unwanted toxicities associated with the treatment. However, the half-life of antibodies is typically as long as 14 days or more. Therefore, the half-lives of VHH-Fc variants were tested to observe and measure any shortening of the half-life.

[0401] Twenty-eight (28) 8-week-old male B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ (Tg32 hom, JAX stock#014565) mice were assigned to 7 groups of 4 mice each, as outlined in the table. Tg32 mice contain humanized FcRn and are generally regarded as a surrogate for human pharmacokinetics of antibodies compared to non-human primates. (See, for example, 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, body weights were measured and the test article was administered IV to all mice at 3 mg / kg and 5 ml / kg. Twenty-five μL blood samples were collected from each mouse at each time interval. Blood samples were collected into 1 μL of K3EDTA, processed into plasma, diluted 1 / 10 in PBS containing 50% glycerol, transferred to a dedicated 96-well storage plate, and stored at -20°C. All plasma samples were evaluated via hIgG ELISA selected for its high sensitivity to all seven test articles.

[0402] As observed in Table 6, introduction of mutations within FcRn generally enabled shortening of the half-life of anti-HER2 VHH-Fc. Interestingly, contrary to the results published in this field, not all Fc variants showed a shortened half-life when incorporated into the tested immunoconjugates, consistent with the previously published results found in the literature. (See, for example, Burvenich IJ et al., "Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor." MAbs. May-Jun 2016;8(4):775-86).

[0403]

[0404]

[0405] As observed in Table 7, introducing mutations within FcRn generally enables shortening the half-life of anti-DLL3 VHH-Fc. Similar to the HER2-binding immunoconjugates and contrary to the published results, not all Fc variants show a shortened half-life, consistent with the previously published results found in the literature.

[0406] Example 11. Complete Mass Analysis of VHH-Fc

[0407] The conjugate was deglycosylated and then analyzed using internal Endo-S enzyme (final concentration of 10 μg / mL) at 37 °C for 1 hour.

[0408] To analyze the complete mass, 8 μL of the sample was injected into a Waters Acquity UPLC-Q-TOF with a UPLC BEH200 SEC 1.7 μM 4.6 x 150 mm column. These samples were eluted with a mobile phase of water / ACN (70 / 30, v / v) with 0.1% TFA and 0.1% FA (formic acid) at a flow rate of 0.25 mL / min for 11 min.

[0409] Example 12. Tracing Bifunctional Chelating Agents

[0410] Several chelating agents are known to those skilled in the art and are pre-functionalized for antibody conjugation. 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 general procedure below.

[0411] Other reagents used in these procedures were purchased from Millipore Sigma, CombiBlocks, Chem-Impex, and Broadpharm. All solvents were obtained from VWR and used as received without treatment for water-free conditions, unless otherwise indicated. Mass spectra were acquired using an Agilent HPLC-MS or Waters HPCS-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 with a properly sized normal-phase silica cartridge, and fractions were collected at 254 nm. The final compounds were purified by Agilent preparative HPLC using an acetonitrile / water (+0.1% TFA) gradient. NMR spectra were acquired using a Bruker 400 MHz NMR instrument and processed using MestReNova v.14. Detailed NMR data were compiled using the multi-analysis function in manual mode.

[0412] Figure 5 The synthesis of PEG5-DOTA is shown, which includes the compounds numbered (2)-(5) as described below. Compound 3 was prepared by HATU coupling followed by TFA deprotection. It was used without chromatographic purification.

[0413] 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]butyric acid; tetra(trifluoroacetic acid): Compound 2 (100 mg, 0.143 mmol) was dissolved in DMF (2 mL), HATU (65.1 mg, 0.171 mmol) was added, and then DIPEA (0.099 mL, 73.8 mg, 0.57 mmol) was added. After 3 min, a solution of Boc-NH-PEG5-amine (65.1 mg, 0.17 mmol) was added to the reaction. After stirring for 10 min, HPLC showed the reaction was complete. After 1 h, the reaction was quenched with approximately 5 mL of NaHCO3 (sat), then 5 mL of water was added, and the mixture was extracted with 4 x 30 mL of Et2O. The combined organics were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated in vacuo to give a crude protected intermediate of good purity. m / z found = 1063.6 (M+H).

[0414] The intermediate from the above was directly dissolved in DCM (5 mL), and TFA (5 mL) was added. The reaction was stirred for 24 h until HPLC indicated complete removal of the Boc and tBu esters. The reaction solution was concentrated in vacuo and co-evaporated twice with 25 mL of DCM. The residue was precipitated from DCM with Et2O, and the remaining solid was then thoroughly triturated using sonication (15 - 30 min) to afford the title compound as an off-white powder in good purity (128 mg, 86% over two steps). 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).

[0415] Synthesis of compound (4) bis(2,3,5,6 - tetrafluorophenyl) adipate: 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, and then a solution of 2,3,5,6 - tetrafluorophenol in 20 mL of DCM was added. The conversion of the product was monitored by TLC (Rf = 0.5; 75% DCM / hexane). The reaction mixture was concentrated in vacuo and purified by flash chromatography (0 - 100% DCM / hexane) to afford the title compound as a crystalline white powder (2.48 g, 82%). 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). LCMS indicated poor signal for this compound.

[0416] 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]butyric 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) adipate (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 a preparative HPLC (Agilent, Hanover, CT) with a gradient of 15 - 50% MeCN / water + 0.1% TFA to give the title compound as a white powder (2x TFA salt) (10.6 mg, 50%). 1H NMR (400 MHz, deuterium oxide) δ 7.20 (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).

[0417] Figure 6 Shows the synthesis of PEG5-Py4Pa, which includes compounds numbered (6)-(10), as described below.

[0418] Synthesis of compound (6) tert-butyl 6-[({[4-(benzyloxy)-6-{[bis({6-[(tert-butoxycarbonyl)pyridin-2-yl]methyl}amino)methyl]pyridin-2-yl}methyl)({6-[(tert-butoxycarbonyl)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), and the solution was heated to reflux. After 16 h, the reaction was cooled and the solvent was removed in vacuo. The crude product was dissolved in 200 mL of DCM and washed with 2 x 75 mL of NaHCO3 (sat) and 2 x 75 mL of saturated brine. The DCM layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give a brown crude oil (950 mg) which was used in the next step without further purification. The above intermediate 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, then purged with N2 again, and then 30% Pd / C (50 mg, 0.14 mmol) was added. After purging with N2 again, the reaction was heated to 50 °C and stirred for 6 h, where LCMS indicated completion of the reaction. The reaction mixture was filtered through Celite, washed with 3 x 50 mL of MeOH, and then concentrated in vacuo to a pale yellow oil. The crude product was purified by flash chromatography using a Biotage Sfaramino 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) δ 7.88 (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).

[0419] Synthesis of compound (7) tert-Butyl N-[17-(2-bromoacetamido)-3,6,9,12,15-pentaoxaheptadec-1-yl]carbamate: A solution of tert-butyl N-(17-amino-3,6,9,12,15-pentaoxaheptadec-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 min. The reaction was warmed to room temperature and after 90 min, HPLC showed complete conversion to the product. The reaction was concentrated, partitioned between Et 2 O and water, NaHCO3 (sat) was added, and the mixture was then extracted with 3 x 25 mL of Et 2 O. The combined organics were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude residue was co-evaporated with acetonitrile once to remove water. The title compound was recovered as a slightly brown oil (261 mg, 99%). 1 1H NMR (400 MHz, chloroform-d) δ 3.90 (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).

[0420] 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-pentaoxaheptadec-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, HPLC indicated no starting material remaining. 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%). 1 1H NMR (400 MHz, methanol-d 4)δ 7.89 (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). Observed m / z = 670.0 (M + 2H / 2).

[0421] Synthesis of compound (9) 6 - ({[(4 - {[(17 - amino - 3,6,9,12,15 - pentaoxaheptadec - 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 tert - butyl intermediate remaining. The reaction was concentrated in vacuo and co - evaporated with DCM once. The crude oil was triturated twice with Et 2 O by sonication and collected by filtration to give 100 mg (64%, as the 5x TFA salt) of the title compound as a slightly brown solid. 1 1H NMR (400 MHz, methanol - d 4 )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 = 5.3 Hz, 2H), 3.48 (t, J = 5.6 Hz, 2H), 3.20 - 3.12 (m, 2H). Observed m / z = 1014.3 (M + H).

[0422] Synthesis of compound (10) 6-[({[6-({bis[(6-carboxypyridin-2-yl)methyl]amino}methyl)-4-[({17-[6-oxo-6-(2,3,5,6-tetrafluorophenoxy)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) adipate (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 a preparative HPLC (Agilent, Hanover, CT) with a gradient of 25 - 60% MeCN / H2O + 0.1% TFA to give the title compound as a white powder (3x TFA salt) (57.5 mg, 56%). 1 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).

[0423] 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 of L Li et al., Bioconjugate Chem. 2021, 32, 1348 - 1363. The spectroscopic and LCMS data matched the reported values.

[0424] Example 13. Conjugation of VHH-Fc protein with chelator-linker

[0425] Conjugation can be carried out using many methods that can be used to prepare IgG radiolabeled conjugates and IgG antibody-drug conjugates. For information on the scope of applicable methods, see PW Howard Antibody-Drug Conjugates (ADCs), Protein Therapeutics, 1st Edition, Chapter 9, pp278-279 (2017).

[0426] For typical lysine-based conjugation, the VHH-Fc buffer is exchanged to 0.1M NaHCO3 at pH 8.5-9.5 using a Microsep Advance centrifugal device (Pall 10KMWCO, Cat#: MCP010C41) or a Zeba column (ThermoFisher, Cat#: 87768), and then sterilized using a 0.22μm Costar Spin-X centrifuge tube (Corning, Cat#: 8160). The buffer-exchanged antibody is quantified by BCA assay. An appropriate molar excess (5-20eq) of chelator-linker (50mM in DMSO) is added to VHH-Fc (final concentration 2mg / mL), and the reaction is incubated in a Thermomixer at 25°C for 2h or overnight. After the reaction is complete, the sample is passed through a Zeba column (ThermoFisher, Cat#: 87770) according to the manufacturer's protocol to remove unreacted chelator-linker and buffer-exchanged into PBS (pH 7.4) (LifeTechnologies, Cat#: 10010-023). This VHH-Fc-chelator conjugate (VFCC) is stored at 4°C before analysis and purification.

[0427] Example 14. Purification of VHH-Fc-chelator conjugate (VFCC) using SEC

[0428] To remove high molecular weight substances (HMWS) and low molecular weight substances (LMWS), VHH-Fc was purified by SEC using an AKTA Pure FPLC system 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 and concentrated using a Microsep Advance centrifugal device (Pall 10 kMWCO, Cat#: MCP010C41). The concentrated sample was transferred to a 0.22 μm 0.5 mL Ultrafree-MC GV centrifugal filter (Millipore, Cat#: UFC30GV0S) and spun at 3,000 x g for 3 minutes.

[0429] Example 15. Protein Quantification

[0430] The VHH-Fc protein content was quantified using a Pierce BCA Protein Assay Kit (Thermo, Cat#: 23225) standardized with cetuximab (LIST / E:094822, DIN 02271249, 2 mg / mL).

[0431] Example 16. Chelator to VHH-Fc Ratio (CAR) Analysis

[0432] The chelator loading rate, described herein as CAR, can be analyzed by methods applicable in the field of antibody conjugates. For a review of these methods in the ADC context, see A Wakankar et al., mAbs 3:161 (2011). The CAR of each conjugate was analyzed by DG-SEC-MS.

[0433] The conjugates were analyzed by the deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this context, mass distributions were obtained after spectral deconvolution, enabling the calculation of the average CAR of the formulation.

[0434] The conjugates were analyzed by the deglycosylation and UPLC-Q-TOF procedures described in Example 11. In this context, mass distributions were obtained after spectral deconvolution, enabling the calculation of the average CAR of the formulation.

[0435] Example 17. Binding of VHH-Fc Conjugates to Cells Expressing Target Proteins

[0436] In some cases, conjugation can have a negative impact on the binding of VHH-Fc to the target protein. Thus, similar to that described above, the binding of the VHH-Fc conjugate was tested. Table 8 shows the cell binding data of the VHH-Fc chelator conjugate.

[0437]

[0438]

[0439] As observed in Table 8, binding was observed for both long and short DOTA linkers. Also as shown in Table 8, binding was also observed in the case of increasing chelator VHH-Fc ratios (CAR).

[0440] Example 18. Percent integrity analysis

[0441] The percentage of intact immunoconjugate was determined by HPLC-SEC. 12 μL of the conjugate was added to a glass insert in a standard HPLC vial. 10 μL of the sample was injected onto an Agilent HPLC-SEC with a Wyatt Technology WTC-050S5 SN:0429BNWBD129 column and eluted with 1x PBS (100%) at a flow rate of 0.5 mL / min for 40 min.

[0442] Example 19. Endotoxin level determination

[0443] According to the manufacturing protocol, the endotoxin test was performed using Wako's Limulus Amebocyte Lysate PyrostarTM ES-F single test (Cat#: WPESK-0015). Based on the maximum injection dose designed for each animal in the study, the QC cut-off was set simultaneously in accordance with appropriate animal care and FDA guidelines.

[0444] Example 20. Radiolabeling with In-111

[0445] Each of 40 μg of the 4 test articles was diluted to 100 μL in 500 μL lo-bind Eppendorf tubes with 0.1 M ammonium acetate buffer and 18 - 25 μL (20 - 22 MBq) [111In]InCl3 was added and mixed with a pipette. The reaction mixture was incubated in an incubator at 37 °C for 1 hour. Then the tubes were transferred to a 4 °C refrigerator.

[0446] The incorporation of the radionuclide was determined by spotting 0.5 μL of the sample at the starting point of a 1.5 x 10 cm iTLC strip. The strip was then placed in a 50 mL Falcon tube containing 2 mL of mobile phase (0.1 M sodium acetate buffer, pH 5, containing 25 mM EDTA) until the solvent reached the top of the strip. The strip was removed and exposed to...

Claims

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

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

3. An immunoconjugate comprising a multivalent antibody and a chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula I: A - B - C Wherein: 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 chelator, wherein the multivalent antibody comprises a polypeptide having a structure of Formula II: A - C - B Wherein: A comprises a first antigen-binding domain; B comprises a second antigen-binding domain; and C comprises an Fc domain.

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

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

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

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

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

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

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

12. The immunoconjugate according to any one of claims 1 to 11, wherein the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected, according to EU numbering, from: (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) 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; (lll) A330L; (mmm) P331A or P331S; or (nnn) E233P; (ooo) L234A, L235E, G237A, A330S, and P331S; or (ppp) any combination of (a)-(ppp).; 13. The immunoconjugate according to any one of claims 2 to 11, wherein the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, and P331S according to EU numbering.

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

15. The immunoconjugate according to any one of claims 2 to 14, wherein the alteration of one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to the following amino acid residues selected according to EU numbering: I253A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof.

16. The immunoconjugate according to any one of claims 2 to 14, wherein the alteration of one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to the following amino acid residues selected according to EU numbering: I253A, S254A, H310A, H435Q, Y436A, and combinations thereof.

17. The immunoconjugate according to any one of claims 2 to 14, wherein the alteration of one or more amino acid residues that alters the binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is directed to the following amino acid residues selected according to EU numbering: I253A, H310A, H435Q, and combinations thereof.

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

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

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

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

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

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

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

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

27. The immunoconjugate according to 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 immunoconjugate according to any one of claims 1 to 27, wherein the first antigen-binding domain binds to FOLR1 and comprises: Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO: 1; complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO: 2; and complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:

3.

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

513.

30. The immunoconjugate according to any one of claims 1 to 26, wherein the first antigen-binding domain binds to FOLR1 and comprises: Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:1; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:2; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:3, and wherein said second antigen-binding domain binds DLL3 and comprises: Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:5; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:6; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:7; Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:107; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:110; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:113; Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:207; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:210; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:213; Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:307; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:310; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:313; Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:407; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:410; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:413; or Complementary determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:507; Complementary determining region (CDR) 2 comprising the amino acid sequence shown in SEQ ID NO:510; and Complementary determining region (CDR) 3 comprising the amino acid sequence shown in SEQ ID NO:

513.

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

4.

32. The immunoconjugate according to any one of claims 1 to 31, wherein the first antigen-binding domain or the second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 8, 101-106, 201-206, 301-306, 401-406 or 501-506.

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

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

35. The immunoconjugate according to any one of claims 1 to 33, wherein the chelator is an α-emitter chelator.

36. The immunoconjugate according to any one of claims 1 to 33, wherein the chelator is a β-emitter chelator or a γ-emitter chelator.

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

38. The immunoconjugate according to any one of claims 1 to 37, wherein the chelator is selected from: 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, desferrioxamine, DFO* and combinations thereof.

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

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

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

42. The immunoconjugate according to any one of claims 1 to 37, wherein the chelator is directly conjugated to the antigen-binding region and / or the Fc domain.

43. The immunoconjugate according to any one of claims 1 to 37, wherein the chelator is conjugated to the antigen-binding region and / or the Fc domain through a linker.

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

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

46. An immunoconjugate comprising a multivalent antibody, a chelator, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula I: A-B-C Wherein: A comprises a first VHH domain; B comprises a second VHH domain; and C comprises an Fc domain, wherein the first VHH domain binds to FOLR1 or DLL3; and wherein the second VHH domain binds to FOLR1 or DLL3.

47. An immunoconjugate comprising a multivalent antibody, a chelator, and a radioisotope, wherein the multivalent antibody comprises a homodimer of a polypeptide having the structure of Formula II: A-C-B Wherein: A comprises a first VHH domain; B comprises a second VHH domain; and C comprises an Fc domain, wherein the first VHH domain binds to FOLR1 or DLL3; and wherein the second VHH domain binds to FOLR1 or DLL3.

48. The immunoconjugate according to claim 45, wherein the radioisotope is an α emitter.

49. The immunoconjugate according to claim 48, wherein the radioisotope is an α emitter selected from: 225-Ac, 223-Ra, 224-Ra, 227-Th, 212-Pb, 212-Bi, and 213-Bi.

50. The immunoconjugate according to any one of claims 48 or 49, wherein the radioisotope is 225-Ac.

51. The immunoconjugate according to claim 48, wherein the radioisotope is a β emitter.

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

53. A method of killing tumor cells or cancer cells, the method comprising: contacting the tumor cells or cancer cells with the immunoconjugate according to any one of claims 1 to 52, thereby killing the tumor cells or cancer cells.

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

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

56. A method of treating cancer or a tumor in an individual, the method comprising administering to the individual an immunoconjugate according to any one of claims 1 to 52, thereby treating the cancer or the tumor.

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

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

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

60. The method according to any one of claims 56 to 59, which comprises administering to the individual from 0.5 μCi to 30.0 μCi per kilogram.

61. The method according to any one of claims 56 to 60, which comprises administering to the individual from 10 mCi to 75 mCi per square meter of body surface area.

62. The method according to any one of claims 56 to 61, wherein the cancer or tumor expresses the antigen to which the immunoconjugate specifically binds.

63. Use of an immunoconjugate according to any one of claims 1 to 52 for the method of treating cancer or a tumor in an individual.

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

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

66. The method according to any one of claims 64 or 65, wherein the cancer cells or tumor cells comprise lung cancer cells, breast cancer cells, ovarian cancer cells, or neuroendocrine cancer cells.

67. The method according to any one of claims 64 to 66, wherein the cancer cells or tumor cells express the antigen to which the immunoconjugate specifically binds.

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

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

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

71. The method according to any one of claims 68 to 70, wherein the tumor expresses the antigen to which the immunoconjugate specifically binds.

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

73. An expression vector comprising the nucleic acid according to claim 72.

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

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