Her2 binding agents and uses thereof

Abstract

In some embodiments, HER2 binding agents are provided that comprise a VHH domain that specifically binds to HER2. Also provided are nucleic acids encoding the proteins, vectors and host cells comprising the nucleic acids, and methods of making and using the HER2 binding agents. The present disclosure also provides HER2 binding agent conjugates for therapeutic and / or diagnostic purposes, wherein a protein is conjugated to a detectable marker and HER2 binds to a targeted radiotherapy agent. Also provided are methods of using such HER2 binding agents to detect, monitor and / or treat diseases and conditions, such as cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 344,808, filed on May 23, 2022. The disclosure of this priority application is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The electronic copy of the sequence listing created on May 23, 2023 is named 122878.WO012.xml and is 271,281 bytes in size. Background of the Invention

[0006] Significant progress has been achieved in the development and optimization of HER2-targeted therapies for breast cancer. Examples of FDA-approved drugs with significant clinical efficacy for metastatic disease include the anti-HER2 antibody trastuzumab, the anti-HER2-HER3 dimerization inhibitor pertuzumab, and the antibody-drug conjugate T-DM1 (Krop et al., Lancet Oncol. (2014) 15 (7): 689-99; Slamon et al., NEJM (2001) 344: 783-92). However, many limitations and drug resistance have compromised the effectiveness of these targeted therapies in HER2. + Efficacy in breast cancer, and they are not suitable for treating most breast cancers in which HER2 is weakly overexpressed.

[0007] Brain metastasis is a devastating progression of breast cancer, with an incidence as high as 30-55% reported in patients with HER2-positive breast cancer. Brain metastasis is a significant source of morbidity in patients with HER2-positive breast cancer, with up to 50% of patients dying as a direct consequence of CNS disease progression. + Treatment options for breast cancer brain metastasis (BCBM) are limited, and due to insufficient penetration of antibodies through the blood-brain barrier (BBB), the same anti-HER2 therapy that slows growth throughout the body generally does not control brain metastasis (Lampson, mAbs (2011) 3: 153-60). In addition, clinical data reveal an increased incidence of HER2-positive breast cancer brain metastasis after adjuvant trastuzumab therapy (Olson et al., Ann Oncol. (2013) 24: 1526-33).

[0008] Furthermore, despite adequate drug delivery, the efficacy of small molecules against breast cancer brain metastases is very limited and can only be slightly increased by the addition of further therapeutic modalities (Lin et al., J Clin Oncol. (2008) 26:1993-9; Lin et al., Clin Cancer Res. (2009) 15:1452-9; Bachelot et al., Lancet Oncol. (2013) 14:64-71).

[0009] Hyperproliferative disorders such as cancers characterized by overexpression of HER2 (known as HER2 + Cancer) usually has a poor prognosis or is resistant to many standard therapies. Therefore, new therapies that effectively treat cancers such as HER2-positive cancers and metastatic HER2-positive cancers are needed. The present disclosure meets these needs and also provides other advantages, including the ability to selectively deliver HER2 targeting agents to HER2-positive tumors in the central nervous system without damaging normal brain tissue. Summary of the invention

[0010] The present disclosure provides novel HER2 (human epidermal growth factor receptor 2) binding VHH domains and solves the problems related to the treatment of HER2 + Cancer (including HER2 + The present disclosure provides radiolabeled targeted HER2 binding agents that can be taken up and retained by tumor cells while reducing the amount of radionuclides taken up by normal cells, particularly the kidney, which has been found to be the greatest toxicity limiting problem for targeted radiotherapeutics. The present disclosure includes HER2 binding agents that are radiolabeled with radiohalogen atoms in a manner that minimizes the loss of radiohalogens due to dehalogenation in vivo, and have been found to maintain the biological activity of HER2 binding VHH domains after in vivo administration, maximize retention in diseased cells such as cancer cells, and minimize radioactive retention in normal tissues.

[0011] The present disclosure provides a HER2 binding agent comprising a VHH domain that specifically binds to HER2. Also provided are nucleic acids encoding HER2 binding agents, vectors and host cells comprising nucleic acids, and methods for preparing and using HER2 binding agents. The present disclosure also provides a HER2 binding agent containing a detectable label and a radiotherapy agent targeted by a HER2 binding agent for treatment and / or diagnosis purposes. Also provided are methods for detecting, monitoring and / or treating diseases and conditions such as cancer using HER2 binding agents.

[0012] In some embodiments, the present disclosure relates to:

[0013] [1] A HER2-binding agent comprising a HER2-binding VHH domain, wherein the VHH domain comprises:

[0014] (a) a complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 82-101; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-123; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-157 and 206;

[0015] (b) CDR1, CDR2 and CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 contained in the VHH sequences disclosed in Table 2;

[0016] (c) the VHH amino acid sequences disclosed in Table 2;

[0017] (d) a VHH sequence selected from SEQ ID NOs: 1-81 and 205;

[0018] (e) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VHH sequence disclosed in Table 2;

[0019] (f) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a VHH sequence selected from SEQ ID NOs: 1-81 and 205;

[0020] (g) an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 additions, substitutions or deletions compared to the reference VHH sequence disclosed in Table 2; or

[0021] (h) an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 additions, substitutions or deletions compared to a reference VHH sequence selected from SEQ ID NOs: 1-81 and 205;

[0022] [2] The HER2-binding agent according to [1], wherein the VHH domain comprises:

[0023] (a) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 135 or 136;

[0024] (b) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 135;

[0025] (c) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 136;

[0026] (d) a CDR1 comprising SEQ ID NO: 82; a CDR2 comprising SEQ ID NO: 102; and a CDR3 comprising SEQ ID NO: 124, 145, 146 or 147;

[0027] (e) a CDR1 comprising SEQ ID NO:83; a CDR2 comprising SEQ ID NO:103; and a CDR3 comprising SEQ ID NO:125;

[0028] (f) a CDR1 comprising SEQ ID NO:84; a CDR2 comprising SEQ ID NO:104; and a CDR3 comprising SEQ ID NO:126;

[0029] (g) a CDR1 comprising SEQ ID NO:85; a CDR2 comprising SEQ ID NO:105; and a CDR3 comprising SEQ ID NO:127;

[0030] (h) a CDR1 comprising SEQ ID NO: 86; a CDR2 comprising SEQ ID NO: 106; and a CDR3 comprising SEQ ID NO: 128, 130, 134 or 155;

[0031] (i) a CDR1 comprising SEQ ID NO: 87; a CDR2 comprising SEQ ID NO: 117; and a CDR3 comprising SEQ ID NO: 129;

[0032] (j) a CDR1 comprising SEQ ID NO:88; a CDR2 comprising SEQ ID NO:118; and a CDR3 comprising SEQ ID NO:130;

[0033] (k) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 131 or 157;

[0034] (1) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108; and a CDR3 comprising SEQ ID NO: 131;

[0035] (m) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 156;

[0036] (n) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 157;

[0037] (o) a CDR1 comprising SEQ ID NO:90; a CDR2 comprising SEQ ID NO:109; and a CDR3 comprising SEQ ID NO:132;

[0038] (p) a CDR1 comprising SEQ ID NO:91; a CDR2 comprising SEQ ID NO:119; and a CDR3 comprising SEQ ID NO:137;

[0039] (q) a CDR1 comprising SEQ ID NO:92; a CDR2 comprising SEQ ID NO:120; and a CDR3 comprising SEQ ID NO:138;

[0040] (r) a CDR1 comprising SEQ ID NO:93; a CDR2 comprising SEQ ID NO:113 or 114; and a CDR3 comprising SEQ ID NO:139;

[0041] (s) a CDR1 comprising SEQ ID NO:93, a CDR2 comprising SEQ ID NO:114, and a CDR3 comprising SEQ ID NO:206;

[0042] (t) a CDR1 comprising SEQ ID NO:94; a CDR2 comprising SEQ ID NO:115; and a CDR3 comprising SEQ ID NO:140 or 149;

[0043] (u) a CDR1 comprising SEQ ID NO:95; a CDR2 comprising SEQ ID NO:121; and a CDR3 comprising SEQ ID NO:141;

[0044] (v) a CDR1 comprising SEQ ID NO:96; a CDR2 comprising SEQ ID NO:122; and a CDR3 comprising SEQ ID NO:142;

[0045] (w) a CDR1 comprising SEQ ID NO:97; a CDR2 comprising SEQ ID NO:116; and a CDR3 comprising SEQ ID NO:150;

[0046] (x) a CDR1 comprising SEQ ID NO: 98; a CDR2 comprising SEQ ID NO: 107; and a CDR3 comprising SEQ ID NO: 144, 151, 152 or 153;

[0047] (y) a CDR1 comprising SEQ ID NO: 99; a CDR2 comprising SEQ ID NO: 109; and a CDR3 comprising SEQ ID NO: 132 or 148; or

[0048] (z) a CDR1 comprising SEQ ID NO: 100; a CDR2 comprising SEQ ID NO: 110; and a CDR3 comprising SEQ ID NO: 133 or 154; or

[0049] The HER2-binding agent according to [1], wherein the VHH domain comprises:

[0050] (aa) comprises CDR1 of SEQ ID NO:97; CDR2 of SEQ ID NO:123; and CDR3 of SEQ ID NO:143; or

[0051] (ab) CDR1 comprising SEQ ID NO:101; CDR2 comprising SEQ ID NO:111; and CDR3 comprising SEQ ID NO:136.

[0052] [3] The HER2-binding agent of [1] or [2], wherein the HER2-binding agent is monovalent;

[0053] [4] The HER2-binding agent of [1] or [2], wherein the HER2-binding agent is multivalent (e.g., bivalent, trivalent, or tetravalent);

[0054] [5] The HER2-binding agent of any one of [1] to [4], wherein the HER2-binding agent is monospecific;

[0055] [6] The HER2-binding agent of any one of [1], [2] or [4], wherein the HER2-binding agent is multispecific and specifically binds to HER2 and different antigens;

[0056] [7] The HER2-binding agent of any one of [1]-[6], wherein the VHH domain is humanized;

[0057] [8] The HER2-binding agent of any one of [1] to [7], wherein the HER2-binding agent comprises a fusion protein comprising a HER2-binding VHH domain and a heterologous sequence;

[0058] [9] The HER2-binding agent of any one of [1] to [8], wherein the HER2-binding agent comprises an N-terminal amino acid residue or polypeptide sequence, optionally wherein the N-terminal residue or sequence improves protein expression, solubility, purification, half-life or affinity, the N-terminal residue or sequence such as Met-Ala or an Ala leader peptide;

[0059]

[10] The HER2-binding agent of any one of [1]-[9], wherein the HER2-binding agent comprises a C-terminal amino acid residue or polypeptide sequence, optionally wherein the C-terminal residue or sequence promotes radiolabel selectivity, loading, cellular internalization, half-life or affinity;

[0060]

[11] The HER2 binding agent of any one of [1] to

[10] , wherein the HER2 binding agent comprises a polypeptide sequence having the following structure:

[0061] VHH-(Gly n XA m Gly k ) j

[0062] [i] wherein the VHH is a VHH disclosed in Table 2 or elsewhere herein (e.g., a VHH selected from SEQ ID NOs: 1-81 and 205),

[0063] [ii] n = 0-5,

[0064] [iii] m = 0-5,

[0065] [iv] k = 0-6,

[0066] [v]j=0-8, and

[0067] [vi] Xaa is an amino acid that allows for site-specific conjugation of a radiolabeled prosthetic group or allows for direct radiolabeling;

[0068]

[12] The HER2-binding agent of any one of [8]-

[11] , wherein the fusion protein comprises Fc;

[0069]

[13] The HER2-binding agent of any one of [8]-

[11] , wherein the fusion protein is a chimeric antigen receptor (CAR);

[0070]

[14] An isolated nucleic acid encoding the HER2 binding agent of any one of [1]-

[13] ;

[0071]

[15] A vector comprising the nucleic acid of

[14] , optionally wherein the vector is an expression vector;

[0072]

[16] A host cell comprising the nucleic acid of

[14] or the vector of

[15] ;

[0073]

[17] The host cell of

[16] , wherein the host cell is a eukaryotic cell such as a mammalian cell or a yeast cell or a prokaryotic cell;

[0074]

[18] The HER2-binding agent of any one of [1]-

[13] , wherein the HER2-binding agent is conjugated to a label;

[0075]

[19] The HER2 binding agent of

[18] , wherein the label is a fluorescent dye, a radionuclide, an enzyme, a toxin or a chemotherapeutic agent;

[0076]

[20] The HER2 binding agent of

[18] or

[19] , wherein the label is a radionuclide;

[0077]

[21] The HER2 binding agent of

[19] or

[20] , wherein the radionuclide is a radioactive halogen isotope;

[0078]

[22] The HER2 binding agent of

[19] or

[20] , wherein the radionuclide is a radioactive halogen isotope selected from the following:

[0079] a) 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I; or

[0080] b) 75 Br, 77 Br, 122 I. 124 I. 125 I. 131 I and 211 At;

[0081]

[23] The HER2 binding agent of

[19] or

[20] , wherein the radionuclide is a radioactive metal isotope;

[0082]

[24] The HER2 binding agent of

[19] or

[20] , wherein the radionuclide is a radioactive metal isotope selected from the following:

[0083] a) 44 Sc, 45 Ti, 51 Cr, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 68 Ge, 75 Se, 82 Sr. 86 Y.99 Mo, 99m Tc, 110m In, 111 In, 166 Ho, 186 Re, 195m Pt and 201 Tl; or

[0084] b) 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89 Zr, 90 Y. 111 In, 153 Sm, 149 Tb, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th and 227 Th;

[0085]

[25] The HER2-binding agent of any one of [1]-

[13] and

[18] -

[24] , wherein the HER2-binding agent is conjugated to a label via a chelating moiety;

[0086]

[26] The HER2 binder of

[25] , wherein the chelating moiety is covalently linked to the protein via a lysine or cysteine ​​residue;

[0087]

[27] The HER2 binding agent of

[25] or

[26] , wherein the marker forms a complex with the metal, and wherein the complex is chelated by a chelating moiety;

[0088]

[28] The HER2 binding agent of any one of [1]-

[12] and

[18] -

[27] , wherein the HER2 binding agent is a targeted radiotherapy agent having the following structure:

[0089] VHH-RLC,

[0090] in

[0091] VHH is a protein comprising a HER2-binding VHH domain provided herein (e.g., having a VHH amino acid sequence as shown in Table 2, e.g., a VHH comprising an amino acid sequence selected from SEQ ID NOs: 1-81 and 205, or a derivative thereof), and

[0092] RLC is a radiolabeled chemical used to facilitate the attachment of a radionuclide to a VHH, either directly or indirectly;

[0093]

[29] The HER2 binding agent of

[28] , wherein the targeted radiotherapy agent comprises a prosthetic compound or a radioactive halogen precursor represented by Formula 2:

[0094] MC-Cm-L4-Cm-T

[0095] (Formula 2),

[0096] in:

[0097] MC is a multidentate metal chelating moiety;

[0098] Cm is thiourea, amide or thioether;

[0099] L4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain, optionally having NH, CO or S at one or both ends;

[0100] T is a compound of any of the following:

[0101] (a) a compound in the form of a prosthetic compound or a radioactive halogen precursor represented by Formula 1:

[0102]

[0103] in:

[0104] X is CH or N;

[0105] L1 and L3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain;

[0106] MMCM is the polymer conjugation moiety;

[0107] L2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain comprising at least three oxygen atoms, wherein L2 optionally comprises a brush border enzyme-cleavable peptide;

[0108] CG is selected from guanidine; PO3H; SO3H; one or more charged D-amino acids or L-amino acids selected from arginine, phosphono / sulfophenylalanine, glutamic acid, aspartic acid, and lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and Z-guanidine;

[0109] Z is (CH2) n ;

[0110] n is greater than 1;

[0111] m is 0 to 3; and

[0112] Y is an alkyl metal moiety, a boronic acid moiety, a boronic ester moiety or a moiety selected from 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 131 I and 211 A radioactive halogen selected from the group consisting of At, or a pharmaceutically acceptable salt or solvate thereof;

[0113] b) the compound of a), wherein the compound is a radiohalogen precursor, optionally wherein Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), trimethylsilyl (SiMe3), boronic acid (B(OH)2) or boronic ester (B(OR)2), and optionally wherein R comprises a cyclic moiety or an aliphatic group;

[0114] (c) The compound of (a), wherein the compound is a prosthetic compound, and wherein Y is a radiohalogen selected from the group consisting of: 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 131 I and 211 At;

[0115] (d) The compound of (a), wherein MMCM is an active ester or (Gly)m, wherein m is 1 or greater;

[0116] (e) The compound of (a), wherein the MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS) ester, tetrafluorophenol (TFP) ester, pentafluorophenol (PFP), p-nitrophenol (PNP), an isothiocyanate group or a maleimide group;

[0117] (f) The compound of (a), wherein MMCM is Gly-Gly-Gly;

[0118] (g) The compound of (a), wherein L2 is (CH2) P , where p = 1 to 6;

[0119] (h) The compound of (a), wherein the optional brush border enzyme cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys;

[0120] (i) The compound of (a), which is represented by the following structure:

[0121]

[0122] (j) The compound of i), wherein the compound comprises N-succinimidyl 3-guanidinomethyl-5-[ 131 I] iodobenzoate (isoSGMIB), or N-succinimidyl 3-[ 211 At]astato-5-guanidinomethylbenzoate (isoSAGMB), or maleimidoethyl 2-(guanidinomethyl)-5-iodobenzoate (isoMEGMIB), or maleimidoethyl 3-(guanidinomethyl)-5-astatobenzoate (isoMEGMAB);

[0123] or a pharmaceutically acceptable salt or solvate thereof;

[0124]

[30]

[29] HER2 binder, wherein MC is a macrocyclic structure;

[0125]

[31] The HER2 binder of

[30] , wherein MC is selected from DOTA, TETA, NOTP and NOTA;

[0126]

[32]

[30] The HER2 binder, wherein MC is an acyclic multidentate ligand;

[0127]

[33] The HER2 binder of

[30] , wherein MC is selected from EDTA, EDTMP and DTPA;

[0128]

[34] The HER2 binding agent of

[30] , wherein the compound is a radiohalogen precursor, optionally wherein Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), trimethylsilyl (SiMe3), boronic acid (B(OH)2) or boron ester (B(OR)2), and optionally wherein R comprises a cyclic moiety or an aliphatic group;

[0129] (c) The HER2-binding agent of (a), wherein the compound is a prosthetic compound, and wherein Y is a radiohalogen selected from: 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I.125 I. 131 I and 211 At;

[0130]

[36] The HER2-binding agent of

[30] , further comprising a metal associated with the MC;

[0131]

[37] The HER2 binding agent of

[30] , wherein the metal is a radioactive metal selected from the following: 177 Lu, 64 Cu, 67 Cu, 111 In, 90 Y. 225 Ac, 212 Bi, 213 Bi, 153 Sm, 166 Ho, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 ;

[0132]

[38] A pharmaceutical composition comprising the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] , and a pharmaceutically acceptable carrier;

[0133]

[39] A method for treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] ;

[0134]

[40] The method of

[39] , wherein the disease is cancer.

[0135]

[41] A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] ;

[0136]

[42] A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the targeted radiotherapy agent of any one of

[28] -

[37] ;

[0137]

[43] The method of any one of

[40] -

[42] , wherein the cancer is HER2 + ;

[0138]

[44] Treatment of HER2 +A method for treating cancer, comprising administering to a subject in need thereof an effective amount of a HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or a pharmaceutical composition of

[38] ;

[0139]

[45] The method of any one of

[40] -

[44] , wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial carcinoma, and epidermal-derived cancers and / or metastases arising therefrom;

[0140]

[46] The method of any one of

[40] -

[45] , wherein the cancer is breast cancer and / or metastasis originating therefrom;

[0141]

[47] The method of

[46] , wherein the cancer is breast cancer brain metastasis;

[0142]

[48] ​​The method of any one of

[40] -

[47] , wherein the subject is scored for HER2(2+) or HER2(3+) by immunohistochemistry;

[0143]

[49] Killing HER2 + A method for treating a cell, comprising contacting the cell with the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] ;

[0144]

[50] The method of

[49] , wherein the cell is a cancer cell;

[0145]

[51] The method of

[50] , wherein the cancer cell is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial carcinoma, and epidermal-derived cancers and / or metastases arising therefrom;

[0146]

[52] The method of any one of

[49] -

[51] , wherein the cell is contacted with a HER2-binding agent in vitro or in vivo;

[0147]

[53] The HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] , or the pharmaceutical composition of

[38] , for use in medicine (e.g., in the method of any one of

[39] -

[52] );

[0148]

[54] Use of the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] in the preparation of a medicament for treating a disease in a subject in need thereof (e.g., in the method of any one of

[39] -

[52] );

[0149]

[55] The use of

[54] , wherein the disease is cancer;

[0150]

[56] A method for diagnosing a disease or medical condition in a subject, comprising administering to the subject the HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] ;

[0151]

[57] The method of

[56] , wherein the disease is cancer;

[0152]

[58] Detection of HER2 in subjects + A method for a cell, comprising:

[0153] a) administering to a subject a detectably labeled HER2 binding agent of any one of [1]-13] and

[18] -

[37] or the pharmaceutical composition of

[38] , wherein the HER2 binding agent is detectably labeled; and

[0154] b) detecting the presence of a labeled HER2 binding agent and HER2 in a subject + Binding to cells, where detection of binding indicates the presence of HER2 + cell;

[0155]

[59]

[58] , wherein the labeled HER2 binding agent is detected and the HER2 in the subject + The combination of cells includes imaging HER2+ cells in the subject;

[0156]

[60] A method for imaging HER2+ cells in a subject, comprising administering to the subject a detectably labeled HER2 binding agent of any one of [1]-

[13] and

[18] -

[37] or the pharmaceutical composition of

[38] , wherein the HER2 binding agent is detectably labeled;

[0157]

[61] The method of

[60] , wherein imaging HER2+ cells in the subject comprises performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject;

[0158]

[62] The method of any one of

[58] -

[61] , wherein HER2 + Cells are HER2 + cancer cell;

[0159]

[63] The method of any one of

[39] -

[48] and

[54] -

[62] , wherein the subject is a mammal, such as a human or a non-human primate;

[0160]

[64] The method of any one of

[39] -

[48] and

[54] -

[63] , wherein the subject has cancer;

[0161]

[65] A method for preparing a HER2 binding agent, comprising:

[0162] (a) culturing the host cell of

[16] or

[17] under conditions where a HER2 binding agent is produced; and

[0163] (b) recovering the HER2-binding agent produced by the host cell.

[0164] Other features, objects and advantages of the present invention are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating embodiments and aspects of the present invention, is given only by way of illustration and not limitation. From the detailed description, various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figures 1A-1J is a series of graphs depicting complete ELISA affinity binding curves of exemplary VHH sequences against the human HER2 receptor following protein expression and purification.

[0167] Figure 2A-2I is a series of graphs depicting the specificity of exemplary VHH proteins for other receptors within the ErbB family.

[0168] Figures 3A-3E is a series of graphs depicting the cross-reactivity of exemplary VHH proteins with cross-species HER2 receptors found in other animals.

[0169] Figures 4A-4B is a pair of graphs depicting competitive inhibition of exemplary VHH proteins with (A) Trastuzumab or (B) Pertuzumab in an indirect ELISA assay format.

[0170] Figures 5A-5E Involving use in 131 In vitro cell assays evaluating VHH_128 after radiolabeling with [I]-isoSGMIB. (A) Radiolabeled construct. (B) Affinity for SKOV-3 ovarian cancer cells. (C) Surface binding or (D) internalization of [I]-isoSGMIB in SKOV-3 cells after 2 h incubation. 131 (E) Measurement of [I]-isoSGMIB-VHH128 levels in SKOV-3 cells in the presence of 100-fold trastuzumab 131 I] Internalization.

[0171] Figure 6is a series of graphs depicting the expression of KOV-3 xenograft tumors in female athymic mice. 131 Tissue accumulation levels of 1-isoSGMIB-VHH128 over 24 h.

[0172] Figure 7 is a series of graphs depicting the effects of BT-474 xenograft tumor model in female NOD SCID mice. 131 Tissue accumulation levels of 1-isoSGMIB-VHH128 over 24 h.

[0173] Figures 8A-8C Involving the use of 211 HER2-targeted alpha particle therapy with astatine-labeled VHH. (A) Using isoSAGMB 211 Scheme and structure of At-labeled VHH_128. (B)[ 211 Affinity binding curves of [At]-isoSAGMB-VHH128 against HER2-expressing BT-474 cells. (C) Accumulation levels in BT-474 tumors xenografted in athymic mice.

[0174] Figures 9A-9C This study involves radiolabeling HER2-targeting VHHs using maleimide-based radiolabeling chemistry. (A) Radiolabeling of HER2-targeting VHHs using isoMEGMIB 131 I-labeled scheme and structure of VHH_1041. (B) [ 131 (I) Affinity binding curves of -isoMEGMIB-VHH141 against HER2-expressing BT-474 cells. (C) In vitro cell binding and internalization ratios on BT-474 cells; E: efflux, S: surface bound, I: internalized.

[0175] Fig.10 is a series of graphs depicting the expression of [ 131 Tissue accumulation levels of I]-isoMEGMIB-VHH141 over 48 h. DETAILED DESCRIPTION OF THE INVENTION

[0177] Provided herein is a HER2 binding agent comprising a VHH domain that specifically binds to HER2. Also provided are nucleic acids encoding proteins, vectors and host cells comprising nucleic acids, and methods for preparing and using HER2 binding agents. The disclosure also provides HER2 binding agents containing detectable markers and HER2 binding agent-targeted radiotherapeutic agents for treatment and / or diagnosis purposes. Also provided are methods for detecting, monitoring and / or treating diseases and conditions such as cancer using HER2 binding agents.

[0178] In certain embodiments, the present disclosure relates to HER2 targeted radiotherapeutics. The provided targeted radiotherapeutics exhibit advantageous properties over conventional radiotherapeutics, including greater radionuclide stability in blood circulation, lower uptake and dose exposure to normal tissues including the kidney, higher binding affinity and cellular internalization, and + Greater tumor uptake in tumors and for radionuclides (including 131 Iodine and 211 Other features and advantages of the disclosed compositions and methods will be apparent from the following disclosure, drawings and claims.

[0179] definition

[0180] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the compositions provided, suitable methods and materials are described herein. Each publication, patent application, patent and other references mentioned in the present disclosure are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) will control. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0181] It should be understood that wherever embodiments are provided herein with the language "comprising," otherwise similar embodiments are provided described with the language "containing," "consisting of," and / or "consisting essentially of," etc. However, when used as a transitional phrase in a claim, each phrase should be interpreted individually and in the appropriate legal and factual context (e.g., in a claim, the transitional phrase "comprising" is considered more of an open-ended phrase, while "consisting of" is more exclusive, and "consisting essentially of" achieves a middle ground).

[0182] As used herein, the singular forms "a," "an," and "the" include plural referents unless explicitly stated or clear from the context that such is not intended.

[0183] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0184] As used herein, when applied to one or more values ​​of interest, the terms "about" and "approximately" refer to values ​​similar to the reference value. In some embodiments, the term "about" or "approximately" refers to a numerical range falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the reference value (greater than or less than), unless otherwise specified or obvious from the context (unless such numbers exceed 100% of possible values). For example, when used in the context of a percentage amino acid sequence identity ratio with a reference polypeptide sequence, "about" can mean + / -10% of the value.

[0185] Unless otherwise required, the use of any and all examples or exemplary language herein, such as "such as" or "including" is intended only to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0186] Where embodiments of the present disclosure are described in terms of Markush groups or other groups of alternatives, the disclosed compositions or methods encompass not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group in which one or more group members are absent. The disclosed compositions and methods also contemplate the explicit exclusion of one or more of any group members in the disclosed compositions or methods.

[0187] As used herein, the terms "isolated" and "purified" or their iterations refer to molecules that have been separated from at least some of the components with which they are normally found or produced in nature. For example, a protein is said to be "isolated" when it is separated from at least some of the components of the cell in which it is produced. When a protein is secreted by a cell after expression, physically separating the supernatant containing the protein from the cell in which it is produced is considered to be "isolated" the protein. Similarly, a nucleic acid is said to be "isolated" when it is not part of a larger nucleic acid in which it is normally found in nature (such as, in the case of a DNA nucleic acid, genomic DNA or mitochondrial DNA), or when it is separated from at least some of the components of the cell in which it is produced, such as in the case of an RNA nucleic acid. Thus, a DNA nucleic acid contained in a vector inside a host cell can be referred to as "isolated".

[0188] As used herein, the term "HER2" has its general meaning in the art, and refers to human epidermal growth factor receptor 2 (HER2) protein. HER2 is also referred to as Her-2, Her-2 / Neu, Neu, ErbB-2, CD340 (differentiation cluster 340) or p185, all of which are included in "HER2" herein. The extracellular domain of HER2 includes four domains, domain I (about 1-195 amino acid residues), domain II (about 196-319 amino acid residues), domain III (about 320-488 amino acid residues) and domain IV (about 489-630 amino acid residues) (residue numbering without signal peptide). See Garrett et al., Mol Cell. (2003) 11: 495-505, Cho et al., Nature (2003) 421: 756-60, Franklin et al., Cancer Cell (2004) 5: 317-28, Tse et al., Cancer Treat Rev. (2012) 38 (2): 133-42, or Plowman et al., Proc Natl Acad Sci. (1993) 90: 1746-50. In some embodiments, the HER2 binding agents provided herein bind to the extracellular domain of a HER2 protein having an amino acid sequence disclosed in the following Genbank numbers: NP_001005862, NP_001276865, NP_001276866, NP_001276867, and / or NP_004439 (as of January 1, 2020).

[0189] The terms VHH (variable domain of the heavy chain of a heavy chain antibody) and "single domain antibody" are used interchangeably herein to refer to a single antigen-binding antibody fragment consisting of a polypeptide sequence with three complementary determining regions (CDRs). The basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementary determining regions 1 to 3, respectively. VHH generally has a molecular weight of 12-15 kDa. The VHH binding domain of the HER2 binding agent provided herein can bind to HER2 alone without pairing with the corresponding CDR-containing polypeptide. Unless otherwise specified in the context, the term "VHH binding domain" is used herein to refer to full-length VHH, VHH binding fragments and / or VHH variants that bind to an antigen of interest and / or bind to an antigen of interest (e.g., HER2). The HER2 binding VHH domains provided can be engineered from any species, including but not limited to mice, humans, camels, llamas, alpacas, llamas, guanacos, sharks, goats, rabbits and / or cattle. In some embodiments, the VHH binding domains are derived from camelid species (e.g., camels, llamas, dromedaries, alpacas, llamas and guanacos).

[0190] Unless the context dictates otherwise, the terms "HER2-binding VHH", "HER2-binding VHH domain" and "VHH that specifically binds to HER2" are used interchangeably herein to refer to a HER2-binding VHH or a HER2-binding VHH domain.

[0191] The VHH binding domain of the "humanized" form is a binding domain containing a minimal sequence derived from a non-human immunoglobulin or a non-human germline sequence. In some embodiments, a humanized antibody is a human immunoglobulin (acceptor antibody), wherein the residues of the CDR (defined below) of the acceptor are replaced with CDR residues of non-human species such as mice, rats, rabbits, camels, llamas, alpacas or non-human primates (donor antibodies) with desired specificity, affinity and / or ability. In some cases, the framework ("FR") residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may be included in residues not found in the acceptor antibody or the donor antibody. These modifications may be performed to further improve antibody performance, such as binding affinity, isomerization, immunogenicity, radiolabeling efficiency, catabolism, enzymatic digestion, etc. The number of these amino acid substitutions in FR is generally no more than 6. As will be appreciated, a humanized sequence can be identified by its primary sequence, and does not necessarily represent the process of creating a VHH binding domain. Methods for humanizing VHH binding domains are well known in the art. Typically, the humanizing substitutions are chosen such that the resulting humanized VHH domain still retains the favorable properties of the HER2 binding agents of the present disclosure.

[0192] The terms "specific binding", "specific recognition" and "specific for..." are used interchangeably herein to refer to a measurable and reproducible interaction, such as the binding between a target and an antigen binding protein (such as HER2 and HER2 binding agents provided herein), which determines the presence of a target in the presence of a heterogeneous population of molecules including the target. For example, an antigen binding protein that specifically binds to a target (which may be an epitope) is an antigen binding protein that binds to this target with greater affinity, avidity, greater ease and / or longer duration than it binds to other targets. In some embodiments, the extent of binding of an antigen binding protein to an unrelated target is less than about 10% of the binding of an antigen binding protein (e.g., a HER2 binding agent) to a target (e.g., HER2), as measured, for example, by a radioimmunoassay (RIA). In some embodiments, when the K of the binding is greater than the K of the binding, the binding is greater than the K of the binding. D When the concentration is ≤1 μM, such as ≤100 nM or ≤10 nM, the antibody is said to specifically bind to the antigen. The terms "HER2 binding polypeptide", "HER2 binding protein" and "HER2 binding VHH domain" and variations thereof refer to polypeptides and VHH domains that specifically bind to HER2, respectively.

[0193] "Affinity" refers to the strength of the sum of non-covalent interactions between the binding interface of a ligand (e.g., a HER2 binding molecule or agent) and the binding site on its partner molecule (e.g., a HER2 receptor). The affinity or apparent affinity of a molecule X for its partner Y is often measured using the dissociation constant (K D ) or K D - Apparent expression. Affinity can also be expressed by its kinetics and binding rate (k on ) and its kinetic release rate (k off ) ratio indicates (K D =k off / k on Affinity can be measured by common methods known in the art (such as ELISA K D , KinExA, flow cytometry and / or surface plasmon resonance devices), including those described herein. Such methods include, but are not limited to, or flow cytometry. As used herein, the term " D " refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. As used herein, the term "K D "Including K D and K D -Appearance. In some embodiments, the K of the antigen binding molecule DMeasurements are made by flow cytometry using an antigen-expressing cell line (e.g., cells expressing HER2) and fitting the mean fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (GraphPad Prism Software). In some such embodiments, K D It's K D -Appearance.

[0194] An "affinity matured" VHH-HER2-binding domain refers to a VHH binding domain comprising an amino acid sequence having one or more changes in one or more CDRs and / or one or more FRs, compared to a reference parent VHH binding domain sequence without the changes, such changes resulting in an improvement in the affinity of the VHH-containing polypeptide for HER2. HER2 binder variants include affinity matured variants of the HER2 binding VHH domains provided herein.

[0195] As used herein, "variant" refers to a biologically active polypeptide, such as a HER2 binding VHH domain, having one or more amino acid additions, substitutions, insertions and / or deletions compared to the corresponding sequence of the HER2 binding VHH domain disclosed herein. In some embodiments, the variant is a biologically active polypeptide, such as a HER2 binding VHH domain, which has at least about 80% amino acid sequence identity with a reference amino acid sequence after aligning the sequence and introducing a gap (gap) when necessary to achieve the maximum percentage sequence identity and not considering any conservative substitution as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the HER2 binding domain provided herein. In some embodiments, the variant contains an amino acid sequence having at least about 80%, 90%, 95% or 97% amino acid sequence identity with a reference VHH binding domain. In some embodiments, the variant contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the VHH binding domain disclosed in Table 2. In some embodiments, the variant contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence disclosed in any one of SEQ ID NOs: 1-81 and 205. For purposes of determining percent amino acid sequence identity, alignment can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN. TM(DNASTAR) software. Exemplary computer programs for determining the identity between two sequences include GCG program packages (Devereux et al., Nucleic Acids Research (1984) 12: 387), BLAST, BLASTP (Karlin and Altschul, (1990) Proc Natl Acad Sci. USA 87 (6): 2264-8), BLASTN and FASTA (Atschul et al., J Molec Biol. (1990) 215: 403) using the default parameters of the corresponding programs. Certain embodiments of HER2 binding variants are provided herein.

[0196] The term "vector" is used to describe a nucleic acid that can be or has been engineered to contain a cloned polynucleotide sequence that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, a promoter and / or enhancer) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase). The terms "expression vector," "expression construct," or "expression cassette" are used interchangeably throughout this disclosure and are intended to include any type of genetic construct containing a nucleic acid encoding a gene product, wherein part or all of the nucleic acid coding sequence can be transcribed in a host cell.

[0197] "Host cell" refers to a cell that can be or has been a recipient of a vector and / or heterologous nucleic acid. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic host cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting mammalian host cells encompassed by the present disclosure include, but are not limited to, NSO cells, PER.C Host cells include 293 and CHO cells and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and progeny may not necessarily be identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations. Host cells include cells transfected in vivo with nucleic acids encoding the VHH binding domains provided herein.

[0198] As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that introduces antigen specificity into engineered cells (e.g., T cells, such as initial T cells, central memory T cells, effector memory T cells, or any combination thereof) via an antigen binding domain (e.g., HER2 disclosed herein in combination with VHH domains). This combines the antigen binding properties of the antigen binding domain with the T cell activity (e.g., lysis ability and self-renewal) of the T cell. CAR generally includes an extracellular antigen binding domain (extracellular domain), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain generally includes at least one ITAM signaling domain (e.g., derived from CD3ζ) and optionally at least one costimulatory signaling domain (e.g., derived from CD28 or 4-1BB). In the CAR provided herein, the HER2 provided herein in combination with VHH domains forms an antigen binding domain and is positioned extracellularly when expressed in cells.

[0199] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand (e.g., HER2), inducing or increasing cell death, inducing or increasing cell proliferation, inducing or increasing cellular internalization, inducing or increasing intracellular retention, and inducing or increasing expression of cytokines.

[0200] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to mammals, especially humans. Pharmaceutically acceptable carriers refer to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation adjuvant. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Supplementary biologically active agents (e.g., therapeutic agents) may also be incorporated into the composition.

[0201] The term "radionuclide" refers to a moiety that contains a radioactive isotope of at least one element. Examples of radionuclides that can be used in accordance with the present disclosure include 18 F. 35 S. 75 Br, 76 Br, 77 Br, 89 Zr, 99m Tc, 67Ga, 68 Ga, 64 Cu, 67 Cu, 52 Mn, 90 Y. 111 In, 177 Lu, 153 Sm, 166 Ho, 122 I. 123 I. 124 I. 125 I. 131 I. 211 At 212 Pb, 212 Bi, 213 Bi, 227 Th and 225 Ac. In some embodiments, the radionuclide is an alpha radionuclide (e.g., 211 At or 225 Ac). In some embodiments, the radionuclide is a beta radionuclide (e.g., 131 I or 177 Lu). In some embodiments, the radionuclide is a radiohalogen isotope or a radiometal isotope. In some embodiments, the radionuclide is a radiohalogen isotope. In some embodiments, the radionuclide is a radiohalogen isotope selected from: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I or 75 Br, 77 Br, 122 I. 124 I. 125 I. 131 I and 211 In certain embodiments, the radionuclide is 131 I. 211 At 125 I or 124 1. In some embodiments, the radionuclide is a radioactive metal isotope. In some embodiments, the radionuclide is a radioactive metal isotope selected from: 144 Sc, 45 Ti, 51 Cr, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 68 Ge, 75 Se, 82Sr. 86 Y. 99 Mo, 99m Tc, 110m In, 111 In, 166 Ho, 186 Re, 195m Pt and 201 Tl; or 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89 Zr, 90 Y. 111 In, 153 Sm, 149 Tb, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th and 227 Th. In certain embodiments, the radionuclide is 177 Lu, 131 I. 211 At or 225 Ac. In some embodiments, the radionuclide can be used for therapy. In certain embodiments, the radionuclide is selected from: 75 Br, 77 Br, 122 I. 124 I. 125 I. 131 I and 211 At; 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89 Zr, 90 Y. 111 In, 153 Sm, 149 Tb, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th and227 Th; or 32 P. In some embodiments, the radionuclide can be used for diagnosis. In certain embodiments, the radionuclide is selected from: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I; 44 Sc, 45 Ti, 51 Cr, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 68 Ge, 75 Se, 82 Sr. 86 Y. 99 Mo, 99m Tc, 110m In, 111 In, 166 Ho, 186 Re, 195m Pt and 201 or 11 C. 13 N. 15 O. 24 Na, 32 P and 133 Xe.

[0202] The term "prosthetic group" or "bifunctional labeling agent" refers to a small organic molecule that can be chemically linked to contain a radionuclide, which can be linked to a peptide or protein, for example, a protein containing a HER2 binding VHH domain. Radiolabeled prosthetic agents are compounds or groups that typically include a radiolabel, a charged group (CG), and a macromolecular conjugated moiety (MMCM), which are suitable for attachment to the protein component of the HER2 binding agent. Each of these components can optionally be associated with one or more cleavable (or non-cleavable) linkers.

[0203] The term "chelator ligand" or "chelator" as used herein with respect to radiopharmaceutical chemistries refers to a bifunctional chelator or conjugate (BFC) moiety that covalently links a radiolabeled prosthetic group to a biologically active targeting molecule (e.g., a peptide or protein, such as a protein comprising a HER2 binding VHH domain disclosed herein). BFCs utilize functional groups such as carboxylic acids or activated esters for amide coupling, isothiocyanates for thiourea coupling, and maleimides for thiol coupling.

[0204] The terms "label" and "detectable marker" refer to a polypeptide or portion attached to a protein, such as a HER2 binding VHH domain, that makes a reaction (e.g., binding) between members of a specific binding pair detectable. Thus, the term "labeled binding protein" refers to a protein with an incorporated label that provides identification of the binding protein. In some embodiments, the label is a detectable label that can produce a signal that can be detected by visual or instrumental means, such as an amino acid incorporated with a radioactive label or a biotinyl portion that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods) attached to a protein. Examples of labels for proteins include, but are not limited to, radioisotopes or radionuclides (e.g. 18 F. 35 S. 75 Br, 76 Br, 77 Br, 89 Zr, 99m Tc, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 52 Mn, 90 Y. 111 In, 177 Lu, 153 Sm, 166 Ho, 122 I. 123 I. 124 I. 125 I. 131 I. 211 At 212 Pb, 212 Bi, 213 Bi, 227 Th and 225 Ac); chromogen, fluorescent dye (e.g., FITC, rhodamine, lanthanide phosphors), enzyme label (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent marker; biotinyl; predetermined protein epitope recognized by secondary reporter (e.g., leucine zipper pair sequence, binding site of secondary antibody, metal binding domain, epitope tag); toxin, chemotherapeutic agent and magnetic agent, such as gadolinium chelate. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridinium compounds, and fluorescence-generating moieties, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable when reacted with yet another moiety.

[0205] The terms "subject", "patient", "animal" and "individual" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats and mice, and other animals. Animals include all vertebrates, such as mammals and non-mammals, such as chickens, amphibians and reptiles. As used herein, "mammal" refers to any member of the class mammalia, including but not limited to humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, and other members of the class mammalia. Patients can be male or female and can be of any suitable age, including infants, minors, teenagers, adults and elderly subjects. In some instances, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or condition such as cancer. In a specific embodiment, the disease or condition is HER2 + Cancer. In certain embodiments, the disease or condition is HER2 + Breast cancer or breast cancer brain metastasis. In certain embodiments, the disease or condition is HER2 + Ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, epidermal-derived cancers, and / or metastases arising therefrom (e.g., ovarian cancer brain metastases).

[0206] In some embodiments, the subject receiving treatment may be a patient designated by the fact that the subject has been identified as having a condition relevant to the treatment or being at sufficient risk of contracting the condition. In certain embodiments, the patient is a human.

[0207] The term "biological sample" refers to a quantity of a substance from an organism such as a mammal or a former organism. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, other cells, organs, tissues, bone marrow, lymph nodes, and spleen. In certain embodiments, the biological sample is derived from a tissue biopsy (e.g., HER2 + Tissue and / or cancerous tissue, such as breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, epidermal-derived cancers, and / or metastases arising therefrom (e.g., breast cancer brain metastases)).

[0208] As used herein, the term "treating", "treatment" or "therapy" of a disease, disorder or condition is a method for obtaining a beneficial or desired clinical outcome. As used herein, "treatment" covers any administration or application of disease therapy in mammals (including humans). For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, alleviating the extent of the disease, preventing or delaying disease spread (e.g., metastasis, such as metastasis to the brain, lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, inhibiting the disease or inhibiting disease progression, inhibiting or slowing the disease or its progression, stopping its development, and alleviating (partial or complete). So-called "treatment" also encompasses reducing the pathological consequences of proliferative diseases. The methods provided herein contemplate any one or more of these aspects of treatment. As with the above inhibition, the term "treatment" does not require 100% removal of all aspects of the disease.

[0209] The terms "treatment of a proliferative disorder," "treatment of a hyperproliferative disorder," and iterations thereof are used herein to include maintaining or stabilizing tumor size, inducing tumor regression (partial or complete), inhibiting tumor growth, and / or increasing the life span of a subject with a proliferative disorder. In some embodiments, the proliferative disorder is HER2 + Solid tumors. These tumors include HER2 + Cancer. In some embodiments, the tumor is associated with breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, epidermis-derived cancers, and / or metastases arising therefrom (e.g., breast cancer that has metastasized to the brain).

[0210] Unless otherwise indicated, the terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein and refer to cells that exhibit uncontrolled growth and / or abnormally increased cell survival and / or inhibition of apoptosis that interferes with the normal function of body organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Types of cancer that can be treated with the methods provided herein include solid tumors, disseminated metastatic disease, and brain metastases.

[0211] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used to treat one or more cancers. Exemplary classes of anticancer agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor ectodomain-Fc fusions, and antibodies), radiotherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0212] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a composition containing an active ingredient (e.g., a HER2 binding compound) that, when administered to a patient alone (i.e., as a monotherapy) or in combination with an additional therapeutic agent, produces a statistically significant reduction in disease progression (e.g., by improving or eliminating the symptoms and / or causes of the disease). An effective amount can be an amount that alleviates, reduces, or mitigates at least one symptom or biological response or effect associated with a disease or condition, prevents the progression of a disease or condition, or improves a patient's physical function. With regard to cancer, an effective amount of a drug or composition is sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down, and in some cases stop cancer cell infiltration into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and in some cases stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of a tumor; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. In some embodiments, an effective amount is an amount sufficient to prevent or delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. The effective amount can be administered in one or more administrations. The therapeutically effective amount of a composition containing an active agent can vary according to factors such as individual disease state, age, sex, and body weight and the ability of the active agent to elicit a desired response in an individual. The therapeutically effective amount is also an amount in which any toxic or deleterious effects of the active agent are offset by the beneficial effects of the treatment. The therapeutically effective amount can be administered in one or more administrations.

[0213] The disclosed HER2 binding agents and pharmaceutical compositions containing these agents can be administered alone (i.e., monotherapy) or in combination with another therapeutic agent (i.e., combination therapy). Administration "in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0214] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least a portion of the administration overlaps in time, or wherein the administration of one of the therapeutic agents falls within a short period of time relative to the administration of the other therapeutic agent, or wherein the therapeutic effects of the two therapeutic agents overlap for at least a period of time.

[0215] The term "sequentially" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0216] As used herein, "in combination" refers to administering another treatment modality in addition to one treatment modality. Thus, "in combination" refers to administering the other treatment modality before, during, or after one treatment modality is administered to an individual.

[0217] An "article of manufacture" is any article (e.g., a package or container) or kit that contains at least one agent, such as a drug described herein for treating a disease or condition described herein (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In some embodiments, the article or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0218] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0219] i. HER2 binders

[0220] The present disclosure provides HER2-binding agents comprising a protein comprising a HER2-binding VHH domain.

[0221] In some embodiments, provided HER2-binding agents bind to HER2 and have a ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M or ≤10 -11 The dissociation constant (K d In some embodiments, the HER2-binding agent binds to HER2 and has a K d Total 10 -7 Up to 10 -11 M or 10 -8 Up to 10 -10 M.

[0222] In some embodiments, provided HER2 binding agents bind HER2 with a KD of about 500 nM or less or 1 nm or less. In some embodiments, provided HER2 binding agents bind HER2 with a KD of about 0.002 / s or less. off Binds to HER2. In some embodiments, the HER2-binding agent has a K of about 500 nM or less. D and k of about 0.002 / s or less off Binds to HER2.

[0223] In some embodiments, the HER2 binding agent contains a VHH binding domain, and the VHH binding domain specifically binds to an epitope on a human protein that is conserved in HER2 proteins from different species. In some embodiments, the HER2 binding agent contains a VHH binding domain that specifically binds to HER2 domain I. In some embodiments, the HER2 binding agent contains a VHH binding domain that specifically binds to HER2 domain II. In certain embodiments, the VHH binding domain competes with trastuzumab for binding to HER2. In some embodiments, the HER2 binding agent contains a VHH binding domain that specifically binds to HER2 domain III. In some embodiments, the HER2 binding agent contains a VHH binding domain that specifically binds to HER2 domain IV. In certain embodiments, the VHH binding domain competes with pertuzumab for binding to HER2. In some embodiments, the HER2 binding agent contains two or more HER2 binding domains that specifically bind to different epitopes of HER2. In some embodiments, the HER2 binding agent contains a VHH binding domain that specifically binds to one HER2 domain and a VHH binding domain that specifically binds to different HER2 domains. In certain embodiments, a HER2 binding agent contains a VHH binding domain that specifically binds to HER2 domain II and a VHH binding domain that specifically binds to HER2 domain IV. In some embodiments, specific binding may include but does not require exclusive binding to HER2.

[0224] In some embodiments, the HER2 binding agent comprises an affinity-matured HER2 binding VHH domain. In some embodiments, the HER2 binding agent comprises a humanized VHH binding domain. In some embodiments, the HER2 binding agent comprises a protein consisting of a single polypeptide. In other embodiments, the HER2 binding agent contains more than one polypeptide.

[0225] In some embodiments, the HER2-binding agent comprises a VHH amino acid sequence disclosed in Table 2. In some embodiments, the HER2-binding agent comprises a VHH amino acid sequence selected from SEQ ID NOs: 1-81 and 205.

[0226] In some embodiments, the HER2 binding agent comprises a HER2 binding fragment and / or variant of the HER2 binding amino acid sequence provided herein. In some embodiments, the HER2 binding agent containing the fragment or variant amino acid sequence has a K of about 500 nM or less or about 1 nm or less. D In some embodiments, the HER2-binding agent binds to HER2 with a k of about 0.002 / s or less. off Binds to HER2. In certain embodiments, the HER2 binding agent has a K of about 500 nM or less or about 1 nm or less.D and k of about 0.002 / s or less off Binds to HER2.

[0227] In some embodiments, the HER2 binding agent comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the HER2 binding VHH domain provided herein. Also included is a sequence with at least 50%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity using the default parameters of the BLAST computer program provided by HGMP. In other embodiments, amino acid sequence identity is determined using a suitable sequence alignment algorithm and default parameters such as BLAST P (Karlin and Altschul, PNAS USA (1990) 87 (6): 2264-8).

[0228] In some embodiments, the HER2-binding agent comprises a VHH sequence disclosed in Table 2. In some embodiments, the HER2-binding agent comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VHH sequence disclosed in Table 2. In some embodiments, the HER2-binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2-binding agent comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205.

[0229] In some embodiments, the HER2-binding agent contains a variant protein comprising an amino acid sequence having one or more additions, insertions, substitutions and / or deletions compared to the VHH binding domains disclosed herein.

[0230] Amino acid substitutions include replacing one or more amino acids in a protein with one or more other amino acids, respectively. Exemplary substitutions are shown in Table 1. In some embodiments, conservative substitutions in the provided HER2 binding agent variants require exchanging the original residues in the VHH sequence disclosed in Table 2 with the amino acid residues in the corresponding exemplary substitution list in Table 1.

[0231] Table 1

[0232]

[0233] In addition, amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. In some embodiments, conservative substitutions require exchanging two members within one of these groups. In some embodiments, non-conservative substitutions require exchanging a member of one of these groups with a member from another group.

[0234] In some embodiments, the HER2-binding agent comprises one or more substitutions selected from the group consisting of: substitution of a Lys residue with an Arg or His residue; substitution of an unpaired Cys residue with an Ala or Ser residue; and substitution of a Glu residue with an Asn residue.

[0235] In some embodiments, the HER2 binding agent comprises an amino acid sequence with 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acid residues added, substituted and / or deleted compared to the reference HER2 binding VHH domain provided herein. Amino acid deletions or insertions may also be performed relative to the amino acid sequence of the HER2 binding VHH provided herein. Thus, for example, amino acids that have no substantial effect on the activity of the HER2 binding domain or at least do not eliminate such activity may be deleted. Such deletions may be advantageous because the total length and molecular weight of the polypeptide may be reduced while still retaining activity. This may enable the amount of protein required for a particular purpose to be reduced--for example, the dosage level may be reduced. In some embodiments, one or more amino acid residues are deleted from the amino terminus of the HER2 binding VHH domain. In some embodiments, one or more amino acid residues are deleted from the carboxyl terminus of the HER2 binding VHH domain. In some embodiments, one or more amino acid residues are deleted from both the amino terminus and the carboxyl terminus of the HER2 binding VHH domain. Amino acid insertions relative to the sequences of the HER2 binding VHH domains disclosed herein are also provided. In some embodiments, one or more additional amino acid residues are located at the amino terminus of the HER2 binding VHH domain. In some embodiments, one or more additional amino acid residues are located at the carboxyl terminus of the HER2 binding VHH domain. In some embodiments, one or more additional amino acid residues are located at the amino terminus and carboxyl terminus of the HER2 binding VHH domain. Amino acid changes relative to the sequences of the HER2 binding VHH provided herein can be performed using any suitable technique known in the art, such as by using site-directed mutagenesis.

[0236] In some embodiments, the HER2-binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residue additions, substitutions, and / or deletions compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) additional amino acid residues compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the additional amino acid residues are located at the amino terminus of the HER2 binding VHH domain compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the additional amino acid residues are located at the carboxyl terminus of the HER2 binding VHH domain compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the HER2-binding agent comprises a sequence having an amino-terminal 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) additional amino acid residues at the carboxyl terminus of the HER2 binding VHH domain, and 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) additional amino acid residues at the carboxyl terminus of the HER2 binding VHH domain.

[0237] In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residue substitutions compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the amino acid substitutions are non-conservative. Mixtures of conservative and non-conservative substitutions are also contemplated.

[0238] In some embodiments, the HER2-binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) residue deletions compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the amino acid residues are deleted at the amino terminus of the HER2-binding VHH domain compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the amino acid residues are deleted at the carboxyl terminus of the HER2-binding VHH domain compared to the reference VHH sequences disclosed in Table 2. In some embodiments, the HER2-binding agent comprises a sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residues, and 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residues deleted at the carboxyl terminus of the HER2 binding VHH domain.

[0239] In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) additions, substitutions, and / or deletions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) additions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the additional amino acid residues are located at the amino terminus of the HER2 binding VHH domain compared to a sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the additional amino acid residues are located at the carboxyl terminus of the HER2 binding VHH domain compared to a sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding agent comprises a sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) additional amino acid residues at the amino terminus and 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) additional amino acid residues at the carboxyl terminus.

[0240] In some embodiments, the HER2-binding agent comprises an amino acid sequence having 1-5, 1-3 or 1, 2, 3, 4 or 5 additions, substitutions and / or deletions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205, wherein one or more additions, substitutions and / or deletions are within and / or adjacent to (within 5AA thereof) the VHH CDRs disclosed in Table 2. In specific embodiments, one or more additions, substitutions and / or deletions are located at residue positions within and / or adjacent to (within 5AA thereof) the VHH CDRs, and which may additionally provide potential radiolabeling sites. Such one or more additions, substitutions and / or deletions are advantageous for targeted radionuclide therapy (TRT) applications for several reasons, for example, it ensures that the VHH retains its affinity, specificity and internalization properties after radiolabeling with its therapeutic payload; it allows pre-conjugation of the VHH with a radiolabeling prosthetic group moiety - such an approach maximizes the radiochemical labeling yield, which is critical for radionuclides based on poor reactivity or short half-lives; it allows site-selective radiolabeling (see, e.g., SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, etc.); and it allows for better control of the identity and heterogeneity of the VHH-drug conjugate or VHH radiolabeled conjugate.

[0241] In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid substitutions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the amino acid substitutions are conservative. In some embodiments, the amino acid substitutions are non-conservative. A mixture of conservative and non-conservative substitutions is also contemplated.

[0242] In some embodiments, the HER2-binding agent comprises an amino acid sequence having 1-5, 1-3, or 1, 2, 3, 4, or 5 substitutions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205, wherein one or more substitutions are within and / or adjacent to (within 5 AA) the VHH CDRs disclosed in Table 2. In specific embodiments, one or more substitutions are located at residue positions within and / or adjacent to (within 5 AA) the VHH CDRs, and which may additionally provide potential radiolabeling sites.

[0243] In some embodiments, the HER2 binding agent comprises an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) deletions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the amino acid residues are deleted at the amino terminus of the HER2 binding VHH domain compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the amino acid residues are deleted at the carboxyl terminus of the HER2 binding VHH domain compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the amino acid residues are deleted at the carboxyl terminus of the HER2 binding VHH domain compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. Compared to the reference sequence of NO: 1-81 and 205, the HER2 binding agent comprises the following sequence, which has 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acid residues, and 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acid residues deleted at the carboxyl terminus of the HER2 binding VHH domain.

[0244] In some embodiments, the HER2 binding polypeptide comprises a fragment of a VHH sequence disclosed in any one of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding agent comprises a HER2 binding fragment of a VHH amino acid sequence containing 50-100, 50-175 consecutive amino acids of a sequence disclosed in any one of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding polypeptide comprises a fragment of a sequence disclosed in Table 2. In some embodiments, the HER2 binding agent comprises a HER2 binding fragment of a VHH amino acid sequence containing 50-100, 50-175 consecutive amino acids of a sequence disclosed in Table 2.

[0245] In some embodiments, the HER2 binding polypeptide comprises an amino acid sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VHH sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a VHH having an amino acid sequence of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binding polypeptide comprises a complementary determining region (CDR) 3 sequence disclosed in Table 2. In some embodiments, the HER2 binding agent contains a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-157 and 206.

[0246] In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence and a CDR2 sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence selected from SEQ ID NOs: 124-157 and 206 and a CDR2 sequence selected from SEQ ID NOs: 102-123. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence and a CDR2 sequence from the same VHH sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence and a CDR2 sequence from different VHH sequences disclosed in Table 2.

[0247] In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence, a CDR2 sequence, and a CDR1 sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence selected from SEQ ID NOs: 124-157 and 206, a CDR2 sequence selected from SEQ ID NOs: 102-123, and a CDR1 sequence selected from SEQ ID NOs: 82-101. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence, a CDR2 sequence, and a CDR1 sequence from the same VHH sequence disclosed in Table 2. In some embodiments, the HER2 binding polypeptide comprises a CDR3 sequence, a CDR2 sequence, and a CDR1 sequence from different VHH sequences disclosed in Table 2.

[0248] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 101; a CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 135 or 136. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NOs: 23-42.

[0249] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 101; a CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 135. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NOs: 23, 32, and 34-38. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 32. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 34. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 35. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 36. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 37. In some embodiments, the HER2 binding agent comprises the following amino acid sequence: SEQ ID NO: 38.

[0250] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 101; a CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 136. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NOs: 24-31, 33, and 39-42. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 41 or 42. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 42.

[0251] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 82; a CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124, 145, 146, or 147. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NOs: 1 and 43-49.

[0252] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 82; a CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 124. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 1 or 43.

[0253] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:82; a CDR2 comprising the amino acid sequence of SEQ ID NO:102; and a CDR3 comprising the amino acid sequence of SEQ ID NO:145. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:44 or 45. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:82; a CDR2 comprising the amino acid sequence of SEQ ID NO:102; and a CDR3 comprising the amino acid sequence of SEQ ID NO:146. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:46 or 47. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 82; a CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 147. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 49.

[0254] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 83; a CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 125. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:2.

[0255] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 84; a CDR2 comprising the amino acid sequence of SEQ ID NO: 104; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 126. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:3.

[0256] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 85; a CDR2 comprising the amino acid sequence of SEQ ID NO: 105; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 127. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:4.

[0257] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 128, 130, 134, or 155. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 5, 17, 22, 79, 80, or 81. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 128. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 134. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 22 or 79. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 86; a CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 155. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 80 or 81. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:81.

[0258] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 87; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 129. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:6.

[0259] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 88; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 130. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:7.

[0260] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NOs: 50, 51, and 52. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 51. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 50 or 52. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 52.

[0261] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 131 or 157. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NO: 8, 19, 70, 71, and 72.

[0262] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 157. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NOs: 70, 71, and 72. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 157. In certain embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 157. In certain embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 70 or 72. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 72.

[0263] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 89; a CDR2 comprising the amino acid sequence of SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 8 or 19. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 19.

[0264] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 90; a CDR2 comprising the amino acid sequence of SEQ ID NO: 109; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 132. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:9.

[0265] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 91; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 137. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 10.

[0266] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 92; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 138. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 11.

[0267] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising an amino acid sequence selected from SEQ ID NO: 113 or 114; and a CDR3 comprising an amino acid sequence of SEQ ID NO: 139 or 206. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NO: 12, 56, 57, and 58. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 113; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139. In certain embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 56 or 58. In some embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 56. In some embodiments, the HER2-binding agent comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 114; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 139. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 114; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206. In some embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 12.

[0268] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 94; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising an amino acid sequence selected from SEQ ID NO: 140 or 149. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NO: 13, 59, 60, and 61. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 94; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 140. In certain embodiments, the HER2 binding agent comprises an amino acid sequence of SEQ ID NO: 13 or 59. In some embodiments, the HER2 binding agent comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the HER2 binding agent comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 94; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 149. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 60 or 61. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 61.

[0269] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 95; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 141. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO:14.

[0270] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 96; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 142. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 15.

[0271] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 97; a CDR2 comprising the amino acid sequence of SEQ ID NO: 123; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 143. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 16.

[0272] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 97; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 150. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 62.

[0273] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144, 151, 152, or 153. In certain embodiments, the HER2-binding agent comprises an amino acid sequence selected from SEQ ID NOs: 18 and 63-69.

[0274] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 18 or 63. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 151. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 64 or 65. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 65.

[0275] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 152 or 153. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 152. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 66 or 67. In some embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 98; a CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 153. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 69.

[0276] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 99; a CDR2 comprising the amino acid sequence of SEQ ID NO: 109; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 132 or 148. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NO: 20, 53, 54, 55, and 73-75. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 99; a CDR2 comprising the amino acid sequence of SEQ ID NO: 109; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 132. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NO: 20, 53, and 73. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO:99; a CDR2 comprising the amino acid sequence of SEQ ID NO:109; and a CDR3 comprising the amino acid sequence of SEQ ID NO:148. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NOs:54, 55, 74, and 75. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:74. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO:75.

[0277] In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 100; a CDR2 comprising the amino acid sequence of SEQ ID NO: 110; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 133 or 154. In certain embodiments, the HER2 binding agent comprises an amino acid sequence selected from SEQ ID NO: 21, 76, 77, and 78. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 100; a CDR2 comprising the amino acid sequence of SEQ ID NO: 110; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 133. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 21 or 76. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the HER2 binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 100; a CDR2 comprising the amino acid sequence of SEQ ID NO: 110; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 154. In certain embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 77 or 78. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the HER2 binding agent comprises the amino acid sequence of SEQ ID NO: 78.

[0278] In some embodiments, the HER2-binding agent comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 101; a CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 136. In certain embodiments, the HER2-binding agent comprises the amino acid sequence of SEQ ID NO: 205.

[0279] In some embodiments, HER2 binding agents contain protein, the protein comprises at least one VHH binding domain specifically binding to HER2 provided herein, and the VHH binding domain is fused to a heterologous peptide or protein sequence. In some embodiments, HER2 is fused to an additional N-terminal and / or C-terminal amino acid sequence in conjunction with a VHH domain. Such additional sequences can be provided for various reasons, such as increasing glycosylation, improving protein expression, solubility, enhancing tumor cell internalization or increasing the ease of purification and / or improving labeling (e.g., radiolabeling) selectivity and load.

[0280] In some embodiments, the HER2 binding agent contains a protein containing an additional N-terminal amino acid sequence that improves protein expression, solubility and / or ease of purification. In some embodiments, the HER2 binding VHH domain comprises MetAla or Ala at the N-terminus. In some embodiments, the HER2 binding VHH domain is fused to a heterologous peptide sequence, which is a polyamino acid sequence, such as a plurality of histidine residues or a plurality of lysine residues (suitably, 2, 3, 4, 5 or 6 residues).

[0281] In some embodiments, the HER2 binding agent comprises a protein comprising a C-terminal amino acid sequence that improves or enables conjugation to a site of a radiolabeled prosthetic group or direct labeling (e.g., radiolabeling). In some embodiments, the protein of the HER2 binding agent further comprises a C-terminal amino acid sequence (Gly n XA m Gly k ) j , wherein: VHH is a HER2 binding VHH domain provided herein (e.g., in Table 2), wherein n=0-5, m=0-5, k=0-6, j=0-8, and Xaa is an amino acid residue that enables site conjugation or direct labeling (e.g., direct radiolabeling) with a labeled prosthetic group (e.g., a radiolabeled prosthetic group). In some embodiments, Xaa is a natural amino acid. In certain embodiments, Xaa is lysine, cysteine, glutamine, tyrosine, histidine, phenylalanine, selenocysteine, or pyrrolysine. In some embodiments, Xaa is a non-natural amino acid. In certain embodiments, Xaa is p-acetylphenylalanine, p-azidomethyl-L-phenylalanine, formylglycine, sulfone, maytansine, or furan-amino acid. In some embodiments, the HER2 binder comprises a C-terminal sequence GGC. In some embodiments, the HER2 binder comprises a C-terminal sequence GGCG (SEQ ID NO: 201). In some embodiments, the HER2 binding agent comprises the C-terminal sequence GGKG (SEQ ID NO: 202). In some embodiments, the HER2 binding agent comprises the C-terminal sequence GGKGGKG (SEQ ID NO: 203). In some embodiments, the HER2 binding agent comprises the C-terminal sequence GGKGGC (SEQ ID NO: 204).

[0282] In some embodiments, the HER2 binding agent comprises a protein comprising an additional C-terminal amino acid sequence that enhances tumor cell internalization of the binding agent. In some embodiments, the HER2 binding agent further comprises the structure VHH-IPS, wherein IPS is an internalization peptide sequence. In some embodiments, the HER2 binding agent further comprises the sequence Arg n(R n ), wherein n=4-16. In some embodiments, the HER2 binding agent further comprises the sequence (ArgLys) n ((RK) n ), wherein n=2-8. In some embodiments, the HER2 binding agent further comprises a C-terminal amino acid sequence selected from the group consisting of GRKKRRQRRRPPQ (SEQ ID NO: 158), RQIKIWFQNRRMKWKK (SEQ ID NO: 159), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 160), KLALKLALKALKAALKLA (SEQ ID NO: 161), RRIP (SEQ ID NO: 162), NRRPRR (SEQ ID NO: 163), an amino acid sequence consisting of SEQ ID NOs: 162 and 163, KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 164), PLILLRLLRGQF (SEQ ID NO: 165), PLIYLRLLRGQF (SEQ ID NO: 166), KLWMRWYSPTTRRYG (SEQ ID NO: 167), GINTLQKYYCRVRG (SEQ ID NO: 168), NO:168), RLWMRWYSPRTRAYGC (SEQ ID NO:169), GKCSTRGRKCCRRKK (SEQ ID NO:170) and GRCAVLSCLPKEQI (SEQ ID NO:171).

[0283] In some embodiments, the HER2 binding agent contains a fusion protein comprising two or more VHH domains. In some embodiments, the HER2 binding agent comprises two or more identical VHH binding domains (e.g., homodimers, homotrimers, or homomultimers). In some embodiments, the protein comprises two or more different VHH binding domains (e.g., heterodimers, heterotrimers, or heteromultimers).

[0284] In some embodiments, the disclosure provides a multi-specific HER2 binding agent containing a fusion protein, the fusion protein comprising at least one HER2 binding VHH domain provided herein, and one or more additional binding domains. In some embodiments, one or more additional binding domains are antibodies (e.g., VHH (single domain antibody) or antigen binding fragments of antibodies). In some embodiments, one or more additional binding domains are VHH binding domains.

[0285] The HER2 binding agents provided can be monovalent or multivalent for HER2 and / or different antigens. In some embodiments, the HER2 binding agent is monovalent for HER2 (for example, comprising a HER2 binding domain). In some embodiments, the multivalent HER2 binding agent is multivalent and contains more than one binding domain. In some embodiments, the HER2 binding agent contains two or more copies of the binding domain that specifically binds to HER2. In some embodiments, the multivalent HER2 binding agent contains 2, 3, 4, 5, 6 or more copies of HER2 binding VHH domains. In some embodiments, the HER2 binding agent comprises two or more copies of different binding domains (for example, VHH binding domains) that specifically bind to HER2. In some embodiments, the HER2 binding agent contains 2, 3, 4, 5, 6 or more different VHH domains that specifically bind to HER2. In some embodiments, the HER2 binding agent contains 2, 3, 4, 5, 6 or more different VHH binding domains that specifically bind to different epitopes of HER2.

[0286] The HER2 binding agents provided can also be monospecific or multispecific. In some embodiments, the HER2 binding agents are monospecific to HER2 (i.e., containing one or more binding domains that bind to the same epitope of HER2). In certain embodiments, the HER2 binding agents contain 2, 3, 4, 5, 6 or more copies of the same HER2 binding VHH domain. In some embodiments, the HER2 binding agents are multispecific. In certain embodiments, the HER2 binding agents are multispecific and contain one or more binding sites that bind to different epitopes of HER2. In certain embodiments, the HER2 binding agents are multispecific and contain 2, 3, 4, 5, 6 or more VHH domains that specifically bind to different epitopes or domains (e.g., HER2 domains I, II, III or IV) of HER2. In some embodiments, the HER2 binding agents are multispecific and include different binding domains (e.g., VHH binding domains) that specifically bind to two or more copies of different antigens (e.g., HER2 and EGFR). In certain embodiments, the HER2 binding agent is multispecific and contains 2, 3, 4, 5, 6 or more VHH domains that specifically bind to different antigens (eg, HER2 and EGFR).

[0287] In additional embodiments, the HER2 binding agent provided is monospecific and multivalent, or the HER2 binding agent provided is multispecific and multivalent. In some embodiments, the HER2 binding agent provided is multivalent and monospecific. In certain embodiments, the HER2 binding agent is multivalent and multispecific, and contains 2, 3, 4, 5, 6 or more copies of the same HER2 binding VHH domain. In some embodiments, the HER2 binding agent provided is multivalent and multispecific. In certain embodiments, the HER2 binding agent is multivalent and multispecific, and contains 2, 3, 4, 5, 6 or more VHH domains that specifically bind to different epitopes of HER2. In some embodiments, the HER2 binding agent is multivalent and monospecific, and contains different binding domains of two or more copies that specifically bind to different antigens (e.g., HER2 and EGFR). In certain embodiments, the HER2 binding agent is multispecific and contains 2, 3, 4, 5, 6 or more VHH domains that specifically bind to different antigens.

[0288] In some embodiments, one or more additional binding domains specifically bind to a second antigen different from HER2. In some embodiments, the second antigen is a tumor-associated antigen or a tumor microenvironment-associated antigen. In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen participates in enhancing or inhibiting a signal transduction pathway in an immune cell. In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen participates in enhancing or inhibiting a signal transduction pathway in an immune cell.

[0289] In some embodiments, the HER2-binding agent contains a fusion protein having the following structure: HER2VHH-Linker-HER2VHH, wherein HER2VHH is a HER-2 binding VHH disclosed herein, and the Linker is a linker provided herein or known in the art.

[0290] In some embodiments, the HER2 binding agent contains a fusion protein comprising a HER2 binding VHH domain operably connected to an IgG Fc. In certain embodiments, the fusion protein is bivalent, having two VHH domains that specifically bind to HER2 (e.g., each molecule has 2 HER2 binding VHH domains and has the structure (HER2VHH-joint-HER2VHH-joint-Fc))). In some embodiments, the fusion protein is tetravalent (e.g., each molecule has 4 HER2 binding VHH domains and has the structure HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-Fc) or hexavalent (e.g., each molecule has 6 HER2 binding VHH domains and has the structure HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-HER2VHH-joint-Fc).

[0291] In some embodiments, the HER2 binding agent contains a fusion protein comprising a HER2 binding VHH domain disclosed herein fused to a heterologous amino acid sequence. The fusion of the HER2 binding VHH domain and the heterologous polypeptide sequence can be at any convenient site on the protein, and can be an N-terminal, C-terminal and / or N-terminal / C-terminal fusion. In some embodiments, the heterologous polypeptide sequence is fused to the N-terminal of the HER2 binding VHH domain. In some embodiments, the heterologous polypeptide sequence is fused to the C-terminal of the HER2 binding VHH domain. In certain embodiments, the heterologous polypeptide sequence is fused to the N-terminal of the HER2 binding VHH domain, and the heterologous polypeptide sequence is fused to the C-terminal of the HER2 binding VHH domain. Such fusion proteins can be prepared by any suitable approach, including by recombinant expression, chemical bonding, enzymatic connection (e.g., sortase A) or other methods known in the art.

[0292] In some embodiments, the heterologous amino acid sequence is a toxin. In some embodiments, the heterologous amino acid sequence is a marker.

[0293] In some embodiments, the HER2 binding VHH domain is fused (operably linked) to a biologically active heterologous polypeptide. In some embodiments, the biologically active molecule is a peptide or protein, such as an enzyme, immunoglobulin, cytokine, anticancer agent or a fragment or variant thereof. Alternatively, the biologically active component of the fusion protein can be an anticancer drug, NSAID, steroid, analgesic, toxin or other pharmaceutically active agent. Anticancer drugs can include cytotoxic or cytostatic drugs.

[0294] In some embodiments, the biologically active molecule in the fusion protein is a therapeutic agent, such as an anti-inflammatory agent (e.g., a steroid or nonsteroidal anti-inflammatory drug), an anti-cancer drug, a cytotoxic agent (such as a toxin, such as cholera toxin or a radionuclide containing a radioactive element for therapeutic or diagnostic use), or a cytostatic or analgesic agent.

[0295] In some embodiments, the HER2 binding agent contains a fusion protein comprising a HER2 binding VHH domain provided herein fused to another immunoglobulin variable region or constant region or another VHH domain with specific binding affinity to HER2 or another antigen. In some embodiments, the fusion protein contains a dimer, trimer, tetramer or higher polymer (i.e., pentamer, hexamer, heptamer, octamer, nonamer or decamer or higher) of a VHH binding domain.

[0296] In some embodiments, the HER2 binding VHH domain is fused to a heterologous amino acid sequence comprising an immunoglobulin domain. In some embodiments, the HER2 binding VHH domain is fused to a heterologous amino acid sequence of an antibody or an engineered fragment thereof, including Fab, Fc, F(ab')2 (including chemically linked F(ab')2 chains), Fab', scFv (including its multimeric form, i.e., two scFvs or three scFvs) or BiTE (bispecific T cell engagers). Antibody fragments also include variable domains and fragments thereof, as well as other single domain antibody type fragments. In some embodiments, the fusion protein is a chimeric antigen receptor. In some embodiments, the immunoglobulin Fc region has an IgG isotype selected from the group consisting of: IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.

[0297] In some embodiments, HER2 binds to VHH domains and to heterologous amino acid sequences (such as human Fc) comprising Fc domains or a portion thereof. In certain embodiments, the Fc portion increases the half-life and / or yield of a polypeptide comprising a HER2 binding domain. In some embodiments, the Fc domain or a portion thereof increases the half-life of a polypeptide comprising a HER2 binding domain. In some embodiments, the multispecific HER2 binder further comprises an Fc domain, such as described herein. In some embodiments, the multispecific HER2 binder comprises at least one VHH domain in conjunction with HER2, at least one additional binding domain in conjunction with different epitopes or domains (e.g., HER2 domains I, II, III or IV) of HER2, and an Fc domain. In some embodiments, the multispecific HER2 binder comprises at least one HER2 in conjunction with VHH domains, at least one additional binding domain in conjunction with different antigens, and an Fc domain. In some embodiments, one or more single domain antibodies of the present disclosure may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (e.g., 2 or 3 constant domains that may be used as part of / form the Fc portion), to the Fc portion, and / or to one or more antibody portions, fragments, or domains that confer one or more effector functions to the polypeptides of the present disclosure and / or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, but not limited thereto, one or more additional amino acid sequences may comprise one or more CH2 and / or CH3 domains of an antibody, such as from a heavy chain antibody or from a conventional human chain antibody; and / or the Fc region may be formed, for example, from IgG (e.g., from IgG1, IgG2, IgG3, or IgG4), from IgE, or from another human Ig such as IgA, IgD, or IgM. For example, International Publication No. WO1994 / 04678 describes a heavy chain antibody comprising a camelid VHH domain or a humanized derivative thereof, wherein the camelid CH2 and / or CH3 domains have been replaced with human CH2 and CH3 domains, thereby providing an immunoglobulin consisting of two heavy chains, each heavy chain comprising a single domain antibody and human CH2 and CH3 domains (but without a CH1 domain), the immunoglobulin having effector functions provided by the CH2 and CH3 domains, and can function in the absence of any light chains.

[0298] In some embodiments, a multispecific HER2 binding agent is a bispecific construct comprising at least one HER2 provided herein in combination with a VHH domain and at least one additional binding domain capable of binding to a surface molecule expressed on an immune effector cell (e.g., a T cell). In some embodiments, the surface molecule is an activating component of a T cell, such as a component of a T cell receptor complex. In some embodiments, the surface molecule is an activating T cell antigen, which is expressed on a T cell and can induce T cell activation when interacting with an antigen binding molecule. For example, in some embodiments, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering a signal transduction cascade of a T cell receptor complex. In some embodiments, the activating T cell antigen is CD3 or CD2. In some embodiments, the bispecific HER2 binding agent provided can specifically bind to an activating T cell antigen expressed on a human T cell, such as human CD3 or human CD2. In a specific embodiment, an additional binding domain specific to an activating T cell antigen (e.g., CD3 or CD2) is an antibody (e.g., VHH (single domain antibody)) or an antigen binding fragment of an antibody. In some embodiments, the one or more additional binding domains specific for an activating T cell antigen are a VHH domain or an antigen-binding fragment or variant of a VHH domain.

[0299] In some embodiments, the HER2-binding agent provided is a bispecific antibody T cell engager comprising at least one HER2-binding VHH domain provided herein and an additional binding domain, which additional binding domain is an antibody (e.g., VHH (single domain antibody)) or antigen-binding fragment specific for an activating component of a T cell (e.g., a T cell surface molecule such as CD3, a CD3 complex, or CD2).

[0300] Connectors

[0301] The polypeptide component of the fusion protein contained in the HER2 binding agent provided herein can be directly fused (operably linked) to the other components of the fusion protein via a linker portion. In some embodiments, the linker is a peptide, peptide nucleic acid or polyamide bond.

[0302] In some embodiments, the HER2 binding agent contains a protein as a fusion protein, which comprises a HER2 binding VHH domain fused to one or more polypeptide sequences via one or more polypeptide linkers. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. Suitable peptide linkers include multiple amino acid residues, such as 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acids. In some embodiments, the linker is mainly composed of amino acids glycine and serine, and is represented as a GS linker in this article. The GS linker can have various lengths, such as a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids. In some embodiments, the GS linker comprises an amino acid sequence selected from the group consisting of: GGSGGS (SEQ ID NO 172); GGSGGSGGS (SEQ ID NO 173); GGSGGSGGSGGS (SEQ ID NO 174); and GGSGGSGGSGGSGGS (SEQ ID NO 175). In some embodiments, the linker is a flexible linker comprising a glycine residue, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ ID NO 176), GGGGG (SEQ ID NO 177), and GGGGGG (SEQ ID NO 178). In some embodiments, the linker is (GGGGS)n, wherein n is 1 to 5 (SEQ ID NO 179); (GGGGGS)n, wherein n is 1 to 4 (SEQ ID NO 180); GGGGS (SEQ ID NO 181); GGGGGS (SEQ ID NO 182); GGGGGSGGGGGSGGGGGS (SEQ ID NO 183); GGGGSGGGGSGGGGS (SEQ ID NO 184); GGSGGGGSGGGGSGGGGS (SEQ ID NO 185); or PGGGG (SEQ ID NO 186). In some embodiments, the HER2-binding agent comprises a combination of a GS linker and a glycine linker.

[0303] In some embodiments, the HER2 binding agent contains a protein as a fusion protein, which is fused to the HER2 binding VHH domain of one or more polypeptide sequences via one or more polypeptide linkers, and the polypeptide linker comprises the sequence Gn, wherein n=2-8. In some embodiments, the linker comprises the sequence (GS)n, (GGS)n, (GGGS)n (SEQIDNO 187) or (GGGGS)n (SEQIDNO 188), wherein n=1-16. In some embodiments, the linker comprises the sequence (GGGGS)3 (SEQIDNO 189). In some embodiments, the linker comprises the sequence (EGKSSGSGSESKST)n (SEQIDNO 190) or (KESGSVSSEQLAQFRSLD)n (SEQIDNO 191), wherein n=1-4. In some embodiments, the linker comprises the sequence GGGSLVPRGSGGGS (SEQIDNO 192) or (GAGSAAGSGEF)n (SEQIDNO 193), wherein n=1-5. In some embodiments, the linker comprises the sequence (GAGAGX)n (SEQ ID NO 194), wherein X = A, Y, V or S (SEQ ID NO 195), and n = 1-16. In some embodiments, the linker comprises the sequence (VPGXG)n, wherein X = A, V, G or S (SEQ ID NO 196), and n = 1-40.

[0304] In some embodiments, the HER2 binding agent contains a protein as a fusion protein, which comprises a HER2 binding VHH domain fused to one or more polypeptide sequences via one or more rigid polypeptide linkers. In some embodiments, the linker comprises the sequence (APAP) n (SEQ ID NO 197), where n = 10-34. In some embodiments, the linker comprises the sequence (EAAAK) n (SEQ ID NO 198), where n = 1-3. In some embodiments, the linker comprises the sequence A (EAAAK) 4ALEA (EAAAK) 4A (SEQ ID NO 199). In some embodiments, the linker comprises the sequence (A) 3 (H) 6 (SEQ ID NO 200).

[0305] In some embodiments, the linker is a cross-linkable linker. In certain embodiments, the linker is PEG, POEGMA or a carbonyl chain.

[0306] Chimeric receptors and engineered cells

[0307] In some embodiments, the present disclosure provides a chimeric antigen receptor (CAR) having an extracellular domain, the extracellular domain comprising one or more HER2 binding VHH domains provided herein. The CAR construct provided herein comprises an extracellular domain, a transmembrane domain and an intracellular signaling region including one or more HER2 binding VHH domains provided herein. One or more HER2 binding VHH domains contained in the extracellular antigen binding unit of CAR "bind" or "can bind" HER2 with sufficient affinity, so that CAR can be used for targeting the therapy of cells or tissues expressing HER2.

[0308] CAR is a synthetic receptor that generally contains an extracellular targeting / binding portion, which is associated with one or more signaling domains in a single fusion molecule, and the synthetic receptor is expressed on the surface of immune effector cells (such as T cells). Therefore, CAR combines antigen specificity and T cell activation properties in a single fusion molecule. In some embodiments, CAR contains a signaling domain of a costimulatory molecule (such as CD28 and CD3ζ) to provide dual signaling to guide the combined activation and costimulatory signals. In some embodiments, CAR comprises three or more signaling domains.

[0309] In some embodiments, CAR contains an antigen binding domain comprising two HER2 binding VHH domains, thereby providing a bivalent binding molecule. In certain embodiments, the antigen binding domain comprises two or more identical HER2 binding VHH domains. In certain embodiments, the antigen binding domain comprises two or more different HER2 binding VHH domains. In certain embodiments, the antigen binding domain comprises two or more HER2 binding VHH domains that bind to different epitopes of HER2. In certain embodiments, the antigen binding domain comprises one or more HER2 binding VHH domains and one or more VHH binding domains that specifically bind to different antigens.

[0310] In a specific embodiment, CAR further comprises a hinge or spacer connecting the extracellular antigen binding domain and the transmembrane domain. The hinge or spacer can be used to realize the different lengths and flexibility of the obtained CAR. Examples of hinges or spacers that can be used include but are not limited to the Fc fragment of an antibody or its fragment or derivative, the hinge region of an antibody or its fragment or derivative, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence (eg, a peptide sequence), or a combination thereof. Other hinges or spacers will be apparent to those skilled in the art and can be used. In some embodiments, the hinge is an IgG4 hinge or a CD8A hinge. In some embodiments, the spacer and the transmembrane domain are hinges and transmembrane domains derived from CD8.

[0311] The transmembrane domain of CAR provided herein is a domain: it is usually passed through or can pass through or across the domain of plasma membrane, and directly or indirectly (for example, via a spacer, such as an immunoglobulin hinge sequence) is connected to the endoplasmic part of the extracellular antigen binding domain and the intracellular signaling domain. In some embodiments, the transmembrane domain of CAR is a transmembrane region, an artificial hydrophobic sequence, or a combination thereof of a transmembrane protein (for example, a type I transmembrane protein). In certain embodiments, the transmembrane domain includes a CD3 ζ domain or a CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art, and can be used in combination with the embodiments of CAR provided herein.

[0312] The intracellular signaling region of CAR provided herein contains one or more intracellular signaling domains, which are engaged by the antigen binding domain of CAR, such as when binding antigen, and transmit signals to T cells. In some embodiments, the intracellular region contains an intracellular signaling domain, which is an ITAM signaling domain or contains an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, derived from the following signaling domains: ζ chains of T cell receptor complexes or any homologues thereof (e.g., η chains, FcRIγ and β chains, MB 1 (Iga) chains and B29 (Ig) chains), human CD3 ζ chains, CD3 polypeptides (δ, γ or ε), syk family tyrosine kinases (e.g., Syk or Zap 70), src family tyrosine kinases (e.g., Lck, Fyn or Lyn) and other molecules involved in T cell transduction (such as CD2, CD5, OX40 and CD28). In a specific embodiment, the intracellular signaling region contains an intracellular signaling domain derived from human CD3 ζ chains.

[0313] In some embodiments, the endoplasmic portion of CAR includes a CD3 ζ signaling domain. In some embodiments, the costimulatory signaling domain is derived from CD28 or 4-1BB and retains the activity of T cell costimulatory signaling.

[0314] In some embodiments, the disclosure provides an isolated nucleic acid construct comprising at least one nucleic acid encoding a CAR as provided herein. In some embodiments, the construct is an expression vector for expressing CAR in a cell. In some embodiments, the expression vector is a viral vector (eg, a lentiviral vector). Viral vector technology is well known in the art and is described in, for example, Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). Many viral-based systems for transferring genes into mammalian cells have been developed, and are known in the art.

[0315] In some embodiments, the disclosure provides a cell or cell group separated, which includes one or more nucleic acid constructs as described herein. Also provided is a cell or cell group separated, which is genetically modified to express the CAR provided herein. Therefore, provided herein is a genetically engineered cell, which includes (such as stably expressed) CAR provided herein. In some embodiments, the cell is selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, hematopoietic stem cells and / or pluripotent embryonic stem cells / induced stem cells. In some cases, the cell is a T cell, such as CD4 and / or CD8 T cells. In some embodiments, the cell is autologous for the subject. For example, in some embodiments, T cells (also referred to as primary T cells) can be separated from a patient to be engineered with a CAR nucleic acid construct, such as transfected or transduced.

[0316] In a specific embodiment, primary T cells (CD4 cells or CD8 cells or both) can be purified in vitro and stimulated with TCR / CD28 agonists (such as anti-CD3 / anti-CD28 coated beads). After two or three days of activation, the recombinant expression vector encoding CAR can be stably introduced into primary T cells by standard lentiviral or retroviral transduction schemes or plasmid electroporation strategies. It is possible to, for example, monitor the CAR expression of cells, and the flow cytometry uses anti-epitope tags or antibodies cross-reacting with natural parent molecules. T cells expressing CAR can be enriched by sorting with anti-epitope tag antibodies, or enriched for high or low expression depending on the application.

[0317] The appropriate function of CAR engineered T cells can be determined by a variety of means. In some cases, in vitro cytotoxicity, proliferation or cytokine determination (e.g., IFN-γ expression) can be used to evaluate the function of engineered T cells. An exemplary standard endpoint is the percentage lysis of tumor lines, the proliferation of engineered T cells or the IFN-γ protein expression in the culture supernatant. In some cases, after CAR is stimulated by antigens, the ability of T cells to stimulate activation can be evaluated by, for example, monitoring the expression, proliferation and / or cytokine production of activation markers (such as CD69, CD44 or CD62L).

[0318] II. Nucleic Acids

[0319] In some embodiments, the disclosure provides nucleic acids encoding the HER2-binding agents provided herein.

[0320] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0321] (a) the VHH amino acid sequence disclosed in Table 2; or

[0322] (b) a VHH sequence selected from SEQ ID NOs 1-81 and 205.

[0323] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0324] (a) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VHH sequence disclosed in Table 2; or

[0325] (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a VHH sequence selected from SEQ ID NOs 1-81 and 205.

[0326] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0327] (a) an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 additions, substitutions or deletions compared to the reference VHH sequence disclosed in Table 2; or

[0328] (b) an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 additions, substitutions or deletions compared to a reference VHH sequence selected from SEQ ID NOs 1-81 and 205.

[0329] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0330] (a) a complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 82-101; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 102-123; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 124-157 and 206; or

[0331] (b) CDR1, CDR2 and CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 contained in the VHH sequences disclosed in Table 2.

[0332] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0333] (a) a CDR1 comprising the amino acid sequence of SEQ ID NO 101; a CDR2 comprising the amino acid sequence of SEQ ID NO 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO 135 or 136;

[0334] (b) a CDR1 comprising the amino acid sequence of SEQ ID NO 101; a CDR2 comprising the amino acid sequence of SEQ ID NO 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO 135;

[0335] (c) a CDR1 comprising the amino acid sequence of SEQ ID NO 101; a CDR2 comprising the amino acid sequence of SEQ ID NO 111; and a CDR3 comprising the amino acid sequence of SEQ ID NO 136;

[0336] (d) a CDR1 comprising the amino acid sequence of SEQ ID NO 82; a CDR2 comprising the amino acid sequence of SEQ ID NO 102; and a CDR3 comprising the amino acid sequence of SEQ ID NO 124, 145, 146 or 147;

[0337] (e) a CDR1 comprising the amino acid sequence of SEQ ID NO 83; a CDR2 comprising the amino acid sequence of SEQ ID NO 103; and a CDR3 comprising the amino acid sequence of SEQ ID NO 125;

[0338] (f) a CDR1 comprising the amino acid sequence of SEQ ID NO 84; a CDR2 comprising the amino acid sequence of SEQ ID NO 104; and a CDR3 comprising the amino acid sequence of SEQ ID NO 126;

[0339] (g) a CDR1 comprising the amino acid sequence of SEQ ID NO 85; a CDR2 comprising the amino acid sequence of SEQ ID NO 105; and a CDR3 comprising the amino acid sequence of SEQ ID NO 127;

[0340] (h) a CDR1 comprising the amino acid sequence of SEQ ID NO 86; a CDR2 comprising the amino acid sequence of SEQ ID NO 106; and a CDR3 comprising the amino acid sequence of SEQ ID NO 128, 130, 134 or 155;

[0341] (i) a CDR1 comprising the amino acid sequence of SEQ ID NO 87; a CDR2 comprising the amino acid sequence selected from SEQ ID NO 117; and a CDR3 comprising the amino acid sequence of SEQ ID NO 129;

[0342] (j) a CDR1 comprising the amino acid sequence of SEQ ID NO 88; a CDR2 comprising the amino acid sequence of SEQ ID NO 118; and a CDR3 comprising an amino acid sequence selected from SEQ ID NO 130;

[0343] (k) a CDR1 comprising the amino acid sequence of SEQ ID NO 89; a CDR2 comprising the amino acid sequence of SEQ ID NO 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO 131 or 157;

[0344] (1) a CDR1 comprising the amino acid sequence of SEQ ID NO 89; a CDR2 comprising the amino acid sequence of SEQ ID NO 108; and a CDR3 comprising the amino acid sequence of SEQ ID NO 131;

[0345] (m) a CDR1 comprising the amino acid sequence of SEQ ID NO 89; a CDR2 comprising the amino acid sequence of SEQ ID NO 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO 156;

[0346] (n) a CDR1 comprising the amino acid sequence of SEQ ID NO 89; a CDR2 comprising the amino acid sequence of SEQ ID NO 108 or 112; and a CDR3 comprising the amino acid sequence of SEQ ID NO 157;

[0347] (o) a CDR1 comprising the amino acid sequence of SEQ ID NO 90; a CDR2 comprising the amino acid sequence of SEQ ID NO 109 and a CDR3 comprising the amino acid sequence of SEQ ID NO 132;

[0348] (p) a CDR1 comprising the amino acid sequence of SEQ ID NO 91; a CDR2 comprising the amino acid sequence of SEQ ID NO 119 and a CDR3 comprising the amino acid sequence of SEQ ID NO 137;

[0349] (q) a CDR1 comprising the amino acid sequence of SEQ ID NO 92; a CDR2 comprising the amino acid sequence of SEQ ID NO 120 and a CDR3 comprising the amino acid sequence of SEQ ID NO 138;

[0350] (r) a CDR1 comprising the amino acid sequence of SEQ ID NO 93; a CDR2 comprising the amino acid sequence of SEQ ID NO 113 or 114 and a CDR3 comprising the amino acid sequence of SEQ ID NO 139;

[0351] (s) a CDR1 comprising the amino acid sequence of SEQ ID NO 93; a CDR2 comprising the amino acid sequence of SEQ ID NO 114 and a CDR3 comprising the amino acid sequence of SEQ ID NO 206;

[0352] (t) a CDR1 comprising the amino acid sequence of SEQ ID NO 94; a CDR2 comprising the amino acid sequence of SEQ ID NO 115 and a CDR3 comprising the amino acid sequence of SEQ ID NO 140 or 149;

[0353] (u) a CDR1 comprising the amino acid sequence of SEQ ID NO 95; a CDR2 comprising the amino acid sequence of SEQ ID NO 121 and a CDR3 comprising the amino acid sequence of SEQ ID NO 141;

[0354] (v) a CDR1 comprising the amino acid sequence of SEQ ID NO 96; a CDR2 comprising the amino acid sequence of SEQ ID NO 122; and a CDR3 comprising the amino acid sequence of SEQ ID NO 142;

[0355] (w) a CDR1 comprising the amino acid sequence of SEQ ID NO 97; a CDR2 comprising the amino acid sequence of SEQ ID NO 116; and a CDR3 comprising the amino acid sequence of SEQ ID NO 150;

[0356] (x) a CDR1 comprising the amino acid sequence of SEQ ID NO 98; a CDR2 comprising the amino acid sequence of SEQ ID NO 107; and a CDR3 comprising the amino acid sequence of SEQ ID NO 144, 151, 152 or 153;

[0357] (y) a CDR1 comprising the amino acid sequence of SEQ ID NO 99; a CDR2 comprising the amino acid sequence of SEQ ID NO 109; and a CDR3 comprising the amino acid sequence of SEQ ID NO 132 or 148; or

[0358] (z) a CDR1 comprising the amino acid sequence of SEQ ID NO 100; a CDR2 comprising the amino acid sequence of SEQ ID NO 110; and a CDR3 comprising the amino acid sequence of SEQ ID NO 133 or 154;

[0359] or

[0360] An isolated nucleic acid encoding a HER2-binding VHH domain comprising:

[0361] (ab) CDR1 comprising SEQ ID NO 97; CDR2 comprising SEQ ID NO 123; and CDR3 comprising SEQ ID NO 143; or

[0362] (ab) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO: 136.

[0363] In some embodiments, the nucleic acid sequence encoding provided is as provided herein fusion protein. In some embodiments, the nucleic acid sequence further comprises one or more regulatory sequences (e.g., transcription promoters, operators or enhancers, or mRNA ribosome binding sites) suitable for replication and / or expression of the encoded protein in the selected host cell. In some embodiments, the nucleic acid further encodes a leader sequence for instructing protein secretion, which is usually cut so that it is not present in the secreted protein. The leader sequence can be a natural heavy chain (or VHH) leader sequence or can be a heterologous leader sequence. Recombinant DNA technology known in the art and / or chemical synthesis methods can be used to construct nucleic acids.

[0364] In some embodiments, the disclosure provides a vector comprising a nucleic acid encoding a HER2-binding protein provided herein. The vector generally includes a selectable marker and a replication origin for propagation in a host. The vector provided includes but is not limited to a DNA vector, a phage vector, a viral vector, and a retroviral vector.

[0365] In some embodiments, the disclosure provides expression vectors, which include nucleic acids encoding HER2-binding proteins provided herein. Typically, expression vectors include nucleic acids encoding HER2-binding proteins, which are operably connected to transcription or translation regulatory sequences, which are suitable for host cells in which the protein is to be expressed, such as those derived from mammals, microorganisms, viruses or insect genes. Examples of regulatory sequences include transcription promoters, operators or enhancers, mRNA ribosome binding sites and appropriate sequences for controlling transcription and translation.

[0366] A variety of host-vector expression systems can be used to express the protein components of the provided HER2 binders. These systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., Biotechnology and Applied Biochemistry (1999) 30 (3): 235-44); insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see, e.g., Babe et al., Canadian Journal of Communication (2000) 25 (1): 19); or animal cell systems infected with viral expression vectors (e.g., vaccinia virus, adenovirus, etc.). Those skilled in the art are aware of various techniques for optimizing mammalian expression of proteins: see, for example, Kaufman, Mol Biotechnol. (2000) 16 (2): 151-60; Colosimo et al., Biotechniques (2000) 29 (2): 314-31. Mammalian cells that can be used for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines COS cells (including COS 7 cells); HEK cells; 293 cells (including 293-6E cells); CHO cells (including CHO-S), DG44. Lec13 CHO cells and FUT8 CHO cells; PER.C 6 cells (Crucell); NSO cells W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for recombinantly expressing HER2 binding polypeptides in bacteria, yeast and other invertebrates are known in the art. In some embodiments, a protein comprising a HER2 binding VHH domain is expressed in yeast (e.g., Pichia pastoris). See, e.g., US Publication No. US2006 / 0270045A1. Mammalian host systems for expressing recombinant proteins are also well known to those skilled in the art. The host cell strain can be selected for its specific ability to process the expressed protein or to produce certain post-translational modifications that contribute to providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can also be important for correct insertion, folding, and / or function.Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular mechanisms and characteristic mechanisms for such post-translational activities, and can be selected to ensure correct modification and processing of introduced foreign proteins. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications on polypeptides.

[0367] In some embodiments, the disclosure provides a host cell comprising a nucleic acid as described herein. In some embodiments, the disclosure provides a host cell comprising a vector containing a nucleic acid disclosed herein. In some embodiments, the disclosure provides a host cell comprising an expression vector disclosed herein. In some embodiments, the host cell is a eukaryotic cell such as a mammalian cell. In some embodiments, the host cell is a prokaryotic cell. In certain embodiments, the host cell expresses a HER2 binding polypeptide disclosed herein.

[0368] Introduction of nucleic acids (including vectors) into desired host cell antigens is accomplished by any method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected into a desired host cell according to any suitable method.

[0369] III. Polypeptide Expression and Production

[0370] The protein components of the HER2-binding agents provided herein can be routinely produced by conventional recombinant techniques and / or peptide synthesis methods known in the art.

[0371] In some embodiments, the present disclosure provides a recombinant method for producing a protein comprising a HER2 binding VHH domain as provided herein. In some embodiments, the method includes culturing a host cell containing a nucleic acid encoding a HER2 binding protein under conditions that produce the protein. In some embodiments, the method further includes recovering the HER2 binding protein produced by the host cell.

[0372] In some embodiments, the HER2 binding protein is expressed in prokaryotic cells such as bacterial cells. In some embodiments, the HER2 binding protein is expressed in eukaryotic cells. In certain embodiments, the HER2 binding protein is expressed in fungal cells (e.g., such as yeast), plant cells, insect cells, or mammalian cells. Such expression can be performed, for example, according to procedures known in the art. In some embodiments, the disclosure provides a cell culture medium comprising a HER2 binding protein expressed by a cell. In some embodiments, the protein is purified from the cell culture medium.

[0373] In some embodiments, the HER2 binding VHH domain is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, for example, S Sitaraman et al., Methods Mol Biol. (2009) 498: 229-44; Spirin, Trends Biotechnol. (2004) 22: 538-45; Endo et al., Biotechnol Adv. (2003) 21: 695-713. In some embodiments, the present disclosure provides a cell-free solution comprising a HER2 binding protein. In some embodiments, the protein is purified from the cell-free solution.

[0374] As an alternative to recombinant DNA technology, the protein contained in the HER2 binding agent provided herein, and / or the nucleic acid encoding the protein can be produced by automated polypeptide or nucleic acid synthesis, respectively. In some embodiments, the protein component of the HER2 binding agent provided herein is synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to known protocols, such as described in Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co., Rockford, Ill., pp. 11 and 12; Tam et al., J Am Chem Soc (1983) 105:6442; Merrifield, Science (1986) 232(4748):341-7; and Barany, G. and Merrifield, RB, "The Peptides, Vol. 2", Academic Press, Inc., New York, 1979, pp. 1-284; Gross et al., The Peptides, Analysis, Synthesis, Biology, Vol. 2, Academic Press, 1980, pp. 3-25. The polypeptide components of the HER2 binding agents of the present disclosure can also be synthesized by solid phase techniques using an exemplary peptide synthesizer (such as Model 433A from Applied Biosystems Inc.). The purity of any given polypeptide produced by automated peptide synthesis or by recombinant methods can be determined using reverse phase HPLC analysis. The chemical authenticity of each polypeptide can be established by any method well known to those skilled in the art.

[0375] Proteins can be recovered and purified from recombinant cell cultures and other solutions by methods known in the art, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using protein A), hydroxyapatite chromatography, lectin and / or heparin chromatography. For therapy, nucleic acid constructs (e.g., in the form of recombinant vectors) can be purified by techniques known in the art, such as by column chromatography as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0376] In certain embodiments, it is contemplated that proteins used in the therapeutic methods of the present disclosure may be modified to improve their therapeutic efficacy. Such modifications of therapeutic compounds may be used, for example, to reduce toxicity, increase circulation time, or modify biodistribution. For example, the toxicity of a potentially important therapeutic compound may be significantly reduced by combining it with a variety of drug carrier vehicles that modify biodistribution.

[0377] One strategy to improve drug activity is to use water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve cellular uptake patterns, change permeability through physiological barriers; and modify the rate of clearance from the body. In order to achieve targeted or sustained release effects, water-soluble polymers have been synthesized, which contain drug moieties as end groups, as part of the backbone, or as pendent groups on the polymer chain. For example, polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and ease of modification. It has been demonstrated that the attachment of PEG to various drugs can improve retention time and reduce toxicity.

[0378] IV. Labeled HER2 Binding Agents

[0379] A. Conjugates

[0380] In some embodiments, the HER2 binding agents provided herein contain proteins comprising a HER2 binding VHH domain, which is conjugated to one or more further moieties via covalent or non-covalent interactions to form a HER2 binding agent conjugate. In some embodiments, the HER2 binding agent conjugate comprises 1, 2, 3, 4, 5 or more further moieties. In some embodiments, the conjugated moieties of the HER2 binding agent conjugate are the same. In some embodiments, two or more conjugated moieties of the HER2 binding agent conjugate are different.

[0381] A portion of the provided HER2-binding agent conjugates can be attached to a protein comprising one or more VHH domains that specifically bind HER2 using any of a variety of molecular biology or chemical conjugation and linkage methods known in the art. For example, conjugates provided herein comprising a protein of at least one HER2 binding VHH domain and one or more cytotoxic agents can be routinely prepared using any of a variety of bifunctional protein coupling agents known in the art, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate HCl), active esters (such as disuccinimidyl substrates), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0382] In some embodiments, a linker such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that facilitates a conjugation reaction is used to connect or conjugate a HER2 binding protein to a portion (e.g., an effector portion such as a drug portion or a radionuclide). In some embodiments, one or more portions of the HER2 binding agent conjugate provided are connected to a protein comprising one or more VHH domains that specifically bind to HER2 via a linker comprising amino acid residues. Exemplary amino acid linker components include, but are not limited to, dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline and alanine-phenylalanine. Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline and glycine-glycine-glycine. The amino acid residues that may comprise an amino acid linker component may include those that occur naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. In some embodiments, the amino acid linker component of the HER2 binding agent conjugate provided herein is designed in the following: its selectivity for enzymatic cleavage by a specific enzyme (e.g., tumor-associated proteases, cathepsins B, C and D, and plasmin proteases). Additional exemplary linker components of the provided HER2 binder conjugates include, but are not limited to, 6-maleimidocaproyl "(MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)-pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB").

[0383] In certain embodiments, the linker can be composed of one or more linker components. For covalent attachment of proteins and drug moieties, the linker typically has two reactive functional groups, i.e., divalent in the sense of reaction. Divalent linker reagents for attaching two or more functional or biologically active moieties (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) and methods of using them in conjugation are known in the art.

[0384] In some embodiments, the protein of the HER2-binding agent conjugate comprises at least one HER2-binding VHH domain, and the protein is conjugated to a drug moiety. In certain embodiments, the protein of the HER2-binding agent conjugate is conjugated to about 1 to about 20 drug moieties. In some embodiments, the protein of the HER2-binding agent conjugate comprises at least one HER2-binding VHH domain, and the protein is conjugated to about 1 to about 20 drug moieties via a linker (L). In some embodiments, the HER2-binding agent conjugate comprises the following components: (VHH domain), (L) q and (partial) m , wherein the VHH domain is any HER2-binding VHH domain provided herein; L is a linker for attaching a protein of a HER2-binding agent to a moiety; m is at least 1; q is 0 or greater; and the resulting HER2-binding conjugate binds to HER2. In specific embodiments, m is 1 to 4 and q is 0 to 8.

[0385] In some embodiments, the disclosure provides a HER2 binding agent conjugate comprising at least one HER2-binding VHH domain provided herein, which is conjugated / attached to a therapeutic agent, such as a cytotoxic agent or a cytostatic agent. In some embodiments, the therapeutic agent is a targeting moiety, a small molecule drug (a non-peptide drug of less than 500 Daltons), a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof), a cytostatic agent, a cytotoxic agent, an immunosuppressant, a radioactive isotope (i.e., a radioconjugate), a prodrug activating enzyme, or an agent that increases biological half-life.

[0386] In some embodiments, the HER2 binding agent conjugate is a drug conjugate (i.e., an antibody drug conjugate (ADC)) comprising one or more HER2 binding domains provided herein conjugated to a therapeutic agent that is cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or a fragment thereof of bacterial, fungal, plant, or animal origin), or a radioactive isotope (i.e., a radioactive conjugate).

[0387] In some embodiments, the HER2 binding agent conjugate comprises at least one HER2 binding VHH domain conjugated to a toxin. In some embodiments, the toxin exerts its cytotoxic and / or cytostatic effects by tubulin binding, DNA binding, or topoisomerase inhibition. In some embodiments, the toxin is a bacterial toxin such as diphtheria toxin, a plant toxin such as ricin, a small molecule toxin such as geldanamycin, maytansinoid, or calicheamicin.

[0388] In some embodiments, the HER2-binding agent conjugate comprises at least one HER2-binding VHH domain conjugated to a therapeutic agent.In certain embodiments, the chemotherapeutic agent is selected from daunorubicin, doxorubicin, methotrexate, and vindesine.

[0389] In some embodiments, the HER2 binding antibody drug conjugate of the present disclosure is provided for targeted delivery of the therapeutic agent conjugated thereto to a tumor. In some cases, this results in targeted killing of tumor cells. In some embodiments, the HER2 binding antibody drug conjugate is internalized in a cell. In some embodiments, the conjugated therapeutic agent of ADC has intracellular activity. In certain embodiments, the HER2 binding agent conjugate is internalized, and the therapeutic agent is a cytotoxin that blocks the protein synthesis of the cell. In certain embodiments, the HER2 binding agent conjugate is internalized, and the therapeutic agent is a cytotoxin that triggers cytotoxicity and cytostatic effects through a mechanism selected from tubulin binding, DNA binding and topoisomerase inhibition. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide with ribosome inactivation activity. In certain embodiments, the therapeutic agent is selected from gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin and Pseudomonas exotoxin A, or a variant thereof.

[0390] In some embodiments, the HER2 binding agent contains a HER2 binding protein conjugated directly or indirectly to a marker to facilitate treatment and / or detection of bound or unbound HER2 binding agents. Suitable labeling substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, imaging agents, metal ions, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, 13-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 124 I. 131 I. 177 Lu, 166 Ho, 153 Sm, 18 F. 89Zr, 67 Ga, 68 Ga, 64 Cu and 52 M. For example, in some embodiments, the marker is a radiopaque or radioisotope, such as 3 H. 14 C. 32 P. 35 S. 123 I. 125 I. 131 I. In some embodiments, the label is a radioactive atom used for scintigraphic studies, such as 99 Tc or 123 I, or spin labels for nuclear magnetic resonance (NMR / MRI) imaging, such as 89 Zr, 123 I. 131 I. 111 In, 19 F. 13 C. 15 N. 17 O, gadolinium, manganese, or iron.

[0391] In certain embodiments, the label is a radionuclide. In some embodiments, the radionuclide is a radiohalogen isotope. In certain embodiments, the radionuclide is a radiohalogen isotope selected from: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I or 75 Br, 77 Br, 122 I. 124 I. 125 I. 131 I and 211 In certain embodiments, the radionuclide is 131 I. 211 At 125 I or 124 1. In some embodiments, the radionuclide is a radiometal isotope. In certain embodiments, the radionuclide is a member selected from the following radiometal isotopes: 144 Sc, 45 Ti, 51 Cr, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 68 Ge, 75Se, 82 Sr. 86 Y. 99 Mo, 99m Tc, 110m In, 111 In, 166 Ho, 186 Re, 195m Pt and 201 Tl; or 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89 Zr, 90 Y. 111 In, 153 Sm, 149 Tb, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th and 227 Th. In certain embodiments, the radionuclide is 177 Lu, 131 I. 211 At or 225 Ac.

[0392] In some embodiments, the HER2 binding agent contains a HER2 binding protein conjugated to a marker via a chelating moiety. In some embodiments, the chelating moiety is covalently attached to the protein via a lysine residue. In some embodiments, the marker forms a complex with a metal, and the complex is chelated by the chelating moiety.

[0393] The HER2 binding agent conjugates provided herein can be prepared by any of a variety of methods known in the art (such as organic chemical reactions), conditions and reagents. In some embodiments, the method includes: (1) reacting a nucleophilic group of a protein comprising a HER2 binding VHH domain provided herein with a divalent linker reagent to form a HER2 binding VHH-L via a covalent bond, followed by reaction with a therapeutic agent portion T; and (2) reacting a nucleophilic group of a therapeutic agent portion with a divalent linker reagent to form a TL via a covalent bond, followed by reaction with a nucleophilic group of a protein comprising a HER2 binding VHH domain.

[0394] Nucleophilic groups on proteins include, but are not limited to: (a) N-terminal amine groups, (b) side chain amine groups, such as lysine, (c) side chain thiol groups, such as cysteine, and (d) sugar hydroxyl groups or amino groups where the protein is glycosylated. Amines, thiols and hydroxyls are nucleophilic and can react with electrophilic groups on linker moieties to form covalent bonds, and linker reagents include: (a) active esters, such as NHS esters, HOBt esters, haloformates and acid halides; (b) alkyl and benzyl halides, such as haloacetamides; (c) aldehydes, ketones, carboxyls and maleimide groups. Additional nucleophilic groups can be introduced into proteins by the reaction of lysine with 2-iminothiolane (Traut's reagent), which results in the conversion of amines to thiols. Reactive thiol groups can be introduced into proteins (or fragments thereof) by the introduction of one, two, three, four or more cysteine ​​residues (e.g., preparing mutant proteins comprising one or more non-natural cysteine ​​amino acid residues).

[0395] Conjugates, such as HER2 binding agent conjugates, can also be produced by modifying proteins (such as HER2 binding proteins disclosed herein) to introduce electrophilic moieties, which can react with nucleophilic substituents on linker reagents or therapeutic agents. The sugars of glycosylated proteins can be oxidized, for example, with periodate oxidizing agents to form aldehyde or ketone groups, which can be connected to the amine groups of linker reagents or therapeutic agent moieties. The resulting imine Schiff base group can form a stable linkage, or can be reduced, for example, by a borohydride reagent to form a stable amine linkage. In certain embodiments, the reaction of the carbohydrate moiety of the glycosylated protein with galactose oxidase or sodium metaperiodate can produce carbonyl (aldehyde and ketone) groups in the protein, which can react with appropriate groups on the therapeutic agent moiety (Hermanson, Bioconjugate Techniques). In certain embodiments, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, which results in the generation of aldehydes replacing the first amino acid. Such aldehydes can react with therapeutic agent moieties or linker nucleophiles.

[0396] Likewise, nucleophilic groups on the therapeutic agent moiety can include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that are capable of reacting with electrophilic groups on the linker moiety to form a covalent bond, and linker reagents include: (a) active esters such as NHS esters, HOBti esters, haloformates, and acidhalides; (b) alkyl and benzyl halides such as haloacetamides; and (c) aldehyde, ketone, carboxyl, and maleimide groups.

[0397] The HER2 binding agent conjugates provided herein can be prepared using any method known in the art. See, for example, International Publication Nos. WO 2009 / 067800 and WO 2011 / 133886, and US Publication No. 2014322129, each of which is incorporated herein by reference in its entirety.

[0398] B. Targeted radiotherapy HER2 binders

[0399] In some embodiments, the disclosure provides HER2 for therapeutic and / or diagnostic purposes in combination with targeted radiotherapeutic agents, comprising a HER2 binding agent provided herein. In addition to HER2 in combination with VHH domains as described herein, such targeting agents may also include radiolabeled prosthetic agents / groups and optionally, chelating agents (macrocyclic or acyclic).

[0400] It should be noted that although such agents are described herein as "radiotherapy" agents, the term is intended to cover agents suitable for therapeutic and diagnostic purposes. In addition to HER2 binding agents (i.e., targeting moieties) as described herein, such targeted radiotherapy agents may also include radiolabeled prosthetic agents / groups and, optionally, chelating agents (macrocyclic or acyclic). Radiolabeled prosthetic agents are compounds or radicals that typically include radiolabels, charged groups (groups) (CG) and macromolecular conjugated moieties (MMCM), which are suitable for attachment to HER2 binding agents. Each of these components may optionally be associated with one or more cleavable (or non-cleavable) linkers.

[0401] There is no particular limitation on the adjuvants that can be attached to the HER2 binding agents disclosed herein to provide targeted radiotherapeutic agents. Generally, any adjuvant containing a radiolabel can be used according to the present disclosure, wherein the adjuvant can be appropriately attached to the HER2 binding agents provided herein.

[0402] In some embodiments, the radiolabel may be a radiohalogen. The radiohalogen may include, for example, an alpha emitter, a beta emitter, or an Auger electron emitter. They may include radiohalogens suitable for PET, SPECT, or intraoperative imaging. See, for example, US Patent No. 5,302,700, which is incorporated herein by reference in its entirety. The radiohalogen may, for example, be selected from the group consisting of: 18 F. 75 Br, 76 Br, 77 Br, 123 I. 124 I. 125 I. 131 I and 211 In some embodiments, the radiohalogen is 18F. In some embodiments, the radiohalogen is selected from the group consisting of: 75 Br, 76 Br, 77 Br, 123 I. 124 I. 125 I. 131 I and 211 In some embodiments, the radiohalogen is selected from 123 I. 124 I. 125 I. 131 I and 211 In some embodiments, the radiohalogen is selected from 123 I. 124 I. 125 I and 131 I. In some embodiments, the radiohalogen is 211 At.

[0403] In some embodiments, the radiolabel is a metal radionuclide. Metal radionuclides that can be complexed within an adjuvant (e.g., within a chelating agent as described above) include, but are not limited to 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 90 Y. 111 In, 177 Lu, 212 Pb, 212 Bi, 213 Bihe 225 Ac.

[0404] A charged group (CG) is typically a group that is charged under the physiological conditions of the internal cell environment. In some embodiments, a charged group (CG) includes a guanidine, a PO3H group, or a SO3H group. In some embodiments, a CG is a guanidino-alkyl group containing more than one carbon. In some embodiments, a CG is a guanidino-hydrophilic group (such as an amino or hydroxyl group) and / or an alkoxycarbonylguanidine group. In some embodiments, a CG comprises one or more charged d-amino acids, such as arginine, glutamic acid, aspartic acid, lysine, and / or phosphono / sulfophenylalanine. In some embodiments, a CG comprises a hydrophilic carbohydrate moiety. In some embodiments, a compound may contain one, two, or three CG moieties (and optionally, corresponding linker groups) to increase intracellular trapping in cancer cells. The prosthetic group provided herein may comprise a single CG or may comprise two or more CGs. When more than one CG is associated with a given prosthetic group, each such CG may be the same or different.

[0405] MMCM is a part that can be combined with the HER2 binding agent provided herein. In some embodiments, MMCM is an active ester. Active ester is defined as following ester in this article, usually under mild conditions, under the condition that the biological function loss of the HER2 binding agent is not to be caused, it can be conjugated with the group (such as amine group) present on the HER2 binding agent. Exemplary such MMCM groups include but are not limited to N-hydroxysuccinimide (NHS) or tetrafluorophenol (TFP) ester, pentafluorophenol (PFP), p-nitrophenol (PNP), isothiocyanate group or maleimide group. Such MMCM usually leads to random (non-site-specific) labeling of amine groups on HER2 binding agents. In some embodiments, MMCM is, for example, tripeptide GGG, which provides site-specific conjugation to be performed (such as using enzyme sorting enzyme), which results in conjugation with only one site (N-terminal or C-terminal of HER2 binding agent). Antigens Enzymes used to obtain suitable conjugates include, for example, transglutaminases, lipoic acid ligases, farnesyl transferases, and the like, including, for example, those disclosed in Massa et al., Exp Opin Drug Del. (2016) 13(8): 1149-63; Zhang et al., Chem Soc Rev. (2018) 47: 9106-36; Falck et al., Antibodies (2018) 7(4): 1-19, and van Berkel et al., Drug Disc. Today: Tech. (2018) 30: 3-10, which are not incorporated herein by reference in their entirety. It should be understood that while the prosthetic agent is referred to as comprising an MMCM and the targeted radiotherapeutic agent is generally described as comprising a prosthetic agent attached to a HER2 binding agent, the targeted radiotherapeutic agent will generally not comprise a complete MMCM; rather, it will comprise a portion resulting from the coordination of the MMCM with the HER2 binding agent.

[0406] Certain non-limiting prosthetic groups conjugated to HER2 binding agents to give targeted radiotherapeutic agents contemplated by the present disclosure include, for example, those of Formula 1 and derivatives and variations thereof.

[0407]

[0408] (Formula 1: General structure of type I compounds)

[0409] Some of these type I compounds are generally disclosed in International Publication No. WO2018 / 178936 (Duke University), which is incorporated herein by reference in its entirety. Typically, such prosthetic agents may include homo (X=CH) or hetero (X=N) aromatic rings attached thereto: MMCM for coupling the prosthetic compound / group to the HER2 binding agent, a radioactive halogen (Y) as described above; and one or more CGs. Each of these components may be attached to the aromatic ring via a linker (L1, L2, L3) or may be directly bonded to the aromatic ring (ie, where L1 and / or L2 and / or L3 are bonds). Each of these components shown in Formula 1 is described in further detail below.

[0410] A charged group (CG) is typically present in the auxiliary group of Formula 1, i.e., m is 1 or greater. Typically, m is 1; however, more than one CG may be attached to the ring such that m=2, m=3, and (where X=CH), m may be 4. When more than one CG is attached to the ring, each such CG (and the corresponding L2) may be the same or different. In certain embodiments, as indicated below (as shown in Formula 2), another moiety may be attached to the ring of Formula 1, and, when such additional moiety is charged, m may be 0 (i.e., in some embodiments, the additional moiety may effectively act as a "charged group"). The CG may be directly bound to the aromatic ring of Formula 1 (L2=direct bond) or may be bound to the aromatic ring through a linker (L2). L2 can be a spacer, such as a substituted or unsubstituted alkyl chain (e.g., a simple substituted or unsubstituted alkyl chain, such as a methylene group), a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, a PEG chain of at least three oxygens, or a peptide cleavable by a brush border enzyme, such as any of the foregoing GK, GY or GFK. It should be noted that in certain embodiments, where CG is guanidine and L2 is an unsubstituted alkyl chain, the unsubstituted alkyl chain comprises two or more carbon atoms.

[0411] In some embodiments, a metabolizable spacer or cleavable linker L2 (e.g., a brush border enzyme cleavable linker) is located between the CG and the aromatic ring of Formula 1. Using these formulations, increased uptake and retention of radioactivity in the kidney can be avoided because in the kidney, the CG moiety is cleaved, which eliminates the charge and allows the radioactive species (now neutral or less charged) to escape from the tubular cells in the kidney, where they are then rapidly excreted into the urine. Although radioactive brush border enzyme cleavable linkers have been used before, they have not been used in this manner to form a "charge switch" in which the labeled reagent is charged in the tumor and is therefore retained, but loses its charge in the kidney and is therefore cleared.

[0412] Such linkers include: linker sequences targeting transmembrane peptidase β (meprinβ, a metalloproteinase expressed in the renal brush border membrane) (Jodal et al., PLoS One (2015) 10(4):e0123443); C-terminal lysine linked to the antibody fragment via the ε-amino group of lysine, or C-terminal (N(ε)-amino-1,6-hexane-bisvinyl sulfone) lysine, which reduces renal uptake by utilizing the lysine-specific carboxypeptidase activity of the renal brush border enzyme, which cleaves the radiolabeled peptide linker prior to uptake by proximal tubular cells (Li et al., Bioconjugate Chem. (2002) 13(5):985-95); L-tyrosine O-methyl, L-asparagine, L-glutamine, N-Boc-L-lysine (Akizawa et al., Bioconjugate Chem. (2013) 24:291-9); and glycyllysine (Arano et al., Cancer Res. (1999) 59:128-34); each of which is incorporated herein by reference in its entirety.

[0413] The MMCM may be directly bound to the aromatic ring of Formula 1 (L1 = direct bond) or may be bound to the aromatic ring through a linker (L1). L1 may be, for example, a spacer such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units).

[0414] The positions of these three parts (-L1-MMCM, -L2-CG and -L3-Y) on the aromatic ring of Formula 1 can vary. Where X is CH, these three parts can be placed at any position of the aromatic ring. In some such embodiments, the -L2-CG and -L3-Y parts are respectively located at the 3rd and 4th positions (or respectively at the 4th and 3th positions) relative to the -L1-MMCM part (at the 1st position). In some such embodiments, the -L2-CG and -L3-Y parts are located at the 3rd and 5th positions relative to the -L1-MMCM part, so that the aromatic ring contains the indicated parts at the 1st, 3rd and 5th positions. Where X is N, these three parts can be placed at any of the remaining five positions of the ring, for example, including but not limited to the 2nd, 4th and 6th positions of the ring.

[0415] In certain embodiments, X is CH; L3 is a bond (i.e., the radiohalogen Y is directly attached to the benzene ring); m is 1; L2 is CH2 and CG is Z-guanidine (such that L2-CG is an alkylguanidine, which can alternatively be described as L2 is a bond and CG is an alkyl (e.g., methyl)guanidine); L1 is a bond, and MMCM is a moiety (e.g., an NHS ester) suitable for reacting with an amine on the HER2 binding agent (to form an amide bond in the resulting targeted radiotherapeutic agent).

[0416] Certain prosthetic compounds within the scope of Formula 1 that can be complexed with a HER2 binding agent to provide a targeted radiotherapy agent provided herein include compounds of Formula 1A and derivatives and variants thereof, as shown below. As shown, in Formula 1A, X is CH (i.e., the aromatic ring is a benzene ring), L2 is a methylene group, and three moieties (-L1-MMCM, -L3-Y, and -CH2-CG) are present at positions 1, 3, and 5 of the aromatic ring.

[0417]

[0418] (Formula 1A: General structure of subclass IA compounds)

[0419] Additionally, non-limiting prosthetic groups that can be coordinated to a HER2 binding agent to give a targeted radiotherapeutic agent encompassed by the present disclosure include, for example, those having the general structure shown below in Formula 2 (referred to as "Type II compounds").

[0420] MC-Cm-L4-Cm-T

[0421] (Formula 1: General structure of class II compounds)

[0422] Some of these class II compounds are generally disclosed in International Publication No. WO2018 / 178936 (Duke University), which is incorporated herein by reference in its entirety. Such compounds include a multidentate metal chelating portion (MC), a linker (L4) having a conjugated portion (Cm) at both ends of L4, and a radiohalogenated template (T). T is a cofactor compound and can be, for example, a compound of Formula 1 as shown above or a compound of Formula 1A (compound containing MMCM). In some such embodiments, as described above, m=0, wherein the "MC-Cm-L4-Cm" portion of Formula 2 provides the desired function of the L2-CG portion in Formula 1 above (i.e., the MC-Cm-L4-Cm substituent is a fully "charged group"). In other such embodiments, m=1, 2 or 3, so that the aromatic ring of "T" has at least four substituents, i.e., L1-MMCM, L3-Y, L2-CG and Cm-L4-Cm-MC, and may optionally include one or more additional L2-CG substituents.

[0423] L4 may be as defined above for L1 and L3. Thus, L4 may be a direct bond or may be, for example, a spacer such as a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain or a short polyethylene glycol (PEG) chain (1-10 ethylene glycol units). L4 is again as defined above, but with NH, CO (carbonyl) or S (thioether) at one or both ends.

[0424] Cm can be, for example, thiourea, amide or thioether. For example, in some embodiments, Cm is thiourea (e.g., when the conjugated functional group in the chelating moiety and T is isothiocyanate), amide (when the conjugated functional group in the chelating moiety and T is NHS or TFP active ester or acyl halide) or thioether (when the conjugated functional group in the chelating moiety and T is maleimide).

[0425] T is usually a radiolabeled portion containing MMCM, and the polymer can be coupled to the compound via MMCM to provide a targeted radiotherapy agent. As described above, in some embodiments, T can be a compound / radical of Formula 1 or a compound / radical of Formula 1A. In other embodiments, other radiohalogen templates (T) can be used, including but not limited to, iso-SGMIB (as disclosed in Choi et al., Nucl Med Biol. (2014) 41 (10): 802-12), SIPC (as disclosed in Reist et al., (1997) Nucl Med Biol. 24 (7): 639-48, which is incorporated herein by reference in its entirety) or SDMB (as disclosed in US Patent No. 5,302,700), all of which are incorporated herein by reference in their entirety.

[0426] MC can be any multidentate moiety and can be cyclic or acyclic. The composition of MC can vary. MC can be uncomplexed (lacking metal) or complexed with a metal in a stable (non-radioactive) or radioactive form, such as a trivalent metal (M +3 ) such as lutetium, yttrium, indium, actinium, or gallium and the MC is attached to the linker using one of the free COOH groups present on the MC or via other positions on the MC including one of the MC backbone carbons. Certain specific radiometals that may be complexed with the MC include, but are not limited to, radiometals selected from the group consisting of 177 Lu, 64 Cu, 111 In, 90 Y. 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 It should be noted that this list is not exhaustive, and although these exemplary radiometals are trivalent, certain MCs that may be used in accordance with the present disclosure may incorporate metals of other valencies, and such MCs and radiometals are also included herein.

[0427] In some embodiments, the inclusion of a radioactive metal associated with the MC can eliminate the need for a radioactive atom elsewhere on the molecule (e.g., "Y" when T of Formula 2 = a portion of Formula 1 / 1a). Thus, in compounds of Formula 2, "T" may or may not include a radioactive atom (e.g., a halogen). In some embodiments, T comprises a portion as shown in Formula 1 / 1a above, wherein the "Y" group is a non-radioactive halogen (e.g., non-radioactive bromine or iodine). In other embodiments, compounds of Formula 2 are provided that include both a radioactive halogen (e.g., "Y" when T of Formula 2 = a portion of Formula 1 / 1a) and a radioactive metal (associated with the MC, such as the radioactive metals described above). In certain specific embodiments, such a strategy will allow, for example, the same adjuvant to be used for multiple isotopes. In certain specific examples, compounds of Formula 2 have low energy beta emitters (e.g., 131 I) plus high energy beta emitters (e.g. 90 Y); or alpha emitters (e.g. 225 Ac) metals and beta-emitter halogens (e.g. 131 I); or alpha emitter halogens (e.g. 211 At) and beta-emitting radioactive metals (e.g. 177 Lu).

[0428] In some embodiments, MC is a macrocyclic ligand consisting of a ring containing 8 or more atoms, with at least 3 negatively charged substituents, such as carboxyl or phosphonate groups. Exemplary macrocyclic ligands suitable as MC groups include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP). In other embodiments, MC is MeO-DOTA, as disclosed in Gali et al., Anticancer Research (2001) 21 (4A): 2785-92, which is incorporated herein by reference in its entirety.

[0429] An example of a class II compound suitable for conjugation with a HER2 binding agent to form a targeted radiotherapeutic agent according to the present disclosure is shown below in Formula 2A, wherein MC is a macrocyclic ligand comprising DOTA, and wherein the radiohalogenated template T is a moiety corresponding to Formula 1.

[0430]

[0431] (Formula 2A: Exemplary Class II Compounds with DOTA MC)

[0432] The left square bracket in Formula 2A is intended to express that there is no restriction on the specific site on MC (DOTA) to which the Cm group is bonded, i.e., Cm can be bonded to DOTA at different sites thereon. Similarly, the right square bracket in Formula 2A is intended to express that there is no restriction on the specific site on the "T" ring to which the Cm group is bonded, i.e., Cm can be bonded to T at different sites on the ring. Again, as described above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T in Formula 2A contains four substituents (including connected MC, L2-MMCM, L3-Y, and L2-CG). In other embodiments, the benzene ring of T in Formula 2A contains three substituents (including connected MC, L2-MMCM, and L3-Y). The latter embodiment is particularly relevant when the connected MC is charged, i.e., it can replace the desired function of providing the "L2-CG" substituent.

[0433] In some embodiments, MC is an acyclic ligand consisting of a chain containing 6 or more atoms with at least 3 negatively charged substituents, such as carboxyl or phosphonate groups. Exemplary acyclic ligands suitable as MC groups include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetramethylenephosphonic acid (EDTMP), and ethylenediaminetetraacetic acid (EDTA). Examples of class II compounds are shown in Formula 2B below, where MC is an acyclic ligand comprising DTPA, and where the radiohalogenated template T is a moiety corresponding to Formula 1.

[0434]

[0435] (Formula 2B: Exemplary Class II Compounds with DTPA(acyclic)MC)

[0436] As described above for Formula 2A, the left square bracket in Formula 2B is intended to express that there is no restriction on the specific site on MC (DTPA) to which the Cm group is bonded, i.e., Cm can be bonded to DTPA at different sites thereon. Similarly, the right square bracket in Formula 2A is intended to express that there is no restriction on the specific site on the "T" ring to which the Cm group is bonded, i.e., Cm can be bonded to T at different sites on the ring. Again, as described above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T in Formula 2B contains four substituents (including connected MC, L2-MMCM, L3-Y, and L2-CG). In other embodiments, the benzene ring of T in Formula 2A contains three substituents (including connected MC, L2-MMCM, and L3-Y). The latter embodiment is particularly relevant when the connected MC is charged, i.e., it can replace the desired function of providing the "L2-CG" substituent.

[0437] In some embodiments, targeted radiotherapeutic agents are provided by attaching a HER2 binding agent as described herein to a compound of Formula 2, wherein MC = DOTA, L4 = -NH(CH2)6NH-, T = 3-iodo-5-succinimidyloxycarbonyl-benzoyl, Cm = amide and MMCM = N-hydroxysuccinimide ester (maleimide-containing moiety) or (Gly) n (For site-specific conjugation using sortase (see formula above)).

[0438] Additionally, non-limiting prosthetic groups that can be coordinated to a HER2 binding agent to give a targeted radiotherapeutic agent encompassed by the present disclosure include, for example, those having the general structure shown below in Formula 3 (referred to as "Class III compounds").

[0439] MC-T

[0440] (Formula 3: General structure of class III compounds)

[0441] Some of these class III compounds are generally disclosed in WO 2021 / 096968 (Duke University), which is incorporated herein by reference in its entirety. In such compounds, in some embodiments, MC can be as defined above (and can therefore be cyclic or acyclic, and can be uncomplexed or complexed with a metal). In some embodiments, the disclosed adjuvants and corresponding targeted radiotherapeutic agents include functionalized MC, such as modified DOTA or modified DOTA. An exemplary modified DOTA is DOTA-tris (t-Bu ester), that is, DOTA containing a tert-butyl acetate group associated with each nitrogen atom except the nitrogen atom when it is used to connect to "T". Such compounds suitable for reaction to provide MC-T are also referred to as tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate. Upon removal of the tert-butyl protecting group, a deprotected form is provided, which may be referred to as 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid. Further modified DOTA that may be used in certain embodiments of the present disclosure is an acyclic analog, such as an acyclic analog of DOTA or an acyclic analog of modified DOTA. An exemplary tert-butyl acetate functionalized acyclic DOTA that may be reacted to form MC-T is tert-butyl (2-((2-((2-aminoethyl)(tert-butoxycarbonyl)-amino)ethyl)(tert-butoxycarbonyl)amino)ethyl)glycine ester. Upon removal of the tert-butyl protecting group, a deprotected form is provided, which may be referred to as (2-((2-((2-aminoethyl)(carboxyl)amino)ethyl)(carboxyl)amino)ethyl)glycine. Similarly, in some embodiments, a modified analog of NOTA is employed that includes a tert-butyl acetate group associated with each nitrogen atom except the nitrogen atom when it is used to be attached to "T". Such modified NOTA may be di-tert-butyl 1,4,7-triaza-1,4-dicarboxylate, which may react via the nitrogen atom in the absence of a tert-butyl group attached thereto to give MC-T. It should be understood that in all embodiments herein providing a tert-butyl acetate group associated with a MC group, the deprotected form (comprising an acetate group in place of the tert-butyl acetate group) is also intended to be encompassed herein.

[0442] Certain such modified "MC" groups are shown in the following formulas A-1, A-2 and A-3, respectively, wherein one feasible point of attachment to "T" is shown on each depicted "MC" portion, which provides an associated radiohalogen prosthetic moiety that can be attached to a HER2 binding agent to provide a targeted radiotherapeutic agent comprising the same.

[0443]

[0444] In such embodiments, MC is linked to the remainder of the prosthetic group moiety (which comprises "T") via any relevant atom (e.g., via a carbon or nitrogen (but not limited thereto) associated with MC) to give the corresponding radiohalogen prosthetic group moiety. For the modified DOTA and NOTA groups referred to herein, MC may advantageously be linked to T via a nitrogen present on the modified DOTA / NOTA (as shown in Formulas A-1, A-2, and A-3 above) or via other positions on MC (including, for example, via one of the MC backbone carbons).

[0445] T is a radiohalogenated template, as detailed above. In certain embodiments, T may be a compound of formula 1 or a compound of formula 1A. In some embodiments, T is a radiohalogenated template of formula B below, wherein its connection to MC is shown by a wavy line. T may be directly bonded to MC, i.e., it has a direct bond between the carbon atom shown adjacent to R1 and a portion on MC (e.g., wherein MC is DOTA / NOTA or modified DOTA / NOTA), or the carbon atom shown below may be directly bonded to the nitrogen atom of the backbone DOTA / NOTA structure.

[0446]

[0447] In Formula B, the following definitions apply:

[0448] R1 = H, ester or carboxylic acid.

[0449] A is R2-R3-Y or Y.

[0450] B is H, an alkoxy (eg, methoxy, ethoxy, propoxy, or butoxy) group, or a halogen (eg, a non-radioactive halogen such as selected from the group consisting of Cl, F, I, and Br).

[0451] R2 = a direct bond, an alkyl group, or an oxygen-containing moiety. Some examples of "oxygen-containing moieties" provided herein include, but are not limited to: -O-, -O-CH2-, -O-CH2CH2-, and the like.

[0452] R3 = direct bond, alkyl group or aromatic moiety (eg benzene ring).

[0453] The other definitions are as presented above for compounds of the other formulae.

[0454] In certain embodiments, R2 is -O-CH2- and R3 is a benzene ring, such that R2-R3 is a benzyloxy group attached to the central benzene ring of formula C. The positions of the substituents on the benzene ring relative to each other can vary. The L1-MMCM on the aromatic ring can vary relative to other substituents (A, B, and the connection to MC). In certain embodiments, the L1-MMCM group is in the meta position of the connection to MC. In some embodiments, the central benzene ring of the compound does not contain a substituent in the ortho position of L1-MMCM (so that the carbon connected to L1-MMCM is in the ortho position of two unsubstituted carbon atoms). In a specific embodiment, A is in the ortho position of the carbon connected to MC. Wherein A is in the ortho position of the carbon connected to MC, in some such embodiments, B is in the ortho position of the A substituent, and is H. In a specific embodiment, A is in the meta position of the carbon connected to MC. Wherein A is in the meta position of the carbon connected to MC, in some such embodiments, B is in the ortho position of the carbon connected to MC (and the meta position of the A substituent), and in certain embodiments, B is an alkoxy group (e.g., methoxy).

[0455] Various embodiments within Formula B are shown below. For example, in some embodiments, R1 is an ester group, such as CO2 t Bu, for example, as shown in the following formula B-1. In some embodiments, the central aromatic ring of T is functionalized with an alkoxy group (particularly a methoxy (OCH3) group) as a substituent B, for example, with three other substituents on the aromatic ring, for example, as shown in the following formula B-2. In some embodiments, the halogenation site or precursor portion is placed on an aromatic ring (aromatic ring) attached (directly or indirectly) to a central aromatic ring (aryl ring) (for example, including but not limited to being connected to a central aromatic ring via an O-alkyl-part), as shown in formula B-3, wherein, although the position of the halogen on the benzene ring can vary, the halogen is in the para position of the connection to the rest of the molecule. In some such embodiments as shown in formula C-3, B is H. In certain embodiments, as shown in formula B-4, the halogen site is on an aromatic ring connected to a central aromatic ring, wherein the aromatic ring is directly bonded to the central aromatic ring, as shown. In some such embodiments as shown in formula B-4, B is H. It should be noted that in the embodiment where the halogenation site or precursor moiety is attached to the aromatic ring of the central aromatic ring, the aromatic ring may not include other substituents, or in some embodiments, may include one or more additional substituents at different positions relative to the halogenation site or precursor moiety. For example, in some embodiments, the aromatic ring further includes guanidinomethyl. Therefore, although, for example, the following formulas B-3 and B-4 show aromatic rings substituted only with I (which may alternatively be any "Y" group provided herein), the disclosure is not limited thereto, and in various embodiments, other substituents may be present on the ring. In a specific embodiment, a portion of formula C-3 is provided, wherein the aromatic ring is substituted with I (or other Y) and guanidinomethyl.

[0456]

[0457]

[0458] In some embodiments, the halogenation site / precursor moiety is directly on the central aromatic ring (e.g., connected via a direct bond), as shown explicitly in formulas B-5 and B-6. In some such embodiments as shown in formula B-5, B is H. In formula B-5, the iodination site is adjacent to the chelate. In some such embodiments as shown in formula B-6, B is advantageously OCH3.

[0459]

[0460]

[0461] In a specific embodiment, a DOTA-SIB or NOTA-SIB hybrid radiohalogen prosthetic moiety as shown below is attached to a HER2 binding agent as described herein to provide a targeted radiotherapy agent. Although "I" is shown as a radiohalogen in the structure, it should be understood that various "Y" groups provided herein can be substituted at this position; in addition, the N-hydroxysuccinimidyl ester shown can be replaced by other groups (e.g., TFP ester groups) to attach to the HER2 binding agent. Formulas 3A, 3B, 3B', 3C and 3D depict non-limiting prosthetic moieties with DOTA-SIB (wherein 3B' provides a guanidinomethyl group on the aromatic ring (which can be in the ortho or meta position of the I substituent), and wherein X and Y can be placed in any of the five positions on the aromatic ring; if X is an iodine radioisotope or another radiohalogen (or a precursor thereof), then Y is a guanidinomethyl group, and vice versa). Formula 3E depicts non-limiting prosthetic moieties with NOTA-SIB, respectively,

[0462]

[0463]

[0464]

[0465] Non-limiting examples of structures in which L1-MC is a tetrafluorophenol (TFP) ester are shown in Formulas 3F to 3H below, and these structures can be further modified as described above (e.g., including but not limited to replacing I with other Y groups and adding one or more substituents to the O-alkylphenyl group of Formula 3G or 3H).

[0466]

[0467]

[0468] Although most structures shown in Formula 3 contain ester-containing R1-substituents (e.g., CO2 t Bu group), but in other embodiments, a radiohalogen prosthetic moiety or a precursor thereof is provided wherein R1 is H, as shown in Formula 3I below.

[0469]

[0470] Additional non-limiting radiohalogen prosthetic moieties that can be attached to a HER2 binding agent to give a targeted radiotherapeutic agent encompassed by the present disclosure include, for example, N-succinimidyl 5-[*I]iodo-3-pyridinecarboxyl ([*I]SIPC); SFPC, SAPC or any other radiohalogen derivative; N-succinimidyl-3-[*I]-iodobenzoate ([*I]SIB) and SFB, SAB or other halogen radionuclides; N-succinimidyl-4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB), as well as SAGMB, SFGMB or other radiohalogen derivatives; N-maleimidoethyl yl-3-(guanidinomethyl)-4-[*I]-iodobenzamide ([*I]MEGMIB); compounds comprising acyclic bifunctional chelators, including, for example, p-SCN-Bz-DTPA; tetra-t-Bu-DTPA; mercaptoacetyl triglycine (MAG3); MX-DTPA; CHX-DTPA (wherein CHX can be CHX-A or CHX-B), or macrocyclic bifunctional chelators, including P-SCN-Bn-DOTA; maleimidocysteineamido-DOTA derivatives; and benzyl TETA derivatives.

[0471] definition

[0472] "Cm-Cn alkyl" by itself or in composite expressions such as Cm-Cn haloalkyl, Cm-Cn alkylcarbonyl, Cm-Cn alkylamine, etc., means a straight or branched aliphatic hydrocarbon radical having a specified number of carbon atoms, for example, C1-C4 alkyl means an alkyl radical having 1-4 carbon atoms. C1-C6 alkyl has a corresponding meaning and also includes all straight and branched isomers of pentyl and hexyl. Exemplary alkyl groups for preparing the provided compositions are C1-C6 alkyl groups, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl, in particular C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-butyl and isobutyl. In a particular embodiment, one or more alkyl radicals are methyl and / or isopropyl. Alkyl groups may be unsubstituted or substituted with one or more identical or different substituents, each of which is independently selected from halogen, alkenyl, alkynyl, arene, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH, and -C(=O)O-alkyl. In certain embodiments, unless otherwise specified, alkyl groups are unsubstituted.

[0473] “C2-C n "Alkenyl" means a straight or branched aliphatic hydrocarbon radical containing at least one carbon-carbon double bond and having a specified number of carbon atoms, for example, C2-C4 alkenyl means an alkene radical having 2-4 carbon atoms; C2-C6 alkenyl means an alkene radical having 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl and hexenyl. Alkenyl can be unsubstituted or substituted with one or more substituents that are the same or different, each substituent being independent. In some embodiments, the alkenyl group is independently selected from halogen, alkenyl, alkynyl, arene, cycloalkyl, cyano, hydroxyl, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH and -C(=O)O-alkyl. In certain embodiments, alkenyl is unsubstituted unless otherwise specified.

[0474] “C2-C n"Alkynyl" means a straight or branched aliphatic hydrocarbon radical containing at least one carbon-carbon triple bond and having a specified number of carbon atoms, for example, C2-C4 alkynyl means an alkynyl radical having 2-4 carbon atoms; C2-C6 alkynyl means an alkynyl radical having 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl, pentynyl and hexynyl. Alkynyl can be unsubstituted or substituted with one or more substituents which may be the same or different, each substituent being Independently selected from halogen, alkenyl, alkynyl, arene, cycloalkyl, cyano, hydroxyl, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH and -C(O)O-alkyl. In a specific embodiment, unless otherwise indicated, alkynyl is unsubstituted.

[0475] As used herein, the term “C m -C n "Haloalkyl" means a C group in which at least one C atom is substituted by a halogen such as iodine, bromine or fluorine. m -C n Alkyl, (e.g., C m -C n A haloalkyl group may contain one to three halogen atoms). A typical haloalkyl group is a C1-C2 haloalkyl group, wherein halo suitably represents iodine. Exemplary haloalkyl groups include iodomethyl, diiodomethyl and triiodomethyl. As used herein, only one of the halogens may be radioactive.

[0476] The term "C m -C n "Hydroxyalkyl" means a C group in which at least one C atom is substituted by a hydroxy group. m -C n Alkyl. Typical C m -C n Hydroxyalkyl is a C m -C n Exemplary hydroxyalkyl groups include hydroxymethyl and hydroxyethyl.

[0477] As used herein, the term "C1-C 11 "Alkylene" means a straight or branched divalent alkyl radical having the indicated number of carbon atoms. In certain embodiments, the C m -C nThe alkylene radical is a C1-C3 alkylene. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH(CH3)-, and -CH(CH(CH3)2)-.

[0478] “C m -C n "Alkoxy" means a group C m -C n Alkyl-O-, where C m -C n Alkyl is as defined above. In certain embodiments, C1-C4 alkoxy is C1-C4 alkoxy, including methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-butoxy, sec-butoxy and isobutoxy. In certain embodiments, C m -C n Alkoxy is methoxy or isopropoxy. C1-C6-alkoxy has a corresponding meaning, extending to all straight-chain and branched isomers of pentyloxy and hexyloxy.

[0479] The term "Me" means methyl, and "MeO" means methoxy. The term "amino" means the free radical -NH2. The term "halo" means a halogen free radical, such as fluoro, chloro, bromo, iodo or astatine. Typically, the halo group is iodo, bromo or astatine. The term "aryl" means an aromatic ring, such as phenyl, biphenyl or naphthyl.

[0480] The term "heterocycloalkyl" refers to a stable saturated monocyclic 3-12 membered ring containing 1-4 heteroatoms independently selected from O, S and N. In some embodiments, the stable saturated monocyclic 3-12 membered ring contains 4 N heteroatoms. In a second embodiment, the stable saturated monocyclic 3-12 membered ring contains 2 heteroatoms independently selected from O, S and N. In a third embodiment, the stable saturated monocyclic 3-12 membered ring contains 3 heteroatoms independently selected from O, S, and N. Heterocycloalkyl can be unsubstituted or substituted with one or more identical or different substituents, each of which is independently selected from halogen, alkenyl, alkynyl, arene, cycloalkyl, cyano, hydroxyl, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl), -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. In a specific embodiment, unless otherwise indicated, heterocycloalkyl is unsubstituted.

[0481] The term "heteroaryl" refers to a stable aromatic ring containing 1-4 heteroatoms independently selected from O, S and N. In certain embodiments, heteroaryl moieties useful in the present disclosure have 6 ring atoms. In some embodiments, the stable aromatic ring system contains one heteroatom which is N.

[0482] The term "amino C m -C n "Alkyl" means a C m -C n Alkyl, which is substituted by an amino group, i.e. one of the hydrogen atoms of the alkyl part is replaced by an NH2 group. m -C n "Alkyl" is amino C1-C6 alkyl.

[0483] The term "amino C m -C n "Alkylcarbonyl" means a C m -C n Alkylcarbonyl, in which one of the hydrogen atoms of the alkyl portion is replaced by an NH2 group. m -C n "Alkylcarbonyl" is amino C1-C6 alkylcarbonyl. Amino C m -C n Examples of alkylcarbonyl groups include, but are not limited to, glycyl: C(=O)CH2NH2, alanyl: C(=O)CH(NH2)CH3, valyl: C=OCH(NH2)CH(CH3)2, leucyl: C(=O)CH(NH2)(CH2)3CH3, isoleucyl: C(=O)CH(NH2)CH(CH3)(CH2CH3) and norleucyl: C(=O)CH(NH2)(CH2)3CH3, etc. This definition is not limited to naturally occurring amino acids.

[0484] Relevant terms are to be interpreted accordingly, consistent with the definitions provided above and common usage in the technical field.

[0485] As used herein, the term "(=O)" forms a carbonyl moiety when attached to a carbon atom. It should be noted that an atom can only carry an oxo group when the valency of the atom permits.

[0486] The terms "monophosphate, diphosphate and triphosphate" refer to the following groups:

[0487]

[0488] The terms "thiomonophosphate, thiodiphosphate and thiotriphosphate" refer to the following groups:

[0489]

[0490] As used herein, radical positions on any molecular moiety used in the definitions may be anywhere on that moiety so long as it is chemically stable. When any variable present occurs more than one time in any moiety, each definition is independent.

[0491] Whenever used herein, the phrases "compounds of Formula 1," "compounds of Formula 1A," "compounds of Formula 2," "compounds of Formula 3," or "compounds of the present disclosure," or similar phrases, are meant to include compounds of Formula 1 and subgroups of compounds of Formula 1, compounds of Formula 2 and subgroups of compounds of Formula 2, and compounds of Formula 3 and subgroups of Formula 3, respectively, including possible stereochemically isomeric forms, and pharmaceutically acceptable salts and solvates thereof.

[0492] The term "solvate" encompasses any pharmaceutically acceptable solvates that the compounds of formulae 1, 2 and 3 and their salts are able to form. Such solvates are, for example, hydrates, alcoholates, such as ethanolates, propanolates, etc., especially hydrates.

[0493] Generally, the names of the compounds provided herein were generated using ChemDraw Professional 16.0. In addition, if the stereochemistry of a structure or portion of a structure is not indicated, for example, with bold or dashed lines, then the structure or portion of a structure should be understood to encompass all stereoisomers thereof.

[0494] Joints can also be selected to promote the bonding of corresponding parts and core structures.For example, as discussed in more detail below about the exemplary synthetic route of artificial compounds, representative joints are difunctional alkyl chains (for example-CH2-,-C2H4-,-C3H6- etc.) with 1-6 carbon atoms, in which one of the carbon atoms can be replaced by cyclic (hydrocarbon ring) free or heterocyclic (heterocycle) free radicals.Representational heterocyclic free radicals have at least one nitrogen-atoms in heterocycle.Therefore, the specific examples of this type of heterocyclic radical are diazinyl, diazolyl, triazinyl, triazolyl, tetrazinyl and tetrazolyl. These and other heterocyclic radicals, or other cyclic radicals, may be optionally fused to another cyclic or heterocyclic radical, or additionally fused to another cyclic or heterocyclic radical that is itself part of a fused ring system (e.g., a triazole radical may be fused to an 8-membered cyclic or heterocyclic radical, which itself is fused to two 6-membered rings, as is the case with a triazole radical (or other nitrogen-substituted heterocyclic hydrocarbon radical) fused to a dibenzoazo radical). Thus, a linker containing three or more fused rings (such as hydrocarbon rings, heterocycles, and combinations of these rings) is possible. Representative charged group linkers L2 are divalent substituted or unsubstituted alkyl chains, substituted or unsubstituted alkenyl chains, or substituted or unsubstituted alkynyl chains having 1-6 carbon atoms. Typically, L1, L2, L3 and / or L4 can be (or can include) a substituted or unsubstituted divalent alkyl radical having 1 to 6 carbon atoms, wherein one or more carbon atoms can be substituted and / or replaced by heteroatoms such as NH, O or S, or can be replaced or replaced by another alkyl group (e.g., resulting in the formation of a branched alkyl group) having 1 to 8 carbon atoms. For example, one carbon atom of an alkyl radical can be substituted to provide a carbonyl (C=O) group, and an adjacent carbon atom is substituted by NH, thereby resulting in a peptide / amide linkage -(C=O)-NH-. Therefore, representative linkers L1, L2, L3 and L4 can include divalent alkyl radicals having one or more such peptide linkages, -NH- linkages, -(C=O)- linkages and / or cyclic -C6H4- linkages, including any two, three or four such linkages incorporated into the alkyl chain. Additionally, where L1, L2 and / or L3 are divalent alkyl radicals, carbon-carbon double bonds and / or carbon-carbon triple bonds may be formed between one or more pairs of adjacent carbon atoms to provide divalent, unsaturated (e.g., olefinic) alkyl radicals.

[0495] In some embodiments, the HER2 binding agent is a targeted radiotherapy agent having the following structure:

[0496] VHH-RLC,

[0497] Wherein VHH is a protein comprising a HER2 binding VHH domain provided herein (e.g., having a sequence of any one of SEQ ID NOs 1-81 and 205, or a fragment or variant thereof), and RLC is a radiolabeled chemical used to facilitate direct or indirect attachment of a radionuclide to VHH. In some embodiments, RLC is a class I compound, a class II compound, a class III compound, a compound, an indirect halogenated prosthetic compound, an acyclic bifunctional chelator.

[0498] In some embodiments, the RLC is a class I compound. Examples of certain non-limiting targeted radiotherapeutic agents falling within the scope of the present disclosure are as follows, wherein the VHH is a protein comprising a HER2 binding VHH domain provided herein (e.g., having a sequence of any one of SEQ ID NOs 1-81 and 205, or a fragment or variant thereof):

[0499]

[0500] In some embodiments, RLC is

[0501]

[0502]

[0503] In some embodiments, the RLC is a class II compound. Examples of certain non-limiting targeted radiotherapeutic agents falling within the scope of the present disclosure are as follows, wherein the VHH is a protein comprising a HER2 binding VHH domain provided herein (e.g., a sequence having any one of SEQ ID NOs 1-81 and 205, or a fragment or variant thereof):

[0504] In some embodiments, RLC is a class III compound. Examples of certain non-limiting targeted radiotherapeutic agents falling within the scope of the present disclosure are as follows: wherein VHH is a protein comprising a HER2 binding VHH domain provided herein (e.g., having a sequence of any one of SEQ ID NOs 1-81 and 205, or a fragment or variant thereof),

[0505]

[0506]

[0507] In some embodiments, RLC is a direct radiolabeling using an electrophilic substitution reaction for radiohalogenation. In some embodiments, RLC is a direct radiolabeling using an electrophilic substitution reaction (radiohalogenation using Iodogen, chloragen T, and N-halosuccinimide).

[0508] In some embodiments, RLC is an indirect halogenated auxiliary compound. In some embodiments, RLC is N-succinimidyl 5-[*I] iodo-3-pyridine carboxyl ([*I]SIPC). In some embodiments, RLC is SFPC, SAPC, or any other radioactive halogen derivative known in the art. In some embodiments, RLC is N-succinimidyl-3-[*I]-iodobenzoate ([*I]SIB). In some embodiments, RLC is SFB, SAB, or any other halogen radionuclide known in the art. In some embodiments, RLC is N-succinimidyl-4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB). In some embodiments, RLC is SAGMB, SFGMB, or any other radioactive halogen derivative known in the art. In some embodiments, RLC is N-maleimidoethyl-3-(guanidinomethyl)-4-[*I]-iodobenzamide ([*I]MEGMIB).

[0509] In some embodiments, RLC is an acyclic bifunctional chelator. In some embodiments, RLC is p-SCN-Bz-DTPA, tetra-t-Bu-DTPA, mercaptoacetyl triglycine (MAG3) or MX-DTPA. In some embodiments, RLC is CHX-DTPA (wherein CHX can be CHX-A or CHX-B).

[0510] In some embodiments, RLC is an acyclic bifunctional chelator. In some embodiments, RLC is P-SCN-Bn-DOTA. In some embodiments, RLC is a maleimidocysteine ​​amido-DOTA derivative. In some embodiments, RLC is a benzyl TETA derivative.

[0511] Radioactive halogen prosthetic moieties and corresponding targeted radiotherapeutic agents can be prepared in various ways, for example according to methods known in the art. Targeted radiotherapeutic agents can be provided, for example, by attaching a radionuclide / radioactive halogen prosthetic moiety directly or indirectly to a HER2 binding agent provided herein. Some such methods are described in, for example, International Publication Nos. WO 2018 / 178936 and WO2021 / 096968 (Duke University), each of which is incorporated herein by reference in its entirety.

[0512] For example, in some embodiments, a precursor of a radiohalogen prosthetic moiety is prepared (e.g., comprising a tin or other alkyl metal moiety, or a boronic acid moiety or a boronic ester moiety, such as where Y in the above formula is, for example, a tin or other alkyl metal moiety, or a boronic acid moiety or a boronic ester moiety). The precursor can then be radiolabeled and purified. In some embodiments, such radiolabeling methods can employ an electrophilic substitution reaction, and one or more catalysts can be used, for example, including but not limited to 1,2,3,6-tetrachloro-3α,6α-diphenylglycouril Chloramine-T or N-halosuccinimide is used. The resulting radiohalogen prosthetic moiety is then conjugated to a HER2 binder using standard conditions to give a targeted radiotherapeutic agent. Non-limiting examples of the preparation of certain radioiodinated adjuvants and the use of such radioiodinated adjuvants to label HER2 binders are provided, for example, in Vaidyanathan et al., Nat Prot. (2007) 2: 282-6; and Choi et al., Nucl Med Biol. (2014) 1: 802-12, which are incorporated herein by reference in their entirety; WO2018 / 178936 provides preparation of 211 At-labeled adjuvants and non-limiting examples of using such agents to label HER2-binding agents are provided in the disclosure herein incorporated by reference in its entirety.

[0513] In some embodiments, a "pre-conjugation" method is used. For example, a precursor of a radioactive halogen prosthetic group portion is first prepared and reacted with a HER2 binding agent, and then a HER2 binding agent-precursor conjugate is radiolabeled to give a targeted radiotherapy agent. In some such embodiments, a disadvantage (particularly for radioiodination) is that, in addition to one or more expected prosthetic group sites for radiolabeling (i.e., a portion with an alkyl metal group or a boric acid or boron ester group), the constituent tyrosine, phenylalanine, and even histidine residues that may be present on the HER2 binding agent can also be radioiodinated. The problem with the radioactive halogen on tyrosine is that, due to the action of endogenous deiodinases, once the targeted radiotherapy agent is in vivo, the radioactivity will fall off (come off), and will not be positioned at the targeted cell together with the HER2 binding agent. Although in some embodiments, this can be minimized by performing radioiodination at a lower pH (4-5), it cannot be completely avoided. One method to avoid this potential problem is to first introduce non-radioactive iodine into those tyrosine residues before radioiodination of the macromolecule. It is likely that, under the mediation of these same endogenous deiodinases, non-radioactive iodine on the constituent tyrosine residues will be removed, thereby restoring the original tyrosine structure and maintaining the affinity of the HER2 binder to the envisioned target. Non-radioactive iodination of the HER2 binder can be achieved. In some embodiments, the HER2 binder is treated with excess sodium iodide in the presence of an oxidizing agent (e.g., chloramine-T).

[0514] V. Pharmaceutical Compositions and Formulations

[0515] The present disclosure also provides a pharmaceutical composition comprising a HER2 binding agent or nucleic acid disclosed herein, and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a HER2 binding agent or nucleic acid as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises any suitable and pharmaceutically acceptable carrier, diluent, adjuvant or buffer solution.

[0516] The composition can be formulated for a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for appropriate formulation. Formulations suitable for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science (1990) 249: 1527-33).

[0517] In certain embodiments, pharmaceutical compositions may include formulation materials used to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextran); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, e.g., sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0518] In some embodiments, the pharmaceutical composition may comprise nanoparticles, such as polymer nanoparticles, liposomes, or micelles (See Anselmo et al., Bioeng Transl Med. (2016) 1: 10-29).

[0519] In some embodiments, pharmaceutical compositions can include sustained or controlled delivery formulations. The technology for preparing sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads and depot injection (depot injection), is also known to those skilled in the art. Sustained release formulations can include, for example, porous polymer microparticles or semipermeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained release matrix can include polyesters, hydrogels, copolymers of lactic acid, L-glutamic acid and γ-ethyl-L-glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate or poly-D (-) -3-hydroxybutyric acid. Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0520] The pharmaceutical composition containing the HER2 binding agent disclosed herein can be present in dosage unit form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhaled, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the recombinant human sialidase disclosed herein, recombinant human sialidase fusion protein or antibody conjugate is administered by IV infusion. In certain embodiments, the recombinant human sialidase disclosed herein, recombinant human sialidase fusion protein or antibody conjugate is administered by intratumoral injection. Useful preparations can be prepared by methods known in the pharmaceutical field. For example, see Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990). Suitable formulation components for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or glucose.

[0521] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of production and storage and should be preserved against the microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0522] Intravenous drug delivery 1 can be contained in a syringe, pen or bag. In certain embodiments, the bag can be connected to a channel including a tube and / or a needle. In certain embodiments, the formulation can be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation can be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation can be lyophilized, and 45mg of the lyophilized formulation can be contained in one vial. In certain embodiments, the lyophilized formulation of about 40mg-about 100mg can be contained in one vial. In certain embodiments, the lyophilized formulations from 12, 27 or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation can be a liquid formulation and is stored as about 250mg / vial to about 1,000mg / vial. In certain embodiments, the formulation can be a liquid formulation and is stored as about 600mg / vial. In certain embodiments, the formulation can be a liquid formulation and is stored as about 250mg / vial.

[0523] These compositions can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged as is for use, or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier before administration. The pH of the preparation is generally between 3 and 11, between 5 and 9 or between 6 and 8 in certain embodiments, and between 7 and 8, such as 7 to 7.5 in a specific embodiment. The composition of the resulting solid form can be packaged into multiple single-dose units, each unit containing a fixed amount of one or more of the above-mentioned medicaments. The composition of the solid form can also be packaged in a container with a flexible amount.

[0524] In certain embodiments, the present disclosure provides formulations with extended shelf life comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0525] In certain embodiments, the aqueous formulation of the protein of the present disclosure is prepared in a pH buffer solution. The buffer of the present invention may have a pH ranging from about 4 to about 8, such as about 4.5 to about 6.0, or about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Intermediate ranges of the above pH are also intended to be part of the present disclosure. For example, it is intended to include a range of values ​​using a combination of any of the above values ​​as an upper and / or lower limit. Examples of buffers that will control pH within this range include acetates (e.g., sodium acetate), succinates (such as sodium succinate), gluconates, histidine, citrates, and other organic acid buffers.

[0526] In certain embodiments, formulations include a buffer system containing citrate and phosphate to maintain pH in the range of about 4 to about 8. In certain embodiments, the pH range can be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or in the pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3mg / ml citric acid (e.g. 1.305mg / ml), about 0.3mg / ml sodium citrate (e.g. 0.305mg / ml), about 1.5mg / ml disodium phosphate dihydrate (e.g. 1.53mg / ml), about 0.9mg / ml sodium dihydrogen phosphate dihydrate (e.g. 0.86) and about 6.2mg / ml sodium chloride (e.g. 6.165mg / ml). In certain embodiments, the buffer system includes 1-1.5 mg / ml of citric acid, 0.25 to 0.5 mg / ml of sodium citrate, 1.25 to 1.75 mg / ml of disodium phosphate dihydrate, 0.7 to 1.1 mg / ml of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / ml of sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0527] Acting as a tension agent and can stabilize the polyol of multi-specific binding protein can also be included in the formulation. The polyol is added to the formulation in an amount that can be varied in terms of the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of the polyol added can also be changed in terms of the molecular weight of the polyol. For example, compared with a disaccharide (e.g., trehalose), a smaller amount of monosaccharides (such as mannitol) can be added. In certain embodiments, the polyol that can be used as a tension agent in the formulation is mannitol. In certain embodiments, the concentration of mannitol can be about 5 to about 20 mg / ml. In certain embodiments, the concentration of mannitol can be about 7.5 to about 15 mg / ml. In certain embodiments, the concentration of mannitol can be about 10-14 mg / ml. In certain embodiments, the concentration of mannitol can be about 12 mg / ml. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0528] Detergents or surfactants can also be added to the formulation. Exemplary detergents include nonionic detergents, such as polysorbates (e.g., polysorbates 20, 80, etc.) or poloxamer (e.g., poloxamer 188). The amount of the detergent added is such that it reduces the aggregation of the antibody formulated and / or minimizes the formation of microparticles in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant, which is a polysorbate. In certain embodiments, the formulation may contain detergents polysorbate 80 or tween 80. Tween 80 is a term for describing polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain polysorbate 80 between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0529] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be present in a USP / Ph Eur I type 50R vial at a concentration of 10 mg / mL, the vial being closed with a rubber stopper and sealed with an aluminum crimp seal. The stopper may be made of an elastomer that complies with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with a 0.9% saline solution.

[0530] In certain embodiments, liquid formulations of the present disclosure can be prepared as 10mg / mL concentration solutions combined with stable levels of sugar. In certain embodiments, liquid formulations can be prepared in aqueous carriers. In certain embodiments, stabilizers can be added in an amount not greater than that which may result in an undesirable or unsuitable viscosity for intravenous administration. In certain embodiments, sugar can be a disaccharide, such as sucrose. In certain embodiments, liquid formulations can also include one or more of a buffer, a surfactant, and a preservative.

[0531] In certain embodiments, the pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0532] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0533] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multiple-use (multi-dose) formulations.

[0534] The multispecific binding protein can be lyophilized to produce a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffer, a surfactant, a filler, and / or a preservative.

[0535] The amount of sucrose or maltose used to stabilize the lyophilized drug product can be a weight ratio of at least 1:2 protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be 1:2 to 1:5. In certain embodiments, the pH of the formulation can be set by adding a pharmaceutically acceptable acid and / or base before lyophilization. In certain embodiments, a pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, a pharmaceutically acceptable base can be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present disclosure can be adjusted to between 6 and 8. In certain embodiments, the pH range of the lyophilized drug product can be 7 to 8.

[0536] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0537] The specific dosage can be a uniform dosage for each patient, such as 50-5000 mg of protein. Alternatively, the dosage of the patient can be customized for the approximate body weight or surface area of ​​the patient. Other factors determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further improvements in the calculation required to determine the appropriate dosage for treatment are routinely performed by those skilled in the art, especially in view of the dosage information and determination disclosed herein. The dosage can also be determined by using a known determination for determining the dosage in combination with appropriate dose-response data. As the progress of the disease is monitored, the dosage of individual patients can be adjusted. The blood level of a targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta (2001) 308: 43-53; Steimer et al., Clinica Chimica Acta (2001) 308: 33-41).

[0538] In general, a dosage based on body weight is from about 0.01 μg to about 100 mg / kg body weight, from about 0.01 μg to about 50 mg / kg body weight, from about 0.01 μg to about 10 mg / kg body weight, from about 0.01 μg to about 1 mg / kg body weight, from about 10 μg to about 100 μg / kg body weight, from about 10 μg to about 50 μg / kg body weight, from about 50 μg to about 100 mg / kg body weight, from about 50 μg to about 50 mg / kg body weight, from about 50 μg to about 10 mg / kg body weight, from about 50 μg to about 1 mg / kg body weight, from about 50 μg to about 100 μg / kg body weight, from about 100 μg to about 100 mg / kg body weight, from about 100 μg to about 50 mg / kg body weight, from about 100 μg to about 10 mg / kg body weight, from about 100 μg to about 1 mg / kg body weight, from about 1 mg to about 100 mg / kg body weight, from about 1 mg to about 50 mg / kg body weight, from about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, or about 50 mg to about 100 mg / kg body weight.

[0539] Dosages can be administered once or multiple times daily, weekly, monthly or annually. One of ordinary skill in the art can easily estimate the repetition rate of administration based on the residence time and concentration of the targetable construct or complex in body fluids or tissues measured. Administration of the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion or by direct intralesional injection. This can be administered once or multiple times daily, once or multiple times weekly, once or multiple times monthly and once or multiple times annually.

[0540] The pharmaceutical composition can be applied to the subject as needed. In some embodiments, the pharmaceutical composition of an effective dose is applied to the subject once or multiple times. In various embodiments, the pharmaceutical composition of an effective dose is applied to the subject once a month, less than once a month, such as, for example, once every two months, once every three months, or once every six months. In other embodiments, the pharmaceutical composition of an effective dose is applied more than once a month, such as, for example, once every two weeks, once a week, twice a week, three times a week, once a day, or multiple times a day. The pharmaceutical composition of an effective dose is applied to the subject at least once. In some embodiments, the pharmaceutical composition of an effective dose is applied multiple times, including a time period that lasts at least one month, at least six months, or at least one year. In some embodiments, the pharmaceutical composition is applied to the subject as needed to alleviate one or more symptoms of the condition.

[0541] In some embodiments, the present disclosure provides HER2 binding agents for use in medicine. In some embodiments, the present disclosure provides HER2 binding agents for use in treating diseases. In certain embodiments, the present disclosure provides HER2 binding agents for use in treating cancer.

[0542] In some embodiments, the present disclosure provides the use of a HER2 binding agent in the manufacture of a medicament for treating a disease in a patient in need thereof. In some embodiments, the present disclosure provides the use of a HER2 binding agent in the manufacture of a medicament for treating a cancer in a patient in need thereof. In some embodiments, the present disclosure provides the use of a HER2 binding agent in the manufacture of a medicament for treating a HER2 + Use in Medications for Cancer. In certain embodiments, the disclosure provides for the use of a HER2 binding agent in the preparation of a medicament for treating a disease in a patient in need thereof.

[0543] VI. How to use

[0544] In some embodiments, the present disclosure provides uses of HER2-binding agents (including labeled HER2-binding agents) for treating or diagnosing a disease or condition in a subject, and uses of HER2-binding agents (including labeled HER2-binding agents) in the preparation of a medicament for treating or diagnosing a disease or condition in a subject.

[0545] In some embodiments, the present disclosure provides a method for detecting HER2 in a subject. + A method for treating a cell, the method comprising: a) administering a labeled HER2 binding agent or a pharmaceutical composition provided herein to a subject; and b) detecting the labeled HER2 binding agent and HER2 in the subject. + Binding to cells, where detection of binding indicates the presence of HER2 + In some embodiments, HER2 + Cells are HER2 + Tumor cells. In some embodiments, the subject has cancer. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the non-human primate is a cynomolgus monkey or a rhesus monkey.

[0546] In some embodiments, the present disclosure provides a method for imaging a disease site in a subject, comprising administering to the subject a labeled HER2 binding agent or pharmaceutical composition provided herein. In some embodiments, HER2 in a subject is +Imaging cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, HER2 + Cells are HER2 + Tumor cells. In some embodiments, the subject has cancer. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the non-human primate is a cynomolgus monkey or a rhesus monkey.

[0547] In some embodiments, the disclosure provides a method for diagnosing a disease or medical condition of a subject, comprising administering to the subject a HER2 binding agent or pharmaceutical composition of a label provided herein. In some embodiments, the subject suffers from cancer. In some embodiments, the subject is a human or non-human primate. In some embodiments, the non-human primate is a cynomolgus monkey or a rhesus monkey.

[0548] In some embodiments, the present disclosure provides a method for detecting HER2 in a subject. + A method for detecting HER2 in a cell, the method comprising: a) administering a labeled HER2 binding agent to a subject; and b) detecting the labeled HER2 binding agent and HER2 in the subject. + Binding to cells, where detection of binding indicates the presence of HER2 + In some embodiments, the labeled HER2-binding agent binds to HER2 in the subject. + Cell combinations include HER2 in the subject + In some embodiments, HER2 in a subject is imaged. + Imaging cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, HER2 + Cells are HER2 + In some embodiments, HER2 + Cells are HER2 +Tumor cells. In some embodiments, the detection is performed within about 1 day or less (e.g., about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes or less) after administration. In some embodiments, the method is repeated once or multiple times, such as about 1 to 4 times per year. In some embodiments, the method is repeated about 1 day after the previous administration of the HER2 binding agent. In some embodiments, the method is repeated for more than 1 year. In some embodiments, the method has a sensitivity of about 1nM to about 30nM. In some embodiments, the subject is a human or non-human primate. In some embodiments, the subject is a cynomolgus monkey or a rhesus monkey. In some embodiments, the subject is a human. In some embodiments, the subject suffers from cancer.

[0549] In some embodiments, the HER2 binding agents provided herein are used to investigate the properties of the disease condition of the patient. The HER2 binding agents can be used to prepare images of the site of the disease in the subject using imaging techniques such as X-rays, gamma rays or PET scans or phases. In some embodiments, the HER2 binding agents provided herein are used to image the site of the disease in the subject, the method comprising administering to the subject a suitable detectable labeled HER2 binding agent provided herein and then scanning the subject's body. Alternatively, administering to the subject a HER2 binding agent provided herein can provide test results by analyzing a sample from the subject after administering the molecule. Such embodiments may include a method for diagnosing a disease or medical condition of a subject, comprising administering a HER2 binding agent provided herein. Embodiments include methods for detecting the site of a disease or medical condition and the use of a HER2 binding agent provided herein in such methods.

[0550] In some embodiments, the HER2 binding agents provided herein are suitable for treating a disease or condition. In some embodiments, the disease or condition is proliferative. In some embodiments, the disease or condition is. In some embodiments, the disease or condition is cancer. In certain embodiments, the disease or condition is HER2 +Cancer. In some embodiments, the cancer is selected from: breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, epidermal derived cancers and / or metastasis originating therefrom. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, digestive tract cancer, bladder cancer, and pancreatic cancer expressing HER2. In specific embodiments, the HER2 binding agents provided herein are suitable for treating metastasis. In specific embodiments, the HER2 binding agents provided herein are suitable for treating breast cancer. In certain embodiments, the HER2 binding agents provided herein are suitable for treating breast cancer metastasis. In specific embodiments, the HER2 binding agents are suitable for treating breast cancer that has metastasized to the brain.

[0551] In some embodiments, the HER2-binding agents provided herein are suitable for treating HER2 in patients who are not suitable for treatment with trastuzumab or pertuzumab. + Cancer. Typically, patients are scored for HER2(1+), HER2(2+), or HER2(3+) by immunohistochemistry. Thus, in some embodiments, the present disclosure provides HER2 for treating patients in need thereof. + A method for cancer, comprising the steps of: i) staging a patient for HER2 expression, and ii) when the patient is considered to have HER2 in step i) + or HER2 ++ When the HER2 binding agent provided herein is used, the patient is administered with an effective amount of the HER2 binding agent provided herein. Generally, the test is usually performed simultaneously with the initial biopsy or cancer surgery. Samples of cancerous tissues from previous biopsies or previous surgeries can also be used. Those skilled in the art can easily select the most appropriate method for staging patients for the HER2 expression of the patient. Generally, the two main methods for HER2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). 0-1+ means that a normal amount of HER2 protein is present and the result is HER2 negative. 2+ is known to have a moderate amount of HER2 protein. 3+ is known to have a HER2 protein level higher than the normal level, and the result is HER2 positive. For example, the method disclosed herein generally includes the following steps: i) determining the HER2 level in the tumor sample obtained from the patient, ii) comparing the level determined in step i) with a predetermined reference level, and iii) when the level determined in step i) is lower than a predetermined reference value, administering a therapeutically effective amount of the HER2 binding agent provided herein to the patient.

[0552] In some embodiments, the disclosure provides a HER2 binding agent or a pharmaceutical composition provided herein for use in medicine. In some embodiments, the disclosure provides a HER2 binding agent or a pharmaceutical composition provided herein for use in the preparation of a medicament for treating a disease in a patient in need thereof.

[0553] In some embodiments, the present disclosure provides methods of imaging a site of disease in a subject, comprising administering to the subject a detectably labeled HER2-binding agent or pharmaceutical composition provided herein.

[0554] In some embodiments, the present disclosure provides methods of diagnosing a disease or medical condition in a subject, comprising administering to the subject a HER2-binding agent or pharmaceutical composition provided herein.

[0555] In some embodiments, the present disclosure provides methods of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a HER2-binding agent or pharmaceutical composition provided herein.

[0556] In some embodiments, the present disclosure provides methods for treating HER2 in a subject in need thereof. + Cancer method, comprising administering to a subject a therapeutically effective amount of a HER2 binding agent or pharmaceutical composition provided herein. In some embodiments, the subject is HER2 + or HER2 ++ Subjects.

[0557] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modes. As used herein, the term "combination" administration is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject suffering from the disease, so that the effect of the treatment on the patient overlaps at a point in time. In certain embodiments, when the delivery of the second begins, the delivery of a treatment is still occurring, so that there is overlap in terms of administration. This is sometimes referred to as "simultaneous" or "concurrent delivery" in this article. In other embodiments, the delivery of a treatment ends before the delivery of another treatment begins. In certain embodiments of either case, due to combined administration, treatment is more effective. For example, the second treatment is more effective, for example, compared with the second treatment without the first treatment, the second treatment can be used to see an equivalent effect, or the second treatment has alleviated symptoms to a greater extent, or similar situations can be seen using the first treatment. In certain embodiments, delivery causes the reduction of symptoms or other parameters related to the disease to be greater than the reduction observed by delivering a treatment in the absence of another treatment. The effects of the two treatments can be partially added, completely added, or greater than added (e.g., synergistic). The delivery may be such that when the second treatment is delivered, the effects of the first treatment delivered are still detectable.

[0558] In some embodiments, the present disclosure provides methods of treating a subject by administering a second therapeutic agent in combination with a HER2-binding agent described herein.

[0559] Exemplary therapeutic agents that can be used as part of a combination therapy to treat cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesellarinone, aminoglutethimide, amsacrine, proglumide, Elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozotocin, mitolactin, tegafur, ifosfamide, prednimustine, picibanil, levamisole, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ, colony stimulating factor-1, colony stimulating factor-2, denileukin-2, interleukin-2, luteinizing hormone releasing factor, and variants of the above agents, which may exhibit differential binding to their cognate receptors, as well as an increase or decrease in serum half-life.

[0560] Another class of agents that can be used as part of a combination therapy for treating cancer is an immune checkpoint inhibitor. Checkpoint inhibitors can be, for example, selected from PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0561] VII. Kits and Articles

[0562] Further provided herein are kits or articles of manufacture comprising a HER2 binding agent provided herein, such as a labeled HER2 binding agent. In some embodiments, the kit or article of manufacture comprises instructions for using the HER2 binding agent according to any of the methods described above.

[0563] Also provided herein are methods for preventing and / or treating a disease or disorder (such as cancer) in a subject, comprising administering to the subject an engineered cell comprising a CAR provided herein.

[0564] Unless otherwise defined herein, the scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Although methods and materials similar or equivalent to the methods and materials described herein may also be used in the practice or testing of the present disclosure, exemplary methods and materials are described below. In the event of a conflict, this specification (including definitions) will control. In addition, unless the context otherwise requires, singular terms will include a large number and plural terms will include the singular. Throughout this specification and embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" will be understood to mean including the integer, the group of integers, rather than excluding any other integer, integer or step group. All disclosures and other references described herein are incorporated by reference in their entirety, as if each individual reference is specifically and individually indicated to be incorporated by reference in its entirety. Although many documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term "about" or "approximately" when applied to one or more values ​​of interest refers to a value similar to the reference value. In certain embodiments, the term refers to a numerical range that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) of the reference value, unless otherwise stated or apparent from the context.

[0565] The above description describes multiple embodiments of the present invention. This patent application specifically contemplates all combinations and permutations of the embodiments. According to the present disclosure, the reverse reference in the dependent claims means a direct and clear public abbreviation of each and each combination of the claims indicated by the reverse reference. Any compound disclosed herein can be used in any therapeutic method herein, wherein the individual to be treated is defined as anywhere herein. In addition, the title herein is created for ease of organization and is not intended to limit the scope of the claimed invention in any way.

[0566] In order to better understand the present invention, the following examples are shown. These examples are for illustrative purposes only and should not be interpreted as limiting the scope of the present invention in any way. Example

[0567] Example 1: Generation of HER2-binding VHH domains

[0568] The development of VHH peptides that specifically bind to HER2 is achieved through a variety of approaches. In-house ligand discovery is achieved through proprietary initial Phage screening of llama and alpaca VHH libraries was performed. The library was initially pre-cleared using counter screening against Fc and DDDDK protein sequences displayed on magnetic beads to ensure target specificity.

[0569] A total of 16 different screening conditions were used. Proprietary llama and alpaca VHH phage libraries were screened for the HER2 target protein. The HER2 target protein used was a recombinant form of human HER2 with a C-terminal tag consisting of a human IgG1 Fc domain or a DDDDK (SEQ ID NO: 207) amino acid sequence. The target protein was fixed to the magnetic beads using an Fc or DDDDK (SEQ ID NO: 207) tag by affinity connection. Phage selection was completed in two different blocking solutions: 3% BSA or 3% milk in PBS. The binding interaction between the target protein and the phage library or selection pool was evaluated for different times (15, 60 or 120 minutes). Three rounds of phage selection were performed with each selection strategy, and clones were usually isolated from the third round of phage pools.

[0570] The final separated phage selection pool was evaluated in a 96-well ELISA assay for HER2-Fc and control Fc proteins. A total of 355 positive phagemid clones were identified as positive binders, which were defined as showing a higher binding than background>150%, and were submitted for sequencing. Sequence analysis produced 87 unique VHH sequences with HER2 binding affinity. The plasmids of these positive phagemid clones were then transferred to SS320 Escherichia coli for protein expression and purification to directly assess protein affinity. The purified VHH was then retested in an indirect ELISA format with human HER2-Fc and human HER2-His6, which produced 24 VHHs with avidity stronger than 150nM. Specificity was verified by determining the binding affinity to other human ErBB receptors (including EGFR, HER3 and HER4). Cross-species reactivity was characterized in the HER2-Fc analogs of mice, rats, dogs, rhesus monkeys and cynomolgus monkeys. The top hits were further modified and evolved to generate higher affinity binding proteins and to improve their compatibility and performance as radiolabeled compounds for targeted radiotherapy.

[0571] VHH sequences are also evaluated to identify corresponding framework regions (FR) and complementary determining regions (CDR). Typically, the linear sequence of a VHH protein corresponds to the following structural sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In order to ensure the maximum performance of VHH proteins as targeted drug conjugates (e.g., radioligands or other types of drug conjugates), all amino acids that can provide sites for radiolabel conjugation (or other types of drug conjugation) in the CDR region are mutated to prevent such conjugation. In addition, amino acids in the framework region within five amino acids of CDR are also modified to prevent spatial interference with the binding pocket of VHH after radiolabeling (or any other type of drug conjugation). Such sequence modifications ensure that the ligand maintains the optimal binding and internalization characteristics required as a radioligand. Sequence modifications include: the following substitutions: replacement of lysine (K) residues with arginine (R) or histidine (H) residues; replacement of unpaired cysteine ​​(C) residues with alanine (A) or serine (S) residues; or replacement of glutamine (Q) residues with asparagine (N).

[0572] Definitions of the structural CDR sequences of VHH_106 to VHH_172 are provided in Table 2, along with exemplary C-terminal peptide fusions.

[0573] Table 2: Exemplary HER2 binding sequences

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583] Figures 1A-1J ELISA affinity binding curves of exemplary VHH proteins against human HER2 receptor after protein expression and purification are presented.

[0584] The binding specificity of exemplary VHH proteins for other receptors within the ErbB family is as follows Figure 2A-2I shown.

[0585] exist Figures 3A-3E The cross-reactivity of exemplary VHH proteins to cross-species HER2 receptors found in other animals is presented in .

[0586] Figures 4A-4B Competitive inhibition of exemplary VHH proteins with trastuzumab and pertuzumab is presented in .

[0587] Example 2: Radiolabeling and in vitro assay of HER2 binding VHH domains

[0588] Figure 5A Depicted using [ 131 Structure of VHH radiolabeled with [I]-isoSGMIB.

[0589] Figure 5B-5E Presents the results of an in vitro cell-based assay designed to evaluate the effects of [ 131 (B) Affinity for SKOV-3 ovarian cancer cells. (C) Surface-bound or (D) internalized [I]-isoSGMIB-labeled VHH_128HER2. 131 (E) Measurement of [I]-isoSGMIB-VHH128 levels in SKOV-3 cells in the presence of 100-fold trastuzumab 131 I] Internalization.

[0590] Example 3: In vivo studies

[0591] Figure 6 Depicted is the SKOV-3 xenograft tumor model in female athymic mice. 131 Tissue accumulation levels of 1-isoSGMIB-VHH128 over 24 h.

[0592] Table 3 presents the 131 Complete biodistribution stacking histogram of I]-isoSGMIB-VHH128 in SKOV-3 tumor xenografts in female athymic mice. The injection dose consisted of 5 μCi, prepared at a specific activity of 1 μCi / ng, and was administered in 100 μL intravenously via the tail vein.

[0593] Table 3

[0594]

[0595] Figure 7 Depicted are the results of a BT-474 xenograft tumor model in female NOD SCID mice. 131 Tissue accumulation levels of 1-isoSGMIB-VHH128 over 24 h.

[0596] Table 4 presents the 131 Complete biodistribution stacking histogram of I]-isoSGMIB-VHH128 in the BT-474 tumor model in female NOD SCID mice. The injection dose consisted of 5 μCi, prepared at a specific activity of 1 μCi / ng, and was administered in 100 μL intravenously via the tail vein.

[0597] Table 4

[0598]

[0599] Figures 8A-8C Describes the use 211 HER2-targeted alpha particle therapy with astatine-labeled VHH. (A) Using isoSAGMB 211 Scheme and structure of At-labeled VHH_128. (B)[ 211 Affinity binding curves of [At]-isoSAGMB-VHH128 against HER2-expressing BT-474 cells. (C) Accumulation levels in BT-474 tumors xenografted in athymic mice.

[0600] Table 5 presents the 211 Complete biodistribution stacking histogram of [At]-isoSAGMB-VHH1028 in the BT-474 tumor model in female athymic mice. The injection dose consisted of 5 μCi, prepared at a specific activity of 1 μCi / ng, and was administered intravenously in 100 μL via the tail vein.

[0601] Table 5

[0602]

[0603] Figures 9A-9C Depicted is the radiolabeling of a HER2-targeting VHH using maleimide-based radiolabeling chemistry. (A) Radiolabeling of a HER2-targeting VHH using isoMEGMIB 131 I-labeled scheme and structure of VHH_1041. (B) [ 131 (I) Affinity binding curves of -isoMEGMIB-VHH141 against HER2-expressing BT-474 cells. (C) In vitro cell binding and internalization ratios on BT-474 cells.

[0604] Fig.10 Depicted are the expression of [ 131 Tissue accumulation levels of I]-isoMEGMIB-VHH141 over 48 h.

[0605] Table 6 presents the131 Complete biodistribution stacking histogram of I]-isoMEGMIB-VHH141 in SKOV-3 tumor xenografts in female athymic mice. The injection dose consisted of 5 μCi, prepared at a specific activity of 1 μCi / ng, and was administered in 100 μL intravenously via the tail vein.

[0606] Table 6

[0607]

Claims

1. A HER2-binding agent comprising a HER2-binding VHH domain, wherein the VHH domain comprises: a) a complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 82-101; a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102-123; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-157 and 206; b) CDR1, CDR2 and CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 contained in a VHH sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205; c) a VHH sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205; d) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a VHH sequence selected from SEQ ID NOs: 1-81 and 205; e) an amino acid sequence having 1-25, 1-20, 1-15, 1-10, 1-5 or 1-3 additions, substitutions or deletions compared to a reference VHH sequence selected from SEQ ID NOs: 1-81 and 205.

2. The HER2 binding agent of claim 1, wherein the VHH domain comprises: a) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 135 or 136; b) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 135; c) a CDR1 comprising SEQ ID NO: 101; a CDR2 comprising SEQ ID NO: 111; and a CDR3 comprising SEQ ID NO: 136; d) a CDR1 comprising SEQ ID NO: 82; a CDR2 comprising SEQ ID NO: 102; and a CDR3 comprising SEQ ID NO: 124, 145, 146 or 147; e) a CDR1 comprising SEQ ID NO: 83; a CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and a CDR3 comprising SEQ ID NO: 125; f) a CDR1 comprising SEQ ID NO:84; a CDR2 comprising SEQ ID NO:104; and a CDR3 comprising SEQ ID NO:126; g) a CDR1 comprising SEQ ID NO: 85; a CDR2 comprising SEQ ID NO: 105; and a CDR3 comprising SEQ ID NO: 127; h) a CDR1 comprising SEQ ID NO: 86; a CDR2 comprising SEQ ID NO: 106; and a CDR3 comprising SEQ ID NO: 128, 130, 134 or 155; i) a CDR1 comprising SEQ ID NO: 87; a CDR2 comprising an amino acid sequence selected from SEQ ID NO: 117; and a CDR3 comprising SEQ ID NO: 129; j) a CDR1 comprising SEQ ID NO: 88; a CDR2 comprising SEQ ID NO: 118; and a CDR3 comprising an amino acid sequence selected from SEQ ID NO: 130; k) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 131 or 157; 1) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108; and a CDR3 comprising SEQ ID NO: 131; m) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 156; n) a CDR1 comprising SEQ ID NO: 89; a CDR2 comprising SEQ ID NO: 108 or 112; and a CDR3 comprising SEQ ID NO: 157; o) CDR1 comprising SEQ ID NO:90; CDR2 comprising SEQ ID NO:109 and CDR3 comprising SEQ ID NO:132; p) CDR1 comprising SEQ ID NO:91; CDR2 comprising SEQ ID NO:119 and CDR3 comprising SEQ ID NO:137; q) CDR1 comprising SEQ ID NO:92; CDR2 comprising SEQ ID NO:120 and CDR3 comprising SEQ ID NO:138; r) a CDR1 comprising SEQ ID NO:93; a CDR2 comprising SEQ ID NO:113 or 114 and a CDR3 comprising SEQ ID NO:139; s) CDR1 comprising SEQ ID NO:93; CDR2 comprising SEQ ID NO:114 and CDR3 comprising SEQ ID NO:206; t) a CDR1 comprising SEQ ID NO: 94; a CDR2 comprising SEQ ID NO: 115 and a CDR3 comprising SEQ ID NO: 140 or 149; u) CDR1 comprising SEQ ID NO:95; CDR2 comprising SEQ ID NO:121 and CDR3 comprising SEQ ID NO:141; v) a CDR1 comprising SEQ ID NO:96; a CDR2 comprising SEQ ID NO:122; and a CDR3 comprising SEQ ID NO:142; w) a CDR1 comprising SEQ ID NO:97; a CDR2 comprising SEQ ID NO:116; and a CDR3 comprising SEQ ID NO:150; x) a CDR1 comprising SEQ ID NO:97; a CDR2 comprising SEQ ID NO:123; and a CDR3 comprising SEQ ID NO:143; y) a CDR1 comprising SEQ ID NO: 98; a CDR2 comprising SEQ ID NO: 107; and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 151, 152 and 153; z) a CDR1 comprising SEQ ID NO: 99; a CDR2 comprising SEQ ID NO: 109; and a CDR3 comprising SEQ ID NO: 132 or 148; aa) comprising CDR1 of SEQ ID NO: 100; comprising CDR2 of SEQ ID NO: 110; and comprising CDR3 of SEQ ID NO: 133 or 154; or bb) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO:

136.

3. The HER2 binding agent of claim 1 or 2, wherein the HER2 binding agent is monovalent.

4. The HER2 binding agent of claim 1 or 2, wherein the HER2 binding agent is multivalent, optionally wherein the HER2 binding agent is bivalent, trivalent or tetravalent.

5. The HER2 binding agent of any one of claims 1-4, wherein the HER2 binding agent is monospecific.

6. The HER2 binding agent of any one of claims 1, 2 or 4, wherein the HER2 binding agent is multispecific and specifically binds HER2 and different antigens. E 7 . The HER2 binding agent of any one of E 1 -6 , wherein the VHH domain is humanized.

8. The HER2-binding agent of any one of claims 1-7, wherein the HER2-binding agent comprises a fusion protein comprising the HER2-binding VHH domain and a heterologous sequence.

9. The HER2 binding agent of any one of claims 1-8, wherein the HER2 binding agent comprises an N-terminal amino acid residue or polypeptide sequence, optionally wherein the N-terminal residue or sequence improves protein expression, solubility, purification, half-life or affinity, optionally wherein the N-terminal residue or sequence is Met-Ala or an Ala leader peptide.

10. The HER2 binding agent of any one of claims 1-9, wherein the HER2 binding agent comprises a C-terminal amino acid residue or polypeptide sequence, optionally wherein the C-terminal residue or sequence promotes radiolabel selectivity, loading, cellular internalization, half-life or affinity.

11. The HER2 binding agent of any one of claims 1-10, wherein the HER2 binding agent comprises a polypeptide sequence having the following structure: VHH–(Gly n Xaa m Gly k ) j i) wherein the VHH is selected from the group consisting of SEQ ID NOs: 1-81 and 205, ii) n = 0-5, iii) m = 0-5, iv) k = 0-6, v) j = 0-8, and vi) Xaa is an amino acid which enables site-specific conjugation of a radiolabeled prosthetic group or direct radiolabeling.

12. The HER2 binding agent of any one of claims 8-11, wherein the fusion protein comprises an Fc region.

13. The HER2 binding agent of any one of claims 8-11, wherein the fusion protein is a chimeric antigen receptor (CAR).

14. An isolated nucleic acid encoding the HER2 binding agent of any one of claims 1-13.

15. A vector comprising the nucleic acid of claim 14, optionally wherein the vector is an expression vector.

16. A host cell comprising the nucleic acid of claim 14 or the vector of claim 15, optionally wherein the host cell is a eukaryotic cell or a prokaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell or a yeast cell.

17. The HER2 binding agent of any one of claims 1-13, wherein the HER2 binding agent is conjugated to a label, optionally wherein the label is a fluorescent dye, a radionuclide, an enzyme, a toxin, or a chemotherapeutic agent.

18. The HER2 binding agent of claim 17, wherein the label is a radionuclide.

19. The HER2 binding agent of claim 18, wherein the radionuclide is a) a radioactive halogen isotope, optionally selected from: i) 18 F, 76 Br, 123 I, 124 I, 125 I and 131 I; or ii) 75 Br, 77 Br, 122 I, 124 I, 125 I, 131 I and 211 At; b) a radioactive metal isotope, optionally selected from: i) 44 Sc, 45 Ti, 51 Cr, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 68 Ge, 75 Se, 82 Sr, 86 Y, 99 Mo, 99m Tc, 110m In, 111 In, 166 Ho, 186 Re, 195m Ptsum 201 Tl;or ii) 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89 Zr, 90 Y, 111 In, 153 Sm, 149 Tb, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th and 227 Th.

20. The HER2 binding agent of any one of claims 1-13 and 17-19, wherein the HER2 binding agent is conjugated to a label via a chelating moiety, optionally wherein the chelating moiety is covalently linked to the protein via a lysine or cysteine ​​residue.

21. The HER2 binding agent of claim 20, wherein the label forms a complex with a metal, and wherein the complex is chelated by the chelating moiety.

22. The HER2 binding agent of any one of claims 1-12 and 17-21, wherein the HER2 binding agent is a targeted radiotherapy agent having the following structure: VHH-RLC, in VHH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-81 and 205, and RLC is a radiolabeled chemical used to facilitate the attachment of a radionuclide to a VHH, either directly or indirectly.

23. The HER2 binding agent of claim 22, wherein the targeted radiotherapeutic agent comprises a prosthetic compound or a radiohalogen precursor represented by Formula 2: MC-Cm-L4-Cm-T (Formula 2), in: MC is a multidentate metal chelating moiety; Cm is thiourea, amide or thioether; L4 is selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain, optionally with NH, CO or S at one or both ends; T is a compound of any of the following: a) a compound in the form of a prosthetic compound or a radiohalogen precursor represented by formula 1: in: X is CH or N; L1 and L3 are independently selected from a bond, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain; MMCM is the macromolecular conjugation moiety; L2 is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a polyethylene glycol (PEG) chain comprising at least three oxygen atoms, wherein L2 optionally comprises a brush border enzyme-cleavable peptide; CG is selected from guanidine; PO3H; SO3H; one or more charged D-amino acids or L-amino acids selected from arginine, phosphono / sulfophenylalanine, glutamic acid, aspartic acid, and lysine; a hydrophilic carbohydrate moiety; a polyethylene glycol (PEG) chain; and Z-guanidine; Z is (CH2) n ; n is greater than 1; m is 0 to 3; and Y is an alkyl metal moiety, a boronic acid moiety, a boronic ester moiety or a moiety selected from 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 131 I and 211 A radioactive halogen selected from the group consisting of At, or a pharmaceutically acceptable salt or solvate thereof; b) the compound of a), wherein the compound is a radiohalogen precursor, optionally wherein Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), trimethylsilyl (SiMe3), boronic acid (B(OH)2) or boronic ester (B(OR)2), and optionally wherein R comprises a cyclic moiety or an aliphatic group; c) The compound of a), wherein the compound is a prosthetic compound, and wherein Y is a radiohalogen selected from the group consisting of: 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 131 I and 211 At; d) The compound of a), wherein MMCM is an active ester or (Gly) m , where m is 1 or greater; e) The compound of a), wherein the MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS) ester, tetrafluorophenol (TFP) ester, pentafluorophenol (PFP), p-nitrophenol (PNP), an isothiocyanate group or a maleimide group; f) the compound of a), wherein MMCM is Gly-Gly-Gly; g) the compound of a), wherein, wherein L2 is (CH2) p , where p = 1 to 6; h) the compound of a), wherein the optional brush border enzyme cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys; i) The compound of a), which is represented by the following structure: j) The compound of i), wherein the compound comprises N-succinimidyl 3-guanidinomethyl-5-[ 131 I] iodobenzoate (isoSGMIB), or N-succinimidyl 3-[ 211 At]astato-5-guanidinomethylbenzoate (isoSAGMB), or maleimidoethyl 2-(guanidinomethyl)-5-iodobenzoate (isoMEGMIB), or maleimidoethyl 3-(guanidinomethyl)-5-astatobenzoate (isoMEGMAB); or a pharmaceutically acceptable salt or solvate thereof.

24. The HER2 binding agent of claim 23, wherein MC is a macrocyclic structure.

25. The HER2 binding agent of claim 24, wherein a) MC is selected from DOTA, TETA, NOTP and NOTA; b) MC is an acyclic multidentate ligand; or c) MC is selected from EDTA, EDTMP and DTPA.

26. The HER2 binding agent of claim 24, wherein the compound is a) a radiohalogen precursor, optionally wherein Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), trimethylsilyl (SiMe3), boronic acid (B(OH)2) or boronic ester (B(OR)2), and optionally wherein R comprises a cyclic moiety or an aliphatic group; or b) a prosthetic compound in which Y is selected from 18 F. 75 Br, 76 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 131 I and 211 Radiohalogens of At.

27. The HER2-binding agent of claim 24, further comprising a metal associated with the MC, optionally wherein the metal is a radioactive metal selected from the group consisting of: 177 Lu, 64 Cu, 67 Cu, 111 In, 90 Y. 225 Ac, 212 Bi, 213 Bi, 153 Sm, 166 Ho, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 Th.

28. A pharmaceutical composition comprising the HER2 binding agent of any one of claims 1-13 and 17-27, and a pharmaceutically acceptable carrier.

29. A method of treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a HER2 binding agent of any one of claims 1-13 and 17-27 or a pharmaceutical composition of claim 28, optionally wherein the disease is cancer.

30. A method for treating cancer, comprising administering an effective amount of the HER2 binding agent of any one of claims 1-13 and 17-27 or the pharmaceutical composition of claim 28 to a subject in need thereof.

31. A method of treating cancer comprising administering to a subject in need thereof an effective amount of the targeted radiotherapeutic agent of any one of claims 22-27.

32. The method of any one of claims 29-31, wherein the cancer is HER2 + .

33. Treatment of HER2 + A method for treating cancer, comprising administering an effective amount of the HER2 binding agent of any one of claims 1-13 and 17-27 or the pharmaceutical composition of claim 28 to a subject in need thereof.

34. The method of any one of claims 29-33, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, prostate cancer, lung cancer, NSCLC, bladder cancer, urothelial carcinoma, and epidermal-derived cancers, and / or metastases arising therefrom.

35. The method of any one of claims 29-33, wherein the cancer is breast cancer and / or metastases arising therefrom.

36. The method of claim 35, wherein the cancer is breast cancer brain metastasis.

37. The method of any one of claims 29-36, wherein the subject is scored for HER2(2+) or HER2(3+) by immunohistochemistry.

38. Kill HER2 + A method for treating a cell, comprising contacting the cell with a HER2 binding agent of any one of claims 1-13 and 17-27 or a pharmaceutical composition of claim 28, optionally wherein the cell is a cancer cell.

39. The method of claim 38, wherein the cancer cells are selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain cancer, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, prostate cancer, lung cancer, NSCLC, bladder cancer, urothelial carcinoma, and epidermal-derived cancers, and / or metastases arising therefrom.

40. The method of claim 38 or 39, wherein the cell is contacted with the HER2-binding agent in vitro or in vivo.

41. The HER2 binding agent of any one of claims 1-13 and 17-27 or the pharmaceutical composition of claim 28, for use in the method of any one of claims 29-40.

42. Use of the HER2 binding agent of any one of claims 1-13 and 17-27 or the pharmaceutical composition of claim 28 in the preparation of a medicament for treating a disease in a subject in need thereof, optionally wherein the disease is cancer.

43. Use of the HER2 binding agent of any one of claims 1-13 and 17-27 or the pharmaceutical composition of claim 28 in the preparation of a medicament for use in the method of any one of claims 29-40.

44. A method of diagnosing a disease or medical condition in a subject, comprising administering to the subject a HER2 binding agent of any one of claims 1-13 and 17-27 or a pharmaceutical composition of claim 28, optionally wherein the disease is cancer.

45. Detection of HER2 in subjects + A method for a cell, comprising: a) administering to the subject a detectably labeled HER2-binding agent of any one of claims 1-13 and 17-27 or a pharmaceutical composition of claim 28, wherein the HER2-binding agent has been detectably labeled; and b) detecting the labeled HER2 binding agent and HER2 in the subject + cells, wherein detection of said binding indicates the presence of HER2 + cell, Optionally, wherein the labeled HER2-binding agent is detected to be associated with HER2 in the subject + The binding of cells includes HER2 in the subject + Cells were imaged.

46. ​​HER2 in subjects + A method for imaging cells, comprising administering to the subject a detectably labeled HER2 binding agent of any one of claims 1-13 and 17-27 or a pharmaceutical composition of claim 28, wherein the HER2 binding agent has been detectably labeled, optionally wherein the HER2 in the subject is detected. + Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject.

47. The method of claim 45 or 46, wherein the HER2 + Cells are HER2 + cancer cell.

48. The method of any one of claims 29-37 and 41-47, wherein the subject is a human, a non-human primate, or a mammal.

49. The method of any one of claims 29-37 and 41-48, wherein the subject has cancer.

50. A method for preparing a HER2 binding agent, comprising: a) culturing the host cell of claim 16 under conditions where a HER2 binding agent is produced; and b) recovering the HER2 binding agent produced by the host cell.

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