HER2-Binding Agent and Its Use

Abstract

The present disclosure provides a HER2 binder comprising a VHH domain that specifically binds to HER2. Also provided are nucleic acids encoding the protein, vectors and host cells comprising the nucleic acids, and methods of manufacturing and using the HER2 binder. The present disclosure also provides a HER2 binder conjugate in which the protein is conjugated to a detectable label, and a HER2-binding targeted radiotherapy agent for therapeutic and / or diagnostic purposes. Also provided are methods of using the HER2 binder for detecting, monitoring and / or treating diseases and conditions such as cancer.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Application No. 63 / 344,808, filed May 23, 2022. The disclosure of the priority - claiming application is hereby incorporated by reference in its entirety into this specification.

[0002] (Reference to Sequence Listing) This application includes a Sequence Listing submitted in XML electronic format, which is hereby incorporated by reference in its entirety into this specification. The Sequence Listing is provided as a copy of an electronic file named "122878.WO012.xml" created on May 23, 2023, with a size of 271,281 bytes.

Background Art

[0003] In the development and optimization of HER2 - targeted therapies for breast cancer, significant progress has been made. Examples of FDA - approved drugs with significant clinical effects against metastatic disease include the anti - HER2 antibody trastuzumab, the anti - HER2 - HER3 dimer 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, HER2 + In breast cancer, there are many limitations and resistances that impair the efficacy of these targeted therapies, and they are not suitable for the treatment of the majority of breast cancers with low - level overexpression of HER2.

[0004] Brain metastases are a devastating progression of breast cancer, and a high incidence of 30 - 55% has been reported in HER2 - positive breast cancer patients. Brain metastases are a major cause of death in HER2 - positive breast cancer patients, and up to 50% of patients die as a direct result of the progression of CNS disease. HER2 +Treatment options for breast cancer brain metastases (BCBM) are limited, and anti-HER2 therapies that slow systemic growth generally cannot control brain metastases due to insufficient penetration of antibodies through the blood-brain barrier (BBB) (Lampson, mAbs (2011) 3:153 - 60). In addition, clinical data have shown that the incidence of brain metastases in HER2-positive breast cancer increases after trastuzumab adjuvant therapy (Olson et al., Ann Oncol. (2013) 24:1526 - 33).

[0005] Furthermore, despite sufficient drug delivery, the efficacy of small molecules against breast cancer brain metastases is also very limited and is only slightly increased by adding another therapy (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).

[0006] Cancers characterized by overexpression of HER2 (so-called HER2 + cancers), etc., are often associated with poor prognosis or resistance to many standard therapies. Therefore, there is a need for new therapeutic agents effective in treating cancers such as HER2-positive cancer and metastatic HER2-positive cancer. The present disclosure meets these needs and provides other advantages including the ability to selectively deliver HER2-targeted agents to HER2-positive tumors in the central nervous system without damaging normal brain tissue. SUMMARY OF THE INVENTION

[0007] (SUMMARY OF THE INVENTION) The present disclosure provides a novel HER2 (human epidermal growth factor receptor 2)-binding VHH domain, HER2 + cancers including cancers that have metastasized to the brain +It solves problems related to the treatment of cancer. The present disclosure provides a radiolabeled targeting HER2 binder that is taken up by and can be retained in tumor cells, while reducing the amount of radionuclide taken up by normal cells (especially the kidneys), which has been found to be the greatest concern regarding toxicity limitations for targeted radiotherapy agents. The present disclosure includes a radiolabeled HER2 binder that minimizes the loss of radioactive halogen due to dehalogenation in vivo, maintains the biological activity of the HER2-binding VHH domain after in vivo administration, maximizes retention in diseased cells such as cancer cells, and minimizes retention of radioactivity in normal tissues.

[0008] The present disclosure provides a HER2 binder comprising a VHH domain that specifically binds to HER2. Also provided are a nucleic acid encoding the HER2 binder, a vector and a host cell comprising the nucleic acid, and methods for manufacturing and using the HER2 binder. The present disclosure also provides a HER2 binder comprising a detectable label and a HER2 binder-targeted radiotherapy agent for therapeutic and / or diagnostic purposes. Also provided is a method of using the HER2 binder to detect, monitor and / or treat diseases and conditions such as cancer.

[0009] In some embodiments, the present disclosure relates to the following: [1] A HER2 binder comprising a HER2-binding VHH domain comprising the following: (a) Complementary determining region (CDR) 1 comprising an amino acid sequence selected from SEQ ID NOs: 82-101; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 102-123; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 124-157 and 206; (b) CDR1, CDR2 and CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 contained in the VHH sequences disclosed in Table 2; (c) The VHH amino acid sequences disclosed in Table 2; (d) A VHH sequence selected from SEQ ID NOs: 1-81 and 205; (e) The VHH sequences disclosed in Table 2 and amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity; (f) The VHH sequences selected from SEQ ID NOs: 1 to 81 and 205 and amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity; (g) An amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3 additions, substitutions or deletions as compared with the reference VHH sequence disclosed in Table 2; or (h) An amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3 additions, substitutions or deletions as compared with the reference VHH sequence selected from SEQ ID NOs: 1 to 81 and 205; [2] The HER2 binder according to [1], wherein the VHH domain comprises the following: (a) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO: 135 or 136; (b) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO: 135; (c) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO: 136; (d) CDR1 comprising SEQ ID NO: 82; CDR2 comprising SEQ ID NO: 102; and CDR3 comprising SEQ ID NO: 124, 145, 146 or 147; (e) CDR1 comprising SEQ ID NO: 83; CDR2 comprising SEQ ID NO: 103; and CDR3 comprising SEQ ID NO: 125; (f) CDR1 comprising SEQ ID NO: 84; CDR2 comprising SEQ ID NO: 104; and CDR3 comprising SEQ ID NO: 126; (g) CDR1 comprising SEQ ID NO: 85; CDR2 comprising SEQ ID NO: 105; and CDR3 comprising SEQ ID NO: 127; (h) CDR1 comprising SEQ ID NO: 86; CDR2 comprising SEQ ID NO: 106; and CDR3 comprising SEQ ID NO: 128, 130, 134 or 155; (i) CDR1 containing SEQ ID NO: 87; CDR2 containing SEQ ID NO: 117; and CDR3 containing SEQ ID NO: 129; (j) CDR1 containing SEQ ID NO: 88; CDR2 containing SEQ ID NO: 118; and CDR3 containing SEQ ID NO: 130; (k) CDR1 containing SEQ ID NO: 89; CDR2 containing SEQ ID NO: 108 or 112; and CDR3 containing SEQ ID NO: 131 or 157; (l) CDR1 containing SEQ ID NO: 89; CDR2 containing SEQ ID NO: 108; and CDR3 containing SEQ ID NO: 131; (m) CDR1 containing SEQ ID NO: 89; CDR2 containing SEQ ID NO: 108 or 112; and CDR3 containing SEQ ID NO: 156; (n) CDR1 containing SEQ ID NO: 89; CDR2 containing SEQ ID NO: 108 or 112; and CDR3 containing SEQ ID NO: 157; (o) CDR1 containing SEQ ID NO: 90; CDR2 containing SEQ ID NO: 109; and CDR3 containing SEQ ID NO: 132; (p) CDR1 containing SEQ ID NO: 91; CDR2 containing SEQ ID NO: 119; and CDR3 containing SEQ ID NO: 137; (q) CDR1 containing SEQ ID NO: 92; CDR2 containing SEQ ID NO: 120; and CDR3 containing SEQ ID NO: 138; (r) CDR1 containing SEQ ID NO: 93; CDR2 containing SEQ ID NO: 113 or 114; and CDR3 containing SEQ ID NO: 139; (s) CDR1 containing SEQ ID NO: 93; CDR2 containing SEQ ID NO: 114; and CDR3 containing SEQ ID NO: 206; (t) CDR1 containing SEQ ID NO: 94; CDR2 containing SEQ ID NO: 115; and CDR3 containing SEQ ID NO: 140 or 149; (u) CDR1 containing SEQ ID NO: 95; CDR2 containing SEQ ID NO: 121; and CDR3 containing SEQ ID NO: 141; (v) CDR1 containing SEQ ID NO: 96; CDR2 containing SEQ ID NO: 122; and CDR3 containing SEQ ID NO: 142; (w) CDR1 containing SEQ ID NO: 97; CDR2 containing SEQ ID NO: 116; and CDR3 containing SEQ ID NO: 150; (x) CDR1 containing SEQ ID NO: 98; CDR2 containing SEQ ID NO: 107; and CDR3 containing SEQ ID NO: 144, 151, 152 or 153; (y) CDR1 containing SEQ ID NO: 99; CDR2 containing SEQ ID NO: 109; and CDR3 containing SEQ ID NO: 132 or 148; or (z) CDR1 containing SEQ ID NO: 100; CDR2 containing SEQ ID NO: 110; and CDR3 containing SEQ ID NO: 133 or 154; Or The HER2 binder according to [1], wherein the VHH domain contains the following: (aa) CDR1 containing SEQ ID NO: 97; CDR2 containing SEQ ID NO: 123; and CDR3 containing SEQ ID NO: 143; or (ab) CDR1 containing SEQ ID NO: 101; CDR2 containing SEQ ID NO: 111; and CDR3 containing SEQ ID NO: 136; [3] The HER2 binder according to [1] or [2], wherein the HER2 binder is monovalent; [4] The HER2 binder according to [1] or [2], wherein the HER2 binder is multivalent (e.g., divalent, trivalent or tetravalent); [5] The HER2 binder according to any one of [1] to [4], wherein the HER2 binder is monospecific; [6] The HER2 binder according to any one of [1], [2] or [4], wherein the HER2 binder is multispecific and specifically binds to HER2 and a different antigen; [7] The HER2 binder according to any one of [1] to [6], wherein the VHH domain is humanized; [8] The HER2 binder according to any one of [1] to [7], wherein the HER2 binder comprises a fusion protein comprising a HER2-binding VHH domain and a heterologous sequence; [9] The HER2 binder contains an N-terminal amino acid residue or polypeptide sequence, for example, Met-Ala or an Ala leader peptide, and optionally, the N-terminal residue or sequence improves protein expression, solubility, purification, half-life or avidity, the HER2 binder according to any one of [1] to [8];

[10] The HER2 binder contains a C-terminal amino acid residue or polypeptide sequence, and optionally, the C-terminal residue or polypeptide sequence promotes the selectivity, loading, intracellular internalization, half-life or avidity of a radiolabel, the HER2 binder according to any one of [1] to [9];

[11] The HER2 binder has the following structure: VHH-(Gly n Xaa m Gly k ) j In the structure, [i] VHH is a VHH disclosed in Table 2 or elsewhere in this specification (for example, a VHH selected from SEQ ID NOs: 1 to 81 and 205); [ii] n = 0 to 5; [iii] m = 0 to 5; [iv] k = 0 to 6; [v] j = 0 to 8; and [vi] Xaa is an amino acid that enables site conjugation to a radiolabeled prosthetic group or direct radiolabeling, The HER2 binder according to any one of [1] to

[10] , comprising a polypeptide sequence having

[12] The HER2 binder according to any one of [8] to

[11] , wherein the fusion protein contains an Fc region;

[13] The HER2 binder according to any one of [8] to

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

[14] An isolated nucleic acid encoding a HER2 binder according to any one of [1] to

[13] ;

[15] A vector containing the nucleic acid of

[14] (optionally, the vector may be an expression vector);

[16] A host cell containing the nucleic acid of

[14] or the vector of

[15] ;

[17] The host cell of

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

[18] The HER2 binder according to any one of [1] to

[13] , wherein the HER2 binder is conjugated to a label; ;

[19] The HER2 binder according to

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

[20] The HER2 binder according to

[18] or

[19] , wherein the label is a radionuclide;

[21] The HER2 binder according to

[19] or

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

[22] The radionuclide is as follows: a) 18 F, 76 Br, 123 I, 124 I, 125 I and 131 I; or b) 75 Br, 77 Br, 122 I, 124 I, 125 I, 131 I and 211 At; The HER2 binder according to

[19] or

[20] , which is a radioactive halogen isotope selected from:

[23] The HER2 binder according to

[19] or

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

[24] The radionuclide is one of the following: 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 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; The HER2 binder according to

[19] or

[20] , which is a radioactive metal isotope selected from the above:

[25] The HER2 binder according to any one of [1] to

[13] and

[18] to

[24] , wherein the HER2 binder is conjugated to a label via a chelate moiety;

[26] The HER2 binder according to

[25] , wherein the chelate moiety is covalently bonded to a protein via a lysine residue or a cysteine residue;

[27] The HER2 binder according to

[25] or

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

[28] The HER2 binder is of the following structure: VHH-RLC In the structure, VHH is a protein containing a HER2-binding VHH domain provided herein (for example, a VHH having a VHH amino acid sequence as shown in Table 2, for example, a VHH containing an amino acid sequence selected from SEQ ID NOs: 1 to 81 and 205 or a derivative thereof); RLC is a radiolabeled chemical used to directly or indirectly facilitate the binding of a radionuclide to the VHH, which is a targeted radiotherapy agent having the structure of any one of [1] to

[12] and

[18] to

[27] ;

[29] The targeted radiotherapy agent is of formula 2: MC-Cm-L4-Cm-T (Formula 2) [Wherein, MC is a polydentate metal chelating moiety; Cm is thiourea, amide or thioether; L4 is selected from a linking, substituted or unsubstituted alkyl chain, substituted or unsubstituted alkenyl chain, an alkyl chain having NH, CO or S at one or both ends optionally, a substituted or unsubstituted alkynyl chain, and a polyethylene glycol (PEG) chain; T is any of the following compounds: (a) Formula 1: [Chemical formula] (Wherein, X is CH or N; L1 and L3 are independently selected from a bonded, 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 a polymeric conjugate 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 containing at least 3 oxygen atoms, where L2 may optionally include a brush border cell enzyme-cleavable peptide; CG is selected from guanidine; PO3H; SO3H; one or more charged D- 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 from 0 to 3; and Y is an alkyl metal moiety, a boronic acid moiety, a boronic acid ester moiety, or 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) The HER2 binder according to

[28] , comprising a prosthetic compound or a radioactive halogen precursor represented by: (b) The compound of (a) wherein the compound is a radioactive halogen precursor, and optionally Y is an alkyl metal moiety selected from the group consisting of trimethylstannyl (SnMe3), tri-n-butylstannyl (SnBu3), trimethylsilyl (SiMe3), boric acid (B(OH)2) or boronic acid ester (B(OR)2), and optionally R contains a cyclic or aliphatic group, the compound of (a); (c) the compound is a prosthetic compound and Y is 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, the compound of (a); (d) the MMCM is an active ester or (Gly)m, where m is 1 or more, the compound of (a); (e) the MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS) ester, tetrafluorophenol (TFP) ester, pentafluorophenol (PFP), paranitrophenol (PNP), isothiocyanate group or maleimide group, the compound of (a); (f) the MMCM is Gly-Gly-Gly, the compound of (a); (g) L2 is (CH2) P and p = 1 to 6, the compound of (a); (h) the optional brush border enzyme-cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr and Gly-Phe-Lys, the compound of (a); (i) the following structure:

Chemical formula

[28] , comprising a prosthetic compound or a radioactive halogen precursor represented thereby, or a pharmaceutically acceptable salt or solvate thereof;

[30] The HER2 binder as described in

[29] , wherein MC is a macrocyclic structure;

[31] The HER2 binder as described in

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

[32] The HER2 binder as described in

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

[33] The HER2 binder as described in

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

[34] The HER2 binder as described in

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

[35] The compound is a prosthetic compound, and Y is 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 At, the HER2 binder as described in

[30] ;

[36] The HER2 binder as described in

[30] , further comprising a metal that binds to MC;

[37] The metal is 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 a radioactive metal selected from Th, the HER2 binder according to

[30] ;

[38] A pharmaceutical composition comprising the HER2 binder according to any one of [1] to

[13] and

[18] to

[37] , and a pharmaceutically acceptable carrier;

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

[13] and

[18] to

[37] , or the pharmaceutical composition of

[38] ;

[40] The method according to

[39] , wherein the disease is cancer;

[41] A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the HER2 binders of [1] to

[13] and

[18] to

[37] , or the pharmaceutical composition of

[38] ;

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

[28] to

[37] ;

[43] The cancer is HER2 + The method according to any one of

[40] to

[42] ;

[44] A method of treating HER2 + cancer, comprising administering to a subject in need thereof an effective amount of the HER2 binder according to any one of [1] to

[13] and

[18] to

[37] or the pharmaceutical composition of

[38] ;

[45] The method according to any one of

[40] to

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

[46] The method according to any one of

[40] to

[45] , wherein the cancer is breast cancer and / or metastases arising from breast cancer;

[47] The method according to

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

[48] The method according to any one of

[40] to

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

[49] HER2 + A method for killing HER2 cells, comprising contacting the cells with a HER2 binder according to any one of [1] to

[13] and

[18] to

[37] or a pharmaceutical composition according to

[38] ; + cells;

[50] The method according to

[49] , wherein the cells are cancer cells;

[51] The method according to

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

[52] The method according to any one of

[49] to

[51] , wherein the cells are contacted with a HER2 binder in vitro or in vivo;

[53] A HER2 binder according to any one of [1] to

[13] and

[18] to

[37] or a pharmaceutical composition according to

[38] for use in a medicament (e.g., in any one of the methods of

[39] to

[52] );

[54] Use of any one of [1] to

[13] and

[18] to

[37] or the pharmaceutical composition of

[38] in the manufacture of a medicament for the treatment of a disease in a subject in need thereof (for example, in any one of the methods of

[39] to

[52] );

[55] Use of

[54] , wherein the disease is cancer;

[56] A method for diagnosing a disease or condition in a subject, comprising administering to the subject any one of the HER2 binders described in any one of [1] to

[13] and

[18] to

[37] or the pharmaceutical composition of

[38] ;

[57] The method of

[56] , wherein the disease is cancer;

[58] The following: a) Administering to the subject any one of the HER2 binders of [1] to

[13] and

[18] to

[37] detectably labeled, or the pharmaceutical composition of

[38] in which the HER2 binder is detectably labeled; and b) Detecting the binding of the labeled HER2 binder to HER2 + cells in the subject (wherein the detection of the binding indicates the presence of HER2 + cells), A method for detecting HER2 + cells in a subject;

[59] Detecting the binding of a labeled HER2 binder to HER2 + cells in a subject includes imaging of HER2 + cells in the method of

[58] ;

[60] A method for imaging HER2 + cells in a subject, comprising administering to the subject any one of the HER2 binders of [1] to

[13] and

[18] to

[37] detectably labeled, or the pharmaceutical composition of

[38] in which the HER2 binder is detectably labeled;

[61] HER2 in a subject +The method according to

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

[62] HER2 + wherein the cells are + cancer cells, the method according to any one of

[58] to

[61] ;

[63] The method according to any one of

[39] to

[48] and

[54] to

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

[64] The method according to any one of

[39] to

[48] and

[54] to

[63] , wherein the subject has cancer;

[65] (a) culturing the host cell of

[16] or

[17] under conditions in which a HER2 binder is produced; and (b) recovering the HER2 binder produced by the host cell, A method for producing a HER2 binder, comprising:

[0010] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description is presented by way of illustration only and not limitation, when showing embodiments and aspects of the present invention. Various changes and modifications within the scope of the present invention will be understood by those skilled in the art from the detailed description.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0012] (Detailed Description of the Invention) Provided herein is a HER2 binder comprising a VHH domain that specifically binds to HER2. Also provided are nucleic acids encoding the protein, vectors and host cells comprising the nucleic acids, and methods for producing and using the HER2 binder. The present disclosure also provides HER2 binders comprising a detectable label, as well as HER2 binder-targeted radiotherapeutic agents for therapeutic and / or diagnostic purposes. Also provided are methods of using the HER2 binder for detecting, monitoring and / or treating diseases and conditions such as cancer.

[0013] In certain embodiments, the present disclosure relates to HER2-targeted radiotherapeutic agents. The provided targeted radiotherapeutic agents have advantageous properties over conventional radiotherapeutic agents, such as, for example, higher stabilization of the radionuclide in the blood circulation, lower uptake and dose exposure to normal tissues including the kidney, higher binding affinity and cell internalization, more uptake of the tumor in the HER2 + tumor, and 131 iodine and 211Higher maximum tolerable volumes are provided for whole body targeted radiotherapy delivery of radionuclides including astatine. Other features and advantages of the disclosed compositions and methods will be apparent from the following disclosure, drawings, and claims.

[0014] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present compositions, the preferred methods and materials are described herein. Each publication, patent application, patent, and other reference mentioned in this disclosure is incorporated herein by reference in its entirety. In case of conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0015] When embodiments are provided herein using the term "comprising," it is understood that other similar embodiments are also provided, described in terms of the terms "containing," "consisting of," and / or "consisting essentially of." However, when used in the claims as a transitional phrase, each term should be interpreted separately in the appropriate legal and factual context (e.g., in the claims, "consisting of" is more exclusive, whereas the transitional phrase "containing" is considered a more open-ended phrase, and "consisting essentially of" is intermediate).

[0016] As used herein, the singular forms "a," "an," and "the" include the plural forms unless it is explicitly stated otherwise or the context clearly indicates that such is not intended.

[0017] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, 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.

[0018] As used herein, the terms "about" and "approximately" when applied to one or more desired values refer to values similar to the recited reference value. In some embodiments, the term "about" or "approximately" refers to a range of values that fall 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 (greater or less) from the recited reference value, unless otherwise specified or clear from the context (except when the numerical value exceeds 100% of the possible numerical values). For example, when used in the context of the percent identity of an amino acid sequence to a reference polypeptide sequence, "about" can mean ±10% of the recited value.

[0019] Any examples or illustrative language herein, such as the use of "such as" or "including", are merely intended to better explain the invention and do not limit the scope of the invention unless recited in the claims. No language in this specification should be construed as indicating that any element not recited in the claims is essential to the practice of the invention.

[0020] When embodiments of the present disclosure are described from the perspective of classifying a Markush group or some other group, the disclosed compositions or methods include not only the entire group being enumerated as a whole, but also each member of the group, individually, and all possible subgroups of the main group, as well as main groups in which one or more of the group members are absent. The disclosed compositions and methods also contemplate explicitly excluding any one or more of the group members in the disclosed compositions or methods.

[0021] As used herein, the terms "isolated" and "purified", or iterations thereof, refer to a molecule that has been separated from at least a portion of the components with which it is normally associated in nature or produced. For example, a protein is "isolated" if it has been separated from at least a portion of the components of the cell that produced the protein. If a protein is secreted from a cell after expression, physically separating the supernatant containing the protein from the cell that produced the protein is considered to "isolate" the protein. Similarly, a nucleic acid is "isolated" if it is not part of a larger nucleic acid that normally exists in nature (e.g., genomic DNA or mitochondrial DNA in the case of DNA nucleic acids), or, in the case of an RNA nucleic acid, if it has been separated from at least a portion of the components of the cell in which it was produced. Thus, a DNA nucleic acid contained in a vector within a host cell can be said to be "isolated".

[0022] As used herein, the term "HER2" has its ordinary meaning in the art and refers to the human epidermal growth factor receptor 2 (HER2) protein. HER2 is also known as Her-2, Her-2 / Neu, Neu, ErbB-2, CD340 (cluster of differentiation 340), or p185, and all of these terms are subsumed by "HER2" herein. The extracellular domain of HER2 contains four domains, domain I (amino acid residues approximately 1 to 195), domain II (amino acid residues approximately 196 to 319), domain III (amino acid residues approximately 320 to 488), and domain IV (amino acid residues approximately 489 to 630) (residue numbers excluding the 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 the HER2 protein having the amino acid sequence disclosed in Genbank Reference: NP_001005862, NP_001276865, NP_001276866, NP_001276867, and / or NP_004439 (as of January 1, 2020).

[0023] The terms "VHH" (the variable heavy domain of a heavy-chain antibody) and "single-domain antibody" are used interchangeably herein to refer to a single antigen-binding antibody fragment comprising a polypeptide sequence having three complementarity-determining regions (CDRs). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. The molecular weight of VHH is usually 12 to 15 kDa. The VHH binding domain of the HER2 binder provided herein can bind only to HER2 without pairing with the corresponding CDR-containing polypeptide. The term "VHH binding domain" is used herein to refer to the full-length VHH, VHH binding fragment and / or VHH variant that binds to the antigen of interest, and / or a fragment or variant of VHH that binds to the antigen of interest (e.g., HER2), unless otherwise indicated by context. The provided HER2-binding VHH domain can be recombinantly derived from any species including, but not limited to, mouse, human, camel, llama, alpaca, vicuña, guanaco, shark, goat, rabbit, and / or cow. In some embodiments, the VHH binding domain is obtained from a camelid species (e.g., camel, llama, dromedary, alpaca, vicuña and guanaco).

[0024] 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 HER2-binding VHH domain, unless otherwise indicated by context.

[0025] The "humanized" form of a VHH binding domain is a binding domain that contains a minimal sequence derived from a non-human immunoglobulin or non-human germline sequence. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's CDRs (defined below) are replaced with residues derived from the CDRs of a non-human species (donor antibody) having the desired specificity, affinity and / or ability (e.g., mouse, rat, rabbit, camel, llama, alpaca or non-human primate). Optionally, framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. Additionally, a humanized antibody can contain residues not present in the recipient antibody or donor antibody. These modifications can be made to further improve the performance of the antibody, e.g., binding affinity, isomerization, immunogenicity, radiolabeling efficiency, catabolism, enzymatic degradation. The number of these amino acid substitutions in the FR is typically 6 or less. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the VHH binding domain was made. Methods for humanizing VHH binding domains are well known in the art. Typically, humanizing substitutions are selected such that the resulting humanized VHH domain still retains the advantageous properties of the HER2 binders of the present disclosure.

[0026] As used herein, the terms "specifically bind", "specifically recognize" and "specific" are used interchangeably to refer to a measurable and reproducible interaction such as the binding between a target and an antigen-binding protein (such as HER2 and the HER2 binders provided herein). 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 higher affinity, avidity, more immediate onset, and / or longer duration than it binds to other targets. In some embodiments, the degree of binding of the antigen-binding protein to an irrelevant target is less than about 10% of the binding of the antigen-binding protein (e.g., a HER2 binder) to the target (e.g., HER2), as measured by, for example, a radioimmunoassay (RIA). In some embodiments, an antibody is said to specifically bind to an antigen when the K D is ≤ 1 μM, e.g., ≤ 100 nM or ≤ 10 nM. Terms such as "HER2-binding polypeptide", "HER2-binding protein" and "HER2-binding VHH domain" refer to polypeptides and VHH domains that specifically bind to HER2, respectively.

[0027] "Affinity" means the strength of the sum of non-covalent interactions between the binding surface of a ligand (e.g., a HER2-binding molecule or agent) and the binding site of a partner molecule (e.g., the HER2 receptor). In general, the affinity or apparent affinity of molecule X for partner Y can be represented by the dissociation constant (K D ) or the apparent K D , respectively. Affinity can also be represented by the ratio of the kinetic association rate constant (k on ) to the kinetic dissociation rate constant (k off ) (K D =k off / k on ). Affinity can be determined by common methods known in the art, including those described herein (e.g., ELISA, K Dcan be measured by, for example, KinExA, flow cytometry, and / or surface plasmon resonance apparatus. Such methods include, but are not limited to, methods including BIACAORE (registered trademark), Octet (registered trademark) or flow cytometry. As used herein, the term "K D " refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. As used herein, the term "K D " includes K D and apparent K D . In some embodiments, the K D of an antigen-binding molecule is measured by flow cytometry by fitting the average fluorescence measured at each antibody concentration to a non-linear one-site binding equation (GraphPad Prism Software) using an antigen-expressing cell line (e.g., HER2-expressing cells). In some such embodiments, K D is apparent K D .

[0028] The "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 as compared to a reference parental VHH binding domain sequence (without the changes described below), wherein said changes improve the affinity of the VHH-containing polypeptide for HER2. HER2 binder variants include the affinity matured variants of the HER2 binding VHH domains provided herein.

[0029] 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 as compared to the corresponding sequence of the HER2-binding VHH domain disclosed herein. In some embodiments, a variant is a biologically active polypeptide that, for example, after aligning sequences and introducing gaps as necessary to achieve a maximum percent sequence identity, has at least about 80% amino acid sequence identity with a reference amino acid sequence and does not consider any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the HER2-binding domains provided herein. In some embodiments, a variant comprises 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, a variant comprises 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, a variant comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the amino acid sequence disclosed in any one of SEQ ID NOs: 1-81 and 205. The alignment for the purpose of determining percent amino acid sequence identity is, for example, BLAST, BLAST-2, ALIGN or MEGALIGN TM(DNASTAR) publicly available computer software, such as software, can be achieved in various ways within the skill of the art. Exemplary computer programs for determining identity between one sequence include the GCG program package (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), and the default parameters of the corresponding programs are used. Certain embodiments including HER2 binding variants are provided herein.

[0030] The term "vector" can be genetically engineered to contain a cloned polynucleotide sequence that can be propagated in a host cell or is used to describe a genetically engineered nucleic acid. A vector can contain one or more regulatory sequences (e.g., a promoter and / or enhancer) that regulate the expression of the polypeptide of interest and / or one or more selectable marker genes (e.g., an antibiotic resistance gene and a gene that can be used in a colorimetric assay (e.g., β-galactosidase)). The terms "expression vector", "expression construct" or "expression cassette" are used interchangeably throughout this disclosure and mean any type of genetic construct that contains a nucleic acid encoding a gene product and in which some or all of the nucleic acid coding sequence is transcribable in a host cell.

[0031] "Host cell" refers to a cell that can be or has been a recipient of a vector and / or a heterologous nucleic acid. A host cell can be a prokaryotic cell or a eukaryotic cell. Examples of eukaryotic host cells include mammalian cells such as primate or non-primate animal cells; fungal cells such as yeast; plant cells; and insect cells. Non-limiting mammalian host cells encompassed by the present disclosure include NSO cells, PER.C6® cells (Crucell), 293 cells, and CHO cells, as well as derivatives thereof (e.g., 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells), but are not limited thereto. Host cells also include the progeny of a single host cell, which progeny need not be identical in form or genomic DNA complementarity to the original parent cell due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the nucleic acid encoding the VHH binding domain provided herein.

[0032] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor in which antigen specificity is introduced into a cell (e.g., a T cell such as a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof) via an antigen binding domain (e.g., the HER2 binding VHH domain disclosed herein). Thereby, the antigen binding properties of the antigen binding domain and the T cell activity of the T cell (e.g., lytic ability and self-renewal ability) are combined. A CAR typically includes an extracellular antigen binding domain (ectodomain), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain generally includes at least one ITAM signaling domain derived from, for example, CD3 zeta, and optionally at least one co-stimulatory signaling domain derived from, for example, CD28 or 4-1BB. In the CAR provided herein, the HER2 binding VHH domain provided herein forms the antigen binding domain and is located outside the cell when expressed intracellularly.

[0033] The term "biological activity" refers to one or more biological properties of a molecule, whether naturally occurring as found in vivo or provided or obtained by recombinant means. Biological properties include, but are not limited to, binding to a ligand (e.g., HER2), induction or increase of cell death, induction or increase of cell proliferation, induction or increase of endocytosis, induction or increase of intracellular retention, and induction or increase of cytokine expression.

[0034] "Pharmaceutically acceptable" refers to molecules and compositions that do not cause adverse reactions, allergic reactions, or other harmful reactions when administered to mammals, particularly humans. A pharmaceutically acceptable carrier is a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, regardless of its type. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, preservatives, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are suitable for pharmaceutical administration. Suitable examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, glucose solution and 5% human serum albumin. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference book in the field, the content of which is incorporated herein by reference. Additional biological active agents (e.g., therapeutic agents) can also be incorporated into the composition.

[0035] The term "radionuclide" refers to a moiety containing a radioisotope 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, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 52 Mn, 90Y, 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 are included. In some embodiments, the radionuclide is an α-radionuclide (e.g., 211 At or 225 Ac). In some embodiments, the radionuclide is a β-radionuclide (e.g., 131 I or 177 Lu). In some embodiments, the radionuclide is a radioactive halogen isotope or a radioactive metal isotope. In some embodiments, the radionuclide is a radioactive halogen isotope. In some embodiments, the radionuclide is 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 At selected radioactive halogen isotopes. In certain embodiments, the radionuclide is 131 I, 211 At, 125 I or 124 I. In some embodiments, the radionuclide is a radioactive metal isotope. In some embodiments, the radionuclide is 144 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 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, which is a radioactive metal isotope selected from. In certain embodiments, the radionuclide is 177 Lu, 131 I, 211 At or 225 Ac. In some embodiments, the radionuclide is useful for therapy. In certain embodiments, the radionuclide is selected from the following: 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 and 227 Th; or 32 P. In some embodiments, the radionuclide is useful for diagnosis. In certain embodiments, the radionuclide is selected from the following: 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 Tl; or 11 C, 13 N, 15 O, 24 Na, 32 P and 133 Xe.

[0036] The term "prosthetic group" or "bifunctional label" refers to a small organic molecule that is chemically linkable to include a radionuclide that can be linked to a peptide or protein (e.g., a protein containing a HER2-binding VHH domain). A radiolabeled prosthetic agent is generally a compound or group that includes a radiolabel, a charged group (CG), and a macromolecular conjugate moiety (MMCM) and is suitable for binding to the protein component of a HER2 binder. Each of these components can be linked, if desired, to one or more cleavable (or non-cleavable) linkers.

[0037] As used herein with respect to radiopharmaceutical chemistry, the terms "chelating agent ligand" or "chelating agent" refer to a bifunctional chelating agent or conjugate (BFC) moiety that covalently attaches a radiolabeled prosthetic group to a biologically active target molecule (e.g., a peptide or protein such as a protein comprising a HER2-binding VHH domain disclosed herein). The BFC utilizes functional groups, e.g., carboxylic acid or active ester for an amide bond, isothiocyanate for a thiourea bond, and maleimide for a thiol bond.

[0038] The terms "label" and "detectable label" refer to a polypeptide or moiety that binds to a protein comprising a HER2-binding VHH domain, for example, and enables the detection of a reaction (e.g., binding) between members of a specific binding pair. Thus, the term "labeled binding protein" refers to a protein into which a label that provides for the identification of the binding protein is incorporated. In some embodiments, the label is a detectable marker that can generate a signal detectable by visual or instrumental means, e.g., incorporation of a radiolabeled amino acid, or binding of a biotinylated moiety to a protein that can be detected by a labeled avidin (e.g., streptavidin comprising a fluorescent marker or enzyme activity detectable by an optical or colorimetric method). 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); chromogenic agents; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors); enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined protein epitopes recognized by secondary reporters (e.g., leucine zipper pairing sequences, binding sites of secondary antibodies, metal binding domains, epitope tags); toxins; chemotherapeutic agents; and magnetic substances such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include, for example, a luminescent moiety such as an acridinium compound, and a fluorescent moiety such as fluorescein. In this regard, the moiety itself may not be a detectable label, or may become detectable by reaction with another site.

[0039] The terms "subject", "patient", "animal" and "individual" are used interchangeably and refer to mammalian patients such as human patients and non-human primates, experimental animals such as rabbits, rats and mice, and other animals. Animals include all vertebrates, for example, mammals, and non-mammals such as chickens, amphibians and reptiles. As used herein, "mammal" is not limited to those hereinafter described, but includes humans, non-human primates such as apes and monkeys such as chimpanzees; domestic animals such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; experimental animals including rodents such as mice, rats and guinea pigs; and any member of mammals including other members of mammals. A patient may be male or female and may be of any suitable age including infants, juveniles, young adults, adults, and elderly subjects. In some examples, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder such as cancer. In certain embodiments, the disease or disorder is HER2 + cancer. In certain embodiments, the disease or disorder is HER2 +It is breast cancer or a breast cancer brain metastasis. In certain embodiments, the disease or disorder is HER2 + ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, gastric cancer (e.g., colorectal cancer and colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, skin-derived cancer and / or metastases arising therefrom (e.g., brain metastases of ovarian cancer).

[0040] In some embodiments, the subject to be treated can be a patient who presents the fact that the subject has a disorder associated with the treatment or has a sufficient risk of developing the disorder. In certain embodiments, the patient is human.

[0041] The term "biological sample" refers to an amount of a substance derived from a living or once-living organism such as a mammal. 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, spleen, etc. In certain embodiments, the biological sample is a tissue biopsy (e.g., HER2 + tissue, and / or obtained from breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, gastric cancer (e.g., colorectal cancer, colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, skin-derived cancer, and / or metastases arising therefrom (e.g., brain metastases of breast cancer).

[0042] As used herein, the terms "treatment," "therapy," or "therapeutic" with respect to a disease, disorder, or condition refer to an approach for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein encompasses any administration or application of a therapeutic agent to a disease in a mammal, including a human. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, regression of disease extent, prevention or delay of disease spread (e.g., metastasis such as metastasis to the brain, lung, or lymph nodes), prevention or delay of disease recurrence, delay or retardation of disease progression, improvement of disease state, inhibition of disease or disease progression, inhibition or blunting of disease or its progression, prevention of disease onset, and remission (partial or complete). Further, "treatment" also encompasses reduction of the pathological consequences of a proliferative disease. The methods provided herein are intended for any one or more of these aspects of treatment. Similarly, the term "treatment" does not require complete elimination of all aspects of the disorder.

[0043] As used herein, the terms "treatment of a proliferative disorder," "treatment of a hyperproliferative disorder," and iterations thereof are used to include maintenance or reduction of tumor size, induction of tumor regression (partial or complete), inhibition of tumor growth, and / or prolongation of the lifespan of a subject having a proliferative disorder. In some embodiments, the proliferative disorder is HER2 + a solid tumor. Such tumors include HER2 + cancers. In some embodiments, the tumor is associated with breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, gastric cancer (e.g., colorectal cancer, colon cancer), pancreatic cancer, kidney cancer, prostate cancer, lung cancer (e.g., NSCLC), bladder cancer, urothelial cancer, epidermally-derived cancer, and / or metastases (s) arising therefrom (e.g., breast cancer metastasized to the brain).

[0044] The terms "tumor cell", "cancer cell", "cancer", "tumor" and / or "neoplasm" are used interchangeably herein unless otherwise specified, and refer to cells (or a plurality of cells) that exhibit uncontrolled proliferation and / or enhanced abnormal cell survival and / or inhibition of apoptosis, which inhibit the normal function of the organs and systems of the body. This definition includes benign and malignant cancers, polyps, hyperplasias as well as dormant tumors or micrometastases. Types of cancer that can be treated by the methods provided herein include solid cancers, disseminated metastatic diseases and brain metastases.

[0045] As used herein, the term "anticancer agent" is used in the broadest sense to refer to an agent used in the treatment of one or more cancers. Exemplary classes of anticancer agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (e.g., cytokines, extracellular domain of receptor-Fc fusions and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (antisense oligonucleotides and siRNA) and oncolytic viruses.

[0046] The terms "effective amount" or "therapeutically effective amount" refer to the amount and / or concentration of a composition containing an active ingredient (e.g., a HER2 binding compound) which, when administered to a patient alone (i.e., as monotherapy) or in combination with another therapeutic agent, results in a statistically significant decrease in the progression of a disease, for example, by ameliorating or eliminating symptoms and / or the cause of the disease. An effective amount may be an amount that restores, reduces or alleviates at least one symptom or biological response or effect associated with a disease or disorder, an amount that arrests the progression of a disorder, or an amount that improves the physical function of a patient. With respect to cancer, an effective amount of an agent or composition is an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce the tumor size; (iii) inhibit, delay, blunt to some extent and in some cases arrest the invasion of cancer cells into peripheral organs; (iv) inhibit tumor metastasis (i.e., blunt to some extent and in some cases arrest); (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In some embodiments, the effective amount is an amount sufficient to prevent or delay onset. In some embodiments, the 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 depending on factors such as the individual's disease state, age, gender and weight, as well as the ability of the active agent to elicit the desired response in the individual. The therapeutically effective amount is also an amount at which the toxicity or harmful effects of the active agent are less than the therapeutically effective effects. The therapeutically effective amount can be administered in one or more divided administrations.

[0047] The disclosed HER2 binders and pharmaceutical compositions containing these agents can be administered alone (i.e., as monotherapy) or in combination with another therapeutic agent (i.e., as combination therapy). Administration "in combination with" one or more other therapeutic agents includes simultaneous (parallel) and sequential administration in any order.

[0048] As used herein, the term "simultaneously" is used to refer to the administration of two or more therapeutic agents where at least a portion of the administrations overlap in time, or the administration of one therapeutic agent occurs within a short period relative to the administration of the other therapeutic agent, or the therapeutic effects of both therapeutic agents overlap for at least a certain period of time.

[0049] As used herein, the term "sequentially" is used to refer to the administration of two or more therapeutic agents that do not overlap in time or whose therapeutic effects do not overlap.

[0050] As used herein, "administered in combination" refers to administering one mode of treatment in addition to another mode of treatment. Thus, "administered in combination" refers to administering one mode of treatment before, during, or after the administration of another mode of treatment to an individual.

[0051] "Product" means any article (e.g., a package or container) or kit that contains at least one reagent (e.g., a pharmaceutical described herein for treating a disease or disorder (e.g., cancer) described herein, or a probe for specifically detecting a biomarker described herein). In some embodiments, the article or kit is marketed, distributed, or sold as a unit for performing the methods described herein.

[0052] The term "package insert" refers to the instructions customarily included in the package of a commercially available therapeutic pharmaceutical that contains information regarding indications, usage, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic pharmaceutical.

[0053] I. HER2 Binding Agent The present disclosure provides a HER2 binding agent comprising a protein comprising a HER2 binding VHH domain.

[0054] In some embodiments, the provided HER2 binding agent binds to HER2 and has a dissociation constant of ≤ 10 -7 M, ≤ 10 -8 M, ≤ 10-9 M, ≤ 10 -10 M, or ≤ 10 -11 The dissociation constant (K d ) of M. In some embodiments, the HER2 binder binds to HER2 and ≤ 10 -7 ~≤ 10 -11 M or ≤ 10 -8 ~≤ 10 -10 The K of M d has.

[0055] In some embodiments, the provided HER2 binder binds to HER2 with a K of about 500 nm or less or 1 nm or less D In some embodiments, the HER2 binder binds to HER2 with a k of about 0.002 / s or less off In some embodiments, the HER2 binder binds to HER2 with a K of about 500 nm or less D and a k of about 0.002 / s or less off to bind to HER2.

[0056] In some embodiments, the HER2 binder comprises a VHH binding domain that specifically binds to an epitope on a human protein that is conserved among different species of HER2 proteins. In some embodiments, the HER2 binder comprises a VHH binding domain that specifically binds to HER2 domain I. In some embodiments, the HER2 binder comprises 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 binder comprises a VHH binding domain that specifically binds to HER2 domain III. In some embodiments, the HER2 binder comprises 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 binder comprises two or more HER2 binding domains that specifically bind to different epitopes of HER2. In some embodiments, the HER2 binder comprises a VHH binding domain that specifically binds to one HER2 domain and a VHH binding domain that specifically binds to a different HER2 domain. In certain embodiments, the HER2 binder comprises 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, but need not, include exclusive binding to HER2.

[0057] In some embodiments, the HER2 binder comprises an affinity matured HER2 binding VHH domain. In some embodiments, the HER2 binder comprises a humanized VHH binding domain. In some embodiments, the HER2 binder comprises one or more polypeptides. In another embodiment, the HER2 binder comprises one or more polypeptides.

[0058] In some embodiments, the HER2 binder comprises the VHH amino acid sequences disclosed in Table 2. In some embodiments, the HER2 binder comprises a VHH amino acid sequence selected from SEQ ID NOs: 1-81 and 205.

[0059] In some embodiments, the HER2 binder comprises a HER2-binding fragment and / or variant of the HER2-binding amino acid sequences provided herein. In some embodiments, the HER2 binder comprising a fragment or variant amino acid sequence binds to HER2 with a K D of about 500 nm or less or about 1 nm or less. In some embodiments, the HER2 binder binds to HER2 with a k off of about 0.002 / s or less. In certain embodiments, the HER2 binder binds to HER2 with a K D of about 500 nm or less or about 1 nm or less and a k off of about 0.002 / s or less.

[0060] In some embodiments, the HER2 binder comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the HER2-binding VHH domain provided herein. Also included are sequences having 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, sequence identity is determined using a suitable sequence alignment algorithm, such as BLAST P (Karlin and Altschul, PNAS USA (1990) 87(6):2264-8), and default parameters.

[0061] In some embodiments, the HER2 binder comprises a VHH sequence disclosed in Table 2. In some embodiments, the HER2 binder comprises 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. In some embodiments, the HER2 binder comprises an amino acid sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binder 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 SEQ ID NOs: 1-81 and 205.

[0062] In some embodiments, the HER2 binder contains a variant protein comprising an amino acid sequence having one or more additions, insertions, substitutions and / or deletions compared to the VHH binding domain disclosed herein.

[0063] Amino acid substitution includes substituting each of one or more amino acids in a protein with one or more other amino acids. Exemplary substitutions are shown in Table 1. In some embodiments, a conservative substitution in a provided HER2 binder variant involves exchanging the original residue in the VHH sequence disclosed in Table 2 with an amino acid residue in the corresponding exemplary substitution list in Table 1.

Table 1

[0064] Furthermore, amino acids can be divided into the following groups according to the properties of their common side chains: (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 that affect the orientation of the chain: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. In some embodiments, conservative substitutions involve the exchange of two members within one of these groups. In some embodiments, non-conservative substitutions involve the exchange of a member of one of these groups with a member of another group.

[0065] In some embodiments, the HER2 binder comprises one or more substitutions selected from: substituting a Lys residue with either an Arg residue or a His residue; substituting an unpaired Cys residue with an Ala residue or a Ser residue; and substituting a Glu residue with an Asn residue.

[0066] In some embodiments, the HER2 binder comprises an amino acid sequence having an addition, substitution, and / or deletion of 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to the reference HER2-binding VHH domain provided herein. Deletions or insertions of amino acids can also be made with respect to the amino acid sequence of the HER2-binding VHH provided herein. Thus, for example, amino acids that do not substantially affect the activity of the HER2-binding domain, or at least do not impair such activity, may be deleted. Such deletions can be advantageous as they can reduce the overall length and molecular weight of the polypeptide while retaining activity. This can make it possible to reduce the amount of protein required for a particular purpose - for example, the dosing level can 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 carboxy terminus of the HER2-binding VHH domain. In some embodiments, one or more amino acid residues are deleted from both the amino and carboxy termini of the HER2-binding VHH domain. Amino acid insertions with respect to the sequence of the HER2-binding VHH domain disclosed herein are also provided. In some embodiments, one or more additional amino acid residues are at the amino terminus of the HER2-binding VHH domain. In some embodiments, one or more additional amino acid residues are at the carboxy terminus of the HER2-binding VHH domain. In some embodiments, one or more additional amino acid residues are present at both the amino and carboxy termini of the HER2-binding VHH domain. Changes to the amino acids with respect to the sequence of the HER2-binding VHH provided herein can be made using any suitable technique known in the art (e.g., using site-directed mutagenesis).

[0067] In some embodiments, the HER2 binder comprises an amino acid sequence having from 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the HER2 binder comprises an amino acid sequence having from 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the additional amino acid residues are present at the amino terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the additional amino acid residues are present at the carboxy terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the HER2 binder comprises an amino acid sequence having from 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 of the HER2-binding VHH domain and from 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 carboxy terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2.

[0068] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to the reference VHH sequence disclosed in Table 2. 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.

[0069] In some embodiments, the HER2 binder comprises an amino acid sequence having a deletion of 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the amino acid residues are deleted at the amino terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the amino acid residues are deleted at the carboxy terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2. In some embodiments, the HER2 binder comprises an amino acid sequence having a deletion of 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 at the amino terminus of the HER2-binding VHH domain and a deletion of 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 at the carboxy terminus of the HER2-binding VHH domain as compared to the reference VHH sequence disclosed in Table 2.

[0070] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the additional amino acid residues are present at the amino terminus of the HER2-binding VHH domain as compared to the sequences selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the additional amino acid residues are present at the carboxy terminus of the HER2-binding VHH domain as compared to the sequences selected from SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 carboxy terminus as compared to the sequences selected from SEQ ID NOs: 1-81 and 205.

[0071] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 5, 1 to 3, or 1, 2, 3, 4, or 5 additions, substitutions, and / or deletions as compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205, wherein the addition(s), substitution(s), and / or deletion(s) are present within and / or proximal (within 5 amino acids) to the VHH CDRs as disclosed in Table 2. In certain embodiments, the addition(s), substitution(s), and / or deletion(s) are present within and / or in the vicinity (within 5 amino acids) of the VHH CDRs and can also provide potential radiolabeling sites otherwise. Such additions, substitutions, and / or deletions are advantageous for targeted isotope therapy (TRT) applications, for example, the VHH can reliably maintain its affinity, specificity, and internalization properties even after being radiolabeled with a therapeutic payload; the VHH can be pre-conjugated with a radiopharmaceutical prosthetic moiety - such an approach can maximize the amount of radiochemical labeling and is extremely important for radionuclides with low reactivity and short half-lives; site-selective radiolabeling can be enabled (e.g., SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, etc.); and better control of the stability and versatility of the VHH-drug conjugate or VHH radiolabel conjugate is possible.

[0072] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 as 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.

[0073] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 5, 1 to 3, or 1, 2, 3, 4, or 5 substitutions compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205, wherein the substitution(s) is / are present within and / or in the vicinity (within 5 amino acids) of the VHH CDR as disclosed in Table 2. In certain embodiments, the substitution(s) is / are present at residue positions within and / or proximal (within 5 amino acids) to the VHH CDR and otherwise can also provide potential radiolabeling sites.

[0074] In some embodiments, the HER2 binder comprises an amino acid sequence having 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 carboxy 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 HER2 binder comprises an amino acid sequence in which 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 are deleted at the amino terminus of the HER2-binding VHH domain and 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 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 are deleted at the carboxy terminus of the HER2-binding VHH domain compared to a reference sequence selected from SEQ ID NOs: 1-81 and 205.

[0075] In some embodiments, the HER2-binding polypeptide comprises a fragment of the 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 contiguous amino acids of the sequence disclosed in any one of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2-binding polypeptide comprises a fragment of the 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 contiguous amino acids of the sequence disclosed in Table 2.

[0076] In some embodiments, the HER2-binding polypeptide comprises the amino acid sequence disclosed in Table 2. In some embodiments, the HER2-binding polypeptide comprises an amino acid sequence selected from 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 to 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 to the VHH having the amino acid sequences of SEQ ID NOs: 1-81 and 205. In some embodiments, the HER2-binding polypeptide comprises the complementarity determining region (CDR) 3 sequence disclosed in Table 2. In some embodiments, the HER2-binding agent comprises a protein containing an amino acid sequence selected from SEQ ID NOs: 124-157 and 206.

[0077] In some embodiments, the HER2-binding polypeptide comprises the CDR3 and CDR2 sequences 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.

[0078] In some embodiments, the HER2-binding polypeptide comprises the CDR3, CDR2, and CDR1 sequences 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 as 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.

[0079] 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.

[0080] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 23, 32, and 34-38. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 38.

[0081] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 24-31, 33, and 39-42. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 41 or 42. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 42.

[0082] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 1 and 43-49.

[0083] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 1 or 43.

[0084] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 44 or 45. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 46 or 47. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 48 or 49. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 49.

[0085] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 2.

[0086] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 3.

[0087] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 4.

[0088] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 5, 17, 22, 79, 80, or 81. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 22 or 79. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 80 or 81. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 81.

[0089] In some embodiments, the HER2 binder comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 87; CDR2 comprising the amino acid sequence of SEQ ID NO: 117; and CDR3 comprising the amino acid sequence of SEQ ID NO: 129. In certain embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 6.

[0090] In some embodiments, the HER2 binder comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 88; CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and CDR3 comprising the amino acid sequence of SEQ ID NO: 130. In certain embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 7.

[0091] In some embodiments, the HER2 binder comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 89; CDR2 comprising the amino acid sequence of SEQ ID NO: 108 or 112; and CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain embodiments, the HER2 binder comprises an amino acid sequence selected from SEQ ID NOs: 50, 51, and 52. In some embodiments, the HER2 binder comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 89; CDR2 comprising the amino acid sequence of SEQ ID NO: 108; and CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain specific embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 51. In certain specific embodiments, the HER2 binder comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 89; CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and CDR3 comprising the amino acid sequence of SEQ ID NO: 156. In certain embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 50 or 52. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 52.

[0092] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequences of SEQ ID NOs: 8, 19, 70, 71 and 72.

[0093] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 70, 71 and 72. In some embodiments, the HER2 binder 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 particular embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 71. In certain particular embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 70 or 72. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 72.

[0094] In some embodiments, the HER2 binder 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 certain embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 8 or 19. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 19.

[0095] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 9.

[0096] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 10.

[0097] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 11.

[0098] In some embodiments, the HER2 binder 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 the amino acid sequence of SEQ ID NO: 139 or 206. In certain embodiments, the HER2 binder comprises an amino acid sequence selected from SEQ ID NO: 12, 56, 57 and 58. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 56 or 58. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 12.

[0099] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NO: 13, 59, 60, and 61. In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NO: 13 or 59. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NO: 60 or 61. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 61.

[0100] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 14.

[0101] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 15.

[0102] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 16.

[0103] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 62.

[0104] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NO: 18 and 63 - 69.

[0105] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 18 or 63. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 64 or 65. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 65.

[0106] In some embodiments, the HER2 binder 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 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 66 or 67. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 68 or 69. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 69.

[0107] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 20, 53, 54, 55, and 73-75. In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 20, 53, and 73. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 54, 55, 74, and 75. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 75.

[0108] In some embodiments, the HER2 binder 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 binder comprises an amino acid sequence selected from SEQ ID NOs: 21, 76, 77, and 78. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 21 or 76. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 77 or 78. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the HER2 binder comprises the amino acid sequence of SEQ ID NO: 78.

[0109] In some embodiments, the HER2 binder 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 binder comprises the amino acid sequence of SEQ ID NO: 205.

[0110] In some embodiments, the HER2 binder comprises a protein comprising at least one VHH binding domain provided herein that specifically binds to HER2 fused to a heterologous peptide or protein sequence. In some embodiments, the HER2-binding VHH domain is fused to additional N-terminal and / or C-terminal amino acid sequences. Such additional sequences can be provided for various reasons, such as to enhance glycosylation, improve protein expression, solubility, enhance tumor cell internalization, or increase ease of purification, and / or to improve the selectivity and payload capacity of labels (e.g., radiolabels).

[0111] In some embodiments, the HER2 binder comprises a protein comprising 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 that is a polyamino acid sequence, e.g., a plurality of histidine residues or a plurality of lysine residues (suitably 2, 3, 4, 5, or 6 residues).

[0112] In some embodiments, the HER2 binder comprises a protein containing a C-terminal amino acid sequence that improves or enables site conjugation or direct labeling (e.g., radiolabeling) to a radiolabeled prosthetic group. In some embodiments, the protein of the HER2 binder has a C-terminal amino acid sequence (Gly n Xaa m Glyk) jFurther comprising: The VHH is a HER2-binding VHH domain provided herein (e.g., Table 2), where n = 0-5, m = 0-5, k = 0-6, and j = 0-8, and Xaa is an amino acid residue that enables site conjugation or direct labeling (e.g., direct radioactive labeling) to a labeled prosthetic group (e.g., a radioactively labeled prosthetic group). In some embodiments, Xaa is a natural amino acid. In certain embodiments, Xaa is lysine, cysteine, glutamine, tyrosine, histidine, phenylalanine, selenocysteine, or pyrrolidine. 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 binder comprises a C-terminal sequence GGKG (SEQ ID NO: 202). In some embodiments, the HER2 binder comprises a C-terminal sequence GGKGGKG (SEQ ID NO: 203). In some embodiments, the HER2 binder comprises a C-terminal sequence GGKGGC (SEQ ID NO: 204).

[0113] In some embodiments, the HER2 binder comprises a protein that further comprises an additional C-terminal amino acid sequence that enhances the internalization of the binder into tumor cells. In some embodiments, the HER2 binder further comprises a structure VHH-IPS, wherein the IPS is an internalization peptide sequence. In some embodiments, the HER2 binder further comprises the sequence Arg n (R n ) and n = 4-16. In some embodiments, the HER2 binder comprises the sequence (ArgLys) n ((RK) nFurther comprising [[ID=]], where n = 2 to 8. In some embodiments, the HER2 binder further comprises a C-terminal amino acid sequence selected from the following group: 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), the 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), RLWMRWYSPRTRAYGC (SEQ ID NO: 169), GKCSTRGRKCCRRKK (SEQ ID NO: 170), and GRCAVLSCLPKEQI (SEQ ID NO: 171).

[0114] In some embodiments, the HER2 binder comprises a fusion protein comprising two or more VHH domains. In some embodiments, the HER2 binder comprises two or more identical VHH binding domains (e.g., homodimer, homotrimer, or homo-oligomer). In some embodiments, the protein comprises two or more different VHH binding domains (e.g., heterodimer, heterotrimer, or hetero-oligomer).

[0115] In some embodiments, the present disclosure provides a multispecific HER2 binder containing a fusion protein comprising at least one HER2-binding VHH domain provided herein and one or more additional binding domains. In some embodiments, the one or more additional binding domains are antibodies (e.g., VHH (single-domain antibody) or antigen-binding fragment of an antibody). In some embodiments, one or more within the additional binding domain are VHH binding domains.

[0116] The provided HER2 binder can be monovalent or multivalent with respect to HER2 and / or different antigens. In some embodiments, the HER2 binder is monovalent with respect to HER2 (e.g., contains 1 HER2 binding domain). In some embodiments, the multivalent HER2 binder is multivalent and contains one or more binding domains. In some embodiments, the HER2 binder contains two or more copies of a binding domain that specifically binds to HER2. In some embodiments, the multivalent HER2 binder contains 2, 3, 4, 5, 6 or more copies of a HER2 binding VHH domain. In some embodiments, the HER2 binder contains two or more copies of different binding domains (e.g., VHH binding domains) that specifically bind to HER2. In some embodiments, the HER2 binder contains 2, 3, 4, 5, 6 or more different VHH domains that specifically bind to HER2. In some embodiments, the HER2 binder includes 2, 3, 4, 5, 6 or more different VHH binding domains that specifically bind to different epitopes of HER2.

[0117] The provided HER2 binder can also be monospecific or multispecific. In some embodiments, the HER2 binder is monospecific for HER2 (i.e., contains one or more binding domains that bind to the same epitope with respect to HER2). In certain embodiments, the HER2 binder comprises 2, 3, 4, 5, 6 or more copies of the same HER2-binding VHH domain. In some embodiments, the HER2 binder is multispecific. In certain embodiments, the HER2 binder is multispecific and comprises one or more binding sites that bind to different epitopes of HER2. In certain embodiments, the HER2 binder is multispecific and comprises 2, 3, 4, 5, 6 domains, or VHH domains, that specifically bind to different epitopes or domains of HER2 (e.g., HER2 domains I, II, III or IV). In some embodiments, the HER2 binder is multispecific and comprises two or more copies of different binding domains (e.g., VHH binding domains) that specifically bind to different antigens (e.g., HER2 and EGFR). In certain embodiments, the HER2 binder is multispecific and comprises 2, 3, 4, 5, 6, or VHH domains that specifically bind to different antigens (e.g., HER2 and EGFR).

[0118] In further embodiments, the provided HER2 binder is monospecific and multivalent, or the provided HER2 binder is multispecific and multivalent. In some embodiments, the provided HER2 binder is multivalent and monospecific. In certain embodiments, the HER2 binder is multivalent and multispecific and comprises 2, 3, 4, 5, 6 or more copies of the same HER2-binding VHH domain. In some embodiments, the provided HER2 binder is multivalent and multispecific. In certain embodiments, the HER2 binder is multivalent and multispecific and comprises 2, 3, 4, 5, 6 domains, or VHH domains, that specifically bind to different epitopes of HER2. In some embodiments, the HER2 binder is multivalent and monospecific and comprises two or more copies of different binding domains that specifically bind to different antigens (e.g., HER2 and EGFR). In certain embodiments, the HER2 binder is multispecific and comprises 2, 3, 4, 5, 6 domains, or VHH domains, that specifically bind to different antigens.

[0119] In some embodiments, one or more of the 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 immune regulatory antigen and the antigen is involved in enhancing or reducing a signaling pathway in immune cells. In some embodiments, the second antigen is an immune regulatory antigen and the antigen is involved in enhancing or reducing a signaling pathway in the immune cells.

[0120] In some embodiments, the HER2 binder contains a fusion protein having the following structure: HER2VHH-linker-HER2VHH, wherein the HER2VHH is a HER-2-binding VHH disclosed herein and the linker is a linker provided herein or a linker known in the art.

[0121] In some embodiments, the HER2 binder comprises a fusion protein comprising a HER2-binding VHH domain operably linked to an IgG Fc. In certain embodiments, the fusion protein is bivalent, having two VHH domains that specifically bind to HER2 (e.g., having two HER2-binding VHH domains per molecule and having the structure HER2VHH-linker-HER2VHH-linker-Fc). In some embodiments, the fusion protein is tetravalent (e.g., having four HER2-binding VHH domains per molecule and having the structure HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-Fc) or hexavalent (e.g., having six HER2-binding VHH domains per molecule and having the structure HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-HER2VHH-linker-Fc).

[0122] In some embodiments, the HER2 binder 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 with the heterologous polypeptide sequence can be at any suitable site on the protein and can be an N-terminal, C-terminal, and / or N-terminal / C-terminal fusion(s). In some embodiments, the heterologous polypeptide sequence is fused to the N-terminus of the HER2-binding VHH domain. In some embodiments, the heterologous polypeptide sequence is fused to the C-terminus of the HER2-binding VHH domain. In certain embodiments, the heterologous polypeptide sequence is fused to the N-terminus of the HER2-binding VHH domain and the heterologous polypeptide sequence is fused to the C-terminus of the HER2-binding VHH domain. Such fusion proteins can be produced by any suitable route, such as recombinant expression, chemical ligation, enzymatic ligation (e.g., sortase A), or other methods known in the art.

[0123] In some embodiments, the heterologous amino acid sequence is a toxin. In some embodiments, the heterologous amino acid sequence is a label.

[0124] In some embodiments, the HER2-binding VHH domain is fused (operably linked) to a heterologous polypeptide having biological activity. In some embodiments, the molecule having biological activity is an enzyme, an immunoglobulin, a cytokine, a peptide or protein such as an anti-cancer agent or a fragment or variant thereof. Alternatively, the biologically active component of the fusion protein may be an anti-cancer agent, an NSAID, a steroid, an analgesic, a toxin or other pharmaceutically active agent. Anti-cancer agents include cytotoxic agents or cytostatic agents.

[0125] In some embodiments, the molecule having biological activity in the fusion protein is a therapeutic agent, such as an anti-inflammatory agent (e.g., a steroid or non-steroidal anti-inflammatory agent), an anti-cancer agent, a cytotoxic agent (e.g., a toxin such as cholera toxin, or a radionuclide containing a therapeutic or diagnostic radioactive element), a cytostatic agent or an analgesic.

[0126] In some embodiments, the HER2 binder comprises a fusion protein comprising the HER2-binding VHH domain provided herein fused to another immunoglobulin variable or constant region, or another VHH domain having specific binding affinity for HER2 or another antigen. In some embodiments, the fusion protein comprises a dimer, trimer, tetramer or higher multimer (i.e., pentamer, hexamer, heptamer, octamer, nonamer, decamer or higher) of the VHH-binding domain.

[0127] 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 a genetically engineered fragment thereof, such as Fab, Fc, F(ab')2 (including chemically linked F(ab')2 chains), Fab', scFv (including its multimers, i.e., di-scFv or tri-scFv) or BiTE (bispecific T cell engager). Antibody fragments 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 is of an IgG isotype selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype and IgG4 isotype.

[0128] In some embodiments, the HER2-binding VHH domain is fused to a heterologous amino acid sequence that includes an Fc domain or a portion thereof (such as human Fc). In certain embodiments, the Fc portion increases the half-life and / or production of the polypeptide comprising the HER2-binding domain. In some embodiments, the Fc domain or a portion thereof increases the half-life of the polypeptide comprising the HER2-binding domain. In some embodiments, the multispecific HER2-binding agent further comprises an Fc domain as described herein. In some embodiments, the multispecific HER2-binding agent comprises at least one VHH domain that binds to HER2, at least one additional binding domain that binds to a different epitope or domain of HER2 (e.g., HER2 domain I, II, III, or IV), and an Fc domain. In some embodiments, the multispecific HER2-binding agent comprises at least one VHH domain that binds to HER2, at least one additional binding domain that binds to a different antigen, 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., two or three constant domains that can be used as part of / to form the Fc portion), an Fc portion, and / or one or more antibody moieties, fragments, or domains that can confer one or more effector functions to the polypeptides of the present disclosure and / or the ability to bind to one or more Fc receptors. For example, for this purpose, without limitation hereinafter, one or more additional amino acid sequences may include one or more CH2 and / or CH3 domains of an antibody (e.g., derived from the heavy chain antibody of a conventional humanized antibody); and / or form an Fc region, e.g., derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, or 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 CH2 and / or CH3 domains of the camel are replaced by human CH2 and CH3 domains, and provides an immunoglobulin consisting of two heavy chains each containing a single domain antibody and human CH2 and CH3 domains (but not the CH1 domain), which has effector functions provided by the CH2 and CH3 domains and can function without the presence of a light chain.

[0129] In some embodiments, the multispecific HER2 binder is a bispecific construct comprising at least one HER2-binding VHH domain provided herein 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 the T cell receptor complex. In some embodiments, the surface molecule is an activating T cell antigen expressed on a T cell that can induce activation of the T cell by interaction with an antigen-binding molecule. For example, in some embodiments, the interaction between the antigen-binding molecule and the activating T cell antigen can induce activation of the T cell by initiating the signaling cascade of the T cell receptor complex. In some embodiments, the activating T cell antigen is CD3 or CD2. In some embodiments, the provided bispecific HER2 binder can specifically bind to an activating T cell antigen expressed on a human T cell, such as human CD3 or human CD2. In certain embodiments, the additional binding domain specific for an activating T cell antigen (e.g., CD3 or CD2) is an antibody (e.g., a VHH (single domain antibody) or an antigen-binding fragment of an antibody). In some embodiments, the additional binding domain specific for one or more activating T cell antigens is a VHH domain or an antigen-binding fragment or variant of a VHH domain.

[0130] In some embodiments, the provided HER2 binder is a bispecific antibody T cell engager comprising at least one HER2-binding VHH domain provided herein and an additional binding domain that is an antibody (e.g., a 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, CD2).

[0131] Linker The polypeptide components of the fusion proteins contained in the HER2 binders provided herein can be directly fused (operably linked) via a linker moiety to the other components of the fusion protein. In some embodiments, the linker is a peptide, peptide nucleic acid, or polyamide bond.

[0132] In some embodiments, the HER2 binder contains a protein that is a fusion protein comprising 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 an immobilization linker. Suitable peptide linkers include a plurality of amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker consists mainly of the amino acids glycine and serine and is designated herein as a GS-linker. The GS-linker can be of various lengths, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids in length. 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 containing glycine residues, e.g., 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 where n is 1-5 (SEQ ID NO: 179); (GGGGGS)n where n is 1-4 (SEQ ID NO: 180); GGGGS (SEQ ID NO: 181); GGGGGGGS (SEQ ID NO: 182); GGGGGGGSGGGGGSGGGGGS (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 binder comprises a combination of a GS linker and a glycine linker.

[0133] In some embodiments, the HER2 binder contains a protein that is a fusion protein comprising a HER2-binding VHH domain fused to one or more polypeptide sequences via one or more polypeptide linkers comprising the sequence Gn (where n = 2 to 8). In some embodiments, the linker comprises the sequence (GS)n, (GGS)n, (GGGS)n (SEQ ID NO: 187) or (GGGGS)n (SEQ ID NO: 188) (n = 1 to 16). In some embodiments, the linker comprises the sequence (GGGGS)3 (SEQ ID NO: 189). In some embodiments, the linker comprises the sequence (EGKSSGSGSESKST)n (SEQ ID NO: 190) or (KESGSVSSEQLAQFRSLD)n (SEQ ID NO: 191) (n = 1 to 4). In some embodiments, the linker comprises the sequence GGGSLVPRGSGGGS (SEQ ID NO: 192) or (GAGSAAGSGEF)n (SEQ ID NO: 193) (n = 1 to 5). In some embodiments, the linker comprises the sequence (GAGAGX)n (SEQ ID NO: 194) (where X = A, Y, V or S (SEQ ID NO: 195) and n = 1 to 16). In some embodiments, the linker comprises the sequence (VPGXG)n (where X = A, V, G or S (SEQ ID NO: 196) and n = 1 to 40).

[0134] In some embodiments, the HER2 binder contains a protein that is a fusion protein comprising a HER2-binding VHH domain fused to one or more polypeptide sequences via one or more immobilized polypeptide linkers. In some embodiments, the linker comprises the sequence (APAP)n (SEQ ID NO: 197) (n = 10 to 34). In some embodiments, the linker comprises the sequence (EAAAK)n (SEQ ID NO: 198) (n = 1 to 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).

[0135] In some embodiments, the linker is a cross-linkable linker. In certain embodiments, the linker is PEG, POEGMA or a carbonyl chain.

[0136] Chimeric receptor and genetically modified cell In some embodiments, the present disclosure provides a chimeric antigen receptor (CAR) having an extracellular domain comprising one or more HER2-binding VHH domains provided herein. The CAR constructs provided herein comprise an extracellular domain comprising one or more HER2-binding VHH domains provided herein, a transmembrane domain, and an intracellular signaling region. The one or more HER2-binding VHH domains included in the extracellular antigen-binding unit of the CAR "bind" or "are capable of binding" to HER2 with sufficient affinity such that the CAR is useful for therapies targeting cells or tissues that express HER2.

[0137] A CAR is generally a synthetic receptor that includes an extracellular target / binding portion, binds to one or more signaling domains in a single fusion molecule, and is expressed on the surface of immune effector cells such as T cells. Thus, a CAR combines antigen specificity and T cell activation properties in a single fusion molecule. In some embodiments, the CAR includes a signaling domain of a costimulatory molecule such as CD28 and CD3ζ, providing dual signaling that combines activation and costimulatory signals. In some embodiments, the CAR includes three or more signaling domains.

[0138] In some embodiments, the CAR comprises an antigen-binding domain that includes two HER2-binding VHH domains, i.e., provides a bivalent binding molecule. In certain embodiments, the antigen-binding domain includes two or more identical HER2-binding VHH domains. In certain embodiments, the antigen-binding domain includes two or more different HER2-binding VHH domains. In certain embodiments, the antigen-binding domain includes two or more HER2-binding VHH domains that bind to different epitopes of HER2. In certain embodiments, the antigen-binding domain includes one or more HER2-binding VHH domains and one or more VHH-binding domains that specifically bind to a different antigen.

[0139] In certain embodiments, the CAR further comprises a hinge or spacer region that links the extracellular antigen-binding domain and the transmembrane domain. Using the hinge or spacer region, various lengths and flexibilities of the resulting CAR can be achieved. Examples of hinge or spacer regions that can be used include, but are not limited to, the Fc fragment of an antibody or a fragment or derivative thereof, the hinge region of an antibody or a fragment or derivative thereof, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence (e.g., a peptide sequence), or a combination thereof. Other hinge regions or spacer regions will be apparent to those skilled in the art and may be used. In some embodiments, the hinge is an IgG4 hinge or a CD8A hinge. In some embodiments, the spacer and transmembrane domain are a hinge and transmembrane domain derived from CD8.

[0140] The transmembrane domain of the CAR provided herein is typically capable of traversing the cell membrane, or being capable of traversing or passing through the cell membrane, and is directly or indirectly (e.g., via a spacer such as an immunoglobulin hinge sequence) linked to the endoplasmic reticulum portion containing the extracellular antigen-binding domain and the intracellular signaling domain. In some embodiments, the transmembrane domain of the CAR is the transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. In certain embodiments, the transmembrane domain includes the CD3ζ domain or the CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art and can be used in connection with the CAR embodiments provided herein.

[0141] The intracellular signaling region of the CAR provided herein includes one or more intracellular signaling domains that transmit signals to T cells upon engagement of the antigen-binding domain of the CAR, e.g., upon antigen binding. In some embodiments, the intracellular region is an ITAM signaling domain or contains an intracellular signaling domain that contains an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, signaling domains obtained from either the ζ chain of the T cell receptor complex or a homolog thereof (e.g., η chain, FcRIγ chain and β chain, MB1 (Iga) chain and B29 (Ig) chain), human CD3ζ chain, CD3 polypeptides (delta, gamma or epsilon), syk family tyrosine kinases (e.g., Syk or ZAP70), src family tyrosine kinases (e.g., Lck, Fyn or Lyn) and other molecules involved in T cell transduction (e.g., CD2, CD5, OX40 and CD28). In certain embodiments, the intracellular signaling region contains an intracellular signaling domain obtained from the human CD3ζ chain.

[0142] In some embodiments, the endoplasmic reticulum portion of the CAR contains the CD3ζ signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from CD28 or 4-1BB and retains the activity of T cell co-stimulatory signaling.

[0143] In some embodiments, the present disclosure provides an isolated nucleic acid construct comprising at least one nucleic acid encoding the CAR provided herein. In some embodiments, the construct is an expression vector for the expression of the CAR in cells. In some embodiments, the expression vector is a viral vector (e.g., a lentiviral vector). Viral vector technology is well known in the art and is described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013. Many viral systems have been developed for gene transfer into mammalian cells and are known in the art.

[0144] In some embodiments, the present disclosure provides an isolated cell or cell population comprising one or more nucleic acid constructs described herein. Also provided are isolated cells or cell populations genetically modified to express the CAR provided herein. Thus, provided herein are genetically engineered cells (e.g., stably expressing cells) comprising the CAR provided herein. In some embodiments, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells. In some cases, the cells are T cells such as CD4 and / or CD8 T cells. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells can be isolated from a patient (also referred to as primary T cells) for genetic engineering (e.g., transfection or transduction) with the CAR nucleic acid construct.

[0145] In certain embodiments, primary T cells can be purified ex vivo (either CD4 cells or CD8 cells, or both) and stimulated with a TCR / CD28 agonist (e.g., anti-CD3 / anti-CD28 coated beads). After a 2 - 3 day activation step, a recombinant expression vector encoding the CAR can be stably introduced into the primary T cells by standard lentiviral and retroviral transduction protocols or plasmid electroporation strategies. The cells can be monitored for CAR expression by flow cytometry, for example, using antibodies that cross-react with an anti-epitope tag or the native parental molecule. T cells expressing the CAR can be enriched by sorting with anti-epitope tag antibodies or can be enriched for high or low expression depending on the application.

[0146] CAR gene-modified T cells can be assayed for appropriate function in a variety of ways. In some cases, the function of the genetically engineered T cells can be evaluated using cytotoxicity, proliferation, or cytokine assays (e.g., expression of IFN-γ) in vitro. Examples of standard endpoints are the lysis rate of tumor lines, the proliferative capacity of the gene-modified T cells, or the expression of IFN-γ protein in the culture supernatant. In some cases, the ability to stimulate T cell activation upon CAR stimulation (e.g., via an antigen) can be evaluated by monitoring the expression, proliferation, and / or cytokine production of activation markers such as CD69, CD44, or CD62L.

[0147] II. Nucleic Acids In some embodiments, the disclosure provides nucleic acids encoding the HER2 binders provided herein.

[0148] In some embodiments, the disclosure provides the following: (a) the VHH amino acid sequences disclosed in Table 2; or (b) VHH sequences selected from SEQ ID NOs: 1 - 81 and 205, Provided is an isolated nucleic acid encoding a HER2-binding VHH domain comprising

[0149] In some embodiments, the disclosure provides: (a) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VHH sequences disclosed in Table 2; or (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the VHH sequences selected from SEQ ID NOs: 1-81 and 205, and provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising

[0150] In some embodiments, the disclosure provides: (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 sequences disclosed in Table 2; or (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 the reference VHH sequences selected from SEQ ID NOs: 1-81 and 205, and provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising

[0151] In some embodiments, the disclosure provides: (a) Complementary determining region (CDR) 1 comprising an amino acid sequence selected from SEQ ID NOs: 82-101; CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 102-123; and CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 124-157 and 206; or (b) CDR1, CDR2 and CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3 contained in the VHH sequences disclosed in Table 2, and provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising

[0152] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a HER2-binding VHH domain comprising: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 101; CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and CDR3 comprising the amino acid sequence of SEQ ID NO: 135 or 136; (b) CDR1 comprising the amino acid sequence of SEQ ID NO: 101; CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and CDR3 comprising the amino acid sequence of SEQ ID NO: 135; (c) CDR1 comprising the amino acid sequence of SEQ ID NO: 101; CDR2 comprising the amino acid sequence of SEQ ID NO: 111; and CDR3 comprising the amino acid sequence of SEQ ID NO: 136; (d) CDR1 comprising the amino acid sequence of SEQ ID NO: 82; CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and CDR3 comprising the amino acid sequence of SEQ ID NO: 124, 145, 146 or 147; (e) CDR1 comprising the amino acid sequence of SEQ ID NO: 83; CDR2 comprising the amino acid sequence of SEQ ID NO: 103; and CDR3 comprising the amino acid sequence of SEQ ID NO: 125; (f) CDR1 comprising the amino acid sequence of SEQ ID NO: 84; CDR2 comprising the amino acid sequence of SEQ ID NO: 104; and CDR3 comprising the amino acid sequence of SEQ ID NO: 126; (g) CDR1 comprising the amino acid sequence of SEQ ID NO: 85; CDR2 comprising the amino acid sequence of SEQ ID NO: 105; and CDR3 comprising the amino acid sequence of SEQ ID NO: 127; (h) CDR1 comprising the amino acid sequence of SEQ ID NO: 86; CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and CDR3 comprising the amino acid sequence of SEQ ID NO: 128, 130, 134 or 155; (i) CDR1 comprising the amino acid sequence of SEQ ID NO: 87; CDR2 comprising an amino acid sequence selected from SEQ ID NO: 117; and CDR3 comprising the amino acid sequence of SEQ ID NO: 129; (j) CDR1 comprising the amino acid sequence of SEQ ID NO: 88; CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and CDR3 comprising an amino acid sequence selected from SEQ ID NO: 130; (k) CDR1 containing the amino acid sequence of SEQ ID NO: 89; CDR2 containing the amino acid sequence of SEQ ID NO: 108 or 112; and CDR3 containing the amino acid sequence of SEQ ID NO: 131 or 157; (l) CDR1 containing the amino acid sequence of SEQ ID NO: 89; CDR2 containing the amino acid sequence of SEQ ID NO: 108; and CDR3 containing the amino acid sequence of SEQ ID NO: 131; (m) CDR1 containing the amino acid sequence of SEQ ID NO: 89; CDR2 containing the amino acid sequence of SEQ ID NO: 108 or 112; and CDR3 containing the amino acid sequence of SEQ ID NO: 156; (n) CDR1 containing the amino acid sequence of SEQ ID NO: 89; CDR2 containing the amino acid sequence of SEQ ID NO: 108 or 112; and CDR3 containing the amino acid sequence of SEQ ID NO: 157; (o) CDR1 containing the amino acid sequence of SEQ ID NO: 90; CDR2 containing the amino acid sequence of SEQ ID NO: 109; and CDR3 containing the amino acid sequence of SEQ ID NO: 132; (p) CDR1 containing the amino acid sequence of SEQ ID NO: 91; CDR2 containing the amino acid sequence of SEQ ID NO: 119; and CDR3 containing the amino acid sequence of SEQ ID NO: 137; (q) CDR1 containing the amino acid sequence of SEQ ID NO: 92; CDR2 containing the amino acid sequence of SEQ ID NO: 120; and CDR3 containing the amino acid sequence of SEQ ID NO: 138; (r) CDR1 containing the amino acid sequence of SEQ ID NO: 93; CDR2 containing the amino acid sequence of SEQ ID NO: 113 or 114; and CDR3 containing the amino acid sequence of SEQ ID NO: 139; (s) CDR1 containing the amino acid sequence of SEQ ID NO: 93; CDR2 containing the amino acid sequence of SEQ ID NO: 114; and CDR3 containing the amino acid sequence of SEQ ID NO: 206; (t) CDR1 containing the amino acid sequence of SEQ ID NO: 94; CDR2 containing the amino acid sequence of SEQ ID NO: 115; and CDR3 containing the amino acid sequence of SEQ ID NO: 140 or 149; (u) CDR1 containing the amino acid sequence of SEQ ID NO: 95; CDR2 containing the amino acid sequence of SEQ ID NO: 121; and CDR3 containing the amino acid sequence of SEQ ID NO: 141; (v) CDR1 comprising the amino acid sequence of SEQ ID NO: 96; CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and CDR3 comprising the amino acid sequence of SEQ ID NO: 142; (w) CDR1 comprising the amino acid sequence of SEQ ID NO: 97; CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and CDR3 comprising the amino acid sequence of SEQ ID NO: 150; (x) CDR1 comprising the amino acid sequence of SEQ ID NO: 98; CDR2 comprising the amino acid sequence of SEQ ID NO: 107; and CDR3 comprising the amino acid sequence of SEQ ID NO: 144, 151, 152 or 153; (y) CDR1 comprising the amino acid sequence of SEQ ID NO: 99; CDR2 comprising the amino acid sequence of SEQ ID NO: 109; and CDR3 comprising the amino acid sequence of SEQ ID NO: 132 or 148; or (z) CDR1 comprising the amino acid sequence of SEQ ID NO: 100; CDR2 comprising the amino acid sequence of SEQ ID NO: 110; and CDR3 comprising the amino acid sequence of SEQ ID NO: 133 or 154; or An isolated nucleic acid encoding a HER2-binding VHH domain comprising: (ab) CDR1 comprising SEQ ID NO: 97; CDR2 comprising SEQ ID NO: 123; and CDR3 comprising SEQ ID NO: 143; or (ab) CDR1 comprising SEQ ID NO: 101; CDR2 comprising SEQ ID NO: 111; and CDR3 comprising SEQ ID NO: 136.

[0153] In some embodiments, the provided nucleic acid sequence encodes a fusion protein provided herein. In some embodiments, the nucleic acid sequence further comprises one or more regulatory sequences (e.g., a transcriptional promoter, operator or enhancer, or an mRNA ribosome binding site) appropriate for replication and / or expression of the encoded protein in a selected host cell. In some embodiments, the nucleic acid further encodes a leader sequence that directs secretion of the protein, and this leader sequence is typically cleaved so that it is not present within the secreted protein. The leader sequence may be a native heavy chain (or VHH) leader sequence or a heterologous leader sequence. The nucleic acid can be constructed using recombinant DNA techniques and / or chemical synthesis methods known in the art.

[0154] In some embodiments, the present disclosure provides a vector comprising a nucleic acid encoding a HER2-binding protein provided herein. Vectors generally include a selectable marker and an origin of replication for propagation in a host. Vectors provided include, but are not limited to, DNA vectors, phage vectors, viral vectors, and retroviral vectors.

[0155] In some embodiments, the present disclosure provides an expression vector comprising a nucleic acid encoding a HER2-binding protein provided herein. Generally, an expression vector includes a nucleic acid encoding a HER2-binding protein operably linked to transcriptional or translational regulatory sequences appropriate for the host cell in which the protein is to be expressed, such as those derived from mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include a transcriptional promoter, operator or enhancer, an mRNA ribosome binding site, and appropriate sequences that control transcription and translation.

[0156] To express the protein component of the provided HER2 binder, various host-vector expression systems can be used. These systems include 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 lines infected with virus expression vectors (e.g., baculovirus); plant cell lines transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or plant cell lines transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see, for example, Babe et al., Canadian Journal of Communication (2000) 25(1):19); or animal cell lines infected with virus expression vectors (e.g., vaccinia virus, adenovirus). 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 useful for recombinant protein production include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS7 cells); HEK cells; 293 cells (e.g., 293-6E cells); CHO cells (e.g., CHO-S cells, DG44.Lec13 CHO cells and FUT8 CHO cells); PER.C6 cells (Crucell); NSO cells W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for recombinant expression of HER2 binding polypeptides in bacteria, yeast and other invertebrates are known in the art. In some embodiments, the protein comprising the HER2 binding VHH domain is expressed in yeast (e.g., P. pastoris).For example, see U.S. Publ. US 2006 / 0270045 A1. Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected to provide specific capabilities for processing the expressed protein or specific post-translational modifications that result in useful protein activities. Such modifications to 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 may also be important for accurate insertion, folding, and / or function. Various host cells (e.g., CHO, HeLa, MDCK, 293, WI38, etc.) possess specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure proper modification and processing of the introduced foreign protein. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the polypeptide.

[0157] In some embodiments, the present disclosure provides a host cell comprising the nucleic acid described herein. In some embodiments, the present disclosure provides a host cell comprising a vector containing the nucleic acid disclosed herein. In some embodiments, the present disclosure provides a host cell comprising the 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 the HER2-binding polypeptide disclosed herein.

[0158] Introduction of a nucleic acid (e.g., a vector) into a desired host cell can be achieved 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. Exemplification of non-limiting methods is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid can be transiently or stably transfected into the desired host cell according to any suitable method.

[0159] III. Expression and production of polypeptides The protein component of the HER2 binder provided herein can be routinely produced by conventional recombinant techniques and / or peptide synthesis methods known in the art.

[0160] In some embodiments, the present disclosure provides a recombinant method for producing a protein comprising the HER2-binding VHH domain provided herein. In some embodiments, the method comprises culturing a host cell comprising a nucleic acid encoding a HER2-binding protein under conditions in which the protein is produced. In some embodiments, the method further comprises recovering the HER2-binding protein produced by the host cell.

[0161] In some embodiments, the HER2-binding protein is expressed in prokaryotic cells (e.g., 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., yeast), plant cells, insect cells, or mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. In some embodiments, the present 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.

[0162] In some embodiments, the HER2-binding VHH domain is produced in a cell-free system. Non-limiting examples of cell-free systems are described, for example, in 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.

[0163] As an alternative to recombinant DNA technology, the proteins and / or nucleic acids encoding such proteins included in the HER2 binders provided herein can each be produced by automated polypeptide synthesis or nucleic acid synthesis. In some embodiments, the protein component of the HER2 binders provided herein is synthesized according to conventional techniques in solution or on a solid support. A variety of automated synthesizers are commercially available and can be used according to known protocols such as those described in Stewart and Young, Solid Phase Peptide Synthesis, second 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, R.B., "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 component of the HER2 binders of the present disclosure can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer such as the Model 433 A of Applied Biosystems Inc. The purity of any given polypeptide produced by automated peptide synthesis or 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 of skill in the art.

[0164] Proteins can be recovered or purified from recombinant cell cultures and other solutions by methods known to those of skill in the art, such as 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 therapeutic use, nucleic acid constructs, e.g., in the form of recombinant vectors, can be purified by techniques known in the art, such as column chromatography, as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0165] In certain embodiments, the proteins used in the therapeutic methods of the present disclosure are intended to be modifiable to improve their therapeutic efficacy. Such modifications to therapeutic compounds can be used, for example, to reduce toxicity, increase circulation time, or alter biodistribution. For example, the toxicity of a very important therapeutic compound can be significantly reduced by combination with various pharmaceutical carrier vehicles that modify its biological distribution.

[0166] As a strategy for improving drug efficacy, the use of water-soluble polymers is available. Various water-soluble polymers have been shown to change biodistribution, improve the mode of cellular uptake, change the permeability to pass through physiological barriers, and change the clearance rate from the body. To obtain a targeting effect or a sustained-release effect, water-soluble polymers have been synthesized that contain the drug moiety as a terminal group, as a part of the backbone, or as a pendant group on the polymer chain. For example, polyethylene glycol (PEG) has been widely used as a drug carrier because of its high biocompatibility and ease of modification. It has been shown that by conjugating PEG to various drugs, the residence time is improved and the toxicity is reduced.

[0167] IV. Labeled HER2-binding agent A. Conjugate In some embodiments, the HER2-binding agents provided herein contain a protein comprising a HER2-binding VHH domain that is conjugated to one or more additional moieties via covalent or non-covalent interactions to form a HER2-binding agent conjugate. In some embodiments, the HER2-binding agent conjugate contains 1, 2, 3, 4, 5 or more additional moieties. In some embodiments, the conjugate moieties of the HER2-binding agent conjugate are the same. In some embodiments, two or more conjugate moieties of the HER2-binding agent conjugate are different.

[0168] The portion of the HER2-binding agent conjugate provided can be conjugated to a protein comprising one or more VHH domains that specifically bind to HER2 using any of a variety of molecular biological or chemical conjugation and ligation methods known in the art. For example, conjugates of a protein comprising at least one HER2-binding VHH domain provided herein and a cytotoxic agent can be prepared routinely 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 (e.g., dimethyl adipimidate hydrochloride), active esters (e.g., disuccinimidyl substrates), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate) and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0169] In some embodiments, a linker (e.g., a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that assists a conjugation reaction) is used to link or conjugate a HER2-binding protein to a moiety (e.g., an effector moiety such as a drug moiety or a radionuclide). In some embodiments, one or more portions of the provided HER2-binding agent conjugate are bound, by a linker comprising an amino acid residue, to a protein comprising one or more VHH domains that specifically bind to HER2. 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. Amino acid residues that can constitute the amino acid linker component can include naturally occurring ones, minor amino acids, and non-naturally occurring amino acid analogs (e.g., citrulline). In some embodiments, the amino acid linker component of the HER2-binding agent conjugate provided herein is designed for selectivity against enzymatic cleavage by specific enzymes (e.g., tumor-associated proteases, cathepsins B, C, and D, and plasmin protease). Further exemplary linker components of the provided HER2-binding agent include, but are not limited to, 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzylcarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio)-pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (“SMCC”), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”).

[0170] In certain embodiments, the linker can be composed of one or more linker components. For the covalent attachment of the protein and the agent moiety, the linker typically has two reactive functional groups, i.e., is bivalent in the sense of reactivity. Bivalent linker reagents useful for attaching two or more functional or biologically active components such as peptides, nucleic acids, agents, toxins, antibodies, haptens and reporter groups, and methods of their use in conjugation are known in the art.

[0171] In some embodiments, the protein of the HER2 binding agent conjugate comprises at least one HER2 binding VHH domain, and said protein is conjugated to an agent moiety. In certain embodiments, the protein of the HER2 binding agent conjugate is conjugated to about 1 to about 20 agent moieties. In some embodiments, the protein of the HER2 binding agent conjugate comprises at least one HER2 binding VHH domain, and said protein is conjugated to about 1 to about 20 agent moieties via a linker (L). In some embodiments, the HER2 binding agent conjugate comprises the following components: (VHH domain), (L)q and (moiety)m, where the VHH domain is any of the HER2 binding VHH domains provided herein; L is a linker for linking the protein of the HER2 binding agent to the moiety; m is at least 1; q is 0 or more; and the resulting HER2 binding agent conjugate binds to HER2. In certain embodiments, m is from 1 to 4 and q is from 0 to 8.

[0172] In some embodiments, the present disclosure provides a HER2-binding agent conjugate comprising at least one HER2-binding VHH domain provided herein, wherein the HER2-binding agent conjugate is conjugated / bound to a therapeutic agent such as a cytotoxic agent or a cell growth inhibitor. In some embodiments, the therapeutic agent is a targeting moiety, a small molecule drug (a non-polypeptide drug less than 500 Daltons), a toxin (e.g., an enzymatically active toxin or a fragment thereof of bacterial, fungal, plant or animal origin), a cell growth inhibitor, a cytotoxic agent, an immunosuppressant, a radioisotope (i.e., a radioactive conjugate), a prodrug activating enzyme or an agent that increases the biological half-life.

[0173] 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 provides cytotoxic, cell growth inhibitory, or otherwise 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 radioisotope (i.e., a radioactive conjugate).

[0174] 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 a cytotoxic and / or cell growth inhibitory effect by tubulin binding, DNA binding or topoisomerase inhibition. In some embodiments, the toxin is a bacterial toxin (e.g., diphtheria toxin), a plant toxin (e.g., ricin), a small molecule toxin (e.g., geldanamycin), a maytansinoid or a calicheamicin.

[0175] In some embodiments, the HER2-binding agent conjugate comprises at least one HER2-binding VHH domain conjugated to a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is selected from daunomycin, doxorubicin, methotrexate, and vindesine.

[0176] In some embodiments, the HER2-binding antibody-drug conjugate of the present disclosure provides targeted delivery of its conjugated therapeutic agent to a tumor. In some cases, this leads to targeted killing of tumor cells. In some embodiments, the HER2-binding antibody-drug conjugate is internalized within cells. In some embodiments, the conjugated therapeutic agent of the ADC has intracellular activity. In certain embodiments, the HER2-binding agent conjugate is internalized and the therapeutic agent is a cytotoxin that inhibits protein synthesis in the cell. In certain embodiments, the HER2-binding agent conjugate is internalized and the therapeutic agent is a cytotoxin that induces cytotoxic and cytostatic effects by a mechanism selected from tubulin binding, DNA binding, and topoisomerase inhibition. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating 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 variants thereof.

[0177] In some embodiments, the HER2 binder contains a HER2-binding protein directly or indirectly conjugated to a label to facilitate the treatment and / or detection of bound or unbound HER2 binders. Suitable labeling substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent substances, luminescent substances, imaging agents, metal ions, and radioactive substances. Exemplary suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; exemplary suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; exemplary suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent substance is luminol; and exemplary suitable radioactive substances include the following: 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, 89 Zr, 67 Ga, 68 Ga, 64 Cu and 52 Mn. For example, in some embodiments, the label is 3 H, 14 C, 32 P, 35 S, 123 I, 125 I, 131 I and other radiopaque substances or radioisotopes. In some embodiments, the label is 99 Tc or 123 I and other radioactive atoms for scintigraphic studies, or 89 Zr, 123 I, 131 I,111 In, 19 F, 13 C, 15 N, 17 O, gadolinium, manganese, or iron, etc., are spin labels for nuclear magnetic resonance (NMR / MRI) imaging.

[0178] In certain embodiments, the label is a radionuclide. In some embodiments, the radionuclide is a radioactive halogen isotope. In certain embodiments, the radionuclide is 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 At, and is a radioactive halogen isotope selected from. In certain embodiments, the radionuclide is 131 I, 211 At, 125 I or 124 I. In certain embodiments, the radionuclide is a radioactive isotope. In some embodiments, the radionuclide is the following: 144 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 47 Sc, 52 Mn, 64 Cu, 67 Cu, 67 Ga, 89Zr, 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 radioisotopes of members selected from Th. In certain embodiments, the radionuclide is 177 Lu, 131 I, 211 At or 225 Ac.

[0179] In some embodiments, the HER2 binder contains a HER2-binding protein conjugated to a label via a chelating moiety. In some embodiments, the chelating moiety is covalently attached to the protein via a lysine residue. In some embodiments, the label forms a complex with a metal, and the complex is chelated by the chelating moiety.

[0180] The HER2 binder conjugates provided herein can be prepared by any of a variety of methods, such as organic chemical reactions, conditions, and reagents known in the art. In some embodiments, the method comprises the following steps: (1) reacting a nucleophile of a protein comprising the HER2-binding VHH domain provided herein with a divalent linker reagent to form a HER2-binding VHH-L via a covalent bond, and then reacting with T of the therapeutic agent moiety; and (2) reacting a nucleophile of the therapeutic agent moiety with a divalent linker reagent to form a T-L via a covalent bond, and then reacting with a nucleophile of a protein comprising the HER2-binding VHH domain.

[0181] Nucleophilic groups on the protein include, but are not limited to, the following: (a) the N-terminal amine group, (b) side-chain amine groups (e.g., lysine), (c) side-chain thiol groups (e.g., cysteine), and (d) the hydroxyl or amino groups of sugars (when the protein is glycosylated). Amine groups, thiol groups, and hydroxyl groups are nucleophilic and can react with electrophilic groups on the linker moiety and linker reagent (e.g., (a) active esters such as NHS ester, HOBt ester, haloformate, and acid halide; (b) alkyl and benzyl halides such as haloacetamide; (c) aldehyde, ketone, carboxyl, and maleimide groups) to form covalent bonds. Additional nucleophilic groups can be introduced into the protein by reacting lysine with 2-iminothiolane (Traut reagent) to convert the amine to a thiol. Reactive thiol groups can be introduced into the protein (or a fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., producing a mutant protein containing one or more non-natural cysteine amino acid residues).

[0182] Conjugates, such as HER2 binding agent conjugates, can also be produced by introducing an electrophilic moiety through modification of a protein, such as the HER2 binding proteins disclosed herein, which moiety can react with a nucleophilic substituent on a linker reagent or therapeutic agent. The sugars of glycosylated proteins can be oxidized, for example, with a periodate oxidizing reagent to form aldehyde and ketone groups and reacted with the amine groups of linker reagents and therapeutic agents. The Schiff base groups of the resulting imines can form stable bonds or can be reduced, for example, with a borohydride reagent to form stable amine bonds. In certain embodiments, reacting the carbohydrate portion of a glycosylated protein with either galactose oxidase or sodium metaperiodate results in the formation of carbonyl (aldehyde and ketone) groups in the protein, which carbonyl groups can react with suitable groups of the therapeutic agent moiety (Hermanson, Bioconjugate Techniques). In certain embodiments, a protein containing an N-terminal serine or threonine residue can react with sodium metaperiodate, resulting in the formation of an aldehyde in place of the first amino acid. Such aldehydes can be reacted with a therapeutic agent moiety or linker nucleophile.

[0183] Similarly, nucleophilic groups on the therapeutic agent moiety include amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazide groups that can react with electrophilic groups on a linker moiety to form a covalent bond, as well as linker reagents including, but not limited to: (a) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (b) alkyl and benzyl halides such as haloacetamides; and (c) aldehyde, ketone, carboxyl, and maleimide groups.

[0184] The HER2-binding agent conjugates provided herein can be manufactured using any method known in the art. See, for example, International Publication Nos. WO2009 / 067800 and WO2011 / 133886 and U.S. Publication No. 2014322129, each of which is incorporated herein by reference in its entirety.

[0185] B. HER2-Binding Agents for Targeted Radiotherapy In some embodiments, the present disclosure provides HER2-binding targeted radiotherapy agents for therapeutic and / or diagnostic purposes that include the HER2-binding agents provided herein. Such targeted agents can include, in addition to the HER2-binding VHH domains described herein, a radioactively labeled prosthetic agent / group and optionally a chelating agent (either macrocyclic or acyclic).

[0186] Such agents are described herein as “radiotherapy” agents, but it should be noted that this term is intended to cover agents suitable for both therapeutic and diagnostic purposes. Such targeted radiotherapy agents can include, in addition to a HER2-binding agent (i.e., the targeting moiety) as described herein, a radioactively labeled prosthetic agent / group and optionally a chelating agent (macrocyclic or acyclic). The radioactively labeled prosthetic agent is generally a compound or group that includes a radioactive label, a charged group (CG), and a macromolecular conjugate moiety (MMCM) suitable for binding to the HER2-binding agent. Each of these components can be linked, optionally, to one or more cleavable (or non-cleavable) linkers.

[0187] The prosthetic agents that can be conjugated to the HER2-binding agents disclosed herein to provide targeted radiotherapy agents are not particularly limited. Generally, any prosthetic agent that includes a radioactive label (wherein the prosthetic agent can be suitably conjugated to the HER2-binding agents provided herein) can be used in accordance with the present disclosure.

[0188] In some embodiments, the radiolabel can be a radioactive halogen. Radioactive halogens can include, for example, alpha emitters, beta emitters, or Auger electron emitters. These can include radioactive halogens suitable for PET, SPECT, or intraoperative imaging. See, for example, U.S. Patent No. 5,302,700 (incorporated herein by reference in its entirety). The radioactive halogen can be 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At, and can be selected from the group consisting of. In some embodiments, the radioactive halogen is 18 F. In some embodiments, the radioactive halogen is 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At, and can be selected from the group consisting of. In some embodiments, the radioactive halogen is 123 I, 124 I, 125 I, 131 I and 211 At, and can be selected from the group consisting of. In some embodiments, the radioactive halogen is 123 I, 124 I, 125 I and 131 I, and can be selected from. In some embodiments, the radioactive halogen is 211 At.

[0189] In some embodiments, the radiolabel is a metallic radionuclide. Metallic radionuclides that can form complexes within a prosthetic agent (e.g., within a chelating agent as mentioned above) include, but are not limited to, the following: 64 Cu, 67 Cu, 67 Ga, 68Ga, 89 Zr, 90 Y, 111 In, 177 Lu, 212 Pb, 212 Bi, 213 Bi and 225 Ac.

[0190] The charged group (CG) is generally a group that is charged under the physiological conditions of the intracellular environment. In some embodiments, the charged group (CG) includes a guanidine, PO3H group or SO3H group. In some embodiments, the CG is a guanidino-alkyl group containing one or more carbons. In some embodiments, the CG is a guanidino-hydrophilic group (including, for example, groups containing an amino group or a hydroxyl group), and / or an alkyloxycarbonylguanidine group. In some embodiments, the CG includes one or more charged D-amino acids, such as arginine, glutamic acid, aspartic acid, lysine and / or phosphono / sulfophenylalanine. In some embodiments, the CG includes a hydrophilic carbohydrate moiety. The compound may, in some embodiments, include one, two or three CG moieties (and, optionally, corresponding linker groups) to increase intracellular capture in cancer cells. The prosthetic groups provided herein can include one CG or may include two or more CGs. When one or more CGs are associated with a given prosthetic group, each such CG may be the same or different.

[0191] The MMCM is a moiety that can bind to the HER2 binder provided herein. In some embodiments, the MMCM is an active ester. An active ester is defined herein as an ester that can conjugate with a group (e.g., an amine group) present on the HER2 binder under mild conditions, i.e., conditions that do not lose the biological function of the HER2 binder. Examples of such MMCM groups include, but are not limited to, N-hydroxysuccinimide (NHS) or tetrafluorophenol (TFP) esters, pentafluorophenol (PFP), paranitrophenol (PNP), isothiocyanate groups, or maleimide groups. Such MMCMs generally result in random (non-site-specific) labeling of the amine groups on the HER2 binder. In some embodiments, the MMCM is, for example, the tripeptide GGG, whereby site-specific conjugation is effected using, for example, the enzyme sortase, resulting in conjugation to only one moiety (either the N-terminus or the C-terminus of the HER2 binder). Enzymes that can be used to obtain appropriate conjugation include, for example, transglutaminase, lipoic acid ligase, farnesyl transferase, and many others including those disclosed; for example, as described 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, each of which is incorporated herein by reference in its entirety. Prosthetic agents are shown as those containing MMCM, and targeted radiotherapy agents are generally described as those containing a prosthetic agent bound to a HER2 binder, and it is understood that the targeted radiotherapy agents usually do not contain intact MMCM, but rather contain moieties resulting from the coordination of MMCM to the HER2 binder.

[0192] To obtain the targeted radiation therapy agents encompassed by the present disclosure, specific non-limiting prosthetic groups conjugated to HER2 binders include, for example, those of Formula 1, as well as derivatives and variants thereof.

Chemical formula

[0193] Certain of said Class I compounds are generally disclosed in International Publication WO2018 / 178936 (Duke University), which is hereby incorporated by reference in its entirety. Generally, such a prosthetic agent can include an MMCM for binding a prosthetic compound / group to a HER2 binder, a radioactive halogen (Y) as described above; and a homo (X=CH) or hetero (X=N) aromatic ring bonded to one or more CGs. Each of these components can also be bonded to the aromatic ring via a linker (L1, L2, L3), or directly bonded to the aromatic ring (i.e., when L1 and / or L2 and / or L3 is a bond). Each of these components shown in Formula 1 will be described in further detail below.

[0194] The charged group (CG) is usually present in the prosthetic group of formula 1 (i.e., m is 1 or more). Usually, m is 1, but one or more CGs can be attached to the ring such that m = 2, m = 3, and m can be 4 (when X = CH). When one or more CGs are attached to the ring, each such CG (and the corresponding L2) can be the same or different. In certain embodiments, as mentioned below (as shown in formula 2), another moiety can be attached to the ring of formula 1, and when such an additional moiety is charged, m can be 0 (i.e., the additional moiety can effectively function as a "charged group" in some embodiments). The CG can be directly attached to the aromatic ring of formula 1 (L2 = direct bond), or can be attached to the aromatic ring via 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 methylene), a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, a PEG chain containing at least three oxygen atoms, or any of the foregoing including a brush border enzyme-cleavable peptide such as GK, GY, or GFK. It should be noted that in certain embodiments, when CG is guanidine and L2 is an unsubstituted alkyl chain, the unsubstituted alkyl chain contains two or more carbon atoms.

[0195] In some embodiments, L2, which is a metabolizable spacer or cleavable linker (e.g., a brush border enzyme-cleavable linker), is disposed between the CG and the aromatic ring of formula 1. In these formulations, the CG moiety is cleaved in the kidney, the charge is removed, and the radionuclide (neutral or low charge at this point) is excluded from the renal tubular cells of the kidney and then rapidly excreted in the urine, thus avoiding an increase in radioactivity uptake and retention in the kidney. Brush border enzyme-cleavable linkers have been used previously with radioactive substances, but have not been used in such a way as to create a "charge switch" where the labeled reagent is retained in the tumor due to being charged, but is excluded from the kidney due to the disappearance of the charge.

[0196] Examples of such linkers include: a linker sequence targeting meprin β, a metalloprotease expressed in the brush border membrane of the kidney (Jodal et al., PLoS One (2015) 10(4):e0123443); a C-terminal lysine linked to an antibody fragment via the ε-amino group of lysine or C-terminal (N(ε)-amino-1,6-hexane-bis-vinylsulfone) lysine that utilizes the lysine-specific carboxypeptidase activity of a brush border membrane enzyme of the kidney to cleave a radiolabeled peptide linker before uptake into proximal tubular cells, showing a decrease in uptake into the kidney (Li et al., Bioconjugate Chem:985-95); L-tyrosine O-methyl, L-asparagine, L-glutamine, N-Boc-L-lysine (Akizawa et al., Bioconjugate Chem. (2013) 24:291-9); and glycyl-lysine (Arano et al., Cancer Res. (1999) 59:128-34); each of which is incorporated herein by reference in its entirety.

[0197] MMCM can also be directly attached to the aromatic ring of Formula 1 (L1 = direct bond), or can be attached to the aromatic ring via a linker (L1). L1 can be a spacer, for example, a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain or a short chain polyethylene glycol (PEG) chain (1 to 10 ethylene glycol units).

[0198] The positions of these three moieties (-L1-MMCM, -L2-CG and -L3-Y) on the aromatic ring of Formula 1 can vary. When X is CH, these three moieties can be located at any position on the aromatic ring. In some such embodiments, the -L2-CG and -L3-Y moieties are located at the 3-position and 4-position (or the 4-position and 3-position, respectively) relative to the -L1-MMCM moiety (position 1). In some such embodiments, the -L2-CG and -L3-Y moieties are located at the 3-position and 5-position relative to the -L1-MMCM moiety, such that the aromatic ring includes reference moieties at positions 1, 3 and 5. When X is N, these three moieties can be located at any of the remaining five positions of the ring, including, but not limited to, positions 2, 4 and 6 of the ring.

[0199] In certain embodiments, X is CH; L3 is a bond (i.e., the radioactive halogen Y is directly attached to the phenyl ring); m is 1; L2 is CH2, and CG is Z-guanidine (L2-CG is an alkylguanidine, or it may be described that L2 is a bond and CG is an alkyl (e.g., methyl) guanidine); L1 is a bond, and MMCM is a moiety suitable for reacting with an amine on a HER2 binder (e.g., an NHS ester) (forming an amide bond in the resulting targeted radiotherapeutic agent).

[0200] Certain prosthetic compounds within the scope of Formula 1 that can form a complex with a HER2 binder to provide the targeted radiotherapeutic agents provided herein include compounds of Formula 1A, as well as 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 the three moieties (-L1-MMCM, -L3-Y and -CH2-CG) are present at positions 1, 3 and 5 of the aromatic ring. [Chemical Formula] (Formula 1A: General structure of compounds of subclass IA type)

[0201] Furthermore, to obtain the targeted radiation therapy agents encompassed by the present disclosure, non-limiting prosthetic groups that can be coordinated with a HER2 binder include, for example, compounds having the general structure of Formula 2 shown below (referred to as "Class II compounds"): MC-Cm-L4-Cm-T (Formula 2: General structure of Class II compounds)

[0202] Certain of said Class II compounds are generally disclosed in International Publication WO2018 / 178936 (Duke University), which is hereby incorporated by reference in its entirety. Such compounds include a multidentate metal chelating moiety (MC), a linker (L4) having conjugate moieties (Cm) at both ends of L4, and a radioactive halogen template (T). T is a prosthetic compound and can be, for example, a compound of Formula 1 or a compound of Formula 1A as shown above (compounds containing MMCM). In some such embodiments, as mentioned above, m = 0, where the "MC-Cm-L4-Cm" portion of Formula 2 provides the desired function of the L2-CG portion of Formula 1 above (i.e., the MC-Cm-L4-Cm substituent is a sufficient "charged group"). In other such embodiments, m = 1, 2, or 3, in which case 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.

[0203] L4 can be as defined above for L1 and L3. Thus, L4 can be a direct bond or a spacer (e.g., a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, a substituted or unsubstituted alkynyl chain, or a short-chain polyethylene glycol (PEG) chain (1 to 10 ethylene glycol units)). Also, L4 is as defined above but has NH, CO (carbonyl), or S (thioether) at one or both ends.

[0204] Cm can be, for example, thiourea, amide or thioether. For example, in some embodiments, Cm is thiourea (e.g., when the chelating moiety and the conjugate functional group of T are isothiocyanate), amide (when the chelating moiety and the conjugate functional group of T are NHS, TFP active ester or acyl halide) or thioether (when the chelating site and the conjugate functional group of T are maleimide).

[0205] T is generally a radiolabeled moiety containing MMCM, through which a polymer can be bound to a compound to provide a targeted radiotherapy agent. As mentioned above, T can be, in some embodiments, the compound / group of formula 1 or the compound / group of formula 1A. In other embodiments, other radioactive halogen 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., Nucl Med Biol. (1997) 24(7):639-48 (both of which are incorporated herein by reference in their entirety); or SDMB as disclosed in U.S. Patent No. 5,302,700, which are incorporated herein by reference in their entirety.

[0206] MC can be any multidentate moiety, cyclic or acyclic. The composition of MC can be varied. MC can be uncomplexed (metal-free) or complexed with a trivalent metal (M +3 ) such as a stable (non-radioactive) or radioactive form of a metal (e.g., lutetium, yttrium, indium, actinium or gallium), and MC is bound to the linker using one of the free COOH groups present on MC or through other positions on MC (e.g., one of the MC backbone carbons). Specific radioactive metals that can form a complex with MC include 177Lu, 64 Cu, 111 In, 90 Y, 225 Ac, 213 Bi, 212 Pb, 212 Bi, 67 Ga, 68 Ga, 89 Zr and 227 Th, although not limited thereto. This list is not exhaustive, and while these exemplified radioactive metals are trivalent, certain MCs used in accordance with the present disclosure are capable of binding metals of other valences, and it should be noted that such MCs and radioactive metals are also encompassed herein.

[0207] In some embodiments, by including a radioactive metal bound to an MC, it may not be necessary to have radioactive atoms at another location on the molecule (e.g., when "Y" in formula 2 is the moiety of formula 1 / 1a). Thus, in the compounds of formula 2, "T" may or may not contain a radioactive atom (e.g., a halogen). In some embodiments, T comprises the moiety represented by formula 1 / 1a above, where the "Y" group is a non-radioactive halogen such as non-radioactive bromine or iodine. In other embodiments, compounds of formula 2 are provided that contain both a radioactive halogen (e.g., when "Y" in formula 2 is the moiety of formula 1 / 1a) and a radioactive metal (MC, e.g., bound to the above radioactive metal). In certain embodiments, such a strategy may, for example, enable the use of the same prosthetic agent for multiple isotopes. In certain embodiments, the compounds of formula 2 have a low energy β emitter (e.g., 131 I) + a high energy β emitter (e.g., 90 Y); or an α emitter (e.g., 225 Ac) metal and a β emitting halogen (e.g., 131 I); or an α emitting halogen (e.g., 211 At) and a β emitting radioactive metal (e.g., 177 Lu).

[0208] In some embodiments, MC is a macrocyclic ligand that includes a ring containing 8 or more atoms and has 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 hereby incorporated by reference in its entirety).

[0209] Examples of class II compounds suitable for conjugation to a HER2 binder to form a targeted radiotherapeutic agent according to the present disclosure are shown in Formula 2A below, where MC in the formula is a macrocyclic ligand containing DOTA, and T of the radioactive halogen template is the moiety corresponding to Formula 1.

Chemical formula

[0210] The left parenthesis in Formula 2A is not intended to limit a specific site on MC(DOTA) to which the Cm group binds, i.e., Cm is intended to be able to bind to DOTA at various sites on MC. Similarly, the right parenthesis in Formula 2A is not intended to limit a specific site on the ring of "T" to which the Cm group binds, i.e., Cm is intended to be able to bind to T at various sites on the ring. Again, as mentioned above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T in Formula 2A contains four substituents (e.g., attached MC, L2-MMCM, L3-Y, L2-CG). In other embodiments, the benzene ring of T in Formula 2A contains three substituents (e.g., linked MC, L2-MMCM, and L3-Y). The latter embodiment is particularly relevant when the attached MC is charged, i.e., it can serve as an alternative when providing the desired function of the "L2-CG" substituent.

[0211] In some embodiments, MC is an acyclic ligand containing a chain of six or more atoms having at least three negatively charged substituents, such as carboxyl groups or phosphonic acid groups. Exemplary acyclic ligands suitable as the MC group include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetramethylenephosphonic acid (EDTMP), and ethylenediaminetetraacetic acid (EDTA). Examples of Class II compounds are shown in Formula 2B below, where MC in the formula is an acyclic ligand containing DTPA, and T of the radioactive halogen template is the moiety corresponding to Formula 1.

Chemical formula

[0212] As mentioned above with respect to Formula 2A, the left parenthesis in Formula 2B does not limit the specific site on MC(DTPA) to which the Cm group binds, i.e., it is intended that Cm can bind to DTPA at various sites on MC. Similarly, the right parenthesis in Formula 2B does not limit the specific site on the ring of "T" to which the Cm group binds, i.e., it is intended that Cm can bind to T at various sites on the ring. Again, as mentioned above, CG-L2 may or may not be present. In some embodiments, the benzene ring of T in Formula 2B contains four substituents (e.g., the attached MC, L2-MMCM, L3-Y, and L2-CG). In other embodiments, the benzene ring of T in Formula 2A contains three substituents (e.g., the linked MC, L2-MMCM, and L3-Y). The latter embodiments are particularly relevant when the attached MC is charged, i.e., it can serve as an alternative in providing the desired function of the "L2-CG" substituent.

[0213] In some specific embodiments, the targeted radiotherapy agent is provided by conjugating the HER2 binder 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 sortase (see the above formula) is (Gly)n for site-specific conjugation.

[0214] Furthermore, to obtain a targeted radiotherapy agent encompassed by the present disclosure, non-limiting prosthetic groups that can be coordinated to the HER2 binder include, for example, compounds having the general structure of Formula 3 shown below (referred to as "Class III type compounds"). MC-T (Formula 3: General structure of Class III compounds)

[0215] Certain such class IIII type compounds are generally disclosed in WO2021 / 096968 (Duke University), which is hereby incorporated by reference in its entirety. In such compounds, the MC can, in some embodiments, be as defined above (and thus can be cyclic or acyclic and can be non-complexed or complexed with a metal). In some embodiments, the disclosed prosthetic agents and corresponding targeted radiotherapy agents include a functionalized MC (e.g., a modified DOTA or a modified DOTA). One exemplary modified DOTA is DOTA-tris(t-Bu ester), i.e., DOTA contains tert-butyl acetate groups that bind to each nitrogen atom other than the nitrogen atom when used to bind to "T". Such compounds suitable for the reaction to provide MC-T are also shown as tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate. Removal of the tert-butyl protecting group provides a deprotected form, which can be shown as 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid. Further modified DOTA for which uses can be found in certain embodiments of the present disclosure are acyclic analogs, e.g., acyclic analogs of DOTA or acyclic analogs of modified DOTA. One exemplary tert-butyl acetate-functionalized acyclic DOTA that can be reacted to form MC-T is tert-butyl (2-((2-(2-aminoethyl)(tert-butoxycarbonyl)-amino)ethyl)(tert-butoxycarbonyl)amino)ethyl)glycinate. Removal of the tert-butyl protecting group provides a deprotected form, which can be shown as (2-((2-((2-aminoethyl)(carboxy)amino)ethyl)(carboxy)amino)ethyl)glycine. Similarly, in some embodiments, modified analogs of NOTA are used, which contain tert-butyl acetate groups that bind to each nitrogen atom other than its nitrogen atom when used to link to "T".Such a modified NOTA may be di-tert-butyl 1,4,7-triazonane-1,4-dicarboxylate, which can be reacted via a nitrogen atom not bonded to a tert-butyl group to obtain MC-T. In all embodiments of the present specification that provide a tert-butyl acetate group that binds to the MC group, it is understood that the deprotected form (including an acetate group instead of the tert-butyl acetate group) is also intended to be included herein.

[0216] Certain such modified "MC" groups are shown below in Formulas A-1, A-2, and A-3, respectively, and a prosthetic site for the relevant radioactive halogen is provided by one executable binding point to the "T" shown in each of the "MC" moieties described, which can bind to a HER2 binder to provide a targeted radiotherapeutic agent containing the same.

Chemical formula

[0217] In such embodiments, MC is linked to the remainder of the prosthetic moiety (including "T") via any corresponding atom, for example, via a carbon or nitrogen (but not limited to this) that binds to MC, providing a corresponding radioactive halogen prosthetic moiety. For the modified DOTA and NOTA groups referred to herein, MC can 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 another position on MC (such as a position via one of the carbons of the MC skeleton).

[0218] As detailed above, T is a radioactive halogen template. In certain embodiments, T can be a compound of Formula 1 or a compound of Formula 1A. In some embodiments, T is a radioactive halogen template of Formula B below, and the bond to MC is indicated by a wavy line. T can be directly bonded to MC; that is, it can have a direct bond between the carbon atom shown adjacent to R1 and a site on MC (e.g., MC is DOTA / NOTA or a modified DOTA / NOTA), or the carbon atom shown below can be directly bonded to a nitrogen atom of the backbone DOTA / NOTA structure. [Chemical formula] In Formula B, the following definitions apply: R1 = H, an ester or a carboxylic acid; A is R2 - R3 - Y or Y; B is H, an alkoxy (e.g., methoxy, ethoxy, propoxy or butoxy) group or a halogen (e.g., a non-radioactive halogen selected from the group consisting of Cl, F, I and Br); R2 = a direct bond, an alkyl group or an oxygen-containing moiety. Specific examples of the "oxygen-containing moiety" provided herein include, but are not limited to: -O-, -O-CH2-, -O-CH2CH2-, etc.

[0219] R3 = a direct bond, an alkyl group or an aromatic moiety (e.g., a phenyl ring).

[0220] Other definitions are as shown above for compounds of other formulas.

[0221] In certain embodiments, R2 is -O-CH2-, R3 is a phenyl ring, and R2-R3 is a benzyloxy group attached to the central phenyl ring of Formula C. The relative positions of the substituents on the phenyl ring with respect to each other can vary. The L1-MMCM on the aromatic ring with respect to another substituent (the bond to A, B, and MC) can vary. In certain embodiments, the L1-MMCM group is in the meta position with respect to the bond to MC. The central phenyl ring of the compound, in some embodiments, does not contain a substituent in the ortho position with respect to L1-MMCM (the carbon to which L1-MMCM is attached is ortho to two unsubstituted carbon atoms). In certain embodiments, A is ortho to the carbon attached to MC. When A is ortho to the carbon attached to MC, in some such embodiments, B is ortho to the A substituent and is H. In certain embodiments, A is meta to the carbon attached to MC. When A is meta to the carbon attached to MC, in some such embodiments, B is ortho to the carbon attached to MC (meta to the A substituent), and in certain embodiments, B is alkoxy (e.g., methoxy).

[0222] Various embodiments within the scope of Formula B are shown below. For example, in some embodiments, R1 is an ester group (e.g., CO2 tIt is (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, as shown in the following formula B-2, it has three other substituents on the aromatic ring. In some embodiments, the halogenated site or the precursor moiety is bonded (directly or indirectly) to the central aryl ring as shown in formula B-3 (wherein the position of the halogen on the phenyl ring can be changed, but the halogen is para to the bond with the rest of the molecule), and is arranged on an aromatic ring (such as being linked to the central aryl ring via an O-alkyl group, but not limited thereto). In some of the above embodiments as shown in formula C-3, B is H. In certain embodiments, as shown in formula B-4, the halogen site is present on an aromatic ring linked to the central aryl ring, and the aromatic ring is directly bonded to the central aryl ring as shown. In some such embodiments as shown in formula B-4, B is H. It should be noted that in embodiments where the halogenated site or the precursor moiety is present on an aromatic ring bonded to the central aryl ring, the aromatic ring may not contain other substituents, or in some embodiments, it may contain one or more additional substituents at various positions relative to the halogenated site or the precursor moiety. For example, in some embodiments, the aromatic ring further contains guanidinomethyl. Therefore, for example, the following formulas B-3 and B-4 are shown using an aromatic ring substituted only with I (or any "Y" group provided herein), but the present disclosure is not limited thereto, and other substituents may be present on the ring in various embodiments. In certain embodiments, a portion of formula C-3 is provided in which the aryl ring is substituted with both I (or other Y) and a guanidinomethyl group.

Chemical formula

Chemical formula

[0223] In some embodiments, the halogenation site / precursor moiety is present directly on the central aryl ring (e.g., linked via a direct bond), as explicitly shown 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 proximal to the chelate. In some such embodiments as shown in Formula B-6, B is preferably OCH3. [Chemistry] [Chemistry]

[0224] In certain embodiments, the DOTA-SIB or NOTA-SIB hybrid radiohalogen prosthetic moieties shown below are conjugated to the HER2-binding agents described herein to provide targeted radiotherapy agents. "I" is shown as the radiohalogen in the structure, but it should be understood that the various "Y" groups as provided herein may be substituted at that position; further, the presented N-hydroxysuccinimidyl esters may be substituted with other groups (e.g., TFP ester groups) for conjugation to the HER2-binding agent. Formulas 3A, 3B, 3B', 3C and 3D represent non-limiting halogen prosthetic moieties having DOTA-SIB [3B' provides a guanidinomethyl group (which can be in the ortho or meta position relative to the I substituent) on the aromatic ring, and X and Y can be located at any of five positions on the aromatic ring; when X is a radioisotope of iodine or another radiohalogen (or a precursor thereof), Y is guanidinomethyl and vice versa]. Formula 3E represents a non-limiting prosthetic moiety each having NOTA-SIB: [Chemical Formula] [Chemical Formula]

[0225] Non-limiting examples of structures where L1-MC is a tetrafluorophenol (TFP) ester are shown in the following Formulas 3F - 3H, which may be further modified as described above (e.g., including substituting I with other Y groups and adding one or more substituents on the O-alkylphenyl group of Formula 3G or 3H, but not limited thereto). [Chemical Formula] [Chemical Formula]

[0226] In most of the structures of Formula 3 shown, the ester-containing R1-substituent (e.g., CO2 t Bu group) is included, but in other embodiments, as shown in Formula 3I below, a radioactive halogen prosthetic moiety or a precursor thereof in which R1 is H is provided. [Chemical Formula]

[0227] Non-limiting examples of additional radioactive halogen prosthetic moieties that can be attached to a HER2 binder to provide a targeted radiotherapeutic agent included in the present disclosure include, for example, N-succinimidyl 5- * I]iodo-3-pyridinecarboxylate( * I]SIPC); SFPC, SAPC or other radioactive halogen derivatives; N-succinimidyl-3- * I]-iodobenzoic acid( * 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 radioactive halogen derivatives; N-maleimidoethyl-3-(guanidinomethyl)-4- * I]iodobenzamide( * I]MEGMIB); compounds containing acyclic bifunctional chelating agents, such as compounds containing p-SCN-Bz-DTPA; tetra-t-Bu-DTPA; mercaptoacetyltriglycine (MAG3); MX-DTPA; CHX-DTPA (CHX in the formula can be CHX-A or CHX-B) or macrocyclic bifunctional chelating agents (e.g., P-SCN-Bn-DOTA); maleimidocysteinamide-DOTA derivatives; and benzyl TETA derivatives.

[0228] (Definition) "C m -C n alkyl", by itself, or C m -C nHaloalkyl, C m -C n Alkylcarbonyl, C m -C n In composite expressions such as alkylamine, it represents a linear or branched aliphatic hydrocarbon group having a predetermined number of carbon atoms. For example, C1-C4 alkyl means an alkyl group having 1 to 4 carbon atoms. C1-C6 alkyl has the corresponding meaning including all linear and branched isomers of pentyl and hexyl. Exemplary alkyl groups for use in manufacturing the provided compositions are C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl), particularly C1-C4 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl, and isobutyl). In certain embodiments, the alkyl group(s) is / are methyl and / or isopropyl. The alkyl group may be substituted or unsubstituted by one or more groups which may be the same or different, and each substituent is independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl)2, -NH(cycloalkyl), -O-C(=O)-alkyl, -O-C(=O)-aryl, -O-C(=O)-cycloalkyl, -C(=O)OH, and -C(=O)O-alkyl. In certain embodiments, the alkyl group is unsubstituted unless otherwise stated.

[0229] “C2-C n"Alkenyl" represents a straight-chain or branched-chain aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having a predetermined number of carbon atoms. For example, C2-C4 alkenyl means an alkenyl group having 2 to 4 carbon atoms; C2-C6 alkenyl means an alkenyl group having 2 to 6 carbon atoms. Non-limiting examples of alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, and hexenyl. The alkenyl group may be unsubstituted or substituted with one or more substituents which may be the same or different, and each substituent is independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl)2, -NH(cycloalkyl), -O-C(=O)-alkyl, -O-C(=O)-aryl, -O-C(=O)-cycloalkyl, -C(=O)OH, and -C(=O)O-alkyl. In certain embodiments, the alkenyl group is unsubstituted unless otherwise specified.

[0230] "C2-C n"Alkynyl" represents a straight-chain or branched aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and having a predetermined number of carbon atoms. For example, C2-C4 alkynyl means an alkynyl group having 2 to 4 carbon atoms; C2-C6 alkynyl means an alkynyl group having 2 to 6 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, propynyl, 2-butynyl, 3-methylbutynyl, pentynyl, and hexynyl. The alkynyl group may be unsubstituted or substituted with one or more substituents which may be the same or different, and each substituent is independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl)2, -NH(cycloalkyl), -O-C(O)-alkyl, -O-C(O)-aryl, -O-C(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. In certain embodiments, the alkynyl group is unsubstituted unless otherwise specified.

[0231] As used herein, the term "C" m -C n "Haloalkyl" means a C in which at least one C atom is substituted with a halogen, such as iodine, bromine, or fluorine m -C n representing alkyl (e.g., C m -C n The haloalkyl group may contain 1 to 3 halogen atoms). A typical haloalkyl group is C1-C2 haloalkyl, where halo preferably represents iodine. Examples of haloalkyl groups include iodomethyl, diiodomethyl, and triiodomethyl. As used herein, only one of the halogens may be radioactive.

[0232] As used herein, the term "C" m -C n "Hydroxyalkyl" means a C in which at least one C atom is substituted with one hydroxy group m -Cn represents alkyl. Typical C m -C n The hydroxyalkyl group is a C in which one C atom is substituted with one hydroxy group m -C n alkyl. Examples of the hydroxyalkyl group include hydroxymethyl and hydroxyethyl.

[0233] As used herein, the term "C m -C n alkylene" represents a straight-chain or branched-chain divalent alkyl group having a predetermined number of carbon atoms. In certain embodiments, the C m -C n alkylene group is C1-C3 alkylene. Non-limiting examples of the alkylene group include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH(CH3)-, and -CH(CH(CH3)2)-.

[0234] "C m -C n alkoxy" represents the group C m -C n alkyl-O-, where C m -C n alkyl is as defined above. In certain embodiments, C m -C n alkoxy is C1-C4 alkoxy, which includes methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy, sec-butoxy, and isobutoxy. In certain embodiments, C m -C n alkoxy is methoxy or isopropoxy. C1-C6 alkoxy has the corresponding meaning extended to include all straight-chain and branched-chain isomers of pentoxy and hexoxy.

[0235] The term "Me" means methyl and "MeO" means methoxy. The term "amino" represents the group -NH2. The term "halo" represents a halogen group such as fluoro, chloro, bromo, iodo or astatine. Typically, the halo group is iodo, bromo or astatine. The term "aryl" represents an aromatic ring, for example, a phenyl, biphenyl or naphthyl group.

[0236] The term "heterocycloalkyl" represents a stable saturated monocyclic 3- to 12-membered ring containing 1 to 4 heteroatoms independently selected from O, S and N. In some embodiments, the stable saturated monocyclic 3- to 12-membered ring contains 4 N heteroatoms. In a second embodiment, the stable saturated monocyclic 3- to 12-membered ring contains 2 heteroatoms independently selected from O, S and N. In a third embodiment, the stable saturated monocyclic 3- to 12-membered ring contains 3 heteroatoms independently selected from O, S and N. The heterocycloalkyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, and each substituent is independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl)2, -NH(cycloalkyl), -O-C(O)-alkyl, -O-C(O)-aryl, -O-C(O)-cycloalkyl, -C(O)OH and -C(O)O-alkyl. In certain embodiments, the heterocycloalkyl group is unsubstituted unless otherwise specified.

[0237] The term "heteroaryl" represents a stable aromatic ring containing 1 to 4 heteroatoms independently selected from O, S and N. In certain embodiments, the heteroaryl moiety useful in the present disclosure has 6 ring atoms. In some embodiments, the stable aromatic ring system contains 1 heteroatom which is N.

[0238] The term "amino C m -C n"Alkyl" is substituted with an amino group (i.e., one hydrogen atom of the alkyl moiety is substituted with an NH2 group) as defined above, C m -C n represents an alkyl group. Typically, "amino C m -C n alkyl" is amino C1-C6 alkyl.

[0239] The term "amino C m -C n alkylcarbonyl" is a C m -C n alkylcarbonyl group where one hydrogen atom of the alkyl moiety is substituted with an NH2- group as defined above. Typically, "amino C m -C n alkylcarbonyl" is amino C1-C6 alkylcarbonyl. Examples of amino C m -C n alkylcarbonyl include, but are not limited to, glycyl: C(=O)CH2NH2, alanyl: C(=O)CH(NH2)CH3, valinyl: C=OCH(NH2)CH(CH3)2, leucinyl: C(=O)CH(NH2)(CH2)3CH3, isoleucinyl: C(=O)CH(NH2)CH(CH3)(CH2CH3), and norleucinyl: C(=O)CH(NH2)(CH2)3CH3. This definition is not limited to naturally occurring amino acids. This definition is not limited to natural amino acids.

[0240] Related terms should be interpreted according to the above definitions and common usage in the art.

[0241] As used herein, the term "(=O)" forms a carbonyl moiety when attached to a carbon atom. It should be noted that an atom can have an oxo group only when the valence of the atom permits it.

[0242] The terms "monophosphate, diphosphate, and triphosphate esters" refer to the group: [Chemistry] refers to.

[0243] The term "thio-monophosphate, thio-diphosphate and thio-triphosphate esters" refers to the group: [Chemistry] refers to.

[0244] As used herein, the position of a group on any molecular moiety used in a definition may be at any position on said moiety, provided it is chemically stable. If any variable present occurs one or more times in any moiety, each definition is independent.

[0245] As used herein, the terms "compound of formula 1", "compound of formula 1A", "compound of formula 2", "compound of formula 3" or "compound of the present disclosure", or similar phrases always mean to include the compound of formula 1 and subgroups of the compound of formula 1, the compound of formula 2 and subgroups of the compound of formula 2, and the compound of formula 3 and subgroups of the compound of formula 3, respectively, which include possible stereochemical isomers, as well as their pharmaceutically acceptable salts and solvates.

[0246] The term "solvate" relates to any pharmaceutically acceptable solvate capable of forming with the compounds of formula 1, formula 2 and formula 3 and their salts. Such solvates are, for example, hydrates, alcoholates such as ethanolates, propanolates, etc., and especially hydrates.

[0247] Generally, the names of the compounds provided herein are created using ChemDraw Professional 16.0. Further, if the stereochemistry of a structure or part of a structure is not indicated, for example, by a bold or dashed line, the structure or part of the structure is to be interpreted as encompassing all of its stereoisomers.

[0248] The linker may also be selected to facilitate the attachment of each moiety to the core structure. For example, with respect to an exemplary synthetic route of a prosthetic compound, as will be discussed in more detail below, representative linkers are bifunctional alkyl chains having 1 to 6 carbon atoms (e.g., -CH2-, -C2H4-, -C3H6-, etc.), in which one carbon atom may be substituted with a cyclic (hydrocarbon ring) group or a heterocyclic (hetero ring) group. Representative heterocyclic groups have at least one nitrogen atom in the heterocycle. Specific examples of such heterocyclic groups are thus diazinyl, diazolyl, triazinyl, triazolyl, tetrazinyl, and tetrazolyl groups. These groups and other heterocyclic groups, or another cyclic group, may optionally be fused to another cyclic group or heterocyclic group, or may be fused to another cyclic group or heterocyclic group that is itself part of a fused ring system (e.g., a triazolyl group can be fused to an 8-membered or heterocyclic group that itself is fused to two 6-membered ring systems when the triazolyl group (or other nitrogen atom-substituted heterocyclic hydrocarbon group) is fused to a dibenzoazocanyl group). Thus, linkers containing three or more fused rings, such as hydrocarbon rings, heterocycles, and combinations of these rings, are also possible. A representative charged group linker, L2, is a divalent substituted or unsubstituted alkyl chain, substituted or unsubstituted alkenyl chain, or substituted or unsubstituted alkynyl chain having 1 to 6 carbon atoms. Generally, L1, L2, L3, and / or L4 may be (or may contain) a substituted or unsubstituted divalent alkyl group having 1 to 6 carbon atoms, where one or more carbon atoms may be substituted and / or replaced with a heteroatom (e.g., NH, O, or S), or may be substituted or replaced with another alkyl group having 1 to 8 carbon atoms that may be linear, branched, or cyclic (e.g., to form a branched alkyl group). For example, substituting one carbon atom of an alkyl group can yield a carbonyl (C=O) group, and substituting an adjacent carbon atom with NH can yield a peptide / amide bond -(C=O)-NH-.Accordingly, representative linkers L1, L2, L3, and L4 can incorporate into the alkyl chain a divalent alkyl group having one or more of the peptide bond, -NH- bond, -(C=O)- bond, and / or cyclic -C6H4- bond (including any combination of two, three, or four of such bonds). Further, in the case of the divalent alkyl group of L1, L2, and / or L3, a carbon-carbon double bond and / or a carbon-carbon triple bond may be formed between one or more pairs of adjacent carbon atoms to provide a divalent unsaturated (e.g., olefinic) alkyl group.

[0249] In some embodiments, the HER2 binder has the following structure: VHH-RLC [wherein VHH is a protein comprising the HER2-binding VHH domain provided herein (e.g., having any one of the sequences of SEQ ID NOs: 1 to 81 and 205 or a fragment or variant thereof), and RLC is a radiolabeling chemical used to facilitate the binding of a radionuclide to the VHH, either directly or indirectly.] It is a targeted radiotherapy agent having. 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 chelating agent.

[0250] In some embodiments, RLC is a Class I compound. Examples of specific non-limiting targeted radiotherapy agents within the scope of the present disclosure are as follows: [Chemical formula] [Chemical formula] (wherein VHH is a protein comprising the HER2-binding VHH domain provided herein (e.g., having any one of the sequences of SEQ ID NOs: 1 to 81 or a fragment or variant thereof).

[0251] In some embodiments, the RLC is as follows. [Chemical formula]

[0252] In some embodiments, the RLC is a Class II compound. Examples of specific non-limiting targeted radiotherapeutic agents that fall within the scope of the present disclosure are as follows, where VHH is a protein comprising a HER2-binding VHH domain provided herein (e.g., having any one of the sequences of SEQ ID NOs: 1-81 and 205, or a fragment or variant thereof): [Chemical formula]

[0253] In some embodiments, the RLC is a Class III compound. Examples of specific non-limiting targeted radiotherapeutic agents that fall within the scope of the present disclosure are as follows: [Chemical formula] [wherein VHH is a protein comprising a HER2-binding VHH domain provided herein (e.g., having any one of the sequences of SEQ ID NOs: 1-81 and 205, or a fragment or variant thereof)].

[0254] In some embodiments, the RLC is a direct radioisotope label that uses an electrophilic substitution reaction for radiohalogenation. In some embodiments, the RLC is a direct radioisotope label that uses an electrophilic substitution reaction with iodosogen, chloramine T, and N-halosuccinimide for radiohalogenation.

[0255] In some embodiments, the RLC is an indirect halogenated prosthetic compound. In some embodiments, the RLC is N-succinimidyl 5- *I] Iodo-3-pyridinecarboxyl ( * I] SIPC). In some embodiments, the RLC is SFPC, SAPC, or any other radioactive halogen derivative known in the art. In some embodiments, the RLC is N-succinimidyl-3- * I]-iodobenzoate ( * I] SIB). In some embodiments, the RLC is SFB, SAB, or any other halogen radionuclide known in the art. In some embodiments, the RLC is N-succinimidyl-4-guanidinomethyl-3- * I] iodobenzoate ( * I] SGMIB). In some embodiments, the RLC is SAGMB, SFGMB, or any other radioactive halogen derivative known in the art. In some embodiments, the RLC is N-maleimidoethyl-3-(guanidinomethyl)-4- * I]-iodobenzamide ( * I] MEGMIB).

[0256] In some embodiments, the RLC is an acyclic bifunctional chelating agent. In some embodiments, the RLC is p-SCN-Bz-DTPA, tetra-t-Bu-DTPA, mercaptoacetyltriglycine (MAG3), or MX-DTPA. In some embodiments, the RLC is CHX-DTPA (where CHX can be CHX-A or CHX-B).

[0257] In some embodiments, the RLC is an acyclic bifunctional chelating agent. In some embodiments, the RLC is P-SCN-Bn-DOTA. In some embodiments, the RLC is a maleimidocysteine amide-DOTA derivative. In some embodiments, the RLC is a benzyl TETA derivative.

[0258] Radioactive halogen prosthetic moieties and corresponding targeted radiotherapy agents can be manufactured in a variety of ways, e.g., according to methods known in the art. Targeted radiotherapy agents can be provided, for example, by directly or indirectly conjugating a radionuclide / radioactive halogen prosthetic moiety to a HER2 binder such as those provided herein. Such specific methods are described, for example, in International Publication Nos. WO2018 / 178936 and WO2021 / 096968 (Duke University), each of which is hereby incorporated by reference in its entirety.

[0259] For example, in some embodiments, a precursor of the radioactive halogen prosthetic moiety is produced (e.g., containing a tin or other alkylmetal moiety, or a boronic acid moiety or boronic ester moiety, e.g., Y in the above formula is, for example, a tin or other alkylmetal moiety, or a boronic acid moiety or boronic ester moiety). The precursor can then be radioactively labeled and purified. Such radioactive labeling approaches can use electrophilic substitution reactions in some embodiments, for example, but not limited to, using one or more catalysts including 1,2,3,6-tetrachloro-3α,6α-diphenylglycoluril (Iodo-gen®), chloramine-T, or N-halosuccinimide. The resulting radioactive halogen prosthetic moiety is conjugated to the HER2 binder using standard conditions to obtain the targeted radiotherapy agent. Non-limiting examples regarding the manufacture of specific radioiodinated prosthetic agents and the use of such radioiodinated prosthetic agents for labeling 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 hereby incorporated by reference in their entirety; 211Non-limiting examples regarding the manufacture of At-labeled prosthetic agents and the use of said agents for labeling HER2-binding agents are described in International Publication No. WO2018 / 178936, which is hereby incorporated by reference in its entirety.

[0260] In some embodiments, a “pre-conjugation” approach is used. For example, a precursor of a radioactive halogen prosthetic moiety is first manufactured and reacted with a HER2-binding agent, and then the HER2-binding agent-precursor conjugate is radio-labeled to obtain a targeted radio-therapeutic agent. In some such embodiments, one drawback (especially in the case of radioiodination) is that the tyrosine, phenylalanine, and histidine residues of components that may be present on the HER2-binding agent may also be radioiodinated in addition to the intended prosthetic moiety(ies) for radio-labeling (i.e., moieties having an alkyl metal group, a boronic acid, or a boronic acid ester group). The problem with radiohalogens on tyrosine is that when the targeted radiotherapeutic agent enters the body, the radioactive substance dissociates due to the action of endogenous deiodinase enzymes and does not localize with the HER2-binding agent at the target cells. This aspect can be minimized in some embodiments by performing radioiodination at a low pH (4-5), but it cannot be completely avoided. One approach to avoid this potential problem is to introduce non-radioactive iodine into the tyrosine residues of polymers first, before subjecting them to radioiodination. By these same endogenous iodine-degrading enzymes, the non-radioactive iodine on the tyrosine residues of the components is removed, thereby restoring the original tyrosine structure and likely maintaining the affinity of the HER2-binding agent for the intended target. Non-radioactive iodination of the HER2-binding agent can be carried out. In some embodiments, it is achieved by treating the HER2-binding agent with an excess of sodium iodide in the presence of an oxidizing agent (e.g., chloramine-T).

[0261] V. Pharmaceutical Compositions and Formulations The present disclosure also provides a pharmaceutical composition comprising a HER2 binder 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 binder or nucleic acid described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises any suitable and pharmaceutically acceptable carrier, diluent, adjuvant or buffer.

[0262] The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for appropriate formulation. Suitable formulations for use in the present disclosure are described 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).

[0263] In certain embodiments, the pharmaceutical composition may include formulation materials for modifying, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorptivity, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, tris hydrochloride, citrate, phosphate, or other organic acid salts); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; emulsifying agents; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., 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).

[0264] In some embodiments, the pharmaceutical composition is nanoparticles, such as polymeric nanoparticles, liposomes or micelles (see Anselmo et al., Bioeng Transl Med. (2016) 1:10-29).

[0265] In some embodiments, the pharmaceutical composition can include a sustained release formulation or a controlled release formulation. Techniques for formulating sustained release or release controlled delivery means such as liposomal carriers, biodegradable microparticles or porous beads and depot injections are also known to those skilled in the art. Examples of sustained release formulations include porous polymer microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. The sustained release composition can also include liposomes that can be produced by any of several methods known in the art.

[0266] The pharmaceutical compositions containing the HER2 binding agents disclosed herein can be presented in unit dosage form and can be manufactured by any suitable method. The pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV) administration, intradermal administration, inhalation administration, transdermal administration, topical administration, transmucosal administration, intrathecal administration, and rectal administration. In certain embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by intravenous injection. In certain embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by intratumoral injection. Useful formulations can be manufactured by methods known in the pharmaceutical art. See, e.g., Remington’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation ingredients suitable for parenteral administration include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as EDTA; buffers such as acetate, citrate, phosphate; and agents for adjusting osmotic pressure such as sodium chloride or glucose, among others.

[0267] Suitable carriers for intravenous administration include saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0268] Intravenous drug delivery formulations may be contained in syringes, pens or bags. In certain embodiments, the bag may be connected to a path including a tube and / or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be lyophilized and placed in about 12 to 60 vials. In certain embodiments, the formulation may be lyophilized, and 45 mg of the lyophilized formulation may be placed in one vial. In certain embodiments, about 40 mg to about 100 mg of the lyophilized formulation may be placed in one vial. In certain embodiments, 12, 27 or 45 vials of the lyophilized formulation are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1,000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial.

[0269] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for immediate use or may be lyophilized, and the lyophilized preparation is combined with a carrier of sterile water prior to administration. The pH of the preparation is usually 3 to 11, in certain embodiments 5 to 9 or 6 to 8, and in particularly specific embodiments 7 to 8, for example 7 to 7.5. The resulting solid form composition may be packaged in multiple single-dose units, and each single-dose unit contains a fixed amount of the above drug or drugs. The solid form composition can also be packaged in a container with variable amounts.

[0270] In certain embodiments, the present disclosure provides a formulation containing the protein of the present disclosure, which can be stored for a long time, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water and sodium hydroxide.

[0271] In certain embodiments, an aqueous formulation comprising the protein of the present disclosure is prepared in a pH buffer solution. The buffer of the present invention can have a pH in the range of about 4 to about 8, such as about 4.5 to about 6.0 or about 4.8 to about 5.5, or can have a pH of about 5.0 to about 5.2. Ranges intermediate the above-recited pH ranges are also intended to be part of the present disclosure. For example, ranges of values using any combination of the above-quoted values as upper and / or lower limits are intended to be included. Examples of buffers for controlling pH within this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid salts buffers.

[0272] In certain embodiments, the formulation includes a buffer system comprising citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range may be a pH range of about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or 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 1.3 mg / ml of citric acid (e.g., 1.305 mg / ml), about 0.3 mg / ml of sodium citrate (e.g., 0.305 mg / ml), about 1.5 mg / ml of disodium phosphate dihydrate (e.g., 1.53 mg / ml), about 1.5 mg / ml of disodium phosphate dihydrate (e.g., 1.53 mg / ml), about 0.9 mg / ml of sodium dihydrogen phosphate dihydrate (e.g., 0.86) and about 6.2 mg / ml of sodium chloride (e.g., 6.165 mg / ml). In certain embodiments, the buffer system includes 1 to 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 using sodium hydroxide.

[0273] A polyol that acts as an isotonic agent and can stabilize a multispecific binding protein may also be included in the formulation. The polyol is added to the formulation and the amount may be varied with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added can also be varied with respect to the molecular weight of the polyol. For example, the amount of monosaccharide (e.g., mannitol) added may be reduced compared to disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used in the formulation as an isotonic agent is mannitol. In certain embodiments, the concentration of mannitol can be from about 5 to about 20 mg / ml. In certain embodiments, the concentration of mannitol can be from about 7.5 to about 15 mg / ml. In certain embodiments, the concentration of mannitol can be from about 10 to 14 mg / ml. In certain embodiments, the concentration of mannitol can be about 12 mg / ml. In certain embodiments, sorbitol, which is a polyol, may be included in the formulation.

[0274] A detergent or surfactant may also be added to the formulation. Examples of detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is an amount that reduces aggregation of the formulated antibody and / or minimizes particle formation in the formulation and / or reduces adsorption. In certain embodiments, the formulation may contain a surfactant that is polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is the term used to represent polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain from about 0.1 mg / mL to about 10 mg / mL or from about 0.5 mg / mL to about 5 mg / mL of polysorbate 80. In certain embodiments, about 0.1% of polysorbate 80 may be added to the formulation.

[0275] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation sealed with a rubber stopper and sealed by aluminum crimping may be present at a concentration of 10 mg / mL in any of USP / Ph Eur type I 50R vials. The stopper may be made of an elastomer compliant with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with 0.9% saline.

[0276] In certain embodiments, the liquid formulations of the present disclosure may be manufactured as a solution at a concentration of 10 mg / mL in combination with a stabilizing level of sugar. In certain embodiments, the liquid formulations may be prepared in an aqueous carrier. In certain embodiments, the stabilizer may be added in an amount below that which would result in an undesired or inappropriate viscosity for intravenous administration. In certain embodiments, the sugar may be a disaccharide (e.g., sucrose). In certain embodiments, the liquid formulations may also include one or more of a buffer, a surfactant, and a preservative.

[0277] In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0278] The aqueous carriers contemplated herein are those that may be pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for the preparation of the liquid formulations. Examples of carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solutions.

[0279] A preservative may be added to the formulations herein, if desired, to inhibit the action of bacteria. The addition of a preservative may, for example, facilitate the manufacture of multiple-dose formulations.

[0280] The multispecific binding protein may be lyophilized to produce a lyophilized formulation comprising the protein and a cryoprotectant. The cryoprotectant may be a sugar, e.g., a disaccharide. In certain embodiments, the cryoprotectant may be sucrose or maltose. The lyophilized formulation may include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0281] The amount of sucrose or maltose useful for stabilizing the lyophilized formulation may be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5. In certain embodiments, the pH of the formulation prior to lyophilization can be set by the addition of pharmaceutically acceptable acids and / or bases. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the 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 between 6 and 8. In certain embodiments, the pH range of the lyophilized formulation may be from 7 to 8.

[0282] The actual dosage of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to be an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and is non-toxic to the patient.

[0283] Specific dosages can be made a fixed amount per patient, for example, 50 to 5,000 mg of protein. Alternatively, the dosage for a patient can be adjusted according to the patient's approximate body weight or surface area. Other factors in determining an appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration and the patient's age, gender and medical condition. Further refinement of the calculations necessary to determine an appropriate dosage for treatment is routinely performed by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages can also be determined by using known assays for determining the dosage to be used in combination with appropriate dose-response data. The dosage for an individual patient can be adjusted while monitoring the progression of the disease. The blood concentration of a targetable construct or complex in a patient can be measured to confirm whether it is necessary to adjust the dosage 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).

[0284] Generally, the dosage based on body weight is about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, 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.

[0285] The administration can be carried out once or multiple times daily, weekly, monthly or annually. A person skilled in the art can easily estimate the repetition rate of the administration based on the measured values of the residence time and concentration of the targetable construct or complex in body fluids or tissues. The administration of the present invention can be performed by intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, infusion via a catheter or direct local injection. It can be administered more than once a day, more than once a week, more than once a month and more than once a year.

[0286] The pharmaceutical composition can be administered to a subject as needed. In some embodiments, an effective amount of the pharmaceutical composition is administered to the subject one or more times. In various embodiments, the effective amount of the pharmaceutical composition is administered to the subject once a month, less than once a month, for example, every two months, every three months, or every six months, less than once a month. In other embodiments, the effective amount of the pharmaceutical composition is administered to the subject more than once a month, for example, every two weeks, every week, twice a week, three times a week, daily, or multiple times a day. The effective amount of the pharmaceutical composition is administered to the subject at least once. In some embodiments, the effective amount of the pharmaceutical composition is administered multiple times over a period of, for example, at least one month, at least six months, or at least one year. In some embodiments, the pharmaceutical composition is administered to the subject as needed to alleviate one or more symptoms of the condition.

[0287] In some embodiments, the present disclosure provides a HER2 binder for use in medicine. In some embodiments, the present disclosure provides a HER2 binder for use in the treatment of a disease. In certain embodiments, the present disclosure provides a HER2 binder for use in the treatment of cancer.

[0288] In some embodiments, the present disclosure provides the use of a HER2 binder in the manufacture of a medicament for the treatment of a disease in a patient in need thereof. In some embodiments, the present disclosure provides the use of a HER2 binder in the manufacture of a medicament for the treatment of cancer in a patient in need thereof. In some embodiments, the present disclosure provides a HER2 + The use of a HER2 binder in the manufacture of a medicament for the treatment of cancer is provided. In certain embodiments, the present disclosure provides the use of a HER2 binder in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.

[0289] VI. Method of Use In some embodiments, the present disclosure provides for the use of a HER2 binder (e.g., a labeled HER2 binder) for the treatment or diagnosis of a disease or condition in a subject and for the use of a HER2 binder (e.g., a labeled HER2 binder) in the manufacture of a medicament for the treatment or diagnosis of a disease or condition in a subject.

[0290] In some embodiments, the present disclosure + provides a method of detecting HER2 + cells in a subject, the method comprising the steps of: a) administering to the subject a labeled HER2 binder or pharmaceutical composition provided herein; and b) detecting the binding of the labeled HER2 binder or pharmaceutical composition to HER2 + cells in the subject, the detection of the binding indicating the presence of HER2 + cells in the subject. In some embodiments, the HER2 + cells are HER2

[0291] tumor cells. In some embodiments, the subject has 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. + In some embodiments, the present disclosure provides a method of imaging a disease site in a subject, the method comprising administering to the subject a labeled HER2 binder or pharmaceutical composition provided herein. In some embodiments, 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. In some embodiments, the HER2 + cells are HER2

[0292] In some embodiments, the present disclosure provides a method of diagnosing a disease or medical condition in a subject, comprising administering to the subject a labeled HER2 binder or pharmaceutical composition provided herein. In some embodiments, the subject has 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.

[0293] In some embodiments, the present disclosure + is a method of detecting HER2 + cells in a subject, comprising the steps of: a) administering a labeled HER2 binder to the subject; and b) detecting the binding of the labeled HER2 binder to HER2 + cells in the subject, wherein detection of the binding indicates the presence of HER2 + cells in the subject. In some embodiments, detecting the binding of the labeled HER2 binder to HER2 + cells in the subject comprises imaging the HER2 + cells in the subject. In some embodiments, imaging the 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. In some embodiments, the HER2 + cells are HER2 + T cells. In some embodiments, the HER2 +They are tumor cells. In some embodiments, the detection is performed within about 1 day after administration (e.g., within about 6 hours, within about 4 hours, within about 2 hours, within about 90 minutes, within about 1 hour, within about 30 minutes). In some embodiments, the method is repeated one or more times, e.g., about 1 to 4 times a year. In some embodiments, the method is repeated about 1 day after the pre - administration of the HER2 binder. In some embodiments, the method is repeated for more than 1 year. In some embodiments, the method has a sensitivity of about 1 nM to about 30 nM. In some embodiments, the subject is a human or a 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 has cancer.

[0294] In some embodiments, the HER2 binders provided herein are used to examine the nature of a patient's disease state. The HER2 binders can be used to create an image of a disease site in a subject using imaging techniques, e.g., X - rays, gamma rays, or PET scans. In some embodiments, the HER2 binders provided herein are used in a method of imaging a subject's disease site, the method including administering to the subject an appropriately detectably labeled HER2 binder provided herein and then scanning the subject's body. Alternatively, by administering the HER2 binder provided herein to a subject, test results can be obtained by analyzing a sample from the subject after molecular administration. Such embodiments can include methods of diagnosing a disease or medical condition in a subject, including administering the HER2 binder provided herein. Embodiments include methods of detecting the site of a disease or medical condition and the use of the HER2 binders provided herein in such methods.

[0295] In some embodiments, the HER2 binding agents provided herein are suitable for treating a disease or disorder. In some embodiments, the disease or disorder is a proliferative disease or disorder. In some embodiments, the disease or disorder is cancer. In certain embodiments, the disease or disorder is HER2 + cancer. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, 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 cancer and / or metastases therefrom. In some embodiments, the cancer is selected from HER2-expressing breast cancer, ovarian cancer, gastrointestinal cancer, bladder cancer, and pancreatic cancer. In certain embodiments, the HER2 binding agents provided herein are suitable for treating metastases. In certain 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 metastases. In certain embodiments, the HER2 binding agent is suitable for treating breast cancer that has metastasized to the brain.

[0296] In some embodiments, the HER2 binding agents provided herein are suitable for treating HER2 + cancer in patients who are not eligible for treatment with trastuzumab or pertuzumab. Typically, patients are scored as HER2 (1+), HER2 (2+), or HER2 (3+) by immunohistochemistry. Thus, in some embodiments, the present disclosure provides a method comprising: i) staging a patient for HER2 expression; and ii) administering to the patient an effective amount of the HER2 binding agents provided herein when the patient is determined to be HER2 + or HER2 ++ as determined in step i), for treating a patient in need thereof with HER2 +Provided is a method for treating cancer. Usually, the test is performed concurrently with the initial biopsy or cancer surgery. Cancer tissue samples obtained from past biopsies or past surgeries may also be used. One of ordinary skill in the art can readily select the most appropriate method for staging a patient with respect to HER2 expression. Usually, the two main methods used as HER2 tests are immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH). 0 to 1+ means that normal amounts of HER2 protein are present and the result is HER2 negative. 2+ means that moderate amounts of HER2 protein are present. 3+ means that the HER2 protein is present in amounts greater than normal and the result is HER2 positive. As an example, the method of the present disclosure generally comprises: i) determining the level of HER2 in a tumor sample obtained from a patient; ii) comparing the level determined in step i) with a pre-determined reference level; and iii) administering to the patient a therapeutically effective amount of a HER2 binding agent provided herein if the level determined in step i) is lower than a pre-determined cut-off value.

[0297] In some embodiments, the present disclosure provides a HER2 binding agent or pharmaceutical composition for use in a medicament provided herein. In some embodiments, the present disclosure provides the use of a HER2 binding agent or pharmaceutical composition provided herein in the manufacture of a medicament for the treatment of a disease in a patient in need thereof.

[0298] In some embodiments, the present disclosure provides a method of imaging a disease site in a subject, comprising administering to the subject a detectably labeled HER2 binding agent or pharmaceutical composition provided herein.

[0299] In some embodiments, the present disclosure provides a method of diagnosing a disease or medical condition in a subject, comprising administering to the subject a HER2 binding agent or pharmaceutical composition provided herein.

[0300] In some embodiments, the present disclosure provides a method of treating a disease in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a HER2 binder or pharmaceutical composition provided herein.

[0301] In some embodiments, the present disclosure provides a method of treating HER2 in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a HER2 binder or pharmaceutical composition provided herein. + cancer. In some embodiments, the subject is a HER2 + or HER2 ++ subject.

[0302] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term administered "in combination" is understood to mean that two (or more) different treatments are administered to a subject during the period of time the subject has a disorder, such that the treatment effects on the subject overlap at the time when the treatment is effective. In certain embodiments, the delivery of one therapeutic agent is still present at the time the delivery of the second therapeutic agent is initiated and overlaps during the administration period. This may be referred to herein as "simultaneous" or "co - administration". In other embodiments, the administration of one therapeutic agent ends before the administration of the other therapeutic agent begins. In any case, in certain embodiments, the treatments are more effective for combination administration. For example, the second therapeutic agent is more effective, e.g., the second therapeutic agent shows the same effect with a smaller amount, or the second therapeutic agent reduces symptoms to a greater extent than the effect that would occur if the second therapeutic agent were administered in the absence of the first therapeutic agent, or a similar situation is seen with the first therapeutic agent. In certain embodiments, administration results in a decrease in symptoms or other parameters associated with the disorder that is higher than the parameters seen when the other therapeutic agent is delivered in the absence of the other therapeutic agent. The effects of the two treatments may be partially additive, wholly additive, or greater than additive (e.g., synergistic). This administration is such that the first therapeutic effect administered can still be detected when the second therapeutic agent is administered.

[0303] In some embodiments, the disclosure provides a method of treating a subject by administering a second therapeutic agent in combination with a HER2 - binding agent described herein.

[0304] Examples of therapeutic agents that can be used as part of a combination therapy in treating cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carbocon, pentostatin, nitracrine, dinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thimalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, besnilone, aminoglutethimide, amsacrine, proglymidine, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butosine, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, iprosulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon α, interferon-2α, interferon-β, interferon-γ, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variants of the aforementioned agents (which show differences in binding to their cognate receptors and may have an increased or decreased serum half-life).

[0305] Another class of agents that can be used as part of a combination therapy in cancer treatment are immune checkpoint inhibitors. Checkpoint inhibitors can be selected, for example, 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.

[0306] VII. Kits and Manufactured Articles Also provided herein are kits and products comprising a HER2 binder provided herein, such as a labeled HER2 binder. In some embodiments, the kit or product comprises instructions for use of any of the HER2 binders of the above methods.

[0307] Also provided herein is a method for the prevention and / or treatment of a disease or condition (e.g., cancer) in a subject, comprising administering to the subject a genetically modified cell comprising a CAR provided herein.

[0308] Unless otherwise defined herein, 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. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification (e.g., definitions) will control. Further, unless the context clearly dictates otherwise, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the terms "have", "include", or variations thereof such as "having", "including", or "comprising" are meant to include the recited integer or group of integers, but are not meant to exclude other integers or groups of integers. All publications and other references mentioned herein are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Although a number of documents are cited herein, citation of a document does not admit that any of these documents forms part of the common general knowledge in the art. As used herein, the term "about" or "approximately" as applied to one or more values of an object refers to a value similar to the recited reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the recited reference value, unless otherwise stated or apparent from the context.

[0309] In the above description, a plurality of embodiments of the present invention have been described. This patent application particularly contemplates all combinations and modifications of the embodiments. According to the present disclosure, back references in the dependent claims are meant as shorthand for the direct and explicit disclosure of each combination of the claims indicated by the back reference and all combinations. Any compound disclosed herein can be used in any treatment method herein, where the individual to be treated is as defined anywhere herein. Further, the headers herein are created to facilitate organization and are not intended to limit the scope of the claimed invention in any way.

[0310] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

Examples

[0311] Example 1: Preparation of HER2-Binding VHH Domain The development of VHH polypeptides that specifically bind to HER2 was achieved by multiple methods. The search for internal ligands was achieved by phage screening of unique naive and alpaca VHH libraries against recombinant HER2-Fc and HER2-DDDDK proteins. The libraries were first pre-cleared using counter screening against the Fc and DDDDK protein sequences presented on magnetic beads to ensure target specificity.

[0312] A total of 16 different screening conditions were utilized. The unique llama and alpaca VHH phage libraries were screened against the HER2 target protein. The HER2 target protein used was a recombinant form of human HER2 with a C-terminal tag containing either the human IgG1 Fc domain or the DDDDK (SEQ ID NO: 207) amino acid sequence. The target protein was immobilized on magnetic beads by affinity binding using the Fc or DDDDK (SEQ ID NO: 207) tag. Phage selections were performed in two different blocking solutions: 3% BSA in PBS or 3% milk in PBS. The binding interaction between the target protein and the phage library or selection pool was evaluated at various times (15 minutes, 60 minutes, or 120 minutes). Three rounds of phage selection were performed using each selection strategy, and clones were isolated from the phage pool of the third round of the normal rounds.

[0313] The finally isolated phage selection pool was evaluated in a 96-well ELISA assay against HER2-Fc and control Fc proteins. A total of 355 positive phagemid clones were identified as positive binders (defined as those showing binding more than 150% higher than the background) and sequenced. As a result of sequence analysis, 87 unique VHH sequences were identified to have HER2 binding affinity. The plasmids of these positive phagemid clones were introduced into SS320 Escherichia coli for direct expression and purification of the protein to directly evaluate the protein affinity. The purified VHHs were retested in an indirect ELISA format against human HER2-Fc and human HER2-His6, and as a result, 24 VHHs with an affinity stronger than 150 nM were obtained. Specificity was verified by measuring the binding affinity to other human ErBB receptors (e.g., EGFR, HER3, and HER4). The species cross-reactivity was characterized in mouse, rat, dog, rhesus monkey, and cynomolgus monkey analogs of HER2-Fc. The top hits were further improved to produce more affinity-binding proteins and improve the compatibility and performance as radiolabeled compounds for targeted radiotherapy.

[0314] The VHH sequences were also evaluated to identify the corresponding framework regions (FR) and complementarity determining regions (CDR). Generally, the linear sequence of a VHH protein corresponds to the following structural sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To maximize the performance of the VHH protein as a targeted drug conjugate (e.g., a radioligand or another type of drug conjugate), all amino acids in the CDR regions that could provide a conjugation site for a radiolabel (or another type of drug conjugation) were mutated to prevent such conjugation. Additionally, the amino acids in the framework regions within five amino acids from the CDR were also modified to prevent steric interference with the binding pocket of the VHH after radiolabeling (or another type of drug conjugation). Such sequence modifications allowed the ligand to maintain the optimal binding and internalization properties required of a radioligand. Sequence modifications included substituting lysine (K) residues with either arginine (R) or histidine (H) residues; substituting unpaired cysteine (C) residues with alanine (A) or serine (S) residues; or substituting glutamine (Q) residues with asparagine (N) residues.

[0315] The identification of the structural CDR sequences from VHH_106 to VHH_172 is shown in Table 2, similar to the exemplification of the C-terminal peptide fusions. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

Table 2-6

Table 2-7

Table 2-8

[0316] Figures 1A-1J represent the ELISA affinity binding curves of exemplary VHH proteins against the human HER2 receptor after protein expression and purification.

[0317] The binding specificities of exemplary VHH proteins against other receptors within the ErbB family are shown in Figures 2A-2I.

[0318] The cross-reactivities of exemplary VHH proteins against cross-species HER2 receptors present in other animals are shown in Figures 3A-3E.

[0319] The competitive inhibitions of exemplary VHH proteins by trastuzumab and pertuzumab are shown in Figures 4A-4B.

[0320] Example 2: Radioactive Labeling and In Vitro Assay of HER2-Binding VHH Domains Figure 5A shows 131 the structure of VHH radiolabeled with Figure 5B-5E show 131 the results of in vitro cell assays for evaluating VHH_128 HER2 binding after radiolabeling with 131 131 ​Level of I]-isoSGMIB-VHH128. (E) Measurement of internalization of 131 I] in SKOV-3 cells in the presence of 100-fold trastuzumab.

[0321] Example 3: In Vivo Test Figure 6 shows the tissue accumulation levels of 131 I]-isoSGMIB-VHH128 over 24 hours in a SKOV-3 xenograft tumor model in female athymic mice.

[0322] Table 3 shows the complete biodistribution accumulation tissue panel of 131 I]-isoSGMIB-VHH128 in SKOV-3 tumor xenografts in female athymic mice. The injected dose was 5 μCi prepared at a specific activity of 1 μCi / ng, and 100 μL was administered intravenously via the tail vein. [Table 3]

[0323] Figure 7 shows the tissue accumulation levels of 131 I]-isoSGMIB-VHH128 over 24 hours in a BT-474 xenograft tumor model in female NOD SCID mice.

[0324] Table 4 shows the complete biodistribution accumulation tissue panel of 131 I]-isoSGMIB-VHH128 in the BT-474 tumor model in female NOD SCID mice. The injected dose was 5 μCi prepared at a specific activity of 1 μCi / ng, and 100 μL was administered intravenously via the tail vein. [Table 4]

[0325] Figures 8A - 8C show 211 HER2-targeted α-particle therapy using an astatine-labeled VHH. (A) VHH_128 was labeled with isoSAGMB 211Scheme and structure for labeling with At. (B) 211 Affinity binding curve of At]-isoSAGMB-VHH128 against HER2-expressing BT-474 cells. (C) Accumulation levels in BT-474 tumors xenografted into athymic mice.

[0326] Table 5 shows the 211 Complete biodistribution accumulation tissue panel of At]-isoSAGMB-VHH1028 in a BT-474 tumor model of female athymic mice. The injected dose was 5 μCi prepared at a specific activity of 1 μCi / ng, and 100 μL was administered intravenously via the tail vein.

[0327]

Table 5

[0328] Figures 9A - 9C show the radiolabeling of HER2-targeted VHH using maleimide-based radiolabeling chemistry. (A) Scheme and structure for labeling 131 VHH_1041 with I using isoMEGMIB. (B) 131 Affinity binding curve of I]-isoMEGMIB-VHH141 against HER2-expressing BT-474 cells. (C) In vitro cell binding and internalization ratios against BT-474 cells.

[0329] Figure 10 shows the 131 Tissue accumulation levels of I]-isoMEGMIB-VHH141 over 48 hours in SKOV-3 xenograft tumors of female athymic mice.

[0330] Table 6 shows the 131 Complete biodistribution accumulation tissue panel of I]-isoMEGMIB-VHH141 in SKOV-3 tumor xenografts of female athymic mice. The injected dose was 5 μCi prepared at a specific activity of 1 μCi / ng, and 100 μL was administered intravenously via the tail vein.

Table 6

Claims

1. a) Complementarity Determination Region (CDR) 1 containing an amino acid sequence selected from SEQ ID NOs. 82-101; CDR2 containing an amino acid sequence selected from SEQ ID NOs: 102-123; and CDR3 containing an amino acid sequence selected from SEQ ID NOs: 124-157 and 206; b) CDR1, CDR2, and CDR3 containing the amino acid sequences of CDR1, CDR2, and CDR3 included in a VHH sequence selected from SEQ ID NOs: 1-81 and 205; c) A VHH sequence selected from sequence numbers 1-81 and 205; d) An amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a VHH sequence selected from SEQ ID NOs: 1-81 and 205; or e) Amino acid sequences 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. Includes a HER2-binding VHH domain, The VHH domain has been humanized upon request. HER2 binder.

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

3. HER2 conjugate a) monovalent; or b) Polyvalent, and optionally the HER2 binder is divalent, trivalent, or tetravalent. The HER2 binder according to claim 1.

4. HER2 conjugate a) It is singular; or b) It is multispecific and specifically binds to HER2 and different antigens. The HER2 binder according to claim 1.

5. The HER2 conjugate according to claim 1, wherein the HER2 conjugate comprises a fusion protein containing a HER2-binding VHH domain and a heterologous sequence, wherein the fusion protein optionally contains an Fc region, or optionally the fusion protein is a chimeric antigen receptor (CAR).

6. HER2 conjugate a) comprising an N-terminal amino acid residue or polypeptide sequence, wherein the N-terminal residue or sequence optionally improves protein expression, solubility, purification, half-life or avidity, and optionally the N-terminal residue or sequence is a Met-Ala or Ala leader peptide; and / or b) comprising a C-terminal amino acid residue or polypeptide sequence, wherein the C-terminal residue or sequence optionally promotes the selectivity, support, intracellular location, half-life, or avidity of the radiolabel. The HER2 binder according to claim 1.

7. The HER2 binder has the following structure: VHH-(Gly n Xaa m Gly k ) j [In the formula, i) VHH is selected from sequence numbers 1-81 and 205; ii) n = 0 to 5; iii) m = 0 to 5; iv) k = 0 to 6; v)j=0~8; and vi) Xaa is an amino acid that enables site conjugation or direct radiolabeling to a radiolabeled prosthetic group. A polypeptide sequence having The HER2 binder according to claim 1.

8. An isolated nucleic acid encoding a HER2 binder according to any one of claims 1 to 7.

9. A vector comprising the nucleic acid according to claim 8, wherein the vector is optionally an expression vector.

10. A host cell comprising the nucleic acid described in claim 8, wherein the host cell is optionally a eukaryotic cell or a prokaryotic cell, and further optionally the eukaryotic cell is a mammalian cell or a yeast cell.

11. The HER2 conjugate according to any one of claims 1 to 7, wherein the HER2 conjugate is conjugated with a label, and optionally the label is a fluorescent dye, a radionuclide, an enzyme, a toxin, or a chemotherapeutic agent.

12. The label is a radionuclide, and the radionuclide can be used as desired. a) Upon request, the following: 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; A radioactive halogen isotope selected from; or b) Upon request, the following: 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, Radioactive metal isotopes selected from That is, The HER2 binder according to claim 11.

13. The HER2 binder according to any one of claims 1 to 7, wherein the HER2 binder is conjugated to a label via a chelate moiety, the chelate moiety is optionally covalently bound to a protein via a lysine or cysteine ​​residue, and the label optionally forms a complex with a metal, the complex being chelated by the chelate moiety.

14. The HER2 conjugate has the following structure: VHH-RLC [In the formula, VHH contains an amino acid sequence selected from SEQ ID NOs: 1-81 and 205. RLCs are radiolabeled chemicals used to directly or indirectly promote the binding of radionuclides to VHH. A targeted radiotherapy agent having the following formula 2: MC-Cm-L 4 -Cm-T (Formula 2) [In the formula, MC is the multi-seat metal chelate portion; Cm is a thiourea, amide, or thioether; L4 is selected from bonded, substituted or unsubstituted alkyl chains, substituted or unsubstituted alkenyl chains, substituted or unsubstituted alkynyl chains which may optionally have NH, CO, or S at one or both ends, and polyethylene glycol (PEG) chains; T is one of the following compounds: a) Formula 1: 【Chemistry 1】 (In the formula, X is either CH or N; L1 and L3 are independently selected from bonded, substituted or unsubstituted alkyl chains, substituted or unsubstituted alkenyl chains, substituted or unsubstituted alkynyl chains, and polyethylene glycol (PEG) chains; MMCM is the polymer conjugate portion; 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 containing at least three oxygen atoms, wherein L2 optionally contains a brush-edge enzyme-cleavable peptide; CG is one or more charged D- or L-amino acids selected from guanidine; PO₃H; SO₃H; arginine; phosphono / sulfophenylalanine, glutamic acid, aspartic acid, and lysine; Selected from a hydrophilic carbohydrate moiety; polyethylene glycol (PEG) chain; and Z-guanidine; Z is (CH 2)n; n is greater than 1; m is between 0 and 3; and Y is a radioactive halogen selected from the group consisting of an alkyl metal moiety, a boronic acid moiety, a boronic acid ester moiety, or 18 F, 75 Br, 76 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 131 I, and 211 At. Compounds in the form of prosthetic compounds or radioactive halogen precursors, or their pharmaceutically acceptable salts or solvates: b) a) compound, wherein the compound is a radioactive halogen precursor, where Y optionally 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 acid ester (B(OR)2), and R optionally contains a cyclic moiety or an aliphatic group; c) a) compound, wherein the compound is a prosthetic compound, where Y is a radioactive halogen selected from the group consisting of 18F, 75Br, 76Br, 77Br, 122I, 123I, 124I, 125I, 131I and 211At; d) The compound of a) in which MMCM is an active ester or (Gly) m, and m is 1 or greater; e) The compound of a) in which MMCM is selected from the group consisting of N-hydroxysuccinimide (NHS) ester, tetrafluorophenol (TFP) ester, pentafluorophenol (PFP), p-nitrophenol (PNP), isothiocyanate group, or maleimide group; f) Compound a) in which MMCM is Gly-Gly-Gly; g) Compound a) in which L₂ is (CH₂)P and p=1 to 6 in the formula; h) A compound of a) in which any brush-edge enzyme-cleavable peptide is selected from the group consisting of Gly-Lys, Gly-Tyr, and Gly-Phe-Lys; i) The following structure: 【Chemistry 2】 The compound of a) shown by; j) N-succinimidyl 3-guanidinomethyl-5-[131I]iodobenzoate (isoSGMIB), N-succinimidyl 3-[211At]astato-5-guanidinomethylbenzoate (isoSAGMB), maleimidoethyl 2-(guanidinomethyl)-5-iodobenzoate (isoMEGMIB), or maleimidoethyl 3-(guanidinomethyl)-5-astatobenzoate (isoMEGMAB), or a pharmaceutically acceptable salt or solvate thereof, the compound of i)] The following are examples of prosthetic compounds or radioactive halogen precursors, A HER2 binder according to any one of claims 1 to 7.

15. MC has a large ring structure, and as desired i) MC is selected from DOTA, TETA, NOTP, and NOTA; ii) MC is an acyclic polydentate ligand; or iii) The MC is selected from EDTA, EDTMP, and DTPA; iv) The compound is a radioactive halogen precursor wherein Y is optionally 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 acid ester (B(OR)2), and R is optionally a radioactive halogen precursor containing a cyclic moiety or an aliphatic group; or v) A prosthetic compound in which Y is a radioactive halogen selected from 18F, 75Br, 76Br, 77Br, 122I, 123I, 124I, 125I, 131I and 211At; or vi) The HER binder further comprises a metal bonded to MC, wherein the metal is optionally a radioactive metal selected from 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. The HER2 binder according to claim 14.

16. A pharmaceutical composition comprising a HER2 binder according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition according to claim 16 for treating a disease in a subject requiring treatment, wherein the disease is preferably cancer, and further preferably the cancer is HER2+; Subjects may, if desired, be scored as HER2(2+) or HER2(3+) by immunohistochemistry; or If desired, the cancer may be selected from breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, prostate cancer, lung cancer, NSCLC, bladder cancer, urothelial carcinoma, and epidermal carcinoma, and / or metastatic lesions arising therefrom, and if desired, the cancer may be a brain metastasis of breast cancer. The pharmaceutical composition according to claim 16.

18. A pharmaceutical composition comprising the targeted radiotherapy agent according to claim 14 for treating cancer in a subject requiring treatment.

19. In vitro HER2 + A method for killing cells, comprising contacting cells with a HER2 binder according to any one of claims 1 to 7, wherein the cells are optionally cancer cells, and further optionally the cancer cells are selected from breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, brain tumor, gastric cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, prostate cancer, lung cancer, NSCLC, bladder cancer, urothelial carcinoma, and / or metastatic lesions derived therefrom.

20. A diagnostic agent for diagnosing a disease or medical condition in a subject, comprising a HER2 conjugate according to any one of claims 1 to 7, wherein the disease is preferably cancer.

21. A HER2 binder in a subject comprising the HER2 binder according to any one of claims 1 to 7. + A drug for detecting cells, wherein the HER binding agent contains a detectable label. Here, the detection is for HER2 in the target. + The process includes detecting the binding of a labeled HER2-binding agent to cells, wherein the detection of the binding is determined to be related to HER2 + This indicates the presence of cells. Upon request, HER2 in the target + The detection of the binding of labeled HER2-binding agents to cells is the target of HER2 + Including imaging cells, Drugs.

22. A HER2 binding agent according to any one of claims 1 to 7, comprising HER2 in a subject + A drug for imaging cells, comprising a HER-binding agent with a detectable label, Upon request, HER2 in the subject + Imaging cells includes performing positron emission tomography (PET) scans or positron emission tomography / computed tomography (PET / CT) scans on a subject; If desired, HER2+ cells are HER2+ cancer cells; If desired, the subject may be a human, a non-human primate, or a mammal, and if desired, the subject may also be suffering from cancer. Drugs.

23. The following steps: a) A step of culturing the host cells according to claim 10 under conditions in which a HER2 conjugate is produced; and b) A step of recovering the HER2 binding agent produced by the host cell, A method for producing an HER2 binder containing [the specified ingredient].

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