EGFR-cMET targeting compounds and their uses

Abstract

Compounds, e.g., radioimmunoconjugates, comprising a chelating moiety or a metal complex thereof, a linker, and an antibody or antigen-binding fragment thereof that targets both EGFR and cMET, pharmaceutical compositions of such compounds, and methods of treating a medical condition, e.g., cancer, using such compounds or pharmaceutical compositions.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 291,910, filed December 20, 2021, the entire contents of which are incorporated by reference herein for all purposes. [Background technology]

[0002] Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. With the development of EGFR tyrosine kinase inhibitors (TKIs), the prognosis of advanced NSCLC has improved significantly in certain patients, but long-term survival of metastatic NSCLC patients remains rare. A recent study by Lin et al. showed that the 5-year survival rate of patients with EGFR-mutated metastatic NSCLC treated with erlotinib or gefitinib was only 14.6%. It has long been known that anti-EGFR TKIs are susceptible to resistance, the underlying resistance mechanism of which is the coexistence of EGFR mutations and cMET overexpression. One promising approach being clinically tested is amivantamab, a human EGFR-cMET bispecific antibody approved for the treatment of non-small cell lung cancer. Targeting the EGFR and cMET receptors increases the specificity of the treatment against cancer cells.

[0003] In general, the efficacy of antibodies depends on the number of target cells. Furthermore, drug resistance remains a major challenge facing targeted cancer therapy, with de novo and acquired resistance limiting the long-term efficacy of targeted therapy. Resistance mechanisms include secondary mutations, activation of oncogenic downstream signaling modules, upregulation of ligands, and amplification of alternative growth factor receptors.

[0004] Thus, there remains a need for improved therapeutic agents (eg, cancer therapeutic agents) that can target both EGFR and cMET without the above-mentioned drawbacks. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure relates to compounds (e.g., radioimmunoconjugates) that target both EGFR and cMET, pharmaceutical compositions thereof, and methods of using such compounds or pharmaceutical compositions to treat cancer. Unlike naked antibodies, radioimmunoconjugates do not need to block receptor function to achieve therapeutic efficacy, but instead release radioactive particles (e.g., alpha emitters) that target surrounding tumor cells within a limited range, thereby preventing off-target associated toxicity. EGFR-cMET targeting compounds (e.g., radioimmunoconjugates) used for EGFR and cMET overexpressing cancers exploit the ability of the EGFR-cMET complex to undergo antibody-induced internalization to specifically deliver targeted radionuclides into cancer cells. Monovalent binding of antibodies to either receptor on normal healthy tissues / cells does not result in internalization.

[0006] In certain embodiments, provided compounds (e.g., radioimmunoconjugates) exhibit increased excretion rates (e.g., after administration to a mammal) compared to some currently known radiotherapeutic agents while maintaining therapeutic efficacy. In some embodiments, faster excretion may limit the time the compound remains in the subject, thereby limiting off-target toxicity. Thus, in some embodiments, provided compounds exhibit reduced off-target toxicity.

[0007] In one aspect, a compound is provided that comprises the following structure, or a pharma- ceutically acceptable salt thereof: AL 1 -(L 2 ) n -B Formula I (In the formula, A is a chelating moiety or a metal complex thereof; B is an antibody or an antigen-binding fragment thereof; L 1is a bond, C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer from 1 to 5, inclusive; L 2 each independently represent the formula II: -X 1 -L 3 -Z 1 - Formula II wherein: X 1 is -C(O)NR 1 -*, -NR 1 C(O)-*, -C(S)NR 1 -*, -NR 1 C(S)-*, -OC(O)NR 1 -*, -NR 1 C(O)O-*, -NR 1 C(O)NR 1 -, -CH2-Ph-C(O)NR 1 -*, -NR 1 C(O)-Ph-CH2-*, -CH2-Ph-NH-C(S)NR 1 -*, -NR 1 C(S)-NH-Ph-CH2-*, -O-, or -NR 1 -, where "*" represents L 3 indicates the connection point to R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L 3 is an optionally substituted C-C 50 Alkyl or optionally substituted C-C 50 is heteroalkyl, Z 1 -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -# or -NR 2-#, where "#" indicates the point of attachment to B, and R 2 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; The antibody, or antigen-binding fragment thereof, comprises a first antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR); and a second antigen-binding domain capable of binding to cMET, The first antigen-binding domain comprises: i. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3, or a variant in which one, two or three amino acids in one or more of HCDR1, HCDR2, and HCDR3 are replaced with another amino acid; and ii. A light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6, or a variant in which one, two or three amino acids in one or more of LCDR1, LCDR2 and LCDR3 are replaced with another amino acid).

[0008] In some embodiments, variable A of formula I is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α,α',α",α'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane), DO ...PA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DOPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DOPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DOPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DOPA (1,4,7,10-tetraazacyclododecane-1,4, DOTA-GA anhydride (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1 ,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine -N,N'-diacetic acid), H2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H6phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N",N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), and porphyrin.

[0009] In certain embodiments, variable A of formula I is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or a metal complex thereof.

[0010] In some embodiments, the compound is represented by formula Ia or a metal complex thereof: [ka] (In the formula, Y 1 is -CH2OCH2(L 2) n -B, -C(O)(L 2 ) n -B or -C(S)(L 2 ) n -B and Y 2 is -CH2CO2H; or Y 1 is H and Y 2 , L 1 -(L 2 ) n In certain embodiments, Y 1 is H.

[0011] In some embodiments, L 1 teeth, [ka] and R L is hydrogen or -COH.

[0012] In certain embodiments, X 1 is -C(O)NR 1 -* or -NR 1 C(O)-*, where "*" is L 3 indicates the connection point to R 1 is H.

[0013] In certain embodiments, Z 1 is CH2.

[0014] In some embodiments, L 3 is (CH2CH2O) 2-20 In some embodiments, L 3 is (CH2CH2O) m (CH2) w In the formula, m and w each independently represent an integer of 0 to 10 (inclusive), and at least one of m and w is not 0.

[0015] In some embodiments, the metal complex comprises a metal selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, lanthanides, and actinides. 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 89 Zr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, and 229 The radionuclide includes a radionuclide selected from the group consisting of Th.

[0016] In some embodiments, variable A is a metal complex of a chelating moiety. In some such embodiments, the metal complex comprises a radionuclide. In some embodiments, the radionuclide is an alpha emitter, e.g., astatine-211 ( 211 At), Bismuth-212( 212 Bi), Bismuth-213(213 Bi), Actinium-225( 225 Ac), Radium-223( 223 Ra), lead-212( 212 Pb), Thorium-227( 227 Th), and terbium-149 ( 149 In some embodiments, the radionuclide is an alpha emitter selected from the group consisting of: 68 Ga, 111 In, 177 Lu, or 225 In some embodiments, the radionuclide is 225 Ac or its descendants.

[0017] In some embodiments, the compound of formula I comprises one of the following structures, or a metal complex thereof: [ka]

[0018] In some embodiments, the compound or a pharma- ceutically acceptable salt thereof comprises the following structure, or a metal complex thereof: [ka]

[0019] In some embodiments, with reference to Formula I, variable A is a metal complex of a chelating moiety, and the metal complex comprises a radionuclide. In certain embodiments, the radionuclide is 68 Ga, 111 In, 177 Lu, or 225 In certain embodiments, the radionuclide is 225 In certain embodiments, the radionuclide is astatine-211 ( 211 At), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), Actinium-225( 225 Ac), Radium-223( 223 Ra), lead-212(212 Pb), Thorium-227( 227 Th), and terbium-149 ( 149 In certain embodiments, the alpha emitter is selected from the group consisting of: 225 Ac or its descendants.

[0020] In some embodiments, with reference to an antibody or antigen-binding fragment thereof comprised in a compound of Formula I, the first antigen-binding domain comprises: i. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3; and ii. A light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6.

[0021] In some embodiments, with reference to an antibody or antigen-binding fragment thereof comprised in a compound of Formula I, the first antigen-binding domain comprises: a. a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; and b. A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:16.

[0022] In some embodiments, with reference to an antibody or antigen-binding fragment thereof comprised in a compound of Formula I, the second antigen-binding domain comprises: i. A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 17 HCDR2 having the amino acid sequence of SEQ ID NO: 18 HCDR3 having the amino acid sequence of SEQ ID NO: 19, or a variant in which one, two or three amino acids in one or more of HCDR1, HCDR2, and HCDR3 are replaced with another amino acid; and ii. A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 20 LCDR2 having the amino acid sequence of SEQ ID NO: 21 LCDR3 having the amino acid sequence of SEQ ID NO: 22, or a variant in which one, two or three amino acids in one or more of LCDR1, LCDR2 and LCDR3 are replaced with another amino acid).

[0023] In some embodiments, with reference to an antibody or antigen-binding fragment thereof comprised in a compound of Formula I, the second antigen-binding domain comprises: i. A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 17 HCDR2 having the amino acid sequence of SEQ ID NO: 18 HCDR3 having the amino acid sequence of SEQ ID NO: 19; and ii. A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 20 LCDR2 having the amino acid sequence of SEQ ID NO: 21 LCDR3 having the amino acid sequence of SEQ ID NO:22.

[0024] In some embodiments, with reference to an antibody or antigen-binding fragment thereof comprised in a compound of Formula I, the second antigen-binding domain comprises: a. a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31; and b. A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. A first antigen-binding domain capable of binding to an epidermal growth factor receptor (EGFR), comprising: i. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3; and ii. A light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6; and b. A second antigen-binding domain capable of binding to cMET, comprising: i. A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 17 HCDR2 having the amino acid sequence of SEQ ID NO: 18 HCDR3 having the amino acid sequence of SEQ ID NO: 19; and ii. A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 20 LCDR2 having the amino acid sequence of SEQ ID NO: 21 LCDR3 having the amino acid sequence of SEQ ID NO:22.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. a first heavy chain comprising a VH region of a first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof; b. a first light chain comprising a VL region of a first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof; c. a second heavy chain comprising a VH region of a second antigen-binding domain and a second heavy chain constant (CH) region or a fragment thereof; and d. A second light chain comprising a VL region of a second antigen binding domain and a second light chain constant (CL) region or a fragment thereof.

[0027] In some embodiments, the first and second CH regions each comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:33.

[0028] In some embodiments, the first and second heavy chains form a heterodimer, where optionally, one of the first and second heavy chains comprises a cysteine ​​(C) residue at position 354, a tryptophan (W) residue at position 366, and the other heavy chain comprises a cysteine ​​(C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368, where the numbering of the constant regions is according to the EU index.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof is a. a modified CH region comprising a substitution of a cysteine ​​amino acid for a native non-cysteine ​​amino acid; and b. The corresponding modified CL region, wherein the modified CL comprises a substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid. where i. the first heavy chain comprises a modified CH region and the first light chain comprises a corresponding modified CL region; or ii. the second heavy chain comprises a modified CH region and the second light chain comprises a corresponding modified CL region; Here, the substituted cysteine ​​in the modified CH region and the corresponding substituted cysteine ​​in the modified light chain are capable of forming a disulfide bond.

[0030] In some embodiments, the modified CH region comprises a substitution at position 126 for a native non-cysteine ​​amino acid to a cysteine ​​amino acid; the corresponding modified CL region comprises a substitution at position 121 for a native non-cysteine ​​amino acid to a cysteine ​​amino acid, where the numbering of the constant regions is according to the EU index.

[0031] In some embodiments, the first and / or second CH region comprises a mutation that reduces or abolishes binding of the antibody, or antigen-binding fragment thereof, to one or more Fcγ receptors.

[0032] In some embodiments, the first and / or second CH region comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, where the numbering of the constant regions is according to the EU index.

[0033] In some embodiments, the compound of formula I is characterized by: a. the first CH region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:39; b. the second CH region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:40; c. the first CL region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:41; and d. The second CL region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:34.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. a first heavy chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:35; b. a second heavy chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:36; c. a first light chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:37; and d. A second light chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:38.

[0035] In some embodiments, the compound of formula I is characterized by: a. the first heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:35; b. the second heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:36; c. the first light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:37; and d. the second light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:38.

[0036] In some embodiments, the compound of formula I is characterized by: a. the first antigen-binding domain capable of binding to EGFR is capable of binding to cynomolgus monkey EGFR; b. the first antigen-binding domain capable of binding to EGFR is capable of binding to mouse EGFR; c. the second antigen-binding domain capable of binding to cMET is capable of binding to cynomolgus cMET; d. the first antigen-binding domain is specific for EGFR; e. the second antigen-binding domain is specific for cMET; f. the antibody or antigen-binding fragment thereof is capable of simultaneously binding to EGFR and cMET; g. the antibody or antigen-binding fragment thereof is capable of being internalized into the cell; h. the antibody or antigen-binding fragment thereof has cytotoxic activity as measured by an in vitro cell viability assay; and / or i. The antibody or antigen-binding fragment thereof is capable of blocking ligand-dependent signaling of EGFR and / or cMET.

[0037] In some embodiments, the compound of formula I is characterized in that the first antigen binding domain is capable of binding to human EGFR with an affinity having a Kd of: a.10-100nM; b. 20-80 nM; c. 30 to 75 nM; or d.35~50nM.

[0038] In some embodiments, the compound of formula I is characterized in that the second antigen binding domain is capable of binding to human cMET with an affinity having a Kd of: a. less than 10 nM; or b. Less than 5 nM.

[0039] In some embodiments, the compound of formula I is characterized in that the first antigen-binding domain is capable of binding to human EGFR with an affinity lower than the affinity of an antigen-binding domain comprising the heavy chain variable region sequence and the light chain variable region sequence of antibody QD6 (set forth in SEQ ID NOs: 47 and 48, respectively).

[0040] In some embodiments, the binding affinity is measured by surface plasmon resonance.

[0041] In some embodiments, the compound of formula I has the following structure: [ka] wherein [ka] is an antibody or antigen-binding fragment thereof that is capable of specifically binding to both EGFR and cMET (e.g., the EGFR-cMET monoclonal bispecific antibody RAA22 / B09 DuetMab described below). In some embodiments, the antibody or antigen-binding fragment thereof is linked to AL via the side chain amino group of a lysine residue. 1 -(L 2 ) n - is linked to.

[0042] In another aspect, the present disclosure also relates to pharmaceutical compositions comprising one of the compounds described above and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0043] Further within the scope of the present invention is a method of treating cancer comprising administering to a subject (eg, a human) in need of treatment a therapeutically effective amount of any of the above compounds or their respective compositions.

[0044] In some embodiments, the cancer is a solid tumor cancer selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing's sarcoma, gallbladder cancer, glioma, head and neck cancer (e.g., head and neck squamous cell carcinoma or HNSCC), liver cancer, lung cancer (e.g., non-small cell lung cancer or NSCLC), neuroblastoma, neuroendocrine carcinoma, ovarian cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma or PDAC), gastric cancer, prostate cancer, renal cell carcinoma, salivary adenoid cystic carcinoma, spermatocytic seminoma, and uveal melanoma.

[0045] In some embodiments, the cancer is lung cancer, colon cancer, pancreatic cancer, or head and neck cancer.

[0046] In some embodiments, the treatment methods of the invention further comprise administering an anti-proliferative agent, a radiosensitizer, an immunomodulatory agent, or an immunomodulatory agent to a subject (e.g., a human) in need thereof.

[0047] Also within the scope of the disclosure is a compound or pharmaceutical composition as described above for use in a method for the treatment of cancer.

[0048] The invention further includes the use of the compound or pharmaceutical composition in the manufacture of a medicament for the treatment of cancer. [Brief description of the drawings]

[0049] [Figure 1A-1B] 1A and 1B are graphical representations of the RAA22 / B09-57 and QD6 / B09-57 DuetMabs, respectively. The anti-EGFR RAA22 and QD6 Fabs, the anti-cMETB09-57 Fab, and the hole and knob heavy chains are shown. The structural rendering is a composite of the individual domain structures. [Diagram 2] Figure 2 shows that simultaneous binding studies using antigen capture configuration were performed by Octet analysis. Sensors loaded with human cMET antigen were exposed to sequential association and dissociation interactions first with antibody and then with human EGFR antigen. Ass = association; Diss = dissociation; NI-NTA = nickel nitrilotriacetic acid. [Figure 3A] FIG. 3A shows ELISA results showing cross-reactivity of EGFR and c-Met species. The high affinity monospecific EGFR IgG, QD6 and the monovalent bispecific EGFR / cMET DuetMAb, QD6 / B09 bound human, cynomolgus, and mouse EGFR. The reduced affinity monospecific EGFR IgG, RAA22, bound weaker to human, cynomolgus, and mouse EGFR compared to QD6, and the corresponding monovalent bispecific EGFR / c-Met DuetMAb, RAA22 / B09, bound even weaker to human and cynomolgus EGFR and only marginally to mouse EGFR compared to the bivalent parent IgG, RAA22. The monospecific c-Met IgG, B09, and all bispecific variants showed comparable binding to human and cynomolgus c-Met, but no detectable binding to mouse c-Met. [Figure 3B]Figure 3B shows the results of an ELISA demonstrating EGFR and c-Met family specificity: None of the antibodies tested showed any appreciable binding to any of the EGFR HER family proteins (HER2, HER3, or HER4) or c-Met family members (Ron (CD136) or Semaphorin 3a). [Figure 4] Figure 4 shows that internalization and trafficking of RAA22 / B09 bispecific mAb to acidified intracellular compartments was visualized using antibodies labeled with pHAb pH-sensitive dye (Promega). Control antibodies included the R347 isotype control and monovalent bispecific control antibodies anti-EGFR RAA22 / R347 and anti-cMET B09 / R347. pHAb-labeled antibodies were incubated with NCI-H1975 lung cancer cells at a concentration of 1.25 μg / mL in a humidified incubator at 37°C and 5% CO2. Fluorescence images were captured at the indicated time points with an Operetta High Content Imaging system using a Cy3 filter. An increase in cell fluorescence intensity over time was taken as evidence of internalization and trafficking to acidic intracellular compartments as measured by pH-sensitive fluorescence. [Diagram 5] FIG. 5 shows that internalization and trafficking of RAA22 / B09 bispecific mAb to acidified intracellular compartments was visualized using antibody labeled with pHAb pH-sensitive dye, but cells were treated with a lower concentration of antibody, 0.625 μg / mL. [Figure 6A]Figure 6A shows the kinetics of internalization of QD6 / B09 and RAA22 / B09 monoclonal antibodies (mAbs) in H1975 cells. (a) Image overlays at the start of internalization and 1 hour after labeling cells with CellTracker Blue CMAC for the cytoplasm (blue) and 2.5 μg / mL of QD6 / B09-AlexaFluor647 (magenta, upper panel) or 2.5 μg / mL of RAA22 / B09 AlexaFluor-647 (magenta, lower panel). Cells were labeled with CellTracker Blue CMAC and then bound with mAbs-AlexaFluor6457 at 2-8 °C and subjected to internalization conditions: (37 °C, 70% humidity, 5% CO2). [Figure 6B] Figure 6B shows the kinetics of internalization of QD6 / B09 and RAA22 / B09 monoclonal antibodies (mAbs) in H1975 cells. Time course of internalization of mAb-AlexaFluor647 determined by quantitative analysis of kinetic images using an algorithm (Materials and Methods). Kinetic images taken at 5 min intervals were processed using an algorithm (Vainshtein, 2015) to determine the accumulation of antibody in the cytoplasm. Cytoplasmic antibody signal normalized to intracellular antibody fluorescence (percentage of cytoplasm) is shown for QD6 / B09-AlexaFluor647 (red) vs. RAA22 / B09-AlexaFluor647 (blue) for one of three independent experiments. The internalization rate constant (kint) was calculated from the internalization time course using curve fitting with the equation Fcyt(t) = (1-e-kint.t)Fmax,cyt, where Fmax,cyt is the maximum ratio of cytoplasmic intensity per cell to total intensity per cell. The T1 / 2 calculated from kint was 37.5 ± 10.6 min for QD6 / B09 and 43.2 ± 15.5 min for RAA22 / B09 (n = 3). [Figure 7A] Figure 7A shows the internalization profile of the QD6 / B09 DuetMab and its respective single-arm control antibody. The internalization profile is represented by the time course of the respective membrane and cytoplasmic signals of each construct. The QD6 / B09 set was acquired using an Opera confocal fluorescence microscope. [Figure 7B] FIG. 7B shows the internalization profile of the RAA22 / B09 DuetMab and its respective single-arm control antibody. The internalization profile is displayed by the time course of the respective membrane and cytoplasmic signals of each construct. This set was acquired using a Zeiss spinning disk confocal fluorescence microscope. Identical profiles of QD6 / B09 and QD6 / IgG indicate an internalization mode driven by the EGFR arm of the QD6 / B09 DuetMab, whereas the RAA22 / B09 DuetMab requires the engagement of both the EGFR and c-MET arms for efficient internalization. [Figure 8A] Figure 8A shows the internalization profile of RAA22 / B09 in cells expressing moderate and high target c-MET and EGFR cell surface receptors. Membrane, cytoplasmic, and total signals of RAA22 / B09-AF647 in H1975 cells are shown. One representative experiment of 2 is shown. H1975 cells show a concomitant decrease in total and membrane intensity, indicating dissociation of the antibody from the cell surface. [Figure 8B] Figure 8B is equivalent to Figure 8A, but in HCC827 cells, which have a stable total signal over the course of the experiment. The decrease in membrane signal is due to antibody internalization. [Figure 9A] Figure 9A shows the internalization of RAA22 / B09 single arm control antibody in HCC827 cells. The intensity profile of RAA22 / IgG single arm is approximately 10-fold lower than RAA22 / B09 due to weaker binding to EGFR by the single arm conjugate. [Figure 9B] FIG. 9B shows that the B09 / IgG single arm dissociates from the cell membrane as indicated by the concomitant decrease in total and membrane signals over time. [Figure 10A] FIG. 10A is a schematic diagram showing the general structure of a bifunctional chelate, including a chelate, a linker, and a bridging group. [Figure 10B] FIG. 10B is a schematic diagram showing the general structure of a bifunctional conjugate comprising a chelate, a linker, and a targeting moiety. [Fig. 10C-10D] 10C and 10D are schematic diagrams showing the structures of two exemplary EGFR-cMET radioimmunoconjugates disclosed herein, [177Lu]-DOTA-anti-EGFR-cMET and [225Ac]-DOTA-anti-EGFR-cMET. [Figure 11] 11 is a schematic diagram showing the synthesis of the bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B). The synthesis of Compound B is described in Example 6. [Figure 12] 12 is a schematic diagram showing the synthesis of the bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C). The synthesis of Compound C is described in Example 7. [Figure 13] 13 is a schematic diagram showing conjugation and radiolabeling for the synthesis of [177Lu]-Compound C-Anti-EGFR-cMET conjugates (e.g., Compound E and Compound F below). See Example 8. [Figure 14A-14B] 14A-14B show the binding of Compound E to various cell lines with different expression levels of EGFR and c-MET in the absence (total binding) or presence (non-specific binding) of 4 μM unlabeled antibody. Specific binding was calculated by subtracting non-specific binding from total binding. Values ​​shown are mean ± SEM (n=3). See Example 9. [Figure 15] Figure 15 shows the results of internalization (or in vitro retention) after incubation with compound E (10 nM) for 2 hours and 24 hours in HCC827 cells, HT29 cells, H441 cells, and H1975 cells with different expression levels of EGFR and c-MET. Values ​​shown are mean ± SEM (n=3). See Example 10. [Figures 16A-16E] Figures 16A-16E show plots representing the results of biodistribution studies in various animal models, in which compound E (0.7MBq / 2μg) was injected into female balb / c mice (n=3) with subcutaneous xenografts at specific time points after intravenous injection. The percentage of injected dose per gram of tissue (%ID / g) is plotted on the x-axis and shown for blood, bone, brain, heart, intestine, kidney, lung, liver, pancreas, spleen, stomach, skin, urine and bladder, and tumor at 4, 24, 48, 96, 168 hours. See Example 11. [Figures 17A-17C] 17A-17C show plots representing the results of in vivo efficacy testing in various animal models of female balb / c mice (n=5) after intravenous injection of compound F (50-400 nCi / 2 μg), cold antibody, and vehicle. See Example 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Radioimmunoconjugates are designed to target proteins or receptors that are upregulated in disease states and deliver a radioactive payload to damage and kill cells of interest (radioimmunotherapy). Delivery of the radioactive payload results in targeted emission of alpha, beta, gamma particles, or Auger electrons, which can have direct effects on DNA (such as single- or double-stranded DNA breaks) or indirect effects such as bystander or crossfire effects.

[0051] A radioimmunoconjugate typically contains a biological targeting moiety (e.g., an antibody or antigen-binding fragment thereof capable of specifically binding to both EGFR and cMET), a radionuclide (e.g., an alpha or beta emitter), and a molecule that links the two. The conjugate is formed when a bifunctional chelate is added to the biological targeting moiety such that structural changes are minimized while maintaining target affinity. Once radiolabeled, the final radioimmunoconjugate is formed.

[0052] Bifunctional chelates structurally contain a chelate, a linker, and a bridging group (Figure 10A). When developing new bifunctional chelates, most efforts are focused on the chelating portion of the molecule. Several examples of bifunctional chelates with various cyclic and acyclic structures conjugated to targeting moieties have been described. [Bioconjugate Chem.2000,11,510-519;Bioconjugate Chem.2012,23,1029-1039;Mol Imaging Biol.2011,13,215-221,Bioconjugate Chem.2002,13,110-115.]

[0053] One of the key elements for developing a safe and effective radioimmunoconjugate is to maximize efficacy while minimizing off-target toxicity in normal tissues. Although this is one of the main tenets of new drug development, its application in radioimmunotherapy comes with new challenges. To have a therapeutic effect, radioimmunoconjugates do not need to block receptors, as is required for therapeutic antibodies, nor do they need to release a cytotoxic payload inside cells, as is required for antibody-drug conjugates ("ADCs"). However, the release of toxic particles is an event that occurs as a result of the primary (radioactive) decay and can occur randomly anywhere in the body after administration. When release occurs, damage can occur to surrounding cells within the range of release, which can result in off-target toxicity. Thus, limiting the exposure of normal tissues to these radiations is key to developing new therapeutic radioimmunoconjugates.

[0054] One potential way to reduce off-target exposure is to more effectively remove radioactivity from the body (e.g., normal tissues in the body). One mechanism is to increase the clearance rate of the biological targeting agent. This approach would require identifying ways to reduce the half-life of the biological targeting agent, which has not been well described for biological targeting agents. Regardless of the mechanism, increased drug clearance also negatively impacts pharmacodynamics / efficacy in that the drug is cleared from the body more quickly, reducing the effective concentration at the site of action, which may require a higher total dose, which defeats the desired outcome of reducing the total radioactive dose to normal tissues.

[0055] Other efforts have focused on facilitating metabolism of the portion of the molecule that contains the radioactive moiety. To this end, some efforts have been made to increase the rate of cleavage of radioactivity from biological targeting agents using what are called "cleavable linkers". However, cleavable linkers have been interpreted differently in the context of radioimmunoconjugates. Cornelissen et al. describe a cleavable linker as a bifunctional chelate attached to the biological targeting agent via a reduced cysteine, while other researchers describe the use of an enzyme-cleavable system in which the radioimmunoconjugate must be released in conjunction with a cleaving agent / enzyme [MolCancerTher.2013,12(11),2472-2482;Methods Mol Biol.2009,539,191-211;Bioconjug Chem.2003,14(5),927-33]. These methods are not practical from a drug development point of view (enzymatically cleavable systems) since they either alter the nature of the biological targeting moiety, in the case of cysteine ​​linkage, or, in the case of the cited description, require the administration of two drugs.

[0056] The present disclosure provides, inter alia, compounds, e.g., radioimmunoconjugates, that are more effectively eliminated from the body following catabolism and / or metabolism, thereby more effectively eliminating radioactivity from the body while maintaining therapeutic efficacy. This unexpected advantage is achieved by making modifications to the linker region of the bifunctional chelate.

[0057] The disclosed immunoconjugates, in some embodiments, may achieve reduced total body radioactivity when compared to known bifunctional chelates, for example, by increasing the degree of excretion of catabolic / metabolic products while maintaining the pharmacokinetics of the intact molecule. In some embodiments, this reduction in radioactivity occurs through clearance of catabolic / metabolic by-products without affecting other in vitro and in vivo properties, such as binding specificity (in vitro binding), cellular retention, and in vivo tumor uptake. Thus, in some embodiments, the provided compounds achieve reduced radioactivity in the human body while maintaining on-target activity.

[0058] definition As used herein, the term "bind" or "binding" of a targeting moiety refers to at least a transient interaction or association with a target molecule described herein (e.g., human EGFR-cMET).

[0059] As used herein, the term "bifunctional chelate" refers to a compound comprising a chelate, a linker, and a bridging group. See, e.g., Figure 10A. A "bridging group" is a reactive group capable of linking two or more molecules, e.g., covalently linking a bifunctional chelate to a targeting moiety.

[0060] The term "bifunctional conjugate" as used herein refers to a compound that includes a chelate or metal complex thereof, a linker, and a targeting moiety, such as an antibody or antigen-binding fragment thereof. See, e.g., Figure 10B.

[0061] The term "cancer," as used herein, refers to any cancer caused by the proliferation of malignant tumor cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. In some embodiments, a cancer of the present disclosure comprises cells (e.g., tumor cells) that express EGFR and cMET, such as, but not limited to, lung cancer, colon cancer, pancreatic cancer, or head and neck cancer.

[0062] The term "chelate" as used herein refers to an organic compound or portion thereof that can bind to a central metal or radioactive metal atom at two or more points.

[0063] The term "conjugate," as used herein, refers to a molecule containing a chelating group or a metal complex thereof, a linker group, and optionally a targeting moiety, such as an antibody or antigen-binding fragment thereof.

[0064] As used herein, unless otherwise indicated, the phrase "constant region", when used in reference to an antibody or fragment thereof (e.g., an IgG1, IgG2, or IgG4 constant region), is intended to encompass both wild-type constant regions and variants (e.g., constant regions having at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to a reference sequence of a wild-type constant region).

[0065] As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.

[0066] The compounds listed or described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are contemplated. Compounds containing asymmetrically substituted carbon atoms discussed in this disclosure can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or stereoselective synthesis.

[0067] As used herein, "detection agent" refers to a molecule or atom that is useful for diagnosing disease by locating cells that contain antigens. Various methods of labeling polypeptides with detection agents are known in the art. Examples of detection agents include, but are not limited to, radioisotopes and radionuclides, dyes (such as biotin-streptavidin complexes), contrast agents, luminescent agents (such as fluorescein isothiocyanate or FITC, rhodamine, lanthanide fluorophores, cyanines, and near-infrared dyes), and magnetic agents such as gadolinium chelates.

[0068] As used herein, the term "radionuclide" refers to an atom capable of undergoing radioactive decay (e.g., 3 H, 14 C. 15 N, 18 F, 35 S, 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In,123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, 229 Th, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 Tl). The terms radionuclide, radioisotope, or radioisotope may also be used to describe radionuclides. As described herein, radionuclides may be used as detection agents. In some embodiments, radionuclides may be used as therapeutic agents, e.g., alpha-emitter radionuclides.

[0069] The term "effective amount" of an agent (e.g., any of the conjugates described above), as used herein, is an amount sufficient to produce a beneficial or desired result, such as a clinical result, and thus, "effective amount" varies depending on the context in which it is applied. For example, in therapeutic applications, an "effective amount" may be an amount sufficient to cure or at least partially suppress the symptoms of a disorder and its complications, and / or an amount sufficient to substantially ameliorate at least one symptom associated with a disease or condition. Typically, an "effective amount" in the context of this disclosure is an amount of a compound (e.g., a radioimmunoconjugate) disclosed herein, such as an Ac-225 radioimmunoconjugate, that produces at least some measurable therapeutic response or desired effect in a portion of patients to which it is administered. For example, in the treatment of cancer, an agent or compound that reduces, prevents, delays, inhibits, or stops the symptoms of a disease or condition may be therapeutically effective. A therapeutically effective amount of a drug or compound is not required to cure a disease or condition, but may provide treatment for a disease or condition, such as to delay, hinder, or prevent the onset of the disease or condition, ameliorate the symptoms of the disease or condition, or alter the duration of the disease or condition. For example, the disease or condition may be less severe and / or recovery may be promoted in an individual. An effective amount may be administered by administration of a single dose or multiple (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6) doses.

[0070] The term "immunoconjugate," as used herein, refers to a conjugate that includes a targeting moiety, such as an antibody (or antigen-binding fragment thereof), a nanobody, an affibody, or a consensus sequence from a fibronectin type III domain. In some embodiments, the immunoconjugate includes an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8 conjugates per targeting moiety).

[0071] As used herein, the term "radioconjugate" refers to any conjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

[0072] The term "radioimmunoconjugate" as used herein refers to any immunoconjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein. The radioimmunoconjugates provided in this disclosure typically refer to bifunctional conjugates that include a metal complex formed from a radioisotope or radionuclide.

[0073] The term "radioimmunotherapy" as used herein refers to a method of using a radioimmunoconjugate to produce a therapeutic effect. In some embodiments, radioimmunotherapy involves administration of a radioimmunoconjugate to a subject in need thereof, where administration of the radioimmunoconjugate produces a therapeutic effect in the subject. In some embodiments, radioimmunotherapy involves administration of a radioimmunoconjugate to a cell, where administration of the radioimmunoconjugate kills the cell. Radioimmunotherapy involves selective killing of cells, and in some embodiments, the cells are cancer cells in a subject with cancer.

[0074] The term "pharmaceutical composition" as used herein refers to a composition containing a radioimmunoconjugate as described herein formulated with a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a government regulatory agency as part of a treatment regimen for the treatment of a disease in a mammal. The pharmaceutical composition can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation as described herein.

[0075] "Pharmaceutically acceptable excipient" as used herein refers to any ingredient other than the compounds described herein (e.g., a vehicle in which an active compound can be suspended or dissolved) that has the properties of being non-toxic and non-inflammatory in patients. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, injection agents (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotectants, adsorbents, suspending or dispersing agents, sweeteners, or water of hydration. Representative excipients include, but are not limited to, ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0076] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound described herein that is suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic reaction within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or can be prepared separately by reacting the free base group with a suitable organic acid.

[0077] The compounds of the present invention may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or in the case of the acidic form of the compounds of the present invention, salts may be prepared from inorganic or organic bases. In many cases, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid to form acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, etc. to form base salts. Methods for preparing suitable salts are well established in the art.

[0078] Representative acid addition salts include, inter alia, acetate, adipate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of suitable salts include sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0079] The term "polypeptide" as used herein refers to a series of at least two amino acids linked together by a peptide bond. In some embodiments, a polypeptide comprises at least 3-5 amino acids, each of which is linked to another amino acid by at least one peptide bond. One of skill in the art will appreciate that a polypeptide may include one or more "unnatural" amino acids or other entities that may nevertheless be incorporated into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may include one or more covalently attached sugar moieties. In some embodiments, a single "polypeptide" (e.g., an antibody polypeptide) may include two or more individual polypeptide chains, which may optionally be linked together, e.g., by one or more disulfide bonds or other means.

[0080] "Subject" means a human or non-human animal (eg, a mammal).

[0081] "Substantial identity" or "substantially identical" refers to a polypeptide sequence that has the same polypeptide sequence as a reference sequence, or a polypeptide sequence that has the same specified percentage of amino acid residues at corresponding positions in the reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the reference amino acid sequence. For polypeptides, the length of the comparison sequence will usually be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 consecutive amino acids (e.g., full-length sequence). Sequence identity may be measured using sequence analysis software with default settings (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software can match similar sequences by assigning degrees of homology for various substitutions, deletions, and other modifications.

[0082] As used herein and as well understood in the art, "treating" a condition (e.g., a condition described herein, such as cancer) or "treatment" of a condition is an approach to obtain a beneficial or desired result, such as a clinical result. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable; reduction in the extent of a disease, disorder or condition; a state in which a disease, disorder or condition is stabilized (i.e., not worsening); preventing the spread of a disease, disorder or condition; delaying or slowing the progression of a disease, disorder or condition; amelioration or palliation of a disease, disorder or condition; and remission (whether partial or complete). "Alleviating" a disease, disorder, or condition means that the extent and / or undesirable clinical findings of a disease, disorder, or condition are reduced and / or the course of progression is slowed or prolonged compared to the extent or course in the absence of treatment.

[0083] As used herein, the term "about" or "approximately" when used in reference to a quantitative value includes the stated quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" or "approximately" refers to a ±10% variation from the stated quantitative value, unless otherwise specified or inferred from the context.

[0084] As used herein, the term "targeting moiety" refers to any molecule or any portion of a molecule capable of binding to a given target. The term "EGFR-cMET targeting moiety" refers to a targeting moiety (e.g., an antibody or antigen-binding fragment thereof) capable of binding to both EGFR and cMET, such as an anti-EGFR-cMET antibody.

[0085] As used herein, the term "fragment," when used to refer to an EGFR or cMET fragment, refers to an N-terminally and / or C-terminally truncated EGFR or cMET, or protein domains thereof. Unless otherwise indicated, fragments of EGFR or cMET used in accordance with the embodiments described herein retain the ability of full-length EGFR or cMET to be recognized and / or bound by the EGFR-cMET targeting moieties described in this disclosure.

[0086] Use of the terms "a," "an," and "the," and similar referents in the context of describing this disclosure (particularly in the context of the claims), should be construed to encompass both the singular and the plural, unless otherwise indicated in the specification or clearly contradicted by context.

[0087] Unless otherwise stated or clear from the context, as used herein, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0088] As used herein, the term "and / or" is to be interpreted as a specific disclosure of each of the specified features or components with or without the other.

[0089] The terms "comprise," "have," "include," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The term "consisting of" should be construed as open-ended.

[0090] As used herein, "antibody" refers to a polypeptide, including immunoglobulins and fragments thereof, whose amino acid sequence specifically binds to a designated antigen or fragment thereof. Antibodies according to the invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). One of skill in the art will understand that a characteristic sequence or portion of an antibody can include amino acids found in one or more regions of an antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). Furthermore, one of skill in the art will understand that a characteristic sequence or portion of an antibody can include one or more polypeptide chains and can include sequence elements found in the same polypeptide chain or different polypeptide chains.

[0091] As used herein, an "antigen-binding fragment" refers to a portion of an antibody that retains the specificity of the binding characteristic of the parent antibody.

[0092] The antibodies or antigen-binding fragments thereof of the present disclosure may be isolated and / or substantially purified.

[0093] Compounds such as immunoconjugates or radioimmunoconjugates In one aspect, the disclosure provides a compound, e.g., an immunoconjugate or radioimmunoconjugate, comprising the following structure, or a pharma- ceutically acceptable salt thereof: AL 1 -(L 2 ) n -B Formula I (In the formula, A is a chelating moiety or a metal complex thereof; B is an antibody or antigen-binding fragment thereof capable of binding to both EGFR and cMET, the antibody or antigen-binding fragment thereof having the characteristics disclosed herein; L 1is a bond, C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer from 1 to 5, inclusive; L 2 each independently represent the formula II: -X 1 -L 3 -Z 1 - Formula II wherein: X 1 is -C(O)NR 1 -*, -NR 1 C(O)-*, -C(S)NR 1 -*, -NR 1 C(S)-*, -OC(O)NR 1 -*, -NR 1 C(O)O-*, -NR 1 C(O)NR 1 -*, -CH2-Ph-C(O)NR 1 -*, -NR 1 C(O)-Ph-CH2-*, -CH2-Ph-NH-C(S)NR 1 -*, -NR 1 C(S)-NH-Ph-CH2-*, -O-*, or -NR 1 -*, where "*" is L 3 indicates the connection point to R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, or optionally substituted aryl or heteroaryl; L 3 is an optionally substituted C-C 50 Alkyl or optionally substituted C-C 50 is heteroalkyl, Z 1 -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -# or -NR 2-#, where "#" indicates the point of attachment to B, and R 2 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0094] Exemplary substituents for alkyl, heteroalkyl, aryl, or heteroaryl include halo (e.g., F, Cl, Br, I), OH, CN, nitro, amino, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, C 1-6 Heteroalkyl, C 1-6 These include, but are not limited to, heterocycloalkyl, haloalkyl (eg, CF3), alkoxy (eg, OCH3), alkylamino (eg, NH2CH3), sulfonyl, aryl, and heteroaryl.

[0095] In some embodiments, the compound (e.g., an immunoconjugate or radioimmunoconjugate) has the structure shown below, or a metal complex thereof: [ka] where B is an EGFR-cMET antibody or antigen-binding fragment thereof disclosed herein.

[0096] In some embodiments, the compound of formula I has the following structure: [ka] or a metal complex thereof.

[0097] In some embodiments, provided compounds (e.g., immunoconjugates or radioimmunoconjugates) are capable of binding to different cell lines with different EGFR and cMET expression levels with Kd values ​​of up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 12.5 nM, up to about 10 nM, up to about 7.5 nM, up to about 7 nM, up to about 6.5 nM, up to about 6 nM, up to about 5 nM, up to about 4 nM, up to about 3.5 nM, up to about 3 nM, or up to about 2.5 nM. In some embodiments, provided compounds (e.g., immunoconjugates or radioimmunoconjugates) are capable of binding to different cell lines with different EGFR and cMET expression levels with Kd values ​​of about 15 nM, about 12.5 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4 nM, about 3.5 nM, about 3 nM, or about 2.5 nM.

[0098] In some embodiments, as further described herein, the compound (e.g., an immunoconjugate or radioimmunoconjugate) comprises a chelating moiety or a metal complex thereof, which may comprise a radionuclide. In some such compounds, the average or median ratio of the chelating moiety to the EGFR-cMET targeting moiety (e.g., an EGFR-cMET antibody) is 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or about 1. In some compounds, the average or median ratio of the chelating moiety to the EGFR-cMET targeting moiety (e.g., an EGFR-cMET antibody) is about 1.

[0099] In some embodiments, after a radioimmunoconjugate is administered to a mammal, the percentage of radiation excreted by the intestinal route, the renal route, or both (of the total amount of radiation administered) is greater than the percentage of radiation excreted by a comparable mammal administered a reference radioimmunoconjugate. "Reference immunoconjugate" refers to a known radioimmunoconjugate that differs from a radioimmunoconjugate described herein in at least (1) having a different linker; (2) having a targeting moiety of a different size; and / or (3) lacking a targeting moiety. In some embodiments, a reference radioimmunoconjugate is a known radioimmunoconjugate that differs from a radioimmunoconjugate described herein in at least (1) having a different linker; (2) having a targeting moiety of a different size; and / or (3) lacking a targeting moiety. 90Y]-Ibritumomab tiuxetan (Zevalin ( 90 Y)) and [ 111 In]-Ibritumomab tiuxetan (Zevalin ( 111 In) is selected from the group consisting of

[0100] In some embodiments, the fraction of radiation excreted by a given route or set of routes is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the fraction of radiation excreted by the same route by a comparable mammal administered a reference radioimmunoconjugate. In some embodiments, the fraction of radiation excreted is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the fraction of radiation excreted by a comparable mammal administered a reference radioimmunoconjugate. The extent of excretion can be measured by methods known in the art, for example, by measuring radioactivity in urine and / or feces and / or by measuring total body radioactivity over a period of time, see also, for example, WO 2018 / 024869.

[0101] In some embodiments, the extent of excretion is measured at least or about 12 hours after administration, at least or about 24 hours after administration, at least or about 2 days after administration, at least or about 3 days after administration, at least or about 4 days after administration, at least or about 5 days after administration, at least or about 6 days after administration, or at least or about 7 days after administration.

[0102] In some embodiments, after the compound (e.g., an immunoconjugate or radioimmunoconjugate) is administered to a mammal, the compound (e.g., an immunoconjugate or radioimmunoconjugate) exhibits reduced off-target binding effects (e.g., toxicity) compared to a reference compound (e.g., a reference conjugate, e.g., a reference immunoconjugate such as a reference radioimmunoconjugate). In some embodiments, this reduced off-target binding effect is characteristic of a compound (e.g., an immunoconjugate or radioimmunoconjugate) that also exhibits a higher excretion rate, as described herein.

[0103] targeting part A targeting moiety includes any molecule or any portion of a molecule capable of binding (e.g., capable of specifically binding, specifically binding, etc.) to a given target (e.g., EGFR or cMET, or EGFR-cMET). In some embodiments, the targeting moiety comprises a protein or polypeptide. In some embodiments, the targeting moiety is selected from the group consisting of an antibody or antigen-binding fragment thereof, a nanobody, an affibody, and a consensus sequence from a fibronectin type III domain (e.g., centrin or adnectin). In some embodiments, a moiety is both a targeting moiety and a therapeutic moiety, i.e., a moiety is capable of binding to a given target and also confers a therapeutic benefit.

[0104] In some embodiments, the targeting moiety has a molecular weight of at least 50 kDa, at least 75 kDa, at least 100 kDa, at least 125 kDa, at least 150 kDa, at least 175 kDa, at least 200 kDa, at least 225 kDa, at least 250 kDa, at least 275 kDa, or at least 300 kDa.

[0105] In some embodiments, the targeting moiety is capable of specifically binding to and inhibiting both EGFR and cMET. By "inhibit," it is meant that the targeting moiety at least partially inhibits one or more functions of EGFR, cMET, or both. In some embodiments, the targeting moiety impairs downstream signaling of EGFR, cMET, or both, e.g., inhibiting the proliferation of tumor cells with different expression levels of EGFR and cMET.

[0106] target EGFR Human EGFR (also known as proto-oncogene c-ErbB-1, receptor tyrosine protein kinase erbB-1 and EC 2.7.10.1) is a protein identified by UniProt P00533. Alternative splicing of the mRNA encoded by the human EGFR gene (also known as ERBB, ERBB1 and HER1) gives rise to four isoforms: isoform 1 (UniProt: P00533-1, v2 (last sequence update: November 1, 1997)); isoform 2 (UniProt: P00533-2, v1) (containing substitutions F404L and L405S relative to isoform 1 and lacking the amino acid sequence corresponding to positions 406-1210 in isoform 1); isoform 3 (UniProt: P00533-3, v1) (containing substitutions at positions 628-705 in isoform 1 and lacking the amino acid sequence corresponding to positions 706-1210 in isoform 1); and isoform 4 (UniProt: P00533-4) (containing substitution C628S relative to isoform 1 and lacking the amino acid sequence corresponding to positions 629-1210 in isoform 1).

[0107] The structure and function of EGFR are reviewed, for example, in Ferguson, Annu Rev Biophys. (2008) 37:353-373. EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family). The receptor contains a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain, and a C-terminal regulatory region. EGFR binding to a ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal regulatory region of EGFR.

[0108] Aberrant EGFR expression / activity has been implicated in many diseases, including nervous system disorders and many cancers.

[0109] As used herein, "EGFR" refers to any species of EGFR, including any species of EGFR isoforms, fragments, variants, or homologs.

[0110] cMet Human cMET, also known as hepatocyte growth factor receptor (HGFR) or tyrosine protein kinase Met, is a protein identified by UniProt P08581. Alternative splicing of the mRNA encoded by the human MET gene gives rise to three isoforms: isoform 1 (UniProt: P08581-1, v4 (last sequence update: July 7, 2009)); isoform 2 (UniProt: P08581-2) (inserted with the amino acid sequence "STWWKEPLNIVSFLFCFAS" at position 755 of isoform 1); and isoform 3 (UniProt: P08581-3), also known as soluble met variant 4, (which replaces the amino acid sequence corresponding to positions 755-764 of isoform 1 with "RHVNIALIQR" and further lacks the amino acid sequence corresponding to positions 765-1390 of isoform 1).

[0111] The structure of cMET has been reviewed, for example, in Gherardi, 2003, which is incorporated herein by reference in its entirety. cMET is a heterodimer composed of a disulfide-linked alpha (50 kDa) and beta (145 kDa) chain. cMET contains an N-terminal Sema domain that mediates binding to hepatocyte growth factor (HGF) and an intracellular kinase domain. Ligand binding at the cell surface induces autophosphorylation of cMET on its intracellular domain, which provides a docking site for downstream signaling molecules and activation of several signaling cascades.

[0112] cMET is expressed in normal tissues on the surface of epithelial cells. Overexpression of cMET has been observed in many human tumors and cancers and is often associated with a metastatic phenotype and poor prognosis. Examples of cancers in which high levels of cMET expression have been observed include non-small cell lung cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, breast cancer, and esophagogastric cancer. Co-expression of EGFR and cMET is commonly observed in these cancers.

[0113] Antibody or antigen-binding fragment thereof The term "antibody" describes an immunoglobulin, whether naturally produced or partially or wholly synthetically produced. The antibody may be a human or humanized antibody. The antibody is preferably a monoclonal antibody. Examples of antibodies include immunoglobulin isotypes, such as immunoglobulin G (IgG), their isotypic subclasses, such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof.

[0114] The term "antibody", as used herein, therefore includes antibody fragments, so long as they are shown to bind to the relevant target molecule. Examples of antibody fragments include Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies and single domain antibodies (e.g., VhH). Unless the context requires otherwise, the term "antibody", as used herein, therefore corresponds to "antibody or fragment thereof".

[0115] Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing the CDRs or variable regions of one antibody into a different antibody (EP-A-184187, GB2188638A, and EP-A-239400).

[0116] In view of current techniques related to monoclonal antibody technology, antibodies against most antigens can be prepared. The antigen-binding domain may be a portion of an antibody (e.g., a Fab fragment) or may be a synthetic antibody fragment (e.g., a single chain Fv fragment (scFv)). Monoclonal antibodies suitable for a selected antigen may be prepared by known techniques, for example those described in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies; Techniques and Applications", JGR Hurrell (CRC Press, 1982). Chimeric antibodies are discussed in Neuberger, 1988.

[0117] The antibody according to the present disclosure comprises an antigen-binding domain. "Antigen-binding domain" describes the part of a molecule that binds to all or part of a target antigen. If the antigen is large, the antibody may bind only to a specific part of the antigen, which part is called an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. The antigen-binding site of an antibody preferably comprises a light chain variable (VL) region and a heavy chain variable (VH) region. The VH and VL regions of an antigen-binding domain together constitute an Fv region.

[0118] An antigen-binding domain typically contains six complementarity determining regions (CDRs): three in the VH region (HCDR1, HCDR2, and HCDR3) and three in the VL region (LCDR1, LCDR2, and LCDR3). Together, the six CDRs define the paratope of the antigen-binding domain (the portion of the antigen-binding domain that binds to the target antigen).

[0119] The VH and VL regions comprise framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, the VH region is composed of the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0120] There are several different rules for defining the CDRs and FRs of an antibody, including those described in Kabat, 1991, Chothia, 1987, the IMGT numbering described in LeFranc, 2015, and VBASE2 described in Retter, 2005. The CDRs and FRs of the VH and VL regions of the antibodies described herein were defined according to Kabat (Kabat, 1991).

[0121] Antibodies that comprise at least two antigen-binding domains, each of which is capable of binding to a different target, may be referred to as "bispecific antibodies." In contrast, antibodies that bind only to a single target (e.g., EGFR or cMET) are referred to as "monospecific antibodies." The present invention provides compounds (e.g., radioimmunoconjugates) that comprise a bispecific antibody or an antigen-binding fragment thereof, comprising a first antigen-binding domain capable of binding to EGFR and a second antigen-binding domain capable of binding to cMET.

[0122] Anti-EGFR antigen-binding domain An antigen-binding domain capable of binding to EGFR comprises the CDRs of an antibody capable of binding to EGFR. In some embodiments, an antigen-binding domain capable of binding to EGFR further comprises the FRs of an antibody capable of binding to EGFR. That is, in some embodiments, an antigen-binding domain capable of binding to EGFR comprises the VH and VL regions of an antibody capable of binding to EGFR.

[0123] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VH region and a VL region that is or is derived from the VH / VL region of the EGFR-binding antibody clone RAA22.

[0124] In some embodiments, an antigen binding domain capable of binding to EGFR comprises the three HCDRs or three LCDRs, preferably the three VH CDRs and the three VL CDRs, of the anti-EGFR antibody clone RAA22. The VH and VL domain sequences of antibody RAA22 are described herein and therefore the three VH and three VL domain CDRs of said antibody can be determined from said sequences.

[0125] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VH region according to (1): (1) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3.

[0126] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VH region according to (1), wherein the VH region further comprises a FR according to (2) below: (2) HFR1 having the amino acid sequence of SEQ ID NO:7 HFR2 having the amino acid sequence of SEQ ID NO:8 HFR3 having the amino acid sequence of SEQ ID NO:9 HFR4 having the amino acid sequence of SEQ ID NO: 10, or a variant in which one or two or three amino acids in one or more of HFR1, HFR2, HFR3, or HFR4 are replaced with another amino acid.

[0127] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VH region according to (3) below: (3) A VH region comprising a CDR according to (1) and a FR according to (2).

[0128] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VH region according to (4): (4) A VH region comprising an amino acid sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 15, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0129] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VL region according to (5) below: (5) a VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6.

[0130] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VL region according to (5) above, wherein the VL region further comprises a FR according to (6) below: (6) LFR1 having the amino acid sequence of SEQ ID NO: 11 LFR2 having the amino acid sequence of SEQ ID NO: 12 LFR3 having the amino acid sequence of SEQ ID NO: 13 LFR4 having the amino acid sequence of SEQ ID NO: 14, or a variant in which one, two or three amino acids in one or more of LFR1, LFR2, LFR3, or LFR4 are replaced with another amino acid.

[0131] In some embodiments, the antigen-binding domain capable of binding to EGFR comprises a VL region comprising a CDR according to (5) above and a FR according to (6) above.

[0132] In some embodiments, the antigen binding domain capable of binding to EGFR comprises a VL region according to (7) below: (7) A VL region comprising an amino acid sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 16, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0133] In some embodiments, the antigen-binding domain capable of binding to EGFR comprises a VH region according to any one of (1) to (4) above, and a VL region according to any one of (5) to (7) above. In some embodiments, the antigen-binding domain comprises a VH region according to any one of (1), (3), and (4), and a VL region according to any one of (5) and (7).

[0134] Anti-cMET antigen-binding domain An antigen-binding domain capable of binding to cMET comprises the CDRs of an antibody capable of binding to cMET. In some embodiments, an antigen-binding domain capable of binding to cMET further comprises the FRs of an antibody capable of binding to cMET. That is, in some embodiments, an antigen-binding domain capable of binding to cMET comprises the VH and VL regions of an antibody capable of binding to cMET.

[0135] In some embodiments, the antigen-binding domain capable of binding to cMET comprises a VH region and a VL region that are or are derived from the VH / VL region of a cMET-binding antibody clone described herein (i.e., anti-cMET antibody clone B09-GL).

[0136] In some embodiments, an antigen-binding domain capable of binding to cMET comprises the three HCDRs or three LCDRs, preferably the three VH CDRs and the three VL CDRs, of the cMET-binding antibody clone B09-GL. The VH and VL domain sequences of antibody B09-GL are described herein, and thus the three VH and three VL domain CDRs of said antibody can be determined from said sequences.

[0137] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VH region according to (8) below: (8) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 17 HCDR2 having the amino acid sequence of SEQ ID NO: 18 HCDR3 having the amino acid sequence of SEQ ID NO:19.

[0138] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VH region according to (8) above, wherein the VH region further comprises a FR according to (9) below: (9) HFR1 having the amino acid sequence of SEQ ID NO: 23 HFR2 having the amino acid sequence of SEQ ID NO: 24 HFR3 having the amino acid sequence of SEQ ID NO: 25 HFR4 having the amino acid sequence of SEQ ID NO: 26, or a variant in which one or two or three amino acids in one or more of HFR1, HFR2, HFR3, or HFR4 are replaced with another amino acid.

[0139] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VH according to (10) below: (10) A VH region comprising CDRs according to (8) and FRs according to (9).

[0140] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VH region according to (11) below: (11) A VH region comprising an amino acid sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 31, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0141] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VL region according to (12) below: (12) A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 20 LCDR2 having the amino acid sequence of SEQ ID NO: 21 LCDR3 having the amino acid sequence of SEQ ID NO:22.

[0142] In some embodiments, the antigen binding domain capable of binding to cMET comprises a VL region according to (12) above, wherein the VL region further comprises a FR according to (13) below: (13) LFR1 having the amino acid sequence of SEQ ID NO: 27 LFR2 having the amino acid sequence of SEQ ID NO: 28 LFR3 having the amino acid sequence of SEQ ID NO: 29 LFR4 having the amino acid sequence of SEQ ID NO: 30, or a variant in which one, two or three amino acids in one or more of LFR1, LFR2, LFR3, or LFR4 are replaced with another amino acid.

[0143] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VL region according to (14) below: (14) A VL region comprising CDRs according to (12) and FRs according to (13).

[0144] In some embodiments, an antigen binding domain capable of binding to cMET comprises a VL region according to (15) below: (15) A VL region comprising an amino acid sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 32, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0145] In some embodiments, the antigen-binding domain capable of binding to cMET comprises a VH region according to any one of (8) to (11) above and a VL region according to any one of (12) to (15) above.

[0146] Antigen-binding domains of bispecific antibodies The present invention provides compounds (e.g., radioimmunoconjugates) that comprise an antibody (i.e., bispecific antibody) or antigen-binding fragment thereof conjugated to a radioactive payload, where the antibody or antigen-binding fragment thereof of the radioimmunoconjugate comprises a first antigen-binding domain that comprises the CDRs of an antigen-binding domain capable of binding to EGFR, and a second antigen-binding domain that comprises the CDRs of an antigen-binding domain capable of binding to cMET. In some embodiments, the first antigen-binding domain comprises the CDRs and FRs of an antigen-binding domain capable of binding to EGFR, and the second antigen-binding domain comprises the CDRs and FRs of an antigen-binding domain capable of binding to cMET. That is, in some embodiments, the antibody or antigen-binding fragment of the compound (e.g., radioimmunoconjugate) comprises a first antigen-binding domain that comprises the VH and VL regions of an antigen-binding domain capable of binding to EGFR, and a second antigen-binding domain that comprises the VH and VL regions of an antigen-binding domain capable of binding to cMET.

[0147] In some embodiments, a first antigen-binding domain capable of binding to EGFR comprises a VH region and a VL region that are or are derived from the VH / VL region of the EGFR-binding antibody clone RAA22, and a second antigen-binding domain capable of binding to cMET comprises a VH region and a VL region that are or are derived from the VH / VL region of the cMET-binding antibody clone B09-GL. Such a bispecific antibody may be referred to as "RAA22 / B09" or a "RAA22 / B09 bispecific antibody."

[0148] In some embodiments, the first antigen-binding domain comprises: a VH region according to any one of (1) to (4) above, and a VL region according to any one of (5) to (7) above; and the second antigen-binding domain comprises: It comprises a VH region according to any one of (8) to (11) above, and a VL region according to any one of (12) to (15) above.

[0149] In certain embodiments, the compounds (e.g., radioimmunoconjugates) of the invention have the following structure: a first antigen-binding domain capable of binding to the epidermal growth factor receptor (EGFR); and A second antigen-binding domain capable of binding to cMET. and wherein the first antigen-binding domain comprises: (i) a heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3; and (ii) a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6.

[0150] In certain embodiments, the first antigen-binding domain comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 15; and A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 16. and / or the second antigen-binding domain comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 31; and It comprises a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:32.

[0151] Constant region In some embodiments, the antibody or antigen-binding fragment thereof of the compounds (e.g., radioimmunoconjugates) described herein comprises an immunoglobulin heavy chain constant (CH) region. In some embodiments, the CH is or is derived from the heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0152] In some embodiments, the CH region is the constant region of human immunoglobulin G1 (IGHG1; UniProt: P01857-1(v1); SEQ ID NO: 33) or a fragment thereof.

[0153] In some embodiments, the CH region comprises an amino acid sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 39 or 40, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) comprises a heavy chain that comprises, or consists of, a VH region described herein and a CH region described herein.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) described herein comprises an immunoglobulin light chain constant (CL) region or fragment thereof. In some embodiments, the CL region is or is derived from the kappa CL region set forth in SEQ ID NO: 34. In some embodiments, the CL region is or is derived from the lambda CL region set forth in SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) comprises: a first CL region that is or is derived from the kappa CL region set forth in SEQ ID NO: 34; and a second CL region that is or is derived from the lambda CL region set forth in SEQ ID NO: 41.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof of the compounds (e.g., radioimmunoconjugates) described herein comprises: a first heavy chain comprising a VH region of a first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof; a first light chain comprising a VL region of a first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof; a second heavy chain comprising a VH region of a second antigen-binding domain and a second heavy chain constant (CH) region or a fragment thereof; and A second light chain comprising a VL region of a second antigen-binding domain and a second light chain constant (CL) region or a fragment thereof.

[0157] It will be understood that when an antibody or antigen-binding fragment thereof comprises a first VH region and a first CH region, these regions together form the first heavy chain of the antibody or antigen-binding fragment thereof, i.e. the first VH region and the first CH region are linked to each other. Similarly, the second VH region and the second CH region form the second heavy chain of the antibody or antigen-binding fragment thereof; the first VL region and the first CL region form the first light chain of the antibody or antigen-binding fragment thereof; and the second VL region and the second CL region form the second light chain of the antibody or antigen-binding fragment thereof.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) comprises a first and a second heavy chain, wherein: (i) the first heavy chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of the heavy chain set forth in SEQ ID NO: 35; (ii) the second heavy chain comprises an amino acid sequence having at least 70% sequence identity, and more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of the heavy chain set forth in SEQ ID NO: 36.

[0159] In some embodiments, an antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) described herein comprises a light chain that comprises, or consists of, a VL region described herein and a CL region described herein.

[0160] In some embodiments, the antibody or antigen-binding fragment thereof of the compounds (e.g., radioimmunoconjugates) described herein comprises a first and a second light chain, wherein: (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of the light chain set forth in SEQ ID NO: 37; (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity, and more preferably at least any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of the light chain set forth in SEQ ID NO: 38.

[0161] The CH, CL, heavy chain and / or light chain of the antibody or antigen-binding fragment thereof of the compounds (e.g., radioimmunoconjugates) described herein may contain one or more modifications, e.g., to suppress or reduce Fc effector function, promote the formation of heterodimeric antibodies or antigen-binding fragments thereof, increase the efficiency of pairing of cognate heavy and light chains, and / or aid in conjugate formation as described in more detail below. The modified CH, CL, heavy chain, and light chain may be referred to as modified CH, CL, heavy chain, and light chain, respectively.

[0162] The antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) may contain mutations in the CH region of the heavy chain to reduce or inhibit binding of the antibody or antigen-binding fragment thereof to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and / or complement. Such mutations inhibit or reduce Fc effector function. Mutations that reduce or inhibit binding of an antibody to one or more Fcγ receptors and complement are known, including, for example, the L234F / L235E / P331S "triple mutation" or "TM" described in Organesyan, 2008. Other mutations known to modulate antibody effector function are described, for example, in Wang, 2018.

[0163] Examples of CH regions containing a triple mutation are SEQ ID NOs: 39 and 40. Thus, in some embodiments, one of the first and second heavy chains comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 39, and the other heavy chain comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 40, wherein one or both (preferably both) of the CH regions comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, where numbering is according to the EU index.

[0164] Examples of heavy chains of the disclosure that include a CH region containing a triple mutation are SEQ ID NOs: 35 and 36. Thus, in some embodiments, one of the first and second heavy chains has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 35, and the other heavy chain has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 36, and one or both (preferably both) of the heavy chains have chains comprising a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, where numbering is according to the EU index.

[0165] The VL and CL regions and the VH and CH1 regions of the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) together constitute the Fab region. The remainder of the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) constitutes the Fc region.

[0166] Unless otherwise indicated, amino acid residue positions of constant domains, including amino acid sequences, positions of substitutions, deletions and insertions as described herein, are numbered according to the EU numbering system (Edelman, 2007).

[0167] Bispecific configuration The present invention provides asymmetric IgG-like bispecific antibodies or antigen-binding fragments thereof within the compounds (e.g., radioimmunoconjugates) disclosed herein. Asymmetric bispecific molecules are typically monovalent for each target. For example, as described in Klein 2012, the concept of monovalent bispecific IgGs is believed to have a unique therapeutic niche in that they (i) do not undergo receptor homodimerization, (ii) potentially reduce toxicity of non-target tissues by losing the avidity of each antigen, and (iii) have better selectivity when both antigens are either selectively restricted or abundantly expressed on target cells. Thus, in some embodiments, the antibody or antigen-binding fragment thereof is an asymmetric IgG-like bispecific antibody or antigen-binding fragment thereof.

[0168] Asymmetric IgG-like bispecific antibodies involve the heterodimerization of two different heavy chains and the correct pairing of cognate light and heavy chains. Heavy chain heterodimerization can be addressed by several techniques, such as knobs-into-holes, electrostatic steering of CH3, CH3 chains exchanged with modified domains and leucine zippers. Correct light and heavy chain pairing can be ensured using one of these heavy chain heterodimerization techniques along with the use of a common light chain, domain crossover between CH1 and CL, joining heavy and light chains with a linker, assembling heavy-light chain dimers in vitro from two separate monoclonals, interface modification of the entire Fab domain, or disulfide modification of the CH1 / CL interface.

[0169] A particular representative configuration of asymmetric IgG-like bispecific antibodies is referred to as "DuetMab". DuetMab antibodies use KIH technology to heterodimerize two different heavy chains, and increase the efficiency of pairing cognate heavy and light chains by replacing one natural disulfide bond in the CH1-CL interface with an engineered disulfide bond. Disclosures regarding DuetMab can be found, for example, in U.S. Pat. No. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entirety.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) comprises: (a) a modified heavy chain comprising a substitution of a cysteine ​​amino acid for a native non-cysteine ​​amino acid; and (b) a corresponding modified CL region, wherein the modified CL comprises a substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid. Where: (i) the first heavy chain comprises a modified CH region and the first light chain comprises a corresponding modified CL region; or (ii) the second heavy chain comprises a modified CH region and the second light chain comprises a corresponding modified CL region.

[0171] In some embodiments, a substituted cysteine ​​in a modified CH region resulting from the substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid and a corresponding substituted cysteine ​​in a modified CL region resulting from the substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid can form a disulfide bond.

[0172] In some embodiments, the modified CH region comprises a substitution of a native non-cysteine ​​amino acid at position 126 with a cysteine ​​amino acid; the corresponding modified CL region comprises a substitution of a native non-cysteine ​​amino acid at position 121 with a cysteine, where the numbering of the constant regions is according to the EU index.

[0173] In some embodiments, the modified CH region comprises a substitution of a native non-cysteine ​​amino acid at position 126 with a cysteine ​​amino acid, and a substitution of the native cysteine ​​amino acid at position 219 with a non-cysteine ​​amino acid, e.g., valine, and the corresponding modified CL region comprises a substitution of a native non-cysteine ​​amino acid at position 121 with a cysteine, and a substitution of the native cysteine ​​amino acid at position 214 with a non-cysteine ​​amino acid, e.g., valine, where the numbering of the constant regions is according to the EU index.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof comprises a second CH region and a corresponding second light chain, wherein the second CH region and the corresponding second CL do not contain a substitution of a naturally occurring non-cysteine ​​amino acid to a cysteine ​​amino acid and do not contain a substitution of a naturally occurring cysteine ​​to a non-cysteine ​​amino acid.

[0175] Functional properties of the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) binding affinity The antibodies or antigen-binding fragments thereof of the radioimmunoconjugates described herein may be characterized by an antigen-binding domain capable of binding to EGFR with a particular affinity for EGFR, and / or an antigen-binding domain capable of binding to cMET with a particular affinity for cMET. The binding affinity of the antibody or antigen-binding fragment thereof of the compound (e.g., radioimmunoconjugate) to a cognate antigen, such as human, mouse, or cynomolgus EGFR or cMET, can be determined, for example, by surface plasmon resonance (SPR) using a Biacore. The binding affinity can be determined as part of an antibody or antigen-binding fragment thereof, for example, a bispecific antibody comprising a first antigen-binding domain capable of binding to EGFR and a second antigen-binding domain capable of binding to cMET. Alternatively, the binding affinity can be determined using an antibody or antigen-binding fragment thereof that is monospecific for EGFR or cMET. In some embodiments, the binding affinity is determined using a BIACore as provided herein or known in the art.

[0176] Binding affinity is typically measured by Kd (the equilibrium dissociation constant between antigen-binding domain and its antigen).As is well understood, the lower the Kd value, the higher the binding affinity of antigen-binding domain.For example, an antigen-binding domain that can bind to a target with Kd of 10nM will bind to said target with higher affinity than an antigen-binding domain that can bind to the same target with Kd of 100nM.

[0177] Reference to human EGFR may refer to a polypeptide comprising the extracellular domain of EGFR, such as a polypeptide having the amino acid sequence set forth in SEQ ID NO: 42. Reference to mouse EGFR may refer to a polypeptide produced from a molecule available from SinoBiological under catalog number 51091-M08H. Reference to cynomolgus monkey EGFR may refer to the amino acid sequence set forth in SEQ ID NO: 46. Reference to human cMET may refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 43. Reference to mouse cMET may refer to a polypeptide produced from a molecule available from SinoBiological under catalog number 50622-M08H. Reference to cynomolgus monkey cMET may refer to the amino acid sequence set forth in SEQ ID NO: 44.

[0178] EGFR affinity The antibodies or antigen-binding fragments thereof of the compounds described herein (e.g., radioimmunoconjugates) may comprise an antigen-binding domain capable of binding to EGFR with low affinity. As used herein, "low affinity" refers to a first antigen-binding domain capable of binding to human EGFR with a dissociation constant (Kd) of 10 nM or greater. Antibodies or antigen-binding fragments of the compounds described herein (e.g., radioimmunoconjugates) comprising such a low affinity EGFR antigen-binding domain may exhibit reduced on-target toxicity in normal tissues, such as skin toxicity, and thus may have an improved safety profile compared to conjugates comprising an EGFR antigen-binding domain capable of binding to human EGFR with "higher affinity". As used herein, "higher affinity" or "high affinity" refers to a first antigen-binding domain capable of binding to human EGFR with a Kd of less than 10 nM.

[0179] The antigen-binding domain capable of binding to EGFR may bind to human EGFR with an affinity having a Kd of 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, or 40 nM or more. Alternatively, the antigen-binding domain capable of binding to EGFR may bind to human EGFR with a Kd of 10-100 nM, 20-100 nM, 30-100 nM, 40-100 nM, 10-80 nM, 20-80 nM, 30-80 nM, 40-80 nM, 10-70 nM, 20-70 nM, 30-70 nM, 40-70 nM, 10-60 nM, 20-60 nM, 30-60 nM, 40-60 nM, 10-50 nM, 20-50 nM, 30-50 nM, or 40-50 nM.

[0180] The antigen-binding domain capable of binding to EGFR may bind to human EGFR with an affinity lower than that of an antigen-binding domain comprising the heavy chain variable (VH) region sequence and the light chain variable (VL) region sequence of antibody QD6 (set forth in SEQ ID NOs: 47 and 48, respectively).

[0181] For example, an antigen binding domain capable of binding to EGFR may bind to human EGFR with an affinity having a Kd that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold higher than the Kd for binding to human EGFR by an antigen binding domain comprising the heavy and light chain sequences of antibody QD6 (set forth in SEQ ID NOs: 47 and 48, respectively). For example, an antigen-binding domain capable of binding to EGFR may bind to human EGFR with an affinity having a Kd that is 2 to 10 times higher, 3 to 10 times higher, 4 to 10 times higher, 5 to 10 times higher, 6 to 10 times higher, 7 to 10 times higher, 2 to 9 times higher, 3 to 9 times higher, 4 to 9 times higher, 5 to 9 times higher, 6 to 9 times higher, 7 to 9 times higher, 2 to 8 times higher, 3 to 8 times higher, 4 to 8 times higher, 5 to 8 times higher, 6 to 8 times higher, or 7 to 8 times higher than the Kd with which an antigen-binding domain comprising the heavy chain sequence and light chain sequence of antibody QD6 (set forth in SEQ ID NOs: 47 and 48, respectively) binds to human EGFR.

[0182] An antigen binding domain capable of binding to EGFR may bind to human EGFR with an affinity similar to that of an antigen binding domain comprising the heavy and light chain variable region sequences of antibody RAA22 (set forth in SEQ ID NOs: 15 and 16, respectively). For example, an antigen binding domain capable of binding to EGFR may bind to human EGFR with an affinity having a Kd that is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1-fold, or less than 0.5-fold different than the Kd for binding to human EGFR by an antigen binding domain comprising the heavy and light chain variable region sequences of antibody RAA22 (set forth in SEQ ID NOs: 15 and 16, respectively).

[0183] The antigen-binding domain capable of binding to EGFR may also be capable of binding to cynomolgus monkey EGFR. For example, the antigen-binding domain capable of binding to EGFR may bind to cynomolgus monkey EGFR with an affinity having a Kd of less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, or less than 250 nM. Alternatively, the antigen-binding domain capable of binding to EGFR may bind to cynomolgus monkey EGFR with an affinity having a Kd of 100-700 nM, 100-600 nM, 100-500 nM, 100-400 nM, 100-300 nM, 150-250 nM, or 100-200 nM. The antigen-binding domain capable of binding to EGFR may bind to cynomolgus monkey EGFR with a Kd that is 10, 9, 8, 7, 6, 5, 4, or 3 times higher than the Kd with which the antigen-binding domain binds to human EGFR.

[0184] The antigen-binding domain capable of binding to EGFR may also be capable of binding to mouse EGFR. For example, the antigen-binding domain capable of binding to EGFR may bind to mouse EGFR with an affinity having a Kd of less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, or less than 650 nM. Alternatively, the antigen-binding domain capable of binding to EGFR may bind to mouse EGFR with a Kd of 100 nM to 1 μM, 200 to 900 nM, 300 to 800 nM, 400 to 700 nM, 400 to 600 nM, or 450 to 550 nM.

[0185] Preferably, the antigen-binding domain capable of binding to EGFR is capable of binding to human EGFR and cynomolgus EGFR. This cross-reactivity is advantageous since it allows for dosage and safety testing of antibodies and conjugates in cynomolgus monkeys during preclinical development. More preferably, the antigen-binding domain capable of binding to EGFR is capable of binding to human EGFR, cynomolgus EGFR, and mouse EGFR. For example, the antigen-binding domain capable of binding to EGFR may be capable of binding to human EGFR, cynomolgus EGFR, and mouse EGFR with Kd values ​​as described above (e.g., human EGFR with a Kd of 10 nM to 100 nM, cynomolgus EGFR with a Kd of 100 nM to 700 nM, and mouse EGFR with a Kd of 100 nM to 1 μM).

[0186] cMET affinity An antigen-binding domain capable of binding to cMET may bind to human cMET with an affinity having a Kd of less than 20 nM, 15 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2.5 nM. Alternatively, an antigen-binding domain capable of binding to cMET may bind to human cMET with an affinity having a Kd of 1-20 nM, 1-15 nM, 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, 1-3 nM, 1-2.5 nM, or 2-2.5 nM.

[0187] The antigen-binding domain capable of binding to cMET may be capable of binding to cynomolgus EGFR. For example, the antigen-binding domain capable of binding to cMET may bind to cynomolgus cMET with an affinity having a Kd of less than 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2.5 nM. Alternatively, the antigen-binding domain capable of binding to cMET may bind to cynomolgus cMET with an affinity having a Kd of 1-20 nM, 1-15 nM, 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, 1-3 nM, 1-2.5 nM, or 2-2.5 nM. An antigen binding domain capable of binding to cMET may bind to cynomolgus monkey cMET with an affinity having a Kd that is 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 fold or less higher than the Kd with which the antigen binding domain binds to human cMET.

[0188] Preferably, the antigen-binding domain capable of binding to cMET is capable of binding to human cMET and cynomolgus cMET. This cross-reactivity is advantageous because it allows for dosage and safety testing of the antibody in cynomolgus during preclinical development. For example, the antigen-binding domain capable of binding to cMET may be capable of binding to human cMET and cynomolgus cMET with Kd values ​​as described above (e.g., human cMET with a Kd of 1 nM to 20 nM, and cynomolgus cMET with a Kd of 1 nM to 20 nM).

[0189] specific binding The antibodies or antigen-binding fragments thereof of the compounds described herein (e.g., radioimmunoconjugates) may comprise an antigen-binding domain capable of specifically binding to EGFR. The antibodies or antigen-binding fragments thereof of the compounds described herein (e.g., radioimmunoconjugates) may comprise an antigen-binding domain capable of specifically binding to cMET. The antibodies or antigen-binding fragments thereof of the compounds described herein (e.g., radioimmunoconjugates) may comprise a first antigen-binding domain capable of specifically binding to EGFR, the first antigen-binding domain capable of specifically binding to EGFR, and a second antigen-binding domain capable of specifically binding to cMET, the first antigen-binding domain capable of specifically binding to cMET.

[0190] The term "specific" may refer to the state in which an antigen-binding domain does not exhibit any significant binding to molecules other than its specific binding partner (here, EGFR or cMET). Such molecules are called "non-target molecules." The term "specific" also applies when an antibody or antigen-binding fragment thereof is specific for a particular epitope, such as an epitope on EGFR or cMET that is carried by multiple antigens, in which case the antibody or antigen-binding fragment thereof can bind to a variety of antigens carrying the epitope.

[0191] In some embodiments, an antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) is considered not to exhibit any significant binding to a non-target molecule if the extent of binding to the non-target molecule is less than about 10% of the extent of binding of the antibody or antigen-binding fragment thereof to the target, e.g., as measured by ELISA, SPR, biolayer interferometry (BLI), microscale thermophoresis (MST), or by radioimmunoassay (RIA). Alternatively, binding specificity may be reflected in terms of binding affinity, where an antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) described herein is capable of binding to EGFR and / or cMET with an affinity that is at least 0.1 order of magnitude greater than its affinity to another non-target molecule. In some embodiments, an antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) of the present disclosure is capable of binding to EGFR and / or cMET with an affinity that is at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 orders of magnitude greater than its affinity for another non-target molecule.

[0192] EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR, HER2, HER3, and HER4. The RAA22 antigen binding domain shows no binding to HER2, HER3, and HER4, and it has been shown that this antigen binding domain specifically binds to EGFR. Thus, in certain embodiments, the antigen binding domain capable of binding to EGFR does not bind or shows no significant binding to HER2, HER3, or HER4.

[0193] cMET is a member of the subfamily of receptor tyrosine kinases, which includes Ron and Sema 4a. The B09-GL antigen-binding domain did not show binding to Ron and Sema4a, indicating that this antigen-binding domain specifically binds to cMET. Thus, in certain embodiments, an antigen-binding domain capable of binding to cMET does not bind or does not show significant binding to Ron, Sema4a.

[0194] Concurrent meetings The antibody or antigen-binding fragment of the compound (e.g., radioimmunoconjugate) disclosed herein, which comprises a first antigen-binding domain capable of binding to EGFR and a second antigen-binding domain capable of binding to cMET, may be characterized by the ability of both antigen-binding domains to simultaneously bind to their respective EGFR and cMET targets. Since many tumors are known to co-express both EGFR and cMET and thus can be targeted by the compounds disclosed herein, it is expected that the antibody or antigen-binding fragment of the compound (e.g., radioimmunoconjugate) disclosed herein, which has the ability to simultaneously bind to EGFR and cMET, is advantageous. Thus, in some embodiments, the antibody or antigen-binding fragment of the compound (e.g., radioimmunoconjugate) disclosed herein can simultaneously bind to EGFR and cMET.

[0195] Further details of these methods of measuring co-association can be found in the Examples.

[0196] Internalization of antibodies (or antigen-binding fragments thereof) Antibodies or antigen-binding fragments thereof of the compounds described herein (eg, radioimmunoconjugates) may be characterized by their ability to mediate efficient internalization.

[0197] Internalization of an antibody or antigen-binding fragment thereof (or a compound (e.g., a radioimmunoconjugate) comprising said antibody or antigen-binding fragment thereof) by a cell can be analyzed by contacting a live cell with the antibody or antigen-binding fragment thereof and detecting the antibody (or antigen-binding fragment thereof) or compound (e.g., a radioimmunoconjugate) after a period of time sufficient for internalization has elapsed. Internalization can be determined by detecting localization of the antibody (or antigen-binding fragment thereof) or compound (e.g., a radioimmunoconjugate). If the antibody (or antigen-binding fragment thereof) or compound (e.g., a radioimmunoconjugate) remains at the cell surface (e.g., is detected at the cell surface and / or is not detected inside the cell), the antibody (or antigen-binding fragment thereof) or compound (e.g., a radioimmunoconjugate) is determined not to have been internalized. If the antibody (or antigen-binding fragment thereof) or compound (e.g., radioimmunoconjugate) is detected intracellularly (e.g., localized in the cytoplasm or organelles), the antibody (or antigen-binding fragment thereof) or compound (e.g., radioimmunoconjugate) is determined to be internalized.

[0198] An exemplary method for visualizing whether an antibody (or its antigen-binding fragment) or compound (e.g., radioimmunoconjugate) can mediate efficient internalization includes labeling the antibody with a pH-sensitive dye that fluoresces at acidic pH and adding these labeled antibodies or conjugates to cells. Internalization into cells can be detected by monitoring fluorescence. If the fluorescence observed is greater than that of a labeled, unbound control antibody (or its antigen-binding fragment) or compound (e.g., radioimmunoconjugate) over a certain period of time, e.g., 48 hours, the antibody (or its antigen-binding fragment) or compound (e.g., radioimmunoconjugate) is deemed to be capable of mediating internalization and delivery to lysosomes. Details of this method for visualizing antibody (or its antigen-binding fragment) internalization can be found in the Examples.

[0199] An antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) comprising a first antigen-binding domain capable of binding to EGFR and a second antigen-binding domain capable of binding to cMET may be characterized by its ability to mediate more efficient internalization when compared to EGFR or cMET monospecific controls. An antibody or antigen-binding fragment thereof of a compound (e.g., a radioimmunoconjugate) exhibiting this property is expected to be advantageous as it may exhibit greater selectivity for tumor cells that co-express both targets and minimize the effect of the antibody or antigen-binding fragment thereof in normal tissues that do not exhibit significant levels of co-expression.

[0200] In some embodiments, provided herein is a compound (e.g., an immunoconjugate or radioimmunoconjugate) comprising an antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable domain comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are of the antibodies set forth in the Sequence Appendix set forth below.

[0201] In some embodiments, compounds (e.g. immunoconjugates or radioimmunoconjugates) are provided that comprise an antibody or antigen-binding fragment that is a variant of an antibody shown in the Sequence Appendix, such antibodies or antigen-binding fragments having CDR sequences that differ by no more than three amino acid residues per CDR (e.g. three or two or one amino acid residues) from the CDR sequences of the antibodies set forth in the Sequence Appendix. In some embodiments, compounds (e.g. immunoconjugates or radioimmunoconjugates) are provided that comprise an antibody or antigen-binding fragment that is a variant of an antibody shown in the Sequence Appendix, such antibodies or antigen-binding fragments having a set of six CDRs whose sequences differ by a total of no more than three amino acid residues (e.g. three or two or one amino acid residues) from the CDRs of the antibodies set forth in the Sequence Appendix.

[0202] In some embodiments, a compound (e.g., an immunoconjugate or radioimmunoconjugate) is provided that comprises an antibody or antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain, including the heavy chain variable domain and light chain variable sequences of the antibody set forth in the Sequence Appendix. In some embodiments, compounds (e.g., immunoconjugates or radioimmunoconjugates) are provided that comprise an antibody or antigen-binding fragment that is a variant of an antibody shown in the Sequence Appendix, such antibody or antigen-binding fragment having (1) a heavy chain domain that comprises an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain variable domain of an antibody set forth in the Sequence Appendix; and (2) a light chain domain that comprises an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the light chain variable domain of the same antibody set forth in the Sequence Appendix.

[0203] Sequence Appendix CDR sequence of anti-EGFR antibody clone RAA22 HCDR1-DNDFS (SEQ ID NO: 1) HCDR2 - AIVAVFRTETYAQKFQD (SEQ ID NO: 2) HCDR3 - RLMSAISGPGAPLLM (SEQ ID NO: 3) LCDR1-TGTSSDVGGYNYVS (SEQ ID NO: 4) LCDR2-DVSKRPS (SEQ ID NO:5) LCDR3-SSYTSSDTLEI (SEQ ID NO: 6) FR sequence of anti-EGFR antibody clone RAA22 HFR1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 7) HFR2-WVRQAPGQGLEWMG (SEQ ID NO: 8) HFR3-RVKITADISTRTTYMELSSLRSEDTAVYYCAR (SEQ ID NO: 9) HFR4-WGQGTLVTVSS (SEQ ID NO: 10) LFR1-QSALTQPRSVSGSPGQSVTISC (SEQ ID NO: 11) LFR2-WYQQHPGKAPKLMIY (SEQ ID NO: 12) LFR3-GVPDRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 13) LFR4-FGGGTKLTVL (SEQ ID NO: 14) Amino acid sequence of the heavy chain variable (VH) region of anti-EGFR antibody clone RAA22 (SEQ ID NO: 15) [ka] Amino acid sequence of the light chain variable (VL) region of anti-EGFR antibody clone RAA22 (SEQ ID NO: 16): [ka] CDR sequence of anti-c-Met antibody clone B09-GL HCDR1-DYYIH (SEQ ID NO: 17) HCDR2 - WMNPNSGNTGYAQKFQG (SEQ ID NO: 18) HCDR3-GQGYTHS (SEQ ID NO: 19) LCDR1-RASEGIYHWLA (SEQ ID NO: 20) LCDR2-KASSLAS (SEQ ID NO: 21) LCDR3-QQYSNYPPT (SEQ ID NO: 22) FR sequence of anti-c-Met antibody clone B09-GL HFR1-QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 23) HFR2-WVRQATGQGLEWMG (SEQ ID NO: 24) HFR3-RVTMTRDTSISTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 25) HFR4-WGQGTMVTVSS (SEQ ID NO: 26) LFR1-DIQMTQSPSTLSASVGDRVTITC (SEQ ID NO: 27) LFR2-WYQQKPGKAPKLLIY (SEQ ID NO: 28) LFR3-GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 29) LFR4-FGGGTKLEIK (SEQ ID NO: 30) Amino acid sequence of the heavy chain variable (VH) region of anti-c-Met antibody clone B09-GL (SEQ ID NO:31): [ka] Amino acid sequence of the light chain variable (VL) region of anti-c-Met antibody clone B09-GL (SEQ ID NO:32): [ka] Amino acid sequence of human immunoglobulin G1 heavy chain constant (CH) region (SEQ ID NO:33): [ka] Amino acid sequence of the wild-type human immunoglobulin kappa constant region (SEQ ID NO:34): [ka] Amino acid sequence of the anti-EGFR heavy chain in the "EGFR-cMET TM" antibody (SEQ ID NO:35): The following substitutions are shown in bold and underlined: triple mutation (TM; L234F, L235E, and P331S), "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of anti-cMET in the "EGFR-cMET TM" antibody (SEQ ID NO:36): The following substitutions are shown in bold and underlined: triple mutation (TM; L234F, L235E, and P331S); "hole" mutations (T366S, L368A, and Y407V) and a stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the anti-EGFR light chain in the "EGFR-cMET TM" antibody (SEQ ID NO:37): The following substitutions are shown in bold and underlined: S121C and C214V (numbering follows the EU index). [ka] Amino acid sequence of the light chain of anti-cMET in the "EGFR-cMET TM" antibody (SEQ ID NO:38): [ka] Amino acid sequence of human immunoglobulin G1 CH region without cysteine ​​insertion and modified by TM to include a "knob" mutation, a cysteine ​​that forms a stabilizing disulfide bridge (SEQ ID NO:39): The following substitutions are shown in bold and underlined: triple mutation (TM; L234F, L235E, and P331S), "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region without cysteine ​​insertion and modified by TM to include a "hole" mutation, a cysteine ​​that forms a stabilizing disulfide bridge (SEQ ID NO: 40): The following substitutions are shown in bold and underlined: triple mutation (TM; L234F, L235E, and P331S); "hole" mutations (T366S, L368A, and Y407V) and a stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of a human immunoglobulin lambda constant region modified to include S121C and C214V substitutions (SEQ ID NO:41): [ka] Amino acid sequence of human EGFR extracellular domain (SEQ ID NO:42): [ka] Amino acid sequence of human c-Met extracellular domain (SEQ ID NO:43): [ka] Amino acid sequence of cynomolgus c-Met extracellular domain (SEQ ID NO:44): [ka] Amino acid sequence of the cynomolgus monkey EGFR extracellular domain (SEQ ID NO:46): [ka] Amino acid sequence of the VH region of anti-EGFR antibody clone QD6 (SEQ ID NO:47): [ka] Amino acid sequence of the VL region of anti-EGFR antibody clone QD6 (SEQ ID NO:48): [ka]

[0204] Chelate moiety or its metal complex Chelate part Examples of suitable chelating moieties include DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α,α',α",α'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane), -1-yl)acetic acid), DOTA-GA anhydride (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8 ,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid) ), H2dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H6phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N",N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecane dimethane phosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), octacoordinate HOPO (octacoordinate hydroxypyridinone), or porphyrin.

[0205] Preferably, the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α,α',α",α'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and HP-DO3A (10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid).

[0206] In some embodiments, the chelating moiety is DOTA.

[0207] In some embodiments, the compounds include metal complexes of chelating moieties. For example, chelating groups can be used in metal chelate combinations with metals such as manganese, iron, and gadolinium, and isotopes such as any of the radioisotopes and radionuclides discussed herein (e.g., isotopes in the general energy range of 60-10,000 keV).

[0208] In some embodiments, chelating moieties are useful as detection agents, and thus compounds containing such detectable chelating moieties can be used as diagnostic or therapeutic agents.

[0209] Radioisotopes and Radionuclides In some embodiments, the metal complex comprises a radionuclide. Examples of suitable radioisotopes and radionuclides include: 3 H, 14C. 15 N, 18 F, 35 S, 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 66 Ga, 67 Ga, 67 Cu, 68 Ga, 75 Br, 76 Br, 77 Br, 82 Rb, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 117m Sn, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227Th , and 229Th These include, but are not limited to:

[0210] In some embodiments, the metal complex is 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu,64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 89 Zr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 Pm, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227Th , and 229Th The radionuclide includes a radionuclide selected from the group consisting of:

[0211] In certain embodiments, the metal complex is 68 Ga, 89 Zr, 90 Y, 111 In, 177 Lu, and 225 In certain embodiments, the metal complex comprises a radionuclide selected from: 177 Lu or 225 Contains radioactive nuclides of Ac.

[0212] In some embodiments, the radionuclide is an alpha emitter, such as astatine-211 ( 211 At), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), Actinium-225( 225 Ac), Radium-223(223 Ra), lead-212( 212 Pb), Thorium-227( 227Th ), or terbium-149( 149 In some embodiments, the alpha emitter is actinium-225 ( 225 Ac) or their descendants.

[0213] In certain embodiments, the metal complex is 225 Includes alpha emitters of Ac or their progeny.

[0214] Linker The compounds of the present invention have the structure of Formula I: AL 1 -(L 2 ) n -B Formula I where each variable is defined in the Summary section above.

[0215] In some embodiments, the compound of formula I is -L 1 -(L 2 ) n -, wherein L 1 is a bond, C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer from 1 to 5, inclusive; Each L 2 are independently of the formula: -X 1 -L 3 -Z 1 - Formula II wherein: X 1 is -C(O)NR 1 -*, -NR 1 C(O)-*, -C(S)NR 1 -*, -NR 1 C(S)-*, -OC(O)NR 1-*, -NR 1 C(O)O-*, -NR 1 C(O)NR 1 -*, -CH2-Ph-C(O)NR 1 -*, -NR 1 C(O)-Ph-CH2-*, -CH2-Ph-NH-C(S)NR 1 -*, -NR 1 C(S)-NH-Ph-CH2-*, -O-*, or -NR 1 -*, where "*" is L 3 indicates the connection point to R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, or optionally substituted aryl or heteroaryl; L 3 is an optionally substituted C-C 50 Alkyl or optionally substituted C-C 50 Heteroalkyl (e.g., CH2CH2O) 2-20 ) and Z 1 -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -# or -NR 2 -#, where "#" indicates the point of attachment to B, and R 2 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0216] In some embodiments, L 1 is optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl. In certain embodiments, L 1 is a substituted C1-C6 alkyl or a substituted C1-C6 heteroalkyl, where the substituent comprises a heteroaryl group (e.g., a nitrogen-containing 6-membered heteroaryl). 1 is C1-C6 alkyl. For example, L1 In some embodiments, L 1 is a bond. In some embodiments, L 1 teeth, [ka] where R L is hydrogen or -COH.

[0217] In some embodiments, X 1 is -C(O)NR 1 -*, -NR 1 C(O)-* or -NR 1 - and "*" is L 3 indicates the connection point to R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, X 1 is -C(O)NR 1 -*, where "*" is L 3 indicates the connection point to R 1 is hydrogen.

[0218] In some embodiments, L 3 is an optionally substituted C-C 50 Alkyl (e.g., C3-C 30 Alkyl, C3-C 25 Alkyl, C3-C 20 Alkyl, C3-C 15 Alkyl, C3-C 10 Alkyl, C5-C 30 Alkyl, C5-C 25 Alkyl, C5-C 20 Alkyl, C5-C 15 Alkyl, and C5-C 10 alkyl) or optionally substituted C-C 50 Heteroalkyl (e.g., C3-C 30 Heteroalkyl, C3-C 25 Heteroalkyl, C3-C 20Heteroalkyl, C3-C 15 Heteroalkyl, C3-C 10 Heteroalkyl, C5-C 30 Heteroalkyl, C5-C 25 Heteroalkyl, C5-C 20 Heteroalkyl, C5-C 15 Heteroalkyl and C5-C 10 Heteroalkyl). Exemplary C1-C 50 Heteroalkyl is C5-C 30 Polyethylene glycol (e.g., C5-C 25 Polyethylene glycol, C5-C 20 Polyethylene glycol, C5-C 15 In certain embodiments, L 3 is C5-C 25 Polyethylene glycol, C5-C 20 Polyethylene glycol, or C5-C 15 It is polyethylene glycol.

[0219] In some embodiments, L 3 is an optionally substituted C-C 50 Heteroalkyl (e.g., C1-C 40 Heteroalkyl, C1-C 30 Heteroalkyl, C1-C 20 Heteroalkyl, C2-C 18 Heteroalkyl, C3-C 16 Heteroalkyl, C4-C 14 Heteroalkyl, C5-C 12 Heteroalkyl, C6-C 10 Heteroalkyl, C8-C 10 Heteroalkyl, C4 heteroalkyl, C6 heteroalkyl, C8 heteroalkyl, C 10 Heteroalkyl, C 12 Heteroalkyl, C 16 Heteroalkyl, C 20 Heteroalkyl, or C 24 heteroalkyl).

[0220] In some embodiments, L3 is an optionally substituted C1-C olefin copolymer comprising a polyethylene glycol (PEG) moiety containing 1 to 20 oxyethylene (-O-CH2-CH2-) units (e.g., 2 oxyethylene units (PEG2), 3 oxyethylene units (PEG3), 4 oxyethylene units (PEG4), 5 oxyethylene units (PEG5), 6 oxyethylene units (PEG6), 7 oxyethylene units (PEG7), 8 oxyethylene units (PEG8), 9 oxyethylene units (PEG9), 10 oxyethylene units (PEG10), 12 oxyethylene units (PEG12), 14 oxyethylene units (PEG14), 16 oxyethylene units (PEG16), or 18 oxyethylene units (PEG18)). 50 It is heteroalkyl.

[0221] In certain embodiments, L 3 is an optionally substituted C 1 -C 2 ... 1-50 For example, L 3 is the following: [ka] As shown in Figure 1, it contains PEG3.

[0222] In some embodiments, L 3 is (CH2CH2O) m (CH2) w In the formula, m and w each independently represent an integer of 0 to 10 (inclusive), and at least one of m and w is not 0.

[0223] In some embodiments, L 3 is the substitution C1-C 50 Alkyl or substituted C1-C 50 When it is a heteroalkyl, the substituents include a heteroaryl group (eg, a nitrogen-containing 6-membered heteroaryl).

[0224] In some embodiments, Z 1is CH2, C=O, or NR 1 where R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0225] In certain embodiments, AL 1 -(L 2 ) n -B is of formula Ia: [ka] or a metal complex thereof, wherein Y 1 is -CH2OCH2(L 2 ) n -B, -C(O)(L 2 ) n -B or -C(S)(L 2 ) n -B and Y 2 is -CH2CO2H; or Y 1 is H and Y 2 , L 1 -(L 2 ) n -B.

[0226] crosslinking group In some embodiments, the compounds (e.g., radioimmunoconjugates) are synthesized using a bifunctional chelate that includes a chelate, a linker, and a crosslinking group. Once the compound (e.g., radioimmunoconjugate) is formed, the crosslinking group may be absent from the compound (e.g., radioimmunoconjugate).

[0227] In some embodiments, the compound (e.g., a radioimmunoconjugate) includes a crosslinking group instead of or in addition to a targeting moiety (e.g., in some embodiments, B of Formula I includes a crosslinking group).

[0228] A bridging group is a reactive group that can link two or more molecules by a covalent bond. Bridging groups can be used to attach linkers and chelating moieties to therapeutic or targeting moieties. Bridging groups can also be used to attach linkers and chelating moieties to targets in vivo. In some embodiments, the bridging group is an amino-reactive, methionine-reactive or thiol-reactive bridging group, or includes a sortase recognition sequence. In some embodiments, the amino-reactive or thiol-reactive bridging group includes an active ester, such as a hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenol ester, a 4-nitrophenol ester, or an imidate, an anhydride, a thiol, a disulfide, a maleimide, an azide, an alkyne, a strained alkyne, a strained alkene, a halogen, a sulfonate, a haloacetyl, an amine, a hydrazide, a diazirine, a phosphine, a tetrazine, an isothiocyanate, or an oxaziridine. In some embodiments, the sortase recognition sequence is composed of terminal glycine-glycine-glycine (GGG) and / or LPTXG amino acid sequences, where X is any amino acid. One of skill in the art will appreciate that the use of cross-linking groups is not limited to the specific configurations disclosed herein, but may include other known cross-linking groups.

[0229] Pharmaceutical Compositions In one aspect, the present disclosure provides pharmaceutical compositions comprising the compounds disclosed herein. Such pharmaceutical compositions can be formulated for use in various drug delivery systems. For proper formulation, one or more physiologically acceptable excipients or carriers can also be included in the pharmaceutical composition. Non-limiting examples of suitable formulations suitable for use in the present disclosure include those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th Edition, 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science. 249: 1527-1533, 1990).

[0230] The pharmaceutical compositions may be formulated for any of the various routes of administration discussed herein (see, for example, the "Administration and Dosage" subsection herein). Sustained release administration is contemplated by means such as depot injection or erodible implants or components. Thus, the present disclosure provides pharmaceutical compositions comprising an agent (e.g., a radioimmunoconjugate) disclosed herein dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, such as water, buffered water, saline, or PBS. In some embodiments, the pharmaceutical compositions contain pharma- ceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or surfactants, among others. In some embodiments, the pharmaceutical compositions are formulated for oral delivery and may optionally contain inactive ingredients, such as binders or fillers, for formulation into unit dosage forms such as tablets or capsules. In some embodiments, the pharmaceutical compositions are formulated for topical administration and may optionally contain inactive ingredients, such as solvents or emulsifiers, for formulation into creams, ointments, gels, pastes, or eye drops.

[0231] In some embodiments, the pharmaceutical compositions provided may be sterilized by conventional sterilization techniques, e.g., sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized. The lyophilized formulations may, for example, be combined with a sterile aqueous carrier prior to administration. The pH of the formulations is typically 3-11, more preferably 5-9 or 6-8, most preferably 6-7, e.g., 6-6.5. The resulting solid compositions may be packaged in a number of single-dose units, each containing a fixed amount of the agent, e.g., a sealed package of tablets or capsules. The solid pharmaceutical compositions may also be packaged in flexible quantity containers, such as squeezable tubes designed for topically applied creams or ointments.

[0232] Treatment method In one aspect, the disclosure provides a method of treatment comprising administering a compound disclosed herein (e.g., a radioimmunoconjugate) to a subject in need thereof.

[0233] subject In some disclosed methods, a therapy (e.g., including a therapeutic agent) is administered to a subject. In some embodiments, the subject is a mammal, e.g., a human.

[0234] In some embodiments, the subject has cancer or is at risk of developing cancer. For example, the subject may be one who has been diagnosed with cancer. For example, the cancer may be primary cancer or metastatic cancer. The subject may have cancer at any stage, such as stage I, stage II, stage III, stage IV, with or without lymph node involvement, and with or without metastasis. The provided compounds (e.g., radioimmunoconjugates) and compositions may prevent or reduce further growth of the cancer and / or ameliorate the cancer (e.g., prevent or reduce metastasis). In some embodiments, the subject does not have cancer, but has been determined to be at risk of developing cancer due to the presence of one or more risk factors, such as, for example, environmental exposure, the presence of one or more genetic mutations or variants, family history, etc. In some embodiments, the subject has not been diagnosed with cancer.

[0235] In some embodiments, the cancer is any cancer that comprises cells that express EGFR and cMET, hi certain embodiments, the cancer is lung cancer, colon cancer, pancreatic cancer, or head and neck cancer.

[0236] Administration and Dosage The compounds (eg, radioimmunoconjugates) and pharmaceutical compositions thereof disclosed herein can be administered by any of a variety of routes of administration, including systemic and local routes of administration.

[0237] Systemic routes of administration include parenteral and enteral routes. In some embodiments, the compound (e.g., radioimmunoconjugate) or pharmaceutical composition thereof is administered by a parenteral route, e.g., intravenously, intraarterially, intraperitoneally, subcutaneously, intracranially, or intradermally. In some embodiments, the compound (e.g., radioimmunoconjugate) or pharmaceutical composition thereof is administered intravenously. In some embodiments, the compound (e.g., radioimmunoconjugate) or pharmaceutical composition thereof is administered by an enteral route, e.g., gastrointestinal administration, or oral administration.

[0238] Local routes of administration include, but are not limited to, peritumoral and intratumoral injection.

[0239] The pharmaceutical composition can be administered for radiation therapy planning, diagnosis, and / or treatment. When administered for the purpose of radiation therapy planning or diagnosis, the compound (e.g., radioimmunoconjugate) can be administered to the subject in an amount effective to determine a diagnostically effective dose and / or a therapeutically effective amount. In therapeutic applications, a subject (e.g., a human) already suffering from an existing disease state (e.g., cancer) can be administered a pharmaceutical composition in an amount sufficient to cure or at least partially suppress the symptoms of the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount", which is an amount of the compound sufficient to substantially ameliorate at least one symptom associated with the disease or disease state. For example, in the treatment of cancer, an agent or compound that reduces, prevents, delays, inhibits, or stops the symptoms of the disease or disease state can be therapeutically effective. A therapeutically effective amount of an agent or compound is not necessary to cure the disease or disease state, but may provide treatment for the disease or disease state, for example, to delay, hinder, or prevent the onset of the disease or disease state, to ameliorate the symptoms of the disease or disease state, or to alter the duration of the disease or disease state. For example, a disease or condition may be reduced in severity and / or recovery may be promoted in an individual. In some embodiments, a subject is administered a first dose of a compound (e.g., a radioimmunoconjugate) or composition in an amount effective for a radiation treatment regimen, followed by a second dose or set of doses of a therapeutically effective amount of the compound (e.g., a radioimmunoconjugate) or composition.

[0240] For treating cancers involving cells expressing EGFR and cMET, the methods of the invention typically involve administering to a subject (e.g., a human) in need thereof an initial dose of the compound or composition in an amount effective for a radiation therapy regimen, followed by subsequent doses of the compound or composition in a therapeutically effective amount.

[0241] In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose are the same.

[0242] In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose are different.

[0243] Therapeutically effective amounts may vary depending on the severity of the disease or condition and other characteristics of the subject, such as body weight. Therapeutically effective amounts of the disclosed compounds (e.g., radioimmunoconjugates) and compositions for a subject (e.g., a mammal, such as a human) can be determined by one of skill in the art, taking into account individual differences (e.g., differences in age, weight, and condition of the subject).

[0244] In some embodiments, the disclosed compounds (e.g., radioimmunoconjugates) exhibit enhanced ability to target cancer cells. In some embodiments, the effective amount of the disclosed compounds (e.g., radioimmunoconjugates) is less than the equivalent dosage for therapeutic efficacy of the unconjugated and / or non-radiolabeled targeting moiety (e.g., less than about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% or less).

[0245] Single or multiple administrations of the pharmaceutical compositions disclosed herein comprising an effective amount can be carried out with dose levels and patterns selected by the treating physician. Dosage and administration schedules can be determined and adjusted based on the severity of the subject's disease or condition, and can be monitored throughout the course of treatment according to methods commonly practiced by clinicians or as described herein.

[0246] The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. EXAMPLES

[0247] Example 1 - Design and construction of RAA22 / B09 DuetMab This example describes the generation of a bispecific antibody molecule capable of binding to both EGFR and c-Met.

[0248] 1.1 Isolation and identification of anti-cMET antibody 0021U3-B09 cMET-specific scFv antibodies were isolated from a large naive human scFv phage display library in a series of iterative panning selection cycles against recombinant mammalian-expressed biotinylated monomeric human cMET (MedImmune) essentially as described (Vaughan, 1996). scFvs from round 2 of the selection output were expressed in the bacterial periplasm and screened with HGF ligands for their ability to inhibit binding of the human cMET receptor in an HGF:cMET HTRF® (homogeneous time-resolved fluorescence) ligand-receptor inhibitory binding assay. Top hits showing strong inhibitory effects were selected and subjected to DNA sequencing. The unique genes were then converted into human immunoglobulin G2 (IgG2) antibodies and produced in mammalian cells essentially as described (Persic; 1997). The purified antibodies were then ranked based on their inhibitory effects in the HGF:cMET HTRF® binding assay. The most potent antibody, 0021U3-B09, was selected for further characterization.

[0249] 1.2 Optimization of anti-cMET antibody 0021U3-B09. To minimize potential immunogenicity, non-Vernier framework residues (Foote and Winter 1992) in the variable framework regions of 0021U3-B09 were specifically targeted and modified to match the closest human germline sequence. In the VH region, seven amino acid residues were mutated to match the reference human germline sequence IGHV1-8*01. In the VL region, three residues were mutated to match the reference human germline sequence IGKV1-5*03. All residues in the VH and VL regions were successfully changed to germline residues without loss of activity. 0021U3-B09 was affinity optimized using hybridization-based mutagenesis essentially as described (Kunkel 1985). A large-scale scFv library derived from the 0021U3-B09 sequence was created by oligonucleotide-directed mutagenesis of the VH complementarity-determining region 3 (CDR3) using standard molecular biology techniques. The library was subjected to affinity-based solution-phase selection to select variants with higher affinity for human and cynomolgus cMET antigens. Crude scFv-containing periplasmic extracts from the CDR-targeted selection output were screened for improved inhibitory activity in the HGF:cMET HTRF® binding assay. Variants showing significantly improved inhibitory effect compared to the parent 0021U3-B09 were subjected to DNA sequencing and unique genes were converted to human IgG2. The purified antibodies were then ranked based on their inhibitory effect. The most potent antibody, B09-57, was selected for further characterization.

[0250] 1.3 Isolation and characterization of the anti-EGFR antibody Tdev-0004. EGFR-specific scFv antibodies were isolated from a large naive human scFv phage display library in a series of iterative panning selection cycles against recombinant mammalian-expressed biotinylated monomeric human EGFR (MedImmune) essentially as described (Vaughan, 1996). ScFv-displaying phages from round 3 of the selection output were screened for their binding to human and cynomolgus EGFR in ELISA. Top hits showing cross-reactivity were selected and subjected to DNA sequencing. Unique genes were then converted to human immunoglobulin G1 (IgG1) antibodies and produced in mammalian cells essentially as described (Persic; 1997). Purified antibodies were then ranked based on binding to the EGFR-expressing cell line A431 by flow cytometry. Antibody Tdev-0004 showing specific cell binding was selected for further characterization.

[0251] 1.4 Optimization of the anti-EGFR antibody Tdev-0004. Variants RAA22 and QD6 were obtained by optimizing the anti-EGFR Tdev-0004 mAb. The VL framework of Tdev-0004 matched 100% with the reference human germline sequence IGLV2-11*01 / IGLJ2 ​​(see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGLV2-11*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p), while the VH framework showed 79% homology with the closest human germline sequence IGHV1-69*01 / JH4 (see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGHV1-69*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p). To minimize potential immunogenicity, the VH region was first fully germlined by mutating all 13 non-germline framework residues. Germlined binding of the fully germlined variant to cynomolgus monkey EGFR was significantly impaired. Four non-germline residues, K68, I73, R76, and T78, were selectively backmutated to restore binding to cynomolgus monkey EGFR. Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu 1991). The resulting partially germlined variant (designated H4) was used as a template sequence for affinity optimization. Variant H4 was affinity optimized by simple mutagenesis of all six CDRs using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer's instructions. Single amino acid mutated VH and VL libraries were expressed in bacteria as Fab fragments and screened for improved binding to human and cynomolgus EGFR by ELISA. Variants showing improved binding compared to the parental H4 were subjected to DNA sequencing and unique genes were converted to human IgG1.Variant RAA22 was identified by a single mutation in CDRH3. To further improve affinity, individual positive mutations were combined to generate a combinatorial library to screen for variants with enhanced binding to human and cynomolgus EGFR. Variant QD6 was identified by four combined mutations in CDRL2, CDRL3, and CDRH3.

[0252] 1.5 Generation of monovalent bispecific anti-EGFR / cMET DuetMab antibodies. The variable domains of anti-cMET mAb B09-57 and anti-EGFR mAb RAA22 and QD6 were utilized to construct a monovalent bispecific anti-EGFR / cMET antibody on the backbone of the DuetMab platform (Mazor, 2015). Specifically, the VH gene of anti-cMET B09-57 was inserted into a human gamma 1 constant heavy chain carrying a "knob" mutation (T366W) and alternative interchain cysteine ​​mutations (F126C and C219V). The VL gene of B09-57 was inserted in frame into a human kappa constant domain carrying the corresponding alternative interchain cysteine ​​mutations (S121C and C214V) designed to pair with the "knob" heavy chain. Similarly, the VH genes of anti-EGFR RAA22 and affinity-optimized QD6 were inserted into a human gamma 1 constant heavy chain carrying the "hole" mutations (T366S, L368A, and Y407V), and the VL genes of RAA22 and B09-57 were inserted in frame into a human lambda constant domain designed to pair with the "hole" heavy chain. Additionally, two residues in the CH3 domains of the "knob" and "hole" heavy chains were mutated to cysteines (S354C in "knob" and Y349C in "hole") to form a stabilizing disulfide bridge. The Fc domain was further engineered to carry a cysteine ​​insertion after serine 239 (C239i / "Maia") to allow site-specific conjugation of maleimide-bearing cytotoxic drugs (Dimasi, 2017). Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu 1991). The assembled monovalent bispecific anti-EGFR / cMET DuetMab antibodies were named RAA22 / B09-57 and QD6 / B09-57 (Figure 1). DuetMab antibodies were generated from mammalian cells as previously described (Mazor, 2017).

[0253] It was also recognized that reducing or eliminating effector functions of the Fc backbone may have the potential to reduce immunotoxicity and improve pharmacokinetics. Therefore, the inventors also introduced "triple mutants (TM)" of L234F / L235E / P331S (EU numbering) that were previously shown to reduce Fc effector functions of antibody molecules (Organesyan, 2008; Hay, 2016). These TM constructs were designed to carry "hole" mutations in the c-MET arm and "knob" mutations in the EGFR arm.

[0254] The generated "EGFR-cMETTM" DuetMab contains the variable regions of RAA22 and B09 with TM engineered into them and has the amino acid sequence shown in the following table.

[0255] [Table 1]

[0256] Example 2 - Biochemical and biophysical properties In this example, various biochemical and biophysical properties of the RAA22, QD6 and B09-57 monoclonal antibodies and the RAA22 / B09-57 and QD6 / B09-57 bispecific antibody molecules are examined, including their binding affinity to EGFR and c-Met, respectively, and their ability to bind both antigens simultaneously.

[0257] 2.1 Binding affinity of DuetMab and parental mAbs to EGFR and cMET. Kinetic rate constants (k on and k off) and equilibrium dissociation constant (Kd) were measured by SPR at 25° C. using an antibody capture assay on a BIAcore T200 instrument (GE Healthcare, Pittsburgh, PA). Mouse anti-human IgG was immobilized on a CM4 sensor chip with a final surface density of approximately 2000 resonance units (RU). A standard flow cell surface was also prepared on this sensor chip using the same immobilization protocol. Test and control antibodies were prepared from 5 to 20 nM in device buffer (HBS-EP buffer; 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% P-20) along with 3-fold serial dilutions of purified EGFR (human 0.27-200 nM, cynomolgus 0.4-900 nM, and mouse 4-1000 nM) or cMET protein (human 0.27-66 nM, cynomolgus 0.27-22 nM) in device buffer. A sequential approach was used for kinetic measurements. First, the antibody was injected over the capture surface at a flow rate of 10 μL / min. Once the binding of the captured antibody had stabilized, a single concentration of analyte was injected over both the capture and reference surfaces at a flow rate of 75 μL / min. Association phase data was obtained from the resulting binding response curves. After injection of the analyte, the flow was then switched back to instrument buffer for 15 min to allow collection of dissociation phase data, followed by a 1 min pulse of 10 mM glycine, pH 1.5 to regenerate the antibody capture surface on the chip. Binding responses to test and control antibody were recorded from duplicate injections of each concentration of analyte. In addition, several buffer injections were distributed throughout the injection series. Select buffer injections were used in conjunction with reference cell responses to correct for injection artifacts and / or non-specific binding interactions in the raw data set (commonly referred to as "double referencing"). The corrected binding data were globally fit to a 1:1 binding model (Biacore T200 Evaluation software 2.0, GE Healthcare, Pittsburgh, PA). Calculated kinetic parameters (k on and k off ) and k off / k on The Kd determined as is shown in Table 1.

[0258] [Table 2]

[0259] [Table 3]

[0260] As can be seen from the data above, the bispecific antibody molecule QD6 / B09-57 binds with high affinity to human cMet (approximately 2 nM Kd) and binds with high affinity to human EGFR (approximately 6 nM Kd), while the bispecific antibody molecule RAA22 / B09-57 binds with a similarly high affinity to human c-Met (approximately 2 nM Kd) but binds to human EGFR with a lower affinity (approximately 45 nM Kd) compared to QD6 / B09-57.

[0261] 2.2 Simultaneous binding of DuetMab to EGFR and cMET. Simultaneous binding studies to recombinant human EGFR and cMET proteins were measured by biolayer interferometry on an Octet384 instrument essentially as described (Mazor, 2015). Briefly, 5 μg / mL of His-tagged cMET antigen in assay buffer [PBS pH 7.2, 3 mg / mL bovine serum albumin (BSA), 0.05% (v / v) Tween 20] was first captured on a NI-NTA biosensor. After a washing step to remove unbound protein, each loaded biosensor was exposed to sequential association and dissociation interactions, first with 66 nM antibody and then with 500 nM EGFR antigen. Association and dissociation curves were calculated from nonlinear fits of the data using Octet384 software v.9.0. As shown in Figure 2, DuetMab was shown to bind both antigens simultaneously, whereas the parental anti-cMET IgG showed specific binding only to cMET, and the two anti-EGFR IgGs showed no binding to the cMET-loaded sensor.

[0262] 2.3 Specificity of EGFR and cMet Specificity for EGFR and cMET species paralogs and closely related family members was determined by ELISA. Briefly, antigen solution was prepared at 1 μg / mL in PBS, and 50 microliters was coated onto half the area of ​​an ELISA assay plate. Plates were washed and blocked with 1% BSA in PBS containing 0.005% Tween-20 (PBS-T) for 1 h at room temperature. Wells were washed 4 times with PBS-T. As shown in Figure 3, the primary antibodies used were R374 (non-binding IgG1 isotype control antibody), B09 (anti-cMET antibody), QD6 (anti-EGFR antibody), RAA22 (anti-EGFR antibody), QD6 / B09 (bispecific EGFR / c-MET DuetMAb), RAA22 / B09 (bispecific EGFR / c-MET DuetMAb), PaniX (anti-EGFR antibody), MetMab (anti-cMET antibody), and Mab11311 (anti-HER4 antibody). Wells were incubated with 50 μL of the indicated primary antibody diluted in PBS-T in a 1:3 dilution series starting at 10 μg / mL and ending at 0.002 μg / mL, except for MAB1131, the HER4-binding mAb control, where the series started at 1 μg / mL. Wells were washed four times with PBS-T, and then 50 μl of goat anti-human Fab HRP-conjugated secondary antibody diluted 1:5000 in PBS-T was added to each well and incubated for 1 hour at room temperature. Fifty microliters of TMB substrate solution was added to all wells and incubated for 5–30 minutes at room temperature until a strong signal was observed in the positive control wells. Fifty microliters of TMB stop solution was added to all wells, and absorbance was read at 450 nm on a SpectraMax M5 microplate reader. Data were analyzed with SoftMax Pro 5 software and plotted using GraphPad Prism 7 graphing software.

[0263] To determine species cross-reactivity, ELISA assays were performed as described above. As shown in Figure 3A, the high affinity EGFR IgG, QD6 and the monovalent bispecific EGFR / cMET DuetMAb, QD6 / B09, bound human, cynomolgus, and mouse EGFR with strong signals in the ELISA assay. In contrast, the reduced affinity EGFR IgG, RAA22, bound weaker to human, cynomolgus, and mouse EGFR compared to QD6. Binding to mouse EGFR was weak but detectable. The corresponding monovalent bispecific EGFR / cMET DuetMAb, RAA22 / B09, bound even weaker to human and cynomolgus EGFR and only slightly to mouse EGFR compared to the bivalent parent IgG, RAA22. cMET IgG, B09, and all bispecific variants showed comparable binding to human and cynomolgus cMET. No binding to mouse cMET was detected with any of the antibodies. These results are consistent with the binding kinetics determined by surface plasmon resonance measurements on a BIAcore instrument (Table 1 above).

[0264] As shown in Figure 3B, neither the parental IgG nor the derived bispecific antibodies showed any appreciable binding to EGFR HER family proteins, HER2, HER3, or HER4. Similarly, none of the antibodies showed significant binding to cMET family members, Ron (CD136), or semaphorin 3a.

[0265] These results demonstrate that the parent IgG and the resulting bispecific antibody bind specifically to their cognate targets, with no detectable binding to closely related family members.

[0266] Example 3 - Monitoring antibody internalization and transport to acidified compartments using pH-sensitive dyes Target expression in normal non-tumor tissues can result in toxicity that reduces the therapeutic window of the antibody conjugate by reducing payload delivery to the target tissue. The design of the bispecific antibody or antigen-binding fragment thereof of the disclosed compounds (e.g., radioimmunoconjugates) is intended to maximize the delivery of the antibody molecule (i.e., payload delivery vehicle) to tumor cells that co-express two targets while minimizing the impact of the conjugate in normal tissues with little or no co-expression of the targets. To evaluate whether dual targeting using the bispecific antibody RAA22 / B09 with reduced EGFR affinity offers a selectivity advantage compared to single target association, the inventors performed a study comparing the internalization efficiency of the bispecific mAb and the monovalent parent antibody containing the bispecific, using antibodies labeled with pH-sensitive dyes.

[0267] Antibody internalization and trafficking to acidified intracellular compartments such as lysosomes and endosomes was facilitated by the use of antibodies labeled with pHAb pH-sensitive dye (Promega). This dye exhibits very weak fluorescence at pH>7, but strong fluorescence at acidic pH, reaching a maximum at approximately pH 5. Briefly, antibodies were labeled with pHAb amine-reactive dye according to the manufacturer's recommendations. The labeled antibodies were the R347 IgG1 isotype control, the monovalent bispecific control antibodies anti-EGFR antibodies RAA22 / R347 and anti-cMET B09 / R347, and the EGFR / cMET monovalent bispecific antibody RAA22 / B09. NCI-H1975 lung cancer cells, which co-express moderate levels of EGFR (relative receptor density approx. 33,000) and cMET (relative receptor density approx. 50,000), were cultured at 2 × 10 5Cells were seeded at a density of 100 microliters / mL into a clear-bottom, black-walled 96-well assay plate. The plates were incubated overnight at 37° C., 5% CO2 in a humidified incubator. The plates were then cooled on ice for 30 minutes, after which various concentrations of pHAb-labeled bispecific and monovalent control antibodies were added in pre-chilled growth medium. The cultures were cooled on ice for an additional 30 minutes, and then fluorescence was read on an Operetta High Content Imaging system using a Cy3 filter, with this first reading designated as time 0. The plates were returned to the 37° C. incubator and additional readings were taken after 3, 6, 24, 30, and 48 hours.

[0268] A representative internalization experiment using pHAb-labeled mAbs at 1.25 μg / mL to treat NCI H1975 cells is shown in Figure 4. The non-binding IgG1 isotype control antibody R347 did not show any detectable fluorescence at any time point. The EGFR / cMET bispecific antibody RAA22 / B09 showed intracellular fluorescence by 3 h, and the fluorescence intensity continued to increase up to 48 h after treatment. The monovalent EGFR-binding control antibody RAA22 / R347 showed very weak fluorescence from the 24 h time point, which did not increase dramatically up to the 48 h time point. The monovalent monospecific cMET-binding control antibody B09 / R347 showed moderate fluorescence at 24 h, which further increased by 48 h. Nevertheless, the intensity of the fluorescent signal of the monospecific cMET antibody was moderate compared to the bispecific RAA22 / B09, suggesting that the bispecific antibody has a higher internalization efficiency than the monospecific parent antibody in the dual target expressing cell line tested here. When cells were treated with pHAb-labeled mAbs at 0.625 μg / mL, the difference between bispecific antibodies and monospecific controls became even more pronounced (Figure 5). While the monospecific antibodies showed almost no fluorescence even after 48 h, the bispecific RAA22 / B09 antibody showed intracellular fluorescence again by 3 h, and the fluorescence intensity continued to increase up to 48 h after treatment. These results are consistent with the hypothesis that dual-targeting bispecific antibodies mediate efficient internalization into cells that co-express both EGFR and cMET. At the same time, the monospecific parent antibody exhibits reduced uptake and fluorescence intensity compared to the bispecific antibody. A logical extension of these conclusions suggests that bispecific antibodies may behave like monospecific antibodies in tissues that express only one target rather than both, which is generally the case for EGFR and cMET. Of particular note is that the reduced affinity EGFR control mAb, RAA22 / R347, labeled with pHAb, showed negligible uptake and fluorescence. Reduced binding to EGFR may minimize the effect of the antibody conjugate in normal tissues such as skin, which express significant levels of EGFR but little or no cMET.

[0269] Example 4 - Monitoring antibody internalization using confocal microscopy The internalization kinetics of labeled DuetMab:RAA22 / B09 and QD6 / B09 antibodies were assessed in vitro using live cell confocal fluorescence microscopy.

[0270] 4.1 Materials and Methods H1975 and HCC827 cells were obtained from ATCC. RAA22 / B09, QD6 / B09, and single arm derivatives were obtained from MedImmune. QD6 / B09 and single arm specific controls QD6 / IgG and B09 / IgG with IgG Fab arms were obtained from non-specific human IgG1 NMGC and obtained from MedImmune. RAA22 / B09 and single arm specific controls RAA22 / IgG and B09 / IgG derived from non-specific human IgG1 R347 were obtained from MedImmune. RPMI (11875-093), HEPES (15630106), sodium pyruvate (11360070), AlexaFluor® 647 (A-20186) monoclonal antibody labeling kit, Zeba™ Spin Desalting columns (87767), and CellTracker™ Blue CMAC (C2110) were obtained from Life Technologies (Carlsbad, CA). Accutase Cell Detachment Solution (423201) was obtained from BioLegend (San Diego, CA). HyClone heat-inactivated fetal bovine serum (SH30071.03HI) was obtained from GE Life Sciences (Marlborough, MA). PBS (21-040) was obtained from Corning Incorporated (Corning, NY). FcR blocking reagent (130-059-901) was obtained from Miltenyi Biotec Inc. (Auburn, CA). Polypropylene round-bottom tubes (352063) were obtained from BD Biosciences (San Jose, CA). CellCarrier 384-well microplates were obtained from PerkinElmer Inc. (catalog number 6007550, Waltham, MA).

[0271] Preparation of AlexaFluor conjugates Monoclonal antibodies were conjugated with AlexaFluor-647 dye using an antibody labeling kit according to the manufacturer's instructions. Briefly, 50-100 micrograms of antibody in sodium bicarbonate buffer, pH=8.3, was incubated with reactive dye reagent with gentle agitation for 1 h at room temperature. Unincorporated dye was removed by size-exclusion chromatography using Zeba™ spin desalting columns with 40K MWCO equilibrated with 1X PBS according to the manufacturer's instructions.

[0272] Cultivation and preparation of cells for staining Adherent H1975 or HCC827 cells were cultured in T-75 flasks in a CO2 incubator using medium RPMI-1640 containing 10% fetal bovine serum (FBS) until 80-90% confluency after initial seeding. On the day of the experiment, the adherent monolayer grown in the T-75 flask was dissociated into a cell suspension using Accutase. The detached cells were washed twice with 1x PBS using centrifugation at 300 x g for 5 min. The cells were then resuspended in phenol-free RPMI at a concentration of 2 x 106 cells / mL and used for staining.

[0273] Cell staining for imaging A cell suspension of 2x106 cells / mL was incubated with 1 μM CellTracker™ Blue CMAC prepared in phenol-free RPMI for 30 minutes at 37°C in a CO2 incubator. Unincorporated CellTracker™ Blue CMAC dye was removed by washing twice with phenol-free RPMI using centrifugation at 300xg for 5 minutes at 4°C. Cells were then cooled on ice and blocked with 10ul FcR blocking reagent per 1x106 cells for 15 minutes. 2x10 5Cells were dispensed into 5 mL round-bottom tubes and incubated with fluorescent antibodies at a final concentration of 2.5 μg / mL. After removing unbound fluorescent reagent by centrifugation at 4 °C, cells were resuspended in phenol-free RPMI containing 100 mM HEPES, 1 mM sodium pyruvate, and 1% FBS. Cells were transferred to multiple wells of a 384-well imaging plate at a density of 5,000 cells per well and briefly centrifuged at 2,200 rpm for 2 min at 4 °C before image acquisition.

[0274] Acquiring cell images using confocal fluorescence microscopy Stained cells in imaging plates (384-well format) were imaged with the Opera confocal fluorescence imaging system as previously described (Vainshtein, 2015) or transferred to a Zeiss Axio Observer.Z1 inverted microscope (Carl Zeiss Microscopy, Thornwood, NY) equipped with a 40X / 1.2NA LCIPlan Apo objective. For experiments using the Zeiss microscope, the imaging environment was maintained at 37°C, 5% CO2, and 70% humidity using an incubator XLmulti S DARK (PeCon Gmbh, Erbach, Germany). Samples were illuminated with 405, 488, 561, and 63 nm solid-state lasers (Carl Zeiss Microscopy, Thornwood, NY). A series of images were acquired at the indicated times using a Yokogawa CSU-X1 Spinning Disk Unit (Yokogawa Electric Corporation, Tokyo) and an Evolve 512 EMCCD (Photometrics, Tuscon, AZ). Prior to image acquisition, an aliquot of stained cells was used to determine exposure parameters, including laser power, exposure time, and camera gain. Images were processed using ZEN 2.3 (Carl Zeiss Microscopy, Thornwood, NY) and analyzed using Columbus software (PerkinElmer, Waltham, MA).

[0275] Algorithms for image analysis of internalization The algorithm used for quantification of antibody internalization has been described previously (Vainsthein, 2015) with the following updates and modifications: The reference channel used for iterative image processing was generated from CellTracker™ Blue CMAC (CTB) staining of the cells. The signal channel was additionally derived from the antibody AlexaFluor-647 channel of the imager. Images were processed by the algorithm using algorithm-defined parameters. These parameters were initially set as default values ​​and then optimized for each cell type and experiment. Cells were identified using the CTB staining of the images using a threshold setting that detects areas on the image that are more intense than their surroundings by excluding areas with a fluorescence intensity signal below a threshold. The remaining identified cell objects were designated as “Total Cell”. Cells were classified according to morphological characteristic areas (120–600 μm 2 Objects were further selected by filtering for size (number of objects) and circularity (>0.5). Then, using parameters defined in the algorithm, "membrane regions" and "cytoplasmic regions" within the allowed cells were constructed around the boundaries of the objects. The fluorescence intensity of each region was used to monitor the antibody-associated AlexaFluor-647 signal. The fluorescence intensity of each region was reported as the average of the sum of all pixels within the allowed cells.

[0276] The accumulation of antibody-associated fluorescence in the cytoplasm was used to quantify the kinetics of antibody internalization. To ensure comparability of results due to variations in cell staining and fluorescence intensity, the cytoplasmic signal was normalized by the total cell signal at each time point and designated as the internalization rate using the following formula: Internalization rate = intensity(cytoplasm) / (intensity(cytoplasm)+intensity(membrane)). The internalization rate constant, k, was calculated from the time course of internalization by curve fitting of the data using the following formula: F(t)=(1-e-k,t)·F, where F is the maximum ratio of cytoplasmic intensity per cell to total intensity per cell. Curve fitting of the data was performed using Graphpad Prism (GraphPad Software, La Jolla, CA). The half-life of internalization (T) was calculated as the ratio of ln(2) to k.

[0277] 4.2 In vitro internalization of RAA22 / B09 and QD6 / B09 Internalization kinetics of AlexaFluor647 (AF647) primary labeled DuetMab:RAA22 / B09 and QD6 / B09 antibodies were evaluated in vitro using the EGFR and c-MET expressing cell line H1975. Each antibody was pre-bound to cells and live cell confocal fluorescence microscopy was used to monitor antibody translocation from the cell surface to the cytoplasm. Figure 6 shows that both antibodies were primarily localized to the cell surface before being subjected to internalization conditions (T=0) and translocated to the cytoplasmic region (blue) after 1 h (T=1h). Kinetic images were taken every 5 min over the course of internalization and processed using a quantification algorithm (see above) to determine the kinetic constants and half-lives of internalization. Figure 6B shows very similar internalization kinetics of QD6 / B09 and RAA22 / B09 with half-lives of 37.5±10.6 min and 43.2±15.5 min, respectively.

[0278] To evaluate the mode of antibody internalization and investigate the contribution of each arm to the overall internalization of DuetMab, we evaluated the internalization of single-arm specific control antibody molecules QD6 / IgG and B09 / IgG for DuetQD6 / B09, and RAA22 / IgG and B09 / IgG for DuetRAA22 / B09 in H1975 cells expressing both EGFR and c-MET. As only one arm is specific for the target receptor, the control antibody can only be internalized via one receptor, ruling out dual receptor targeting and crosslinking as modes of internalization.

[0279] The internalization profiles of QD6 / B09 (Figure 7A) and RAA22 / B09 (Figure 7B) show very similar patterns of a concomitant decrease in membrane mAb-Fl647 signal accompanied by an increase in mAb-AF647 signal in the cytoplasm, which is a typical profile of internalization. However, their single-arm constructs showed very different internalization profiles. The single-arm QD6 / IgG had a nearly identical internalization time course as the QD6 / B09 DuetMab (Figure 7A, left and center), indicating that internalization of the QD6 / B09 Duet is mainly driven by the EGFR arm of the molecule, with minimal contribution from the B09 arm. Indeed, the B09 / IgG construct showed a very low level of internalization (Figure 7B, right). The rapid and widespread decrease in membrane signal corresponded to a very gradual increase in cytoplasmic signal, likely due to widespread dissociation of prebound B09 / IgG from the cell surface c-MET receptor. Dissociation of the antibody then led to moderate internalization of B09 / IgG. These results revealed that internalization of the QD6 / B09 duet was driven primarily by the EGFR arm of the molecule, with minimal contribution from the B09 arm.

[0280] In contrast, the RAA22 / B09 DuetMab showed a very different internalization profile when compared to its single-arm control antibody. As shown in Figure 7B, the cytoplasmic intensity values ​​of the RAA22 / B09 DuetMab were 10.98- and 4.70-fold higher than RAA22-IgG and B09-IgG, respectively. The inefficient internalization of B09 / IgG could be attributed to its significant dissociation (discussed above), whereas RAA22 / IgG was rapidly internalized. However, due to the lower affinity of the EGFR arm, the number of RAA22 / IgG molecules was 10.98-fold lower (based on fluorescence intensity) than the RAA22-B09 DuetMab. In contrast to the single-arm construct, there was a marked increase in the amount of duet RAA22 / B09 mAb that entered the cytoplasm, indicating that both antibody arms must engage the target receptor to promote internalization. This finding indicates that the internalization mechanisms of QD6 / B09 and RAA22 / B09 DuetMabs are distinct, with QD6 / B09 being driven primarily by the EGFR arm, whereas RAA22 / B09 requires both the EGFR and c-MET arms for association.

[0281] 4.3 Internalization of RAA22 / B09 in cell lines with different levels of target receptor Because binding of both EGFR and c-MET arms to the target receptor promoted internalization of RAA22 / B09 receptors in H1975 cells, we investigated whether increasing the number of EGFR and c-MET receptors would affect the internalization properties. The respective receptor levels, determined by Western blotting, were approximately 33,000 for EGFR and 50,000 for c-MET in H1975 cells, and approximately 790,000 for EGFR and 523,000 for c-MET in HCC827 cells. The internalization profile of RAA22 / B09 in H1975 (medium receptor) and HCC827 (high receptor) cells shows a marked increase in internalization in HCC827 cells (Figure 8).

[0282] As expected, binding to HCC827 cells was on average 8.9-fold higher than H1975 (T = 0, 3.1x10 7 MFI vs. 3.5x10 6 MFI). The level of internalization (as judged by peak cytoplasmic intensity) was 21.7-fold higher in HCC827 cells, suggesting that significantly higher concentrations of antibody enter the cytoplasm in cells expressing high levels of the target receptor. Importantly, in addition to the markedly different intensities, the internalization profiles (membrane and cytoplasmic signals over time) were also significantly different between HCC827 and H1975 cells. In high-expressing HCC827 cells, the decrease in RAA22 / B09-AF647 membrane signal corresponded to an inverse increase in RAA22-B09 cytoplasmic signal, while the total RAA22 / B09 signal was maintained over time, indicating strong dual-arm antibody interaction with both receptors and subsequent internalization. In H1975 cells, there was a concomitant decrease in total and membrane intensity, indicating that a portion of the pre-bound antibody may have dissociated from the cell surface and failed to be internalized into the cells. Similar profiles showing dissociation were observed for RAA22 / IgG and B09 / IgG internalization in HCC827 cells, but single-arm association did not result in effective binding and tended to dissociate (Figures 9A and 9B). This data suggests that there is a mixed mode of receptor interaction (single-arm and dual-arm association) when RAA22 / B09 is internalized in H1975 cells. Taken together, these data suggest that the expression level of the target cell receptor is an important factor determining the extent and efficiency of RAA22 / B09 internalization.

[0283] Example 5. General Materials and Methods for Radiopharmaceuticals Lutetium-177 is available from ITM Medical Isotopes as lutetium trichloride in a 0.05N hydrochloric acid solution; indium-111 is available from BWXT as indium trichloride in a 0.05N hydrochloric acid solution; actinium-225 is available from Oak Ridge National Laboratories as actinium-225 trinitrate or from Canadian Nuclear Laboratories as actinium-225 trichloride.

[0284] Analytical HPLC-MS can be performed using a Waters Acquity HPLC-MS system consisting of a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (samples cooled to 10° C.), a Water Acquity Column Manager (column temperature 30° C.), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD equipped with electrospray ionization, and a Waters Acquity BEH C18, 2.1×50 (1.7 μm) column. Preparative HPLC can be performed using a Waters HPLC system consisting of a Waters 1525 Binary HPLC pump, a Waters 2489 UV / Visible detector (monitoring at 254 nm and 214 nm), and a Waters XBridge Prep Phenyl or C18 19×100 mm (5 μm) column.

[0285] HPLC elution method 1: Waters Acquity BEH C18 2.1×50mm (1.7μm) column; Mobile phase A: H2O (0.1%v / v TFA); Mobile phase B: Acetonitrile (0.1%v / v TFA); Flow rate = 0.3 mL / min; initial = 90% A, 3-3.5 min = 0% A, 4 min = 90% A, 5 min = 90% A.

[0286] HPLC dissolution method 2: Waters XBridge Prep Phenyl 19×100 mm (5 μm) cartridge; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: Aspartame (0.1% v / v TFA); flow rate: 10 mL / min, initial stage = 80% A, 13 min = 0% A.

[0287] HPLC dissolution method 3: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) cartridge; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: Acetonitrile (0.1% v / v TFA); flow rate = 0.3 mL / min; initial stage = 90% A, 8 min = 0% A, 10 min = 0% A, 11 min = 90% A, 12 min = 90% A.

[0288] HPLC dissolution method 4: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) cartridge; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: Aspartame (0.1% v / v TFA); flow rate: 10 mL / min, initial stage = 80% A, 3 min = 80% A, 13 min = 20% A, 18 min = 0% A.

[0289] HPLC dissolution method 5: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) cartridge; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: Aspartame (0.1% v / v TFA); flow rate: 10 mL / min, initial stage = 90% A, 3 min = 90% A, 13 min = 0% A, 20 min = 0% A.

[0290] HPLC dissolution method 6: Waters XBridge Prep C18 OBD 19×100 mm (5 μm) cartridge; mobile phase A: H2O (0.1% v / v TFA); mobile phase B: Aspartame (0.1% v / v TFA); flow rate: 10 mL / min, initial stage = 75% A, 13 min = 0% A, 15 min = 0% A.

[0291] HPLC elution method 7: Waters XBridge Prep C18 OBD 19 x 100 mm (5 μm) column; Mobile phase A: H2O (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min, initial = 80% A, 12 min = 0% A, 15 min = 0% A.

[0292] HPLC elution method 8: Waters XBridge Prep C18 OBD 19 x 100 mm (5 μm) column; Mobile phase A: H2O (0.1% v / v TFA); Mobile phase B: Acetonitrile (0.1% v / v TFA); Flow rate: 10 mL / min, initial = 90% A, 12 min = 0% A, 15 min = 0% A.

[0293] Analytical size exclusion chromatography (SEC) can be performed using a Waters system consisting of a Waters 1525 binary HPLC pump, a Waters 2489 UV / Visible detector (monitoring at 280 nm), a Bioscan Flow Count radio detector (FC-3300), and a TOSOH TSKgel G3000SWxl, 7.8×300 mm column. The isocratic SEC method can be performed with a mobile phase of 0.1 M phosphoric acid, 0.6 M NaCl, 0.025% sodium azide, pH=7, and a flow rate of, for example, mL / min.

[0294] MALDI-MS (positive ion) can be performed using a MALDI Bruker Ultraflextreme spectrometer.

[0295] Radio thin layer chromatography (radio-TLC) can be performed by a Bioscan AR-2000 imaging scanner and can be carried out on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plates using citrate buffer (0.1 M, pH 5.5).

[0296] Example 6. Synthesis of 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B) The bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B), can be synthesized according to the scheme provided in FIG. 11. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA-(tBu)4, 50 mg, 0.07 mmol) in ACN (2.0 mL) is added DSC (50 mg, 0.21 mmol) followed by pyridine (0.20 mL, 2.48 mmol). The reaction is stirred at room temperature for 1 h. 11-Aminoundecanoic acid (70 mg, 0.36 mmol) is added to the reaction mixture followed by PBS solution (1.0 mL) at room temperature. The reaction is stirred at room temperature for 72 h. The reaction mixture is filtered through a syringe filter and directly purified by preparative HPLC using Method 6 to give intermediate 2-A.

[0297] To a solution of intermediate 2-A (40 mg, 0.03 mmol), TFP (90 mg, 0.54 mmol) and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL) at room temperature was added pyridine (0.05 mL, 50 mg, 0.62 mmol). The solution was stirred at room temperature for 24 h. The reaction was directly purified by preparative HPLC using Method 7 to give intermediate 2-B as a wax after concentration using a Biotage V10 Rapid Evaporator.

[0298] Intermediate 2-B is dissolved in DCM / TFA (1.0 mL / 2.0 mL) and stirred at room temperature for 24 h. The reaction is concentrated by airflow and purified directly by preparative HPLC using Method 8 to give Compound B as a clear wax after concentration. An aliquot is analyzed by HPLC-MS Elution Method 3.

[0299] 1 H NMR(600MHz,DMSO-d6)δ 7.99-7.88(m,1H),7.82(t,J=5.5Hz,1H),3.78(broad s,4H),3.43(broad s,12H),3.08(broad s,4H),3.00(m,3H),2.93(broad s,3H),2.77(t,J=7.2Hz,2H),2.30(broad s,2H),1.88(broad s,2H),1.66(p,J=7.3Hz,2H),1.36(m,4H),1.32-1.20(m,9H).

[0300] Example 7. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C) The bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C), is synthesized according to the scheme provided in FIG. 12.

[0301] To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA(tBu)4, 100 mg, 0.143 mmol) in ACN (8.0 mL) is added DSC (73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol). The reaction mixture is stirred at ambient temperature for 90 min. This solution is added to a half solution of amino PEG3 acid (63 mg, 0.285 mmol in 1.2 mL DMF) in a 100 mL round-bottom flask. After 4 h at ambient temperature, the reaction is worked up by concentrating to dryness under a stream of air. The crude material is purified by HPLC elution method 2 (crude material is dissolved in 6 mL of 20% ACN / H2O). Fractions containing the product are pooled, concentrated in vacuo, and then co-evaporated with ACN (3 x 2 mL).

[0302] To a vial containing intermediate 1-A (82 mg, 60 μmol), ACN (2 mL), NEt3 (50 μL, 360 μmol, 6 eq.), HBTU (23 mg, 60 μmol, 1 eq.) and TFP solution (50 mg, 300 μmol, 5 eq., dissolved in 250 μL ACN) are added. The resulting clear solution is stirred at ambient temperature for 3 h. The reaction is worked up by concentrating the solution to dryness under a stream of air, then diluting with ACN / H2O (1:1, 3 mL total) and purifying by preparative HPLC using elution method 4. Fractions containing the product are pooled, concentrated under vacuum, and then coevaporated with ACN (3×2 mL). Intermediate 1-B is obtained as a clear residue.

[0303] To the vial containing intermediate 1-B (67 mg, 64 μmol) is added DCM (2 mL) and TFA (2 mL). The resulting solution is stirred at ambient temperature for 16 h. Additional TFA (2 mL) is added and the reaction is stirred at room temperature for 6 h. The reaction is concentrated to dryness under a stream of air and the crude product is finally dissolved in ACN / H2O (1 mL 10% ACN / H2O). The crude reaction solution is then purified by preparative HPLC using elution method 5. Fractions containing the product are pooled, frozen and lyophilized. Compound C is obtained as a white solid. An aliquot is analyzed by HPLC-MS elution method 3.

[0304] 1 H NMR(DMSO-d6,600MHz)δ 7.97-7.91(m,2H),3.77(t,2H,J=6.0Hz),3.58-3.55(m,2H),3.53-3.48(m,8H ),3.44-3.38(m,10H),3.23-3.08(m,11H),3.02(t,2H,J=6.0Hz),2.93(broad s,4H),2.30(broad s,2H),1.87(broad s,2H).

[0305] Example 8. Conjugation and radiolabeling for the synthesis of radioimmunoconjugates containing EGFR-cMET antibodies 8.1. Synthesis of immunoconjugate Compound D [ka] A 133 mL sterile solution container was charged with a PBS (phosphate buffered saline) solution of EGFR-cMET monoclonal bispecific antibody RAA22 / B09 DuetMab (58.8 mL; 5.1 mg / mL; 300 mg), followed by 5.88 mL of carbonate buffer (1.0 M, pH 9.5), and 2.83 mL of a 0.001 M HCl solution of Compound C (7.7 equivalents; 17.3 mg in 3.00 mL; this solution was prepared immediately before use). The pH of the resulting mixture was measured by pH strips to be approximately 9.5-10. The reaction was allowed to react for 2 hours at room temperature. Ammonium acetate (28.1 g) was added to the reaction mixture and the resulting solution was loaded onto a 5 mL Cytiva HiTrap Butyl HP (HIC) cartridge (the HIC cartridge was first pretreated with sodium acetate buffered saline with Tween (SABST; 50 mL) and then with ammonium acetate solution (50 mL; 0.42 g / mL). The HIC cartridge was washed with ammonium acetate solution (10 mL) and then the product was eluted with SABST (~10 mL). The product solution was diluted to ~15 mL with SABST and loaded onto a Cytiva 15 mL HiPrep 26 / 10 desalting cartridge pretreated with SABST (250 mL). The product fraction was eluted with SABST (~16 mL) to give a clear, colorless liquid and analysis using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) indicated the formation of compound D (15.9 mL, 14.8 mg / mL, 234.9 mg, 78%). A chelate-to-antibody ratio (CAR) of 5.3 was determined using matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry and calculated from the difference in mass-to-charge ratio between the EGFR-cMET antibody and immunoconjugate Compound D.

[0306] 8.2 Radiosynthesis of radioimmunoconjugate compound E [ka] In a 1.5 mL Eppendorf tube, add the SABST solution of compound D (0.100 mL; 2.00 mg / mL; 0.200 mg), followed by [ 177A 0.04 M HCl solution (2.2 mCi) of [Lu]LuCl3 was charged. After 20 min at room temperature, iTLC analysis of the reaction mixture (silica gel (SG) plate, 0.02 M citrate buffer (5% MeOH) as mobile phase) showed a radiochemical conversion (RCC) of >97%. Purification was performed using a 1 mL column packed with Sephadex G50 resin (hydrated with SABST). Product fractions were eluted using SABST and combined. A solution of sodium L-ascorbate and calcium diethylenetriaminepentaacetic acid trisodium salt hydrate (DTPA) in SABST was added and the resulting formulation (approximately 10 mM L-sodium ascorbate, 1 mM DTPA) was analyzed by iTLC and SEC-HPLC at the end of synthesis (EOS), which showed formation of compound E (364 μL, 0.431 mg / mL, specific activity of 10.1 mCi / mg, radiochemical purity >95%, chemical purity >99%).

[0307] 8.3 Radiosynthesis of Radioimmunoconjugate Compound F [ka] Add compound D in SABST solution (0.193 mL, 2.07 mg / mL, 0.400 mg) to a 1.5 mL Eppendorf tube, followed by [ 225 A 0.001 M HCl solution (24 μCi) of Ac]Ac(NO3)3 was added. After 120 min at 35°C, the reaction was quenched by the addition of DTPA (2 μL of a 0.1 M solution in SABST). iTLC analysis of the reaction mixture (SG plate, 0.02 M citrate buffer (5% MeOH) as mobile phase) showed an RCC of 99%. Purification was performed using a 1 mL column packed with G50 resin (hydrated with SABST). Product fractions were eluted using SABST and combined. A solution of sodium L-ascorbate and DTPA in SABST was added and the resulting formulation (approximately 10 mM L-sodium ascorbate, 1 mM DTPA) was analyzed by EOS, iTLC and SEC-HPLC, which showed formation of compound F (448 μL, 0.680 mg / mL, 0.053 mCi / mg specific activity, radiochemical purity >99%, chemical purity >99%).

[0308] Example 9. 177 In vitro binding of [Lu]-Compound C-anti-EGFR-cMET conjugate Using four different cell lines: HCC827 cells, HT29 cells, H441 cells, and H1975 cells, 177 A study was conducted to evaluate the receptor binding affinity of the [Lu]-Compound C-anti-EGFR-cMET conjugate (i.e., Compound E) by following the procedure described below.

[0309] The purpose of this assay was to confirm that the radioimmunoconjugates maintained the binding properties of the native antibody in EGFR- and / or cMET-expressing cell lines, including HT29, H441, HT29, HCC827, and H1975. One day prior to the experiment, cells (1.5-2 × 10 5 ) were seeded in 48-well microplates in 500 μL of supplemented medium. At the start of the assay, cells were washed once with PBS and then treated with increasing concentrations of Compound E (0.05 nM to 100 nM) in the presence and absence of 4 μM cold antibody [total binding (TB) and non-specific binding (NSB)], respectively. Plates were incubated at 4°C for approximately 3 hours with gentle shaking. After incubation, cells were washed twice with PBS and lysed with 1% Triton-X-100. Lysates were transferred to gamma counting tubes and analyzed along with Compound E standards in a Wizard 1470 gamma counter, and the amount of radioactivity was measured in counts per minute (CPM) for each lysate. The remaining lysate (25 μL) from each well was used to analyze protein content using a standard protein quantification assay.

[0310] Total, specific, and nonspecific binding values ​​(fmol / mg) were plotted against conjugate concentration as shown in Figures 14A-14B. Kd and Bmax were derived by curve fitting the specific binding data to a single-site hyperbolic model (Graph Pad Prism software, version 9). The binding data revealed desirable values ​​for Kd. The highest Bmax (fmol / mg) was observed for HCC827 (2861 ± 207) and HT29 (2351 ± 237), followed by H441 (1195 ± 153) and H1975 (498 ± 42).

[0311] The EGFR-cMET conjugate, i.e., compound E, was observed to exhibit high binding affinity of about 9.9 nM in HCC827 cells, about 5.7 nM in HT29 cells, about 15.5 nM in H441 cells, and about 6.0 nM in H1975 cells.

[0312] Example 10. 177 Internalization assessment of [Lu]-Compound C-anti-EGFR-cMET conjugate This internalization assay was designed to determine the degree of cellular retention of radiolabeled linker-antibody derivatives. The assay relies on the intrinsic ability of the EGFR-cMET receptor to internalize when bound to an antibody, and the ability to track radiolabeled compounds. Here, a fixed amount of radioimmunoconjugate is incubated with four different cell lines for a set period of time, and the retention rate is determined by calculating the amount of internalized radioactivity as a percentage of total cell-associated activity.

[0313] According to the protocol below, 177 A study was performed to evaluate the internalization of [Lu]-Compound C-anti-EGFR-cMET conjugate, i.e., Compound E, in four different cell lines: HCC827 cells, HT29 cells, H441 cells, and H1975 cells.

[0314] This assay was designed to determine the extent of cellular retention of radioimmunoconjugate Compound E. Briefly, the above cell lines were plated in triplicate in 24-well plates at 3 × 10 5Cells were seeded at a concentration of 1000 x 1000 cells / well (0 h, 2 h, and 24 h incubation time). The next day, the medium was decanted, the cells were washed once with sterile PBS, and treated with compound E (10 nM) for 2-3 h at 37 °C. After incubation, all plates were immediately placed on ice and the medium was discarded into pre-labeled (unbound) gamma counting tubes. Cells were washed once with sterile PBS, gently shaken, and decanted into pre-labeled (unbound) gamma tubes. Strong acid wash buffer (pH 2.5, 500 μL) was added at 0 h for 5 min at 4 °C, and the buffer was then collected into pre-labeled (membrane-bound) gamma counting tubes. Cells were then lysed with 300 μL of 1% Triton X-100 at room temperature for 30 min with gentle shaking. 250 μL of cell lysate was transferred to gamma counting tubes and counted for 10 min. Weak acid wash buffer (pH 4.6, 500 μL) was added to the plates for 2 and 24 hours and left at 4° C. for 15 minutes. The buffer was then collected in a pre-labeled (membrane-bound) gamma counting tube. 1 mL of warm medium was added to the plates and further incubated at 37° C. for 2 and 24 hours, respectively. After the specified incubation time, the plates were placed on ice and processed in the following manner: The medium was decanted and collected in a pre-labeled (discharge) gamma tube. The plates were then washed once with 1 mL of cold PBS and added to the discharge tube. Strong acid wash buffer was added to all wells and the plates were incubated on ice for 5 minutes. The acid wash fraction was then collected in a pre-labeled (recycle) gamma tube. The cells were lysed with 300 uL of 1% Triton X-100 for 30 minutes at room temperature. 250 μL of cell lysate was transferred to a pre-labeled (retain) gamma counting tube and counted for 10 minutes. 25 μL of the cell lysate fraction was transferred to a 96-well plate for protein quantification (Pierce BCA Protein Assay).

[0315] The results of this internalization study are shown in Figure 15. Retention rates were determined as CPM (dissolution) or CPM (excretion + recycling + dissolution). The lowest excretion rates were observed after 24 hours of culture in HCC827, followed by HT29, H441 and H1975 (approximately 25%, 30%, 35% and 45%, respectively). In this case, there appears to be a correlation between the expression levels of EGFR+cMET receptors and cell retention.

[0316] Example 11. Various animal models 177 In vivo biodistribution of [Lu]-Compound C-anti-EGFR-cMET conjugate According to the following protocol, 177 Five different cell line xenograft mouse models were used to evaluate the in vivo biodistribution of the [Lu]-DOTA-anti-EGFR-cMET conjugate (i.e., compound E).

[0317] Tumor inoculation: Cells were washed with PBS and detached with 0.25% trypsin-EDTA. Harvested cells were resuspended in a 1:1 mixture of PBS and Matrigel (BD, Oakville ON) at the following concentrations: HCC827:50×10 6 cells / mL HT29:20×10 6 cells / mL H1975:50×10 6 cells / mL H292:30×10 6 cells / mL H441:50×10 6 cells / mL

[0318] 100 μL of the mixture was injected subcutaneously into the right flank of 5-7 week-old female Balb / cNCI athymic NCr-nu / nu mice (Charles River Laboratories). Radioactive material injections began when tumors reached a volume of 150–200 mm3, approximately 7–10 days after inoculation, except for HCC827 xenografts, which reached the same size 3 weeks after inoculation.

[0319] Biodistribution study: Five groups of three mice bearing subcutaneous tumors (above) were administered approximately 0.74 MBq 177 200 μL of compound E containing Lu (approximately 2 μg of antibody) was injected intravenously via a lateral tail vein. At the indicated time points (4 h, 24 h, 48 h, 96 h, and 168 h) after injection, one group per time point was anesthetized with isoflurane, exsanguinated by cardiac puncture, and then euthanized to collect blood and different organs by dissection. Tumors and organs were rinsed with PBS to remove residual blood, blotted dry, and collected in pre-weighed gamma counting tubes. Radioactivity counts per minute contained in the tissue samples were measured using a gamma counter and then converted to μCi of decay-corrected radioactivity using calibration standards. Activity measurements and sample weights were used to calculate the percentage of injected dose per gram of tissue weight (%ID / g).

[0320] The results are expressed as percentage of injected dose per gram of tissue (%ID / g) and are shown in Figures 16A-16E. Biodistribution studies of compound E showed a typical biodistribution profile for IgG, with acceptable uptake levels in normal organs. The highest tumor uptake rates (%ID / g) were observed in H292>H441>HT29 and H1975 xenografts [approximately 75% (96 h, 168 h), approximately 35% (48 h, 96 h), approximately 29% (48 h, 96 h), and approximately 20% (48 h, 96 h), respectively]. In the HCC827 xenograft model, tumor uptake (%ID / g) peaked at 48 h with approximately 25% compound E, decreasing to approximately 17% at 96 and 168 h after injection. The HCC827 xenograft model was not advanced to therapeutic testing due to its slow tumor growth rate.

[0321] Example 12. Various animal models 225 In vivo efficacy of Ac]-Compound C-anti-EGFR-cMET conjugate A study was designed to evaluate the efficacy of different doses of Compound F, an actinium-225 labeled radioimmunoconjugate, compared to cold antibody clones and / or vehicle controls.

[0322] The efficacy test was carried out using compound F( 225 Ac radiolabeled EGFR-cMET antibody) and compared with cold antibody and / or vehicle control. Therapeutic efficacy studies were performed using H441, HT29 and H1975 tumor xenografts. In each study, six to seven groups of tumor-bearing animals (n=5) were intravenously injected with 200 μL of compound via the lateral tail vein. Compound F was formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween-80 buffer and administered at an activity of 50-400 nanocuries (nCi). As a control, non-radiolabeled unconjugated antibody was administered at a protein mass equivalent corresponding to the highest radioactivity dose of Compound F tested in the study. Tumor measurements were taken in two dimensions using calipers, two to three times per week for at least 60 days. Tumor length was defined as the longest dimension, and width was measured perpendicular to the tumor length. Animals were also weighed at the same time. Overall physical condition and general behavior were assessed daily. Tumor volumes (mm) were calculated as ellipsoids from caliper measurements. Tumor growth was expressed as relative tumor volume (RTV), which was the tumor volume measured on day X divided by the tumor volume measured on the day of dosing. In all models, 200 nCi and 400 nCi caused long-term tumor regression in all mice (Figures 17A-17C). The best response was observed in the H441 model, where both 50 nCi and 100 nCi doses showed tumor inhibition and regression in multiple mice. In the H1975 model, 50 nCi significantly delayed tumor growth in all mice, and the 100 nCi group showed mixed responses, including tumor growth inhibition, tumor inhibition, and regression. In the HT29 model, no efficacy was observed at the 50 nCi or 100 nCi doses, except for one mouse that showed tumor inhibition at 100 nCi.

[0323] References A number of publications have been cited above in order to more fully describe and disclose the present disclosure and the state of the art to which it pertains. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety.

[0324] [Table 4]

[0325] [Table 5]

[0326] [Table 6]

[0327] [Table 7]

[0328] [Table 8]

[0329] [Table 9]

[0330] [Table 10]

[0331] For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press.

[0332] Other embodiments While the invention has been described in relation to particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any change, use, or application which generally follows the principles of the invention, including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and which may conform to the essential characteristics set forth above.

Claims

1. A compound comprising the structure of Formula I, or a pharmaceutically acceptable salt thereof: A-L １ -(L ２ ) ｎ -B Formula I (In the formula, A is a chelating moiety or a metal complex thereof; B is an antibody or antigen-binding fragment thereof; L １ represents a bond, C═O, C═S, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; n is an integer from 1 to 5, inclusive; L ２ are each independently represented by formula II: -X １ -L ３ -Z １ - Formula II (In the formula, X １ is -C(O)NR １ -*, -NR １ C(O)-*,-C(S)NR １ -*, -NR １ C(S)-*, -OC(O)NR １ -*, -NR １ C(O)O-*, -NR １ C(O)NR １ -, -CH ２ -Ph-C(O)NR １ -*, -NR １ C(O)-Ph-CH ２ -*, -CH ２ -Ph-NH-C(S)NR １ -*, -NR １ C(S)-NH-Ph-CH ２ -*, -O-, or -NR １ -, where "*" represents L ３ indicates the connection point to R １ is hydrogen, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L ３ is an optionally substituted C １ -C ５０ Alkyl or optionally substituted C １ -C ５０ is heteroalkyl, Z １ is -CH ２ -#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR ２ C(O)-#, -C(O)NR ２ -# or -NR ２ -#, where "#" indicates the point of attachment to B, and R ２ is hydrogen, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; the antibody or antigen-binding fragment thereof comprises a first antigen-binding domain capable of binding to epidermal growth factor receptor (EGFR); and a second antigen-binding domain capable of binding to c-mesenchymal epithelial transition factor (cMET); The first antigen-binding domain comprises: i: A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO: 1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3 and ii: a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6).

2. The chelating moiety may be DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α,α',α",α'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapro pionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri( methylenephosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N''',N''''-pentaacetic acid), H ４ octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H ２ dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H ６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N",N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecanedimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), and porphyrin.

3. The compound has the formula Ia: 【Chemistry 1】 (In the formula, Y １ is -CH ２ OCH ２ (L ２ ) ｎ -B, -C(O)(L ２ ) ｎ -B, or -C(S)(L ２ ) ｎ -B and Y ２ is -CH ２ CO ２ Is it H? Y １ is H and Y ２ Is, L １ - (L ２ ) ｎ -B; or L 1 is 【Chemistry 2】 wherein R L is hydrogen or —CO 2 H.

3. The compound of claim 2, which is represented by: or a metal complex thereof, or a pharmaceutically acceptable salt thereof.

4. A is a metal complex of a chelating moiety, said metal complex comprising a metal selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, lanthanides, and actinides, said metal complex comprising: ４４ Sc, ４７ Sc, ５５ Co, ６０ Cu, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ６６ Ga, ６７ Ga, ６８ Ga, ８２ Rb, ８６ Y. ８７ Y. ８９ Zr, ９０ Y. ９７ Ru, ９９ Tc, ９９ｍ Tc, １０５ Rh, １０９ Pd, １１１ In, １１７ｍ Sn, １４９ Pm, １４９ Tb, １５３ Sm, １６６ Ho, １７７ Lu, １８６ Re, １８８ Re, １９８ Au, １９９ Au, ２０１ Tl, ２０３ Pb, ２１１ At, ２１２ Pb, ２１２ Bi, ２１３ Bi, ２２３ Ra, ２２５ Ac, ２２７ Th, and ２２９ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, comprising a radionuclide selected from the group consisting of: Th.

5. The radionuclide is ２２５ 5. The compound of claim 4, wherein the compound is: Ac or a progeny thereof, or a pharmaceutically acceptable salt thereof.

6. Y １ is H, or a pharmaceutically acceptable salt thereof.

7. a. X １ -C(O)NR １ -* or -NR １ C(O)-*, where "*" is L ３ indicates the connection point to R １ is H; b) Z 1 is —CH 2 —; c. n is 1 and L 3 comprises (CH 2 CH 2 O) 2-20 ; or d. n is 1 and L 3 is (CH 2 CH 2 O) m (CH 2 ) w , wherein m and w are each independently an integer from 0 to 10, inclusive, and at least one of m and w is not 0; 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof.

8. The compound has the following structure: 【Transformation 3】 or a metal complex thereof, or a pharmaceutically acceptable salt thereof.

9. The compound has the following structure: 【Chemistry 4】 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, comprising: Claim 10: a. the VH region of the first antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; and b. The VL region of the first antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof.

11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein the VH region of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL region of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

16.

12. the second antigen-binding domain comprises: i: A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 17 HCDR2 having the amino acid sequence of SEQ ID NO: 18 HCDR3 having the amino acid sequence of SEQ ID NO: 19; and ii: A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 20 LCDR2 having the amino acid sequence of SEQ ID NO: 21 LCDR3 having the amino acid sequence of SEQ ID NO: 22 10. The compound of claim 1, comprising:

13. A method for producing a medicament for a medicament comprising the steps of: (a) a VH region of the second antigen-binding domain comprising: (i) an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31; and b. The VL region of the second antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 32; 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof.

14. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein the VH region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 31 and the VL region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

32.

15. the antibody or antigen-binding fragment thereof a. a first heavy chain comprising a VH region of a first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof; b. a first light chain comprising a VL region of a first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof; c. a second heavy chain comprising a VH region of a second antigen-binding domain and a second heavy chain constant (CH) region or fragment thereof; and d. A second light chain comprising a VL region of a second antigen-binding domain and a second light chain constant (CL) region or a fragment thereof.

10. The compound of claim 1, comprising:

16. 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein the first and second CH regions each comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

33.

17. the first and second heavy chains form a heterodimer, and optionally one of the first and second heavy chains comprises a cysteine ​​(C) residue at position 354 and a tryptophan (W) residue at position 366, and the other heavy chain comprises a cysteine ​​(C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368; and / or the first and / or second CH region comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331; 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein the numbering of the constant region is according to the EU index.

18. the antibody or antigen-binding fragment thereof a. a modified CH region comprising a substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid; and b. The corresponding modified CL region, wherein the modified CL comprises a substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid. Including, i: the first heavy chain comprises a modified CH region and the first light chain comprises a corresponding modified CL region; or ii: the second heavy chain comprises a modified CH region and the second light chain comprises a corresponding modified CL region; 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein the substituted cysteine ​​in the modified CH region and the substituted cysteine ​​in the corresponding modified light chain are capable of forming a disulfide bond.

19. 19. The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein the modified CH region comprises a substitution at position 126 for a native non-cysteine ​​amino acid with a cysteine ​​amino acid; and the corresponding modified CL region comprises a substitution at position 121 for a native non-cysteine ​​amino acid with a cysteine ​​amino acid, wherein the numbering of the constant regions is according to the EU index.

20. a. the first CH region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:39; b. the second CH region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:40; c. the first CL region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:41; d) The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein the second CL region comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

34.

21. the antibody or antigen-binding fragment thereof a. a first heavy chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 35; b. a second heavy chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 36; c. a first light chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 37; and d. A second light chain comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

38.

10. The compound of claim 1, comprising:

22. a. the first heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:35; b. the second heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:36; c. the first light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:37; d. the second light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 38; 22. The compound of claim 21, or a pharmaceutically acceptable salt thereof.

23. The compound has the following structure: 【Transformation 5】 (In the formula, 【Transformation 6】 is an antibody or antigen-binding fragment thereof as defined in claim 1, or a pharmaceutically acceptable salt thereof.

24. the antibody or antigen-binding fragment thereof is linked to A-L via the side chain amino group of the lysine residue １ - (L ２ ) ｎ 24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, linked to -.

25. A pharmaceutical composition comprising a compound according to any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

26. 26. The pharmaceutical composition of claim 25 for use in the treatment of cancer.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer is a solid tumor cancer selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing's sarcoma, gallbladder cancer, glioma, head and neck cancer, liver cancer, lung cancer, neuroblastoma, neuroendocrine carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary adenoid cystic carcinoma, spermatocytic seminoma, and uveal melanoma.

28. The pharmaceutical composition of claim 26, wherein the use further comprises administering an antiproliferative agent, a radiosensitizer, or an immunomodulator.

29. 25. Use of a compound according to any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

30. The use of claim 29, wherein the cancer is a solid tumor cancer selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial adenocarcinoma, Ewing's sarcoma, gallbladder cancer, glioma, head and neck cancer, liver cancer, lung cancer, neuroblastoma, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary adenoid cystic carcinoma, spermatocytic seminoma, and uveal melanoma.

31. The use of claim 29, wherein the treatment further comprises administering an antiproliferative agent, a radiosensitizer, or an immunomodulatory agent.

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