Patent Application
20240408249
The present specification makes reference to a Sequence Listing (submitted electronically as a .xml file named “FPI_027_Sequence_Listing.xml” on Sep. 29, 2022). The .xml file was generated on Sep. 26, 2022 and is 263 kilobytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.
The epidermal growth factor receptor variant III (EGFRvIII) is amplified, highly expressed and present in 25-64% of glioblastoma multiforme (GBM). EGFRVIII mRNA and protein expression has been detected in a subset of carcinomas of the breast as well as in head and neck squamous cell carcinoma (HNSCC) using multiple complementary techniques. Unlike wild type (wt) epidermal growth factor receptor (EGFR), which is expressed in tissues of epithelial, mesenchymal and neuronal origin and plays a major role in normal cellular processes such as proliferation, differentiation and development, EGFRVIII is not expressed on normal tissues.
The EGFRVIII variant originates from an in-frame deletion of exons 2-7 of the EGFR gene resulting in the removal of a sequence encoding 267 amino acid residues of the extracellular domain. The newly formed splice junction encodes a glycine residue which has no counterpart in wild type human EGFR and therefore forms a neo-epitope. Moreover, numerous studies showed that normal tissues are devoid of EGFRVIII. EGFRVIII thus contains a new tumor specific cell surface epitope that could be exploited for antibody targeted therapies. However, the EGFRvIII neo-epitope is not very immunogenic compared to the remaining of the human sequence, and many of the antibodies generated to date have not been shown to be specifically recognizing EGFRVIII.
Currently known EGFRVIII antibodies include antibody 13.1.2 and ABT-806. Although ABT-806 has been shown to bind preferentially to tumor EGFR in preclinical models, binding of this antibody to wt EGFR present in human skin has been shown to account for the cutaneous toxicity that ABT-806 exhibits in some patients. Antibodies or antigen-binding fragments thereof that specifically target an epitope of EGFRVIII that is absent or not accessible in EGFR-expressing cells would be beneficial for the treatment of cancer patients.
Thus, there remains a need for improved therapeutics (e.g., cancer therapeutics) that can target EGFRvIII.
The present disclosure relates to compounds that target epidermal growth factor receptor variant III (EGFRvIII), pharmaceutical compositions thereof, and methods of treating or preventing cancer using such pharmaceutical compositions. In certain embodiments, provided compounds exhibit an increased excretion rate (e.g., after being administered to a mammal) compared to some currently known radiotherapeutics, while still maintaining therapeutic efficacy. In some embodiments, a faster excretion may limit off-target toxicities by limiting the amount of time that the compound stays in a subject. Thus, in some embodiments, provided compounds exhibit reduced off-target toxicities.
In one aspect, provided are compounds comprising the following structure, or pharmaceutically acceptable salts thereof:
A-L1-(L2)n-B Formula I
wherein
—X1-L3-Z1- Formula II
wherein
In certain embodiments, variable A in Formula I is a chelating moiety 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)-tetra propionic 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(methylene phosphonic acid)), DOTMP (1,4,6,10)-tetraazacyclodecane-1,4,7,10)-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10)-tetraazacyclododecane-1,4,7,10)-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic 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), Hophospa (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 (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), octadentate-HOPO (octadentate hydroxypyridinones), and porphyrin.
In some embodiments, the compound of Formula I is represented by:
wherein Y1 is —CH2OCH2 (L2)n-B, C═O(L2)n-B, or C═S(L2)n-B and Y2 is —CH2CO2H; or wherein Y1 is H and Y2 is L1-(L2)n-B. In certain embodiments, Y1 is H.
In some embodiments, L1 is
and RL is hydrogen or —CO2H.
In certain embodiments, X1 is —C(O)NR1—* or —NR1C(O)—*, “*” indicating the attachment point to L3, and R1 is H.
In certain embodiments, Z1 is —CH2—.
In some embodiments, L3 comprises (CH2CH2O)2-20. In some embodiments, L3 is (CH2CH2O)m(CH2)w, wherein m and w are each independently an integer between 0 and 10 (inclusive), and at least one of m and w is not 0.
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, a lanthanide, and an actinide. In some embodiments, the metal complex comprises a radionuclide selected from the group consisting of 43Sc, +Sc, 47Sc, 55Co, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 66Ga, 67Ga, 68Ga, 82Rb, 86Y, 87Y, 89Zr, 90Y, 97Ru, 99Tc, 99mTc, 105Rh, 109Pd, 111In, 117mSn, 133La, 134Ce, 149Pm, 149Tb, 153Sm, 166Ho, 177Lu, 186Re, 188Re, 198 Au, 199Au, 201Tl, 203Pb, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 227Th, and 229Th.
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., an alpha emitter selected from the group consisting of Astatine-211 (211At), Bismuth-212 (212Bi), Bismuth-213 (213Bi), Actinium-225 (225Ac), Radium-223 (223Ra), Lead-212 (212Pb), Thorium-227 (227Th), and Terbium-149 (149Tb), or a progeny thereof. In some embodiments, the radionuclide is 68Ga, 111In, 177Lu, or 225Ac. In some embodiments, the radionuclide is 225Ac or a progeny thereof.
In some embodiments, A-L-in Formula I comprises one of the following structures, or a metal complex thereof:
In some embodiments, the compound or a pharmaceutically acceptable salt thereof comprises the following structure, or a metal complex thereof:
In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment thereof.
In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII or a fragment thereof comprises:
In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to EGFRVIII or a fragment thereof comprises:
In some embodiments, the antibody or antigen-binding fragment thereof comprises:
In some embodiments, the antibody or antigen-binding fragment thereof comprises:
In certain embodiments, the antibody or antigen-binding fragment thereof comprises:
In some embodiments, the antibody or antigen-binding fragment thereof comprises:
In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, a single chain antibody, or a multispecific antibody. In some embodiments, the antibody is a humanized monoclonal antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 constant region.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG2 constant region.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG4 constant region. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG4 (S228P) constant region comprising a human kappa light chain constant region having the amino acid sequence set forth in SEQ ID NO: 178 and a human IgG4 (S228P) heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 179.
In some embodiments, the antibody or antigen-binding fragment thereof comprises:
In some embodiments, the antigen-binding fragment comprises a scFv, a Fab, a Fab′ or a (Fab′)2.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a CDRL1 having the amino acid sequence set forth in SEQ ID NO:38, a CDRL2 having the amino acid sequence set forth in SEQ ID NO: 39 and a CDRL3 having the amino acid sequence set forth in SEQ ID NO:40 and a heavy chain variable region comprising a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, a CDRH2 having the amino acid sequence set forth in SEQ ID NO:44 and a CDRH3 having the amino acid sequence set forth in SEQ ID NO:45.
In some embodiments, the compound comprises the following structure:
wherein · is an antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII or a fragment thereof. In some embodiments, the antibody or an antigen-binding fragment thereof is linked to A-L-via the side-chain amino group of a lysine residue
In another aspect, the present invention also relates to a pharmaceutical composition comprising one of the compounds described above and a pharmaceutically acceptable carrier, diluent, or excipient.
Still within the scope of this invention is a method of radiation treatment planning and/or radiation treatment of cancer and the method comprising administering to a subject in need thereof one of the compounds set forth above or a pharmaceutical composition comprising the same.
Also within the scope of this invention is a method of treating cancer that comprises expressing EGFRvIII, the method comprising administering to a subject (e.g., a human) in need thereof a compound or pharmaceutical composition provided herein in a therapeutically effective amount.
Further included in this invention is a method of treating or preventing cancer that comprises cells expressing EGFRVIII, the method comprising administering to a subject (e.g., a human) in need thereof a first dose of a compound or pharmaceutical composition provided above in an amount effective for radiation treatment planning, followed by administering subsequent doses of a compound or pharmaceutical composition provided above in a therapeutically effective amount.
In some embodiments, the compound or composition administered in the first dose and the compound or composition administered a subsequent dose are the same.
In some embodiments, the compound or composition administered in the first dose and the compound or composition administered a subsequent are different.
In some embodiments, the cancer that comprises cells expressing EGFRVIII is glioblastoma multiforme or carcinoma.
In some embodiments, the method of treatment further comprises administering to a subject (e.g., a human) in need thereof an antiproliferative agent, radiation sensitizer, an immunoregulatory or immunomodulatory agent.
In some embodiments, the method of treatment comprises administering the compound or pharmaceutical composition described herein in combination with an antiproliferative agent, in the absence or presence of external beam radiation. In certain embodiments, the antiproliferative agent is temozolomide (TMZ).
In some embodiments, the method of treatment comprises administering a therapeutically effective amount of a compound or pharmaceutical composition described herein in multiple doses (e.g., dosing once weekly for 4 cycles or dosing once every two weeks for 2 cycles).
In some embodiments, the method of treatment comprises administering a therapeutically effective amount of a compound or pharmaceutical composition described herein in a single dose.
Radioimmunoconjugates are designed to target a protein or receptor that is upregulated in a disease state to deliver a radioactive payload to damage and kill cells of interest (radioimmunotherapy). The process of delivering such a payload, via radioactive decay, produces an alpha, beta, or gamma particle or Auger electron that can cause direct effects to DNA (such as single or double stranded DNA breaks) or indirect effects such as by-stander or crossfire effects.
Radioimmunoconjugates typically contain a targeting moiety (e.g., a biological targeting moiety (e.g., an antibody or antigen binding fragment thereof)) that is capable of specifically binding to human EGFRVIII, a radioisotope, and a molecule that links the two. Conjugates are formed when a bifunctional chelate is appended to the targeting molecule (e.g., biological targeting molecule) so that structural alterations are minimal while maintaining target affinity. Once radiolabeled, the final radioimmunoconjugate is formed.
Bifunctional chelates structurally contain a chelate, a linker, and a cross-linking group (
One of the key factors of developing safe and effective radioimmunoconjugates is maximizing efficacy while minimizing off-target toxicity in normal tissue. While this statement is one of the core tenets of developing new drugs, the application to radioimmunotherapeutics presents new challenges. Radioimmunoconjugates do not need to block a receptor, as needed with a therapeutic antibody, or release the cytotoxic payload intracellularly, as required by an antibody drug conjugate (“ADC”), in order to have therapeutic efficacy. However, the emission of the toxic particle is an event that occurs as a result of first-order (radioactive) decay and can occur at random anywhere inside the body after administration. Once the emission occurs, damage could occur to surrounding cells within the range of the emission leading to the potential of off-target toxicity. Therefore, limiting exposure of these emissions to normal tissue is the key to developing new therapeutic radioimmunoconjugates.
One potential method for reducing off-target exposure is to remove the radioactivity more effectively from the body (e.g., from normal tissue in the body). One mechanism is to increase the rate of clearance of the biological targeting agent. This approach likely requires identifying ways to shorten the half-life of the biological targeting agent, which is not well described for biological targeting agents. Regardless of the mechanism, increasing drug clearance will also negatively impact the pharmacodynamics/efficacy in that the more rapid removal of drug from the body will lower the effective concentration at the site of action, which, in turn, would require a higher total dose and would not achieve the desired results of reducing total radioactive dose to normal tissue.
Other efforts have focused on accelerating the 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 the radioactivity from the biological targeting agents using what have been termed “cleavable linkers”. Cleavable linkers, however, have been taken on different meaning as it relates to radioimmunoconjugates. Cornelissen, et al. has described cleavable linkers as those by which the bifunctional chelate attaches to the biologic targeting agent through a reduced cysteine, whereas others have described the use of enzyme-cleavable systems that require the co-administration of the radioimmunoconjugate with a cleaving agent/enzyme to release [Mol Cancer Ther. 2013, 12 (11), 2472-2482; Methods Mol Biol. 2009, 539, 191-211; Bioconjug Chem. 2003, 14 (5), 927-33]. These methods either change the nature of the biological targeting moiety, in the case of the cysteine linkage, or are not practical from a drug development perspective (enzyme cleavable systems) since, in the case of the citations provided, require the administration of two agents.
The present disclosure provides, among other things, compounds, e.g., radioimmunoconjugates, that are more effectively eliminated from the body after catabolism and/or metabolism, while maintaining therapeutic efficacy. Disclosed immunoconjugates may, in some embodiments, achieve a reduction of total body radioactivity, for example, by increasing the extent of excretion of the catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule when compared to known bifunctional chelates. In some embodiments, this reduction in radioactivity results from the clearance of catabolic/metabolic by-products without impacting other in vitro and in vivo properties such as binding specificity (in vitro binding), cellular retention, and tumor uptake in vivo. Thus, in some embodiments, provided compounds achieve reduced radioactivity in the human body while maintaining on-target activity.
As used herein, the term “bind” or “binding” of a targeting moiety means an at least temporary interaction or association with or to a target molecule, e.g., to human EGFRvIII and/or a mutational variant of EGFRvIII, e.g., as described herein.
The terms “bifunctional chelate,” as used herein, refers to a compound that comprises a chelate, a linker, and a cross-linking group. See. e.g.,
The term “bifunctional conjugate,” as used herein, refers to a compound that comprises a chelate or metal complex thereof, a linker, and a targeting moiety, e.g., an antibody or antigen-binding fragment thereof. See. e.g.,
The term “cancer,” as used herein, refers to any cancer caused by the proliferation of malignant neoplastic 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) expressing EGFRVIII, such as, but not limited to, glioblastoma multiforme and carcinoma.
The term “chelate,” as used herein, refers to an organic compound or portion thereof that can be bonded to a central metal or radiometal atom at two or more points.
The term “conjugate,” as used herein, refers to a molecule that contains a chelating group or metal complex thereof, a linker group, and which optionally contains a targeting moiety, e.g., an antibody or antigen-binding fragment thereof.
As used herein, unless otherwise noted, the phrase “constant region,” when used in reference to an antibody or a fragment thereof (e.g., an IgG1, an IgG2, or an 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 with a reference sequence for a wild-type constant region.
As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.
The compounds recited or described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds discussed in the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.
As used herein, “detection agent” refers to a molecule or atom which is useful in diagnosing a disease by locating the cells containing the antigen. 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 with the biotin-streptavidin complex), contrast agents, luminescent agents (e.g., fluorescein isothiocyanate or FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.
As used herein, the term “radionuclide,” refers to an atom capable of undergoing radioactive decay (e.g., 3H, 14C, 15N, 18F, 35S, 47Sc, 55Co, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 75Br, 76Br, 77Br, 89Zr, 86Y, 87Y, 90Y, 97Ru, 99Tc, 99mTc, 105Rh, 109Pd, 111In, 123I, 124I, 125I, 131I, 149Pm, 149Tb, 153Sm, 166Ho, 177Lu, 186Re, 188Re, 198Au, 199Au, 203Pb, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 227Th, 229Th, 66Ga, 67Ga, 68Ga, 82Rb, 117mSn, 201Tl). The terms radioactive nuclide, radioisotope, or radioactive isotope may also be used to describe a radionuclide. Radionuclides may be used as detection agents, as described herein. In some embodiments, the radionuclide may be an alpha-emitting radionuclide.
The term an “effective amount” of an agent (e.g., any of the foregoing conjugates), as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in therapeutic applications, an “effective amount” may be an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications, and/or to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of cancer, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed. For example, the disease or condition may become less severe and/or recovery is accelerated in an individual. An effective amount may be administered by administering a single dose or multiple (e.g., at least two, at least three, at least four, at least five, or at least six) doses.
The term “immunoconjugate,” as used herein, refers to a conjugate that includes a targeting moiety, such as an antibody (or antigen-binding fragment thereof), nanobody, affibody, or a consensus sequence from Fibronectin type III domain. In some embodiments, the immunoconjugate comprises 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).
The term “radioconjugate,” as used herein, refers to any conjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.
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. A radioimmunoconjugate provided in the present disclosure typically refers to a bifunctional conjugate that comprises a metal complex formed from a radioisotope or radionuclide.
The term “radioimmunotherapy,” as used herein, refers a method of using a radioimmunoconjugate to produce a therapeutic effect. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate produces a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a cell, wherein administration of the radioimmunoconjugate kills the cell. Wherein radioimmunotherapy involves the selective killing of a cell, in some embodiments the cell is a cancer cell in a subject having cancer.
The term “pharmaceutical composition,” as used herein, represents a composition containing a radioimmunoconjugate described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions 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., as 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 described herein.
A “pharmaceutically acceptable excipient.” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotectants, sorbents, suspending or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
The term “pharmaceutically acceptable salt,” as use herein, represents those salts of the compounds described here that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response. 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 in 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 separately by reacting the free base group with a suitable organic acid.
The compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases. Frequently, the 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, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.
Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, 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, valerate salts, among others. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.
The term “polypeptide,” as used herein, refers to a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides can include one or more “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may comprise two or more individual polypeptide chains, which may in some cases be linked to one another, for example by one or more disulfide bonds or other means.
By “subject” is meant a human or non-human animal (e.g., a mammal).
By “substantial identity” or “substantially identical” is meant a polypeptide sequence that has the same polypeptide sequence, respectively, as a reference sequence, or has a specified percentage of amino acid residues, respectively, that are the same at the corresponding location within a 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 to the reference amino acid sequence. For polypeptides, the length of comparison sequences will generally 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 contiguous amino acids (e.g., a full-length sequence). Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
As used herein, and as well understood in the art, “to treat” a condition or “treatment” of the condition (e.g., the conditions described herein such as cancer) is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
As used herein, the term “about” or “approximately,” when used in reference to a quantitative value, includes the recited quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” or “approximately” refers to a ±10% variation from the recited quantitative value unless otherwise indicated or inferred from the context.
As used herein, the term “targeting moiety” refers to any molecule or any part of a molecule that is capable of binding to a given target. The term, “EGFRvIII targeting moiety” refers to a targeting moiety that is capable of binding to an EGFRVIII molecule, e.g., a human EGFRvIII.
As used herein, unless otherwise specified, “EGFR” refers to human EGFR. Human EGFR means a full-length protein defied by UniProt P00533, or a fragment or variant thereof. EGFR is also known as epidermal growth factor receptor, ErbB-1, and HER1. In some particular embodiments, EGFR of non-human species, e.g., mouse EGFR, is used. The terms “wt EGFR”, “WT EGFR”, “EGFR WT,” and “EGFR wt” are used interchangeably and refer to wild type EGFR.
As used herein, “EGFRvIII” or “vIII” refers to an EGFR variant resulting from in-frame deletion of exons 2-7 of the coding sequence, or a fragment thereof. EGFRvIII is also known as epidermal growth factor receptor variant III, de2-7EGFR, and ΔEGFR.
As used herein, the term “fragment,” when used to refer to an EGFRVIII fragment, refers to N-terminally and/or C-terminally truncated EGFRvIII or protein domains of EGFRvIII. Unless otherwise noted, fragments of EGFRVIII used in accordance with embodiments described herein retain the capability of the full-length EGFRVIII to be recognized and/or bound by an EGFRvIII-targeting moiety as described in the present disclosure. As an illustrative example, the fragment may be an extracellular domain of EGFRvIII, such as amino acid residues 1 to 76 of EGFRVIII (SEQ ID NO: 119), amino acid residues 1 to 18 of EGFRVIII (SEQ ID NO:125), or amino acid residues 15 to 37 of EGFRVIII (SEQ ID NO:6).
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Unless specifically stated or obvious from context, as used herein the term “or” is understood to be inclusive and covers both “or” and “and”.
The term “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other.
The terms “comprising”, “having”, “including”, 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” is to be construed as close-ended.
As used herein, the term “native” with respect to a protein such as EGFRvIII or EGFR refers to the natural conformation of the protein and includes proteins that are properly folded and/or functional.
As used herein, the term “denatured” with respect to a protein such EGFRvIII or EGFR refers to a protein that has lost its natural conformation and may entail, for example, a loss in the tertiary and secondary structure.
As used herein, the expression “a peptide comprising or consisting of an EGFRvIII fragment” means that the peptide may comprise a portion other than the EGFRVIII fragment or that it consists of the EGFRVIII fragment.
As used herein, a targeting moiety (e.g., an antibody or antigen-binding domain) “binds to an epitope comprising amino acid residues” means that said amino acid residues are either part of the epitope or that it is necessary for the binding of the targeting moiety.
As used herein, when used in reference to a targeting moiety (e.g., an antibody or antigen-binding domain), the term “fails to bind to” a peptide or protein means that the targeting moiety (e.g., antibody or antigen binding fragment) a) does not bind significantly to the peptide or protein when expressed recombinantly or in cells, b) does not bind to the peptide or protein with detectable affinity, c) has similar binding property as a negative control molecule, d) does not bind specifically to the peptide or protein, or e) binds with a value between 0% and 15% as determined by flow cytometry experiments known in the field.
As used herein the term “autologous” refers to materials derived from the same individual.
As used herein, the term “antigen-binding domain” refers to the domain of an antibody or of an antigen-binding fragment which allows specific binding to an antigen.
As used herein, the term “antibody” encompasses monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, human antibody, single domain antibody (such as a VHH, VH, VL, nanobody, or any camelid or llama single domain antibody), multispecific antibody (e.g., bispecific antibodies) etc. The term “antibody” encompasses molecules that have a format similar to those occurring in nature (e.g., human IgGs, etc.). The term “antibody”, also referred to in the art as “immunoglobulin” (Ig), as used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA. IgD. IgE. IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while the heavy chain folds into a variable (VH) and three constant (CH1. CH2. CH3) domains. Interaction of the heavy and light chain variable domains (VH and VL) results in the formation of an antigen-binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art.
Typically, an antibody is constituted from the pairing of two light chains and two heavy chains. Different antibody isotypes exist, including IgA, IgD, IgE, IgG and IgM. Human IgGs are further divided into four distinct sub-groups namely; IgG1, IgG2, IgG3 and IgG4. Therapeutic antibodies are generally developed as IgG1 or IgG2 or lgG4.
In an exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG1 constant region or a fragment thereof. In another exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG2 constant region or a fragment thereof. In another exemplary embodiment, the antibody or antigen-binding fragment of the present disclosure may comprise, for example, a human IgG4 constant region or a fragment or thereof. Constant regions of other antibody subtypes are also contemplated.
The light chain and heavy chain of human antibody IgG isotypes each comprise a variable region having 3 hypervariable regions named complementarity determining regions (CDRs). The light chain CDRs are identified herein as CDRL1 or L1. CDRL2 or L2 and CDRL3 or L3. The heavy chain CDRs are identified herein as CDRH1 or H1. CDRH2 or H2 and CDRH3 or H3. Complementarity determining regions are flanked by framework regions (FR) in the order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in the CDRs which combine to form the antigen-binding site and contribute to binding and recognition of an antigenic determinant. The framework regions may play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen-binding. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat et al (1991) define the “complementarity-determining regions” (CDR) based on sequence variability at the antigen-binding regions of the VH and VL domains. Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the VH and VL domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the Kabat scheme except the CDRH1 loops that is delineated by combining the Kabat and Chothia definitions.
Recombinant DNA technology now allows the design of various antibody format such as single chain antibodies (e.g., single domain), diabody, minibody, nanobody and the like which are encompassed by the present disclosure.
An “antigen-binding fragment,” as referred to herein, may include any suitable antigen-binding fragment known in the art. The antigen-binding fragment may be a naturally-occurring fragment or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of VL and VH connected with a peptide linker), Fab, F(ab) 2, single-domain antibody (sdAb; a fragment composed of a single VL or VH), and multivalent presentations of any of these. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches and various approaches for their construction.
Antigen-binding fragments thereof of the present disclosure encompass molecules having an antigen-binding site comprising amino acid residues that confer specific binding to an antigen (e.g., one or more CDRs).
Exemplary embodiments of antigen-binding fragments disclosure thus includes without limitation (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR), e.g., VH CDR3.
Specific embodiments of antigen-binding fragments may include for example, a scFv, a Fab, a Fab′ or a (Fab′)2.
The term “humanized antibody” encompasses fully humanized antibody (i.e., frameworks are 100% humanized) and partially humanized antibody (e.g., at least one variable region contains one or more amino acids from a human antibody, while other amino acids are amino acids of a non-human parent antibody). Typically, a “humanized antibody” contains CDRs of a non-human parent antibody (e.g., mouse, rat, rabbit, non-human primate, etc.) and frameworks that are identical to those of a natural human antibody or of a human antibody consensus. In such instance, those “humanized antibodies” are characterized as fully humanized. A “humanized antibody” may also contain one or more amino acid substitutions that have no correspondence to those of the human antibody or human antibody consensus. Such substitutions include, for example, back-mutations (e.g., re-introduction of non-human amino acids) that may preserve the antibody characteristics (e.g., affinity, specificity etc.). Such substitutions are usually in the framework region. A “humanized antibody” usually also comprises a constant region (Fc) which is typically that of a human antibody. Typically, the constant region of a “humanized antibody” is identical to that of a human antibody. A humanized antibody may be obtained by CDR grafting (Tsurushita et al, 2005; Jones et al, 1986; Tempest et al, 1991; Riechmann et al, 1988; Queen et al, 1989). Such antibody is considered as fully humanized.
The term “chimeric antibody” refers to an antibody having a constant region from an origin distinct from that of the parent antibody. The term “chimeric antibody” encompasses antibodies having a human constant region. Typically, a “chimeric antibody” is composed of variable regions originating from a mouse antibody and of a human constant region.
The term “hybrid antibody” refers to an antibody comprising one of its heavy or light chain variable region (its heavy or light chain) from a certain type of antibody (e.g., humanized) while the other of the heavy or light chain variable region (the heavy or light chain) is from another type (e.g., murine, chimeric).
Antibodies and/or antigen-binding fragments of the present disclosure may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies. Antibodies or antigen-binding fragment of the present disclosure may include for example, a synthetic antibody, a non-naturally occurring antibody, an antibody obtained following immunization of a non-human mammal etc.
Antibodies or antigen-binding fragments thereof of the present disclosure may be isolated and/or substantially purified.
In one aspect, this disclosure provides compounds, e.g., radioimmunoconjugates, comprising the following structure, or pharmaceutically acceptable salts thereof:
A-L1-(L2)n-B Formula I
wherein
—X1-L3-Z1— Formula II
wherein
Typical substituents of alkyl, heteroalkyl, aryl, or heteroaryl include, but are not limited to halo (e.g., F, Cl, Br, I), OH, CN, nitro, amino, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C1-6 heteroalkyl, C1-6 heterocycloalkyl, haloalkyl (e.g., CF3), alkoxy (e.g., OCH3), alkylamino (e.g., NH2CH3), sulfonyl, aryl, and heteroaryl.
In some embodiments, the compound has or comprises the structure shown below:
wherein B is an EGFRvIII targeting moiety (e.g., an EGFRvIII antibody or antigen-binding fragment thereof).
In some embodiments, A-L-comprises one of the following structures, or a metal complex thereof:
In some embodiments, as further described herein, the compound (e.g., radioimmunoconjugate) comprises a chelating moiety or a metal complex thereof, which metal complex may comprise a radionuclide. In some such compounds, the average ratio or median ratio of the chelating moiety to the EGFRVIII targeting moiety is eight or less, seven or less, six or less, five or less, four or less, three or less, two or less, or about one. In some compounds, the average ratio or median ratio of the chelating moiety to the EGFRvIII targeting moiety is about one.
In some embodiments, after a radioimmunoconjugate is administered to a mammal, the proportion of radiation (of the total amount of radiation that is administered) that is excreted by the intestinal route, the renal route, or both is greater than the proportion of radiation excreted by a comparable mammal that has been administered a reference radioimmunoconjugate. By “reference immunoconjugate” it is meant a known radioimmunoconjugate that differs from a radioimmunoconjugate described herein at least by (1) having a different linker; (2) having a targeting moiety of a different size and/or (3) lacking a targeting moiety. In some embodiments, the reference radioimmunoconjugate is selected from the group consisting of [Y]-ibritumomab tiuxetan (Zevalin (90Y)) and [111In]-ibritumomab tiuxetan (Zevalin (111In)).
In some embodiments, the proportion 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 proportion of radiation excreted by the same route(s) by a comparable mammal that has been administered a reference radioimmunoconjugate. In some embodiments, the proportion 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 proportion of radiation excreted by a comparable mammal that has been administered a reference radioimmunoconjugate. The extent of excretion can be measured by methods known in the art, e.g., by measuring radioactivity in urine and/or feces and/or by measuring total body radioactivity over a period time. See also, e.g., International Patent Publication WO 2018/024869.
In some embodiments, the extent of excretion is measured at a time period of 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.
In some embodiments, after a compound (e.g., radioimmunoconjugate) has been administered to a mammal, the compound (e.g., radioimmunoconjugate) exhibits decreased off-target binding effects (e.g., toxicities) as compared to a reference compound (e.g., reference conjugate, e.g., a reference immunoconjugate such as a reference radioimmunoconjugate). In some embodiments, this decreased off-target binding effect is a feature of a compound (e.g., radioimmunoconjugate) that also exhibits a greater excretion rate as described herein.
Targeting moieties include any molecule or any part of a molecule that is capable of binding (e.g., capable of specifically binding, specifically binds to, etc.) to a given target, e.g., EGFRvIII. In some embodiments, the targeting moiety comprises a protein or polypeptide. In some embodiments, the targeting moiety is selected from the group consisting of antibodies or antigen binding fragments thereof, nanobodies, affibodies, and consensus sequences from Fibronectin type III domains (e.g., Centyrins or Adnectins). In some embodiments, a moiety is both a targeting and a therapeutic moiety, i.e., the moiety is capable of binding to a given target and also confers a therapeutic benefit. In some embodiments, the targeting moiety comprises a small molecule.
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.
Typically, the targeting moiety is capable of binding to EGFRVIII, or a fragment thereof. In some embodiments, the targeting moiety is capable of binding to human EGFRVIII, or a fragment thereof.
In some embodiments, the targeting moiety is capable of binding specifically to EGFRvIII (e.g., is capable of binding to EGFRVIII while exhibiting comparatively little or no binding to wt EGFR).
In some embodiments, the targeting moiety is capable of binding to an extracellular region of EGFRvIII, e.g., domain III (L2) of EGFRVIII, domain IV (CRII) of EGFRvIII, amino acid residues 1 to 76 of EGFRVIII (SEQ ID NO:119), amino acid residues 1 to 18 of EGFRVIII (SEQ ID NO: 125), or amino acid residues 15 to 37 of EGFRVIII (SEQ ID NO: 6).
In some embodiments, the targeting moiety does not bind to wild type EGFR with detectable affinity. By “detectable affinity,” it is generally meant that the binding ability between a targeting moiety and its target, reported by a KD, EC50, or IC50 value, is at most about 105 M or lower. A binding affinity, reported by a KD, EC50, or IC50 value, higher than 105 M is generally no longer measurable with common methods such as ELISA and flow cytometry, and is, therefore, of secondary importance.
In some embodiments, the targeting moiety inhibits EGFRVIII. By “inhibits,” it is meant that the targeting moiety at least partially inhibits one or more functions of EGFRvIII (e.g., human EGFRvIII). In some embodiments, the targeting moiety impairs signaling downstream of EGFRvIII, e.g., results in the suppressed growth of EGFRvIII-positive tumor cells. In some embodiments, the targeting moiety blocks ligand binding to EGFRvIII and/or receptor dimerization of EGFRvIII.
Generally, a targeting moiety of the present disclosure may be able to bind to a peptide comprising an EGFRVIII fragment consisting of amino acid residues 1 to 76 of EGFRVIII (SEQ ID NO:119). In some embodiments, the targeting moiety is able to bind to amino acid residues 1 to 18 of EGFRVIII (SEQ ID NO:125). In some other embodiments, the targeting moiety is able to bind amino acid residues 15 to 37 of EGFRVIII (SEQ ID NO:6).
Embodiments of EGFRvIII targeting moieties encompassed by the present disclosure includes, for example:
Some particular EGFRVIII targeting moieties (e.g., EGFRVIII antibodies or antigen-binding fragments thereof) encompassed by the present disclosure include those that do not require the presence of amino acid residues 1-2 of EGFRvIIII for binding. Particularly contemplated are EGFRVIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) that are capable of binding to one or more EGFRVIII fragments of amino acid residues 19-76 (SEQ ID NO: 138), amino acid residues 19-62 (SEQ ID NO:139), amino acid residues 19-49 (SEQ ID NO:140), amino acid residues 19-45 (SEQ ID NO: 141), amino acid residues 28-45 (SEQ ID NO:143), amino acid residues 28-37 (SEQ ID NO:144), amino acid residues 19-37 (SEQ ID NO:142), amino acid residues 3-45 (SEQ ID NO: 127), amino acid residues 3-49 (SEQ ID NO:126), amino acid residues 3-37 (SEQ ID NO: 128), amino acid residues 6-49 (SEQ ID NO:130), amino acid residues 6-45 (SEQ ID NO:131), amino acid residues 6-37 (SEQ ID NO:132), amino acid residues 10-49 (SEQ ID NO:133), amino acid residues 10-45 (SEQ ID NO:134), amino acid residues 10-37 (SEQ ID NO:135), amino acid residues 15-49 (SEQ ID NO:136), amino acid residues 15-45 (SEQ ID NO:137), or amino acid residues 15-37 (SEQ ID NO:6) of EGFRvIII.
In some embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) provided herein and/or used in accordance with the present disclosure are able to bind to a peptide comprising an EGFRVIII fragment consisting of amino acid residues 3 to 37 of EGFRVIII (SEQ ID NO: 128), such as F260-5G6 (referred herein also as 5G6), F263-1A8 (referred herein also as 1A8), F263-4B3 (referred herein also as 4B3), F263-4E11 (referred herein also as 4E11), F263-5D8 (referred herein also as 5D8) and F265-9C9 (referred to herein also as 9C9) antibody.
In some embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments) provided herein are able to bind to a peptide comprising an EGFRvIII fragment consisting of amino acid residues 1 to 33 of EGFRVIII (SEQ ID NO: 124).
In some embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) of the present specifically bind to EGFRVIII (SEQ ID NO: 5) and are capable of binding to an EGFRVIII fragment selected from the group consisting of:
In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) of the present disclosure may be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 165, and combination thereof.
In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) may be capable of binding to a peptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO:160.
In some embodiments, provided herein are targeting moieties (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to EGFRVIII (SEQ ID NO:5) and that are capable of binding to an EGFRvIII fragment selected from the group consisting of:
In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) of the present disclosure may be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 165, and combination thereof. In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) may be capable of binding to a peptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO:154. In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) may be capable of binding to a peptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 159.
In some other embodiments, provided targeting moieties (e.g., antibodies or antigen-binding fragments thereof) specifically bind to EGFRvIII (SEQ ID NO:5) and are capable of binding to an EGFRVIII fragment selected from the group consisting of:
In some embodiments, the targeting moieties (e.g., antibodies or antigen-binding fragments thereof) may bind to;
In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) may be capable of binding to a peptide comprising or consisting of an amino acid sequence selected from the group consisting SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 165, and combination thereof. In some embodiments, the targeting moiety (e.g., antibody or antigen-binding fragment thereof) may be capable of binding to a peptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO:149.
Also provided are EGFRVIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) that are able to bind an epitope comprising or involving amino acid residue Cys20 in said peptide. These include, for example, EGFRvIII antibodies or antigen-binding fragments thereof that bind EGFRvIII and/or a peptide comprising an EGFRvIII fragment consisting of the amino acid sequence set forth in SEQ ID NO:6 but are not able to bind a peptide comprising or consisting of the amino acid sequence SCVRAAGADSYEMEEDGVRKCKK (SEQ ID NO:149). Such antibodies or antigen binding fragments thereof encompass, for example, the 4B3, 5D8, and 4E11 antibodies.
Also specifically encompassed by the present disclosure are EGFRVIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) that are able to bind an epitope comprising or involving amino acid residue Cys35 in said peptide. These include, for example, EGFRvIII antibodies or antigen-binding fragments thereof that bind EGFRvIII and/or a peptide comprising an EGFRVIII fragment consisting of the amino acid sequence set forth in SEQ ID NO:6 but are not able to bind a peptide comprising or consisting of the amino acid sequence SCVRACGADSYEMEEDGVRKAKK (SEQ ID NO: 163). Such antibodies or antigen binding fragments thereof encompass, for example, the 4B3, 5D8, 9C9 and 4E11 antibodies.
Further encompassed by the present disclosure are EGFRVIII targeting moieties (e.g., EGFRVIII antibodies or antigen-binding fragments thereof) that are able to bind an epitope in a peptide comprising or involving amino acid residue Cys20 and Cys35 in said peptide. These include, for example, EGFRvIII antibodies or antigen-binding fragments thereof that bind EGFRvIII and/or a peptide comprising or consisting of an EGFRvIII fragment set forth in SEQ ID NO:6 but are not able to bind a peptide comprising or consisting of the amino acid sequence selected from SCVRAAGADSYEMEEDGVRKCKK (SEQ ID NO:149) or SCVRACGADSYEMEEDGVRKAKK (SEQ ID NO: 163). Such antibodies or antigen binding fragments thereof encompass, for example, the 4B3, 5D8 and 4E11 antibodies.
In some embodiments, in addition to amino acid residues Cys20 and/or Cys35, the epitope to which the EGFRVIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) of the present disclosure bind or which are involved in their binding may further include amino acid residues Glu26, Asp30, Gly31, and/or Arg33. In some embodiments, the epitope may further include Asp23 and/or Val32.
For example, in some embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) of the present disclosure bind to an epitope comprising or involving:
In some embodiments, in addition to amino acid residues Cys20 and/or Cys35, the epitope to which the EGFRVIII targeting moieties (e.g., EGFRVIII antibodies or antigen-binding fragments thereof) bind or involved in their binding may further include Arg18 and/or Gly21. In some embodiments, the epitope may further include Glu26 and/or Gly31.
For example, in some embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) of the present disclosure bind to an epitope comprising or involving:
In some other embodiments, EGFRvIII targeting moieties (e.g., EGFRvIII antibodies or antigen-binding fragments thereof) of the present disclosure bind to an epitope comprising or involving:
Antibodies typically comprise two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody-binding specificities of each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions. Light chains typically comprise one variable region (VL) and one constant region (CL). An IgG heavy chain includes a variable region (VH), a first constant region (CHI), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).
Antibodies suitable for use with the present disclosure can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. In some embodiments, the antibody or antigen-binding fragment thereof is humanized. In some embodiments, the antibody or antigen-binding fragment thereof is chimeric. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR). In some embodiments, an “antigen binding fragment” comprises a heavy chain variable region and a light chain variable region. These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.
Antibodies or antigen-binding fragments described herein can be produced by any method known in the art for the synthesis of antibodies (See. e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324: WO 98/46645). Chimeric antibodies can be produced using the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.
Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147:60 (1991), or any of the molecules described herein.
“Avimer” relates to a multimeric binding protein or peptide engineered using, for example, in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in greater affinity and specificity compared to single epitope immunoglobin domains.
“Nanobodies” are antibody fragments consisting of a single monomeric variable antibody domain. Nanobodies may also be referred to as single-domain antibodies. Like antibodies, nanobodies are capable of binding selectively to a specific antigen. Nanobodies may be heavy-chain variable domains or light chain domains. Nanobodies may occur naturally or be the product of biological engineering. Nanobodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display). “Affibodies” are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may be considered to mimic certain functions of antibodies.
Affibodies may be engineered variants of the B-domain in the immunoglobulin-binding region of staphylococcal protein A. Affibodies may be engineered variants of the Z-domain, a B-domain that has lower affinity for the Fab region. Affibodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).
Affibody molecules showing specific binding to a variety of different proteins (e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2 and EGFR) have been generated, demonstrating affinities (Kd) in the μM to pM range. “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See for example Hudson et al., (2003). Single-chain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all, or a portion of the light chain variable domain of an antibody. Antibody fragments can be made by various techniques including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant hosts (e.g., E. coli or phage) as described herein.
In certain embodiments, the antibody or antigen-binding fragment thereof is a multispecific, e.g. bispecific. Multispecific antibodies (or antigen-binding fragments thereof) include monoclonal antibodies (or antigen-binding fragments thereof) that have binding specificities for at least two different sites.
The antibodies or antigen-binding fragments thereof that specifically bind to EGFRVIII may be an EGFRVIII antibody antigen-binding fragment thereof described in WO2020191485A1, which is incorporated by reference in its entirety.
In some embodiments, provided herein are EGFRvIII antibodies or antigen-binding fragments thereof selected from the group consisting of:
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 4E11 antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 5G6 antibody,
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 1A8 antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 4B3 antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 5D8 antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 9C9 antibody.
In some embodiments, the antibody or antigen-binding fragment thereof comprise the CDRs of the 11B1 or of the 11C8 antibody.
In some embodiments, the present disclosure provides EGFRVIII antibodies or antigen-binding fragments thereof selected from the group consisting of:
In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to EGFRVIII and which may comprise for example:
In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to EGFRVIII and which may comprise:
In certain embodiments, antibodies or antigen-binding fragments thereof having light chain at least 80%, 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% identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 118 may have CDRs identical to those of SEQ ID NO: 115 or SEQ ID NO: 118 respectively.
In certain embodiments, antibodies or antigen-binding fragments thereof having heavy chain at least 80%, 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% identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 116 may have CDRs identical to those of SEQ ID NO: 62 or SEQ ID NO: 116 respectively.
In some embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to EGFRVIII and which may comprise:
In various embodiments, the light chain variable regions, light chains, heavy chain variable regions or heavy chains which may comprise an amino acid sequence at least 80%, 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% identical to that of given antibody may have CDRs that are identical to that antibody. In some embodiments, the VL and VH sequences of the antibodies and antigen-binding fragments provided in the present disclosure may comprise a sequence substantially identical to the VL and VH sequences provided herein, or may comprise a sequence having at least 80%, 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% sequence identity, wherein sequence variation is preferably outside the CDRs of the VL and VH sequences provided.
In further embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof, which specifically binds to EGFRVIII and which may comprise:
In some embodiments, the present disclosure further provides anti-EGFRvIII antibodies or antigen-binding fragments thereof, which may comprise:
In accordance with the present disclosure, the antibody or antigen-binding fragment thereof set forth above may have CDRs identical or substantially identical to those set forth in SEQ ID NOs: 38, 39, 40, 43, 44 and 45.
In some embodiments, the present disclosure also provides EGFRvIII antibodies or antigen-binding fragments thereof, comprising light chain sequences that comprise the signal sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO: 113) at the N-terminus, and heavy chain sequences that comprise the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO:114) at the N-terminus. In certain embodiments, each of the light chain sequences set forth in SEQ ID NOs: 180, 181, and 182 comprises the signal sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO:113) at the N-terminus. In certain embodiments, each of the heavy chain sequences set forth in SEQ ID NOs: 183, 184, and 185 comprises the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO: 114) at the N-terminus.
In accordance with the present disclosure, the antibody or antigen-binding fragment thereof may have, for example, an affinity to EGFRVIII of less than 100 nM, such as an affinity to EGFRvIII of 50 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less.
Exemplary embodiments of the present disclosure include antibodies or antigen-binding fragments thereof which may comprise a human IgG constant region. Antibodies or antigen-binding fragments of the present disclosure may comprise, for example and without limitation, a human IgG1 constant region or a human IgG2 constant region or a human IgG4 constant region, or a mutational variant thereof.
In an exemplary embodiment, the antigen-binding agents disclosed herein may comprise humanized framework regions.
In accordance with the present disclosure, the antibody or antigen-binding fragment thereof may be monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, a single chain antibody, or a multispecific antibody (e.g., a bispecific antibody).
Bispecific antibodies or antigen-binding fragments thereof of the present disclosure includes those that may comprise a first targeting moiety that specifically binds to a first human EGFRvIII epitope and a second targeting moiety that specifically binds to a second (non-overlapping) human EGFRvIII epitope (e.g., a biparatopic antibody).
Additional embodiments of bispecific antibodies or antigen-binding fragments thereof of the present disclosure includes those that may comprise a first targeting moiety that specifically binds to a first human EGFRvIII epitope and a second targeting moiety that specifically binds to another antigen.
The bispecific antibody or antigen-binding fragment thereof of the present disclosure include bispecific immune cell engagers such as those comprising a first targeting moiety that specifically binds to human EGFRVIII and a second targeting moiety that specifically binds to CD3.
In accordance with the present disclosure, the antigen-binding fragment of an EGFRVIII antibody may comprise, for example, a scFv, a Fab, a Fab′ or a (Fab′)2.
Polypeptides include, for example, any of a variety of hematologic agents (including, for instance, erythropoietin, blood-clotting factors, etc.), interferons, colony stimulating factors, antibodies, enzymes, and hormones. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest can be a polypeptide in the present methods.
A reference polypeptide described herein can include a target-binding domain that is capable of binding to a target of interest (e.g., is capable of binding to an antigen, e.g., EGFRvIII). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor).
Polypeptides suitable for use with compositions and methods of the present disclosure may have a modified amino acid sequence. Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the reference polypeptide). In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g., binding to EGFRVIII). The modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize a characteristic of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.
Modifications include those by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristovlation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.
A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy-terminus of a polypeptide herein can facilitate conjugation of these polypeptides by, e.g., disulfide bonding. For example, a polypeptide can be modified to include a single cysteine residue at the amino-terminus or a single cysteine residue at the carboxy-terminus. Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).
Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH2 (CH2) COOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated “exemplary substitutions” in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened.
Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and/or (c) the bulk of the side chain.
Examples of suitable chelating moieties include, but are not limited to, 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-tetra propionic 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(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacy clotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic 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-v1]-methylamino]ethane), Hophospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-v1]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), octadentate-HOPO (octadentate hydroxypyridinones), or porphyrins.
Preferably, the chelating moiety is selected from 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-tetraazacyclododecan-1-yl) acetic acid), DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic 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).
In some embodiments, the chelating moiety is DOTA.
In some embodiments, compounds comprise a metal complex of a chelating moiety. For example, chelating groups may be used in metal chelate combinations with metals, such as manganese, iron, and gadolinium and isotopes (e.g., isotopes in the general energy range of 60 to 10,000 keV), such as any of the radioisotopes and radionuclides discussed herein.
In some embodiments, chelating moieties are useful as detection agents, and compounds comprising such detectable chelating moieties can therefore be used as diagnostic or theranostic agents.
In some embodiments, variable A of Formula I is a macrocyclic chelating moiety comprising one or more heteroaryl groups (e.g., six-membered nitrogen-containing heteroaryl). Examples of such macrocyclic chelating moiety include, but are not limited to:
In some embodiments, the metal complex comprises a radionuclide. Examples of suitable radioisotopes and radionuclides include, but are not limited to, 3H, 14C, 15N, 18F, 35S, 43Sc, ++Sc, 47Sc, 55Co, 60Cu, 61Cu, 62Cu, 64Cu, 66Ga, 67Ga, 67Cu, 68Ga, 75Br, 76Br, 77Br, 82Rb, 89Zr, 86Y, 87Y, 90Y, 97Ru, 99Tc, 99mTc, 105Rh, 109Pd, 111In, 123I, 124I, 125I, 131I, 133La, 134Ce, 149Pm, 149Tb, 153Sm, 166Ho, 177Lu, 117mSn, 186Re, 188Re, 198Au, 199Au, 201Tl, 203Pb, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 227Th, and 229Th.
In some embodiments, the metal complex comprises a radionuclide selected from 43Sc, +Sc, 47Sc, 55Co, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 66Ga, 67Ga, 68Ga, 82Rb, 86Y, 87Y, 89Zr, 90Y, 97Ru, 99Tc, 99mTc, 105Rh, 109Pd, 111In, 117mSn, 133La, 134Ce, 149Pm, 149Tb, 153Sm, 166Ho, 177Lu, 186Re, 188Re, 198Au, 199Au, 201Tl, 203pb, 211At, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 227Th, and 229Th. In certain embodiments, the metal complex comprises a radionuclide selected from 68Ga, 89Zr, 90Y, 111In, 177Lu, and 225Ac. In certain embodiments, the metal complex comprises a radionuclide being 177Lu or 225Ac.
In some embodiments, the radionuclide is an alpha emitter, e.g., Astatine-211 (211At), Bismuth-212 (212Bi), Bismuth-213 (213Bi), Actinium-225 (225Ac), Radium-223 (223Ra), Lead-212 (212Pb), Thorium-227 (227Th), or Terbium-149 (149Tb), or a progeny thereof. In some embodiments, the alpha-emitter is Actinium-225 (225Ac), or a progeny thereof.
The compounds of this invention generally comprise the structure of Formula I below:
A-L1-(L2)n-B Formula I
wherein each of the variables is defined in the SUMMARY section above.
Each of the compounds of Formula I comprises a linker moiety as -L1-(L2)n-, wherein:
L1 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 between 1 and 5 (inclusive); and each L2, independently, has the structure:
—X1-L3-Z1— Formula II
wherein:
In some embodiments, L1 is optionally substituted C1-C6 alkyl or optionally substituted C1-C6 heteroalkyl. In certain embodiments, L1 is substituted C1-C6 alkyl or substituted C1-C6 heteroalkyl, the substituent comprising a heteroaryl group (e.g., six-membered nitrogen-containing heteroaryl). In some embodiments, L1 is C1-C6 alky. For example, L1 is —CH2CH2—. In some embodiments, L1 is a bond. In some embodiments, L1 is
wherein RL is hydrogen or —CO2H.
In some embodiments, X1 is —C(O)NR1—*, —NR1C(O)—*, or —NR1_“*” indicating the attachment point to L3, and R1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, X1 is —C(O)NR1_*, “*” indicating the attachment point to L3, and R1 is hydrogen.
In some embodiments, L3 is optionally substituted C1-C50 alkyl (e.g., C3-C30 alkyl, C3-C25 alkyl, C3-C20 alkyl, C3-C15 alkyl, C3-C10 alkyl, C5-C30 alkyl, C5-C25 alkyl, C5-C20 alkyl, C5-C15 alkyl, and C5-C10 alkyl) or optionally substituted C1-C50 heteroalkyl (e.g., C3-C30 heteroalkyl, C3-C25 heteroalkyl, C3-C20 heteroalkyl, C3-C15 heteroalkyl, C3-C10 heteroalkyl, C5-C30 heteroalkyl, C5-C25 heteroalkyl, C5-C20 heteroalkyl, C5-C15 heteroalkyl, and C5-C10 heteroalkyl). An exemplary C1-C50 heteroalkyl is C5-C30 polyethylene glycol (e.g., C5-C25 polyethylene glycol, C5-C20 polyethylene glycol, C5-C15 polyethylene glycol). In certain embodiments, L3 is C5-C25 polyethylene glycol, C5-C20 polyethylene glycol, or C5-C15 polyethylene glycol.
In some embodiments, L3 is optionally substituted C1-C50 heteroalkyl (e.g., C1-C40 heteroalkyl, C1-C30 heteroalkyl, C1-C20 heteroalkyl, C2-C18 heteroalkyl, C3-C16 heteroalkyl, C4-C14 heteroalkyl, C5-C12 heteroalkyl, C6-C10 heteroalkyl, C5-C10 heteroalkyl, C4 heteroalkyl, C6 heteroalkyl, C8 heteroalkyl, C10 heteroalkyl, C12 heteroalkyl, C16 heteroalkyl, C20 heteroalkyl, or C24 heteroalkyl).
In some embodiments, L3 is optionally substituted C1-C50 heteroalkyl comprising a polyethylene glycol (PEG) moiety comprising 1-20 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).
In certain embodiments, L3 is optionally substituted C1-50 heteroalkyl comprising a polyethylene glycol (PEG) moiety comprising 1-20 oxyethylene (—O—CH2—CH2—) units or portions thereof. For example, L3 comprises PEG3 as shown below:
In some embodiments, L3 is (CH2CH2O)m(CH2) w, and m and w are each independently an integer between 0 and 10 (inclusive), and at least one of m and w is not 0.
In some embodiments, L3 is substituted C1-C50 alkyl or substituted C1-C50 heteroalkyl, the substituent comprising a heteroaryl group (e.g., six-membered nitrogen-containing heteroaryl).
In some embodiments, Z1 is CH2, C═O, or NR1; wherein R1 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.
In certain embodiments, A-L1-(L2), —B can be represented by the following structure:
In some embodiments, compounds (e.g., radioimmunoconjugates) are synthesized using bifunctional chelates that comprise a chelate, a linker, and a cross-linking group. Once the compound (e.g., radioimmunoconjugate) is formed, the cross-linking group may be absent from the compound (e.g., radioimmunoconjugate).
In some embodiments, compounds (e.g., radioimmunoconjugates) comprise a cross-linking group instead of or in addition to the targeting moiety (e.g., in some embodiments, B in Formula I comprises a cross-linking group).
A cross-linking group is a reactive group that is able to join two or more molecules by a covalent bond. Cross-linking groups may be used to attach the linker and chelating moiety to a therapeutic or targeting moiety. Cross-linking groups may also be used to attach the linker and chelating moiety to a target in vivo. In some embodiments, the cross-linking group is an amino-reactive, methionine reactive or thiol-reactive cross-linking group, or a comprises sortase recognition sequence. In some embodiments, the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine. In some embodiments, the sortase recognition sequence may comprise of a terminal glycine-glycine-glycine (GGG) and/or LPTXG amino acid sequence, where X is any amino acid. A person having ordinary skill in the art will understand that the use of cross-linking groups is not limited to the specific constructs disclosed herein, but rather may include other known cross-linking groups.
In one aspect, the present disclosure provides pharmaceutical compositions comprising compounds disclosed herein. Such pharmaceutical compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in a pharmaceutical composition for proper formulation. Non-limiting examples of suitable formulations compatible for use with the present disclosure include those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, See. e.g., Langer (Science. 249:1527-1533, 1990).
Pharmaceutical compositions may be formulated for any of a variety of routes of administration discussed herein (See. e.g., the “Administration and Dosage” subsection herein), Sustained release administration is contemplated, by such means as depot injections or erodible implants or components. Thus, the present disclosure provides pharmaceutical compositions that include agents disclosed herein (e.g., radioimmunoconjugates) dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others. In some embodiments, pharmaceutical compositions contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others. In some embodiments, pharmaceutical compositions are formulated for oral delivery and may optionally contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule. In some embodiments, pharmaceutical compositions are formulated for local administration and may optionally contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.
In some embodiments, provided pharmaceutical compositions are sterilized by conventional sterilization techniques, e.g., may be sterile filtered. Resulting aqueous solutions may be packaged for use as is, or lyophilized. Lyophilized preparations can be, for example, combined with a sterile aqueous carrier prior to administration. The pH of preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5. Resulting compositions in solid form may be packaged, for example, in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. Pharmaceutical compositions in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
In one aspect, the present disclosure provides methods of treatment comprising administering to a subject in need thereof a compound (e.g., radioimmunoconjugate) as disclosed herein.
In some disclosed methods, a therapy (e.g., comprising a therapeutic agent) is administered to a subject. In some embodiments, the subject is a mammal, e.g., a human.
In some embodiments, the subject has cancer or is at risk of developing cancer. For example, the subject may have been diagnosed with cancer. For example, the cancer may be a primary cancer or a metastatic cancer. Subjects may have any stage of cancer, e.g., stage I, stage II, stage III, or stage IV with or without lymph node involvement and with or without metastases. Provided compounds (e.g., radioimmunoconjugates) and compositions may prevent or reduce further growth of the cancer and/or otherwise ameliorate the cancer (e.g., prevent or reduce metastases). In some embodiments, the subject does not have cancer but has been determined to be at risk of developing cancer, e.g., because of the presence of one or more risk factors such as environmental exposure, presence of one or more genetic mutations or variants, family history, etc. In some embodiments, the subject has not been diagnosed with cancer.
In some embodiments, the cancer is any cancer that comprises cells expressing EGFRvIII. In certain embodiments, the cancer is glioblastoma multiforme or carcinoma.
Compounds (e.g., radioimmunoconjugates) and pharmaceutical compositions thereof disclosed herein may be administered by any of a variety of routes of administration, including systemic and local routes of administration
Systemic routes of administration include parenteral routes and enteral routes. In some embodiments, compounds (e.g., radioimmunoconjugates) or pharmaceutical compositions thereof are administered by a parenteral route, for example, intravenously, intraarterially, intraperitoneally, subcutaneously, intracranially, or intradermally. In some embodiments, compounds (e.g., radioimmunoconjugates) or pharmaceutical compositions thereof are administered intravenously. In some embodiments, compounds (e.g., radioimmunoconjugates) or pharmaceutical compositions thereof are administered by an enteral route of administration, for example, trans-gastrointestinal, or orally.
Local routes of administration include, but are not limited to, peritumoral injections and intratumoral injections.
Pharmaceutical compositions can be administered for radiation treatment planning, diagnostic, and/or therapeutic treatments. When administered for radiation treatment planning or diagnostic purposes, the compound (e.g., radioimmunoconjugate) may be administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose. In therapeutic applications, pharmaceutical compositions may be administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount adequate to accomplish this purpose is defined as a “therapeutically effective amount,” an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of cancer, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but may, for example, provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, such that the disease or condition symptoms are ameliorated, or such that the term of the disease or condition is changed. For example, the disease or condition may become less severe and/or recovery is accelerated in an individual. In some embodiments, a subject is administered a first dose of a compound (e.g., radioimmunoconjugate) or composition in an amount effective for radiation treatment planning, then administered a second dose or set of doses of the compound (e.g., radioimmunoconjugate) or composition in a therapeutically effective amount.
For treating or preventing cancers that comprises cells expressing EGFRVIII, the method typically comprises administering to a subject (e.g., a human) in need thereof a first dose of a compound or composition provided above in an amount effective for radiation treatment planning, followed by administering subsequent doses of a compound or composition provided above in a therapeutically effective amount.
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.
In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose are different.
Therapeutically effective amounts may depend on the severity of the disease or condition and other characteristics of the subject (e.g., weight). Therapeutically effective amounts of disclosed compounds (e.g., radioimmunoconjugates) and compositions for subjects (e.g., mammals such as humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences (e.g., differences in age, weight and the condition of the subject).
In some embodiments, disclosed compounds (e.g., radioimmunoconjugates) exhibit an enhanced ability to target cancer cells. In some embodiments, the effective amount of disclosed compounds (e.g., radioimmunoconjugates) is lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose for a therapeutic effect of the unconjugated, and/or non-radiolabeled targeting moiety.
Single or multiple administrations of compounds or pharmaceutical compositions disclosed herein including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. Dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.
In some embodiments, disclosed methods further include administering an antiproliferative agent, radiation sensitizer, or an immunoregulatory or immunomodulatory agent.
By “antiproliferative” or “antiproliferative agent,” as used interchangeably herein, is meant any anticancer agent, including those antiproliferative agents listed in Table 2, any of which can be used in combination with a radioimmunoconjugate as described herein to treat a condition or disorder. An exemplary antiproliferative agent used in the methods of this disclosure is temozolomide (TMZ), which can be administered in combination with a radioimmunoconjugate set forth above, in the presence or absence of external beam radiation. Antiproliferative agents also include organo-platinum derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.
By “immunoregulatory agent” or “immunomodulatory agent,” as used interchangeably herein, is meant any immuno-modulator, including those listed in Table 2, any of which can be used in combination with a radioimmunoconjugate.
As used herein, “radiation sensitizer” includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiation sensitizers may include, but are not limited to, 5-fluorouracil, analogs of platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF antagonists.
The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Lutetium-177 can be obtained from ITM Medical Isotopes as lutetium trichloride in a 0.05 N hydrochloric acid solution; indium-111, as indium trichloride in a 0.05 N hydrochloric acid solution, can be obtained from BWXT; and actinium-225 can be obtained as actinium-225 trinitrate from Oak Ridge National Laboratories.
Analytical HPLC-MS can be performed using a Waters Acquity HPLC-MS system comprised 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 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 comprised 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.
HPLC elution method 1: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) 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.
HPLC elution method 2: Waters XBridge Prep Phenyl 19×100 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, 13 min=0% A.
HPLC elution method 3: Waters Acquity BEH C18 2.1×50 mm (1.7 μm) 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, 8 min=0% A, 10 min=0% A, 11 min=90% A, 12 min=90% A.
HPLC elution method 4: Waters XBridge Prep C18 OBD 19×100 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, 3 min=80% A, 13 min=20% A, 18 min=0% A.
HPLC elution method 5: Waters XBridge Prep C18 OBD 19×100 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, 3 min=90% A, 13 min=0% A, 20 min=0% A.
HPLC elution method 6: Waters XBridge Prep C18 OBD 19×100 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=75% A, 13 min=0% A, 15 min=0% A.
HPLC elution method 7: Waters XBridge Prep C18 OBD 19×100 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.
HPLC elution method 8: Waters XBridge Prep C18 OBD 19×100 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.
Analytical Size Exclusion Chromatography (SEC) can be performed using a Waters system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiodetector (FC-3300) and TOSOH TSKgel G3000SW×1, 7.8×300 mm column. The isocratic SEC method can have a flow rate of, e.g., mL/min, with a mobile phase of 0.1 M phosphate, 0.6 M NaCl, 0.025% sodium azide, pH=7.
MALDI-MS (positive ion) can be performed using a MALDI Bruker Ultraflextreme Spectrometer.
Radio thin-layer chromatography (radio-TLC) can be performed with 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).
Generation and evaluation of certain EGFRvIII monoclonal antibodies can be referred to WO2020191485A1 (which is incorporated by reference in its entirety).
A 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
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) is added pyridine (0.05 mL, 50 mg, 0.62 mmol) at room temperature. The solution is stirred at room temperature for 24 hours. The reaction is purified directly by Prep-HPLC using method 7 to provide Intermediate 2-B as a wax after concentration using a Biotage V10 Rapid Evaporator.
Intermediate 2-B is dissolved in DCM/TFA (1.0 mL/2.0 mL) and allowed to stir at room temperature for 24 hours. The reaction is concentrated by air stream and purified directly by Prep-HPLC using method 8 to yield Compound B as a clear wax after concentration. An aliquot is analyzed by HPLC-MS elution method 3.
1H NMR (600 MHZ, DMSO-d6) δ 7.99-7.88 (m, 1H), 7.82 (t, J=5.5 Hz, 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=7.2 Hz, 2H), 2.30 (broad s, 2H), 1.88 (broad s, 2H), 1.66 (p, J=7.3 Hz, 2H), 1.36 (m, 4H), 1.32-1.20 (m, 9H).
A 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
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 for 90 min at ambient temperature. This solution is added to a semi-solution of amino-PEG3-acid (63 mg, 0.285 mmol in 1.2 mL of DMF) in a 100 mL round bottom flask. After 4 hours 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 (dissolved the crude in 6 mL of 20% ACN/H2O). The fractions containing product are pooled and concentrated under vacuum and then co-evaporated with ACN (3×2 mL).
To a vial containing Intermediate 1-A (82 mg, 60 umol) is added ACN (2 mL), NEt3 (50 μL, 360 μmol, 6 equiv.), HBTU (23 mg, 60 μmol, 1 equiv) and a TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μL of ACN). The resulting clear solution is stirred at ambient temperature for 3 hours. The reaction is worked up by concentrating the solution to dryness under an air stream and is then diluted with ACN/H2O (1:1, 3 mL total) and purified on preparative HPLC using elution method 4. Fractions containing product are pooled and concentrated under vacuum and then co-evaporated with ACN (3×2 mL). Intermediate 1-B is obtained as a clear residue.
To a 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 hour. Additional, TFA (2 mL) is added, and the reaction is stirred at ambient temperature for 6 hours. The reaction is concentrated to dryness under an air stream, with the crude product being finally dissolved in ACN/H2O (1 mL of 10% ACN/H2O). The crude reaction solution is then purified by preparative HPLC using elution method 5. The fractions containing product are pooled, frozen and lyophilized. Compound C is obtained as a white solid. An aliquot is analyzed by HPLC-MS elution method 3.
1H NMR (DMSO-d6, 600 MHZ) δ 7.97-7.91 (m, 2H), 3.77 (t, 2H, J=6.0 Hz), 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=6.0 Hz), 2.93 (broad s, 4H), 2.30 (broad s, 2H), 1.87 (broad s, 2H).
117Lu-radiolabeled Compound C-anti-EGFRvIII conjugates may be prepared as follows: Compound C (1.4 μmole) is dissolved in a hydrochloric acid solution (0.001 M). An aliquot of Compound C solution (19 μL, 90 nmole) is added to a solution containing anti-EGFRvIII antibody (1.8 nmole) in a carbonate buffer (pH 9.5). After 1 hour at ambient temperature, the resulting immunoconjugate is purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-anti-EGFRvIII is eluted from the column with acetate buffer (pH 6.5). Identities of eluates can be confirmed by, e.g., MALDI-TOF, where typical chelate to antibody ratios (CARs) of 3-4 are obtained.
177Lu (1.6 mCi, 3.9 μL) is added to a solution of Compound C-anti-EGFRvIII (150 μg in acetate buffer (pH 6.5)). After 40 minutes at ambient temperature, the crude product, [177Lu]-Compound C-anti-EGFRvIII, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer.
Two [177Lu]-Compound C-anti-EGFRvIII conjugates, i.e., Conjugates A and B, were prepared using two anti-EGFRvIII antibodies. More specifically, Conjugate A was prepared using EGFRVIII antibody hH2-hL3, comprising a light chain region having the amino acid sequence set forth in SEQ ID NO: 182 and a heavy chain region having the amino acid sequence set forth in SEQ ID NO: 184; Conjugate B was prepared using EGFRvIII antibody hH3-hL1, comprising a light chain region having the amino acid sequence set forth in SEQ ID NO: 180 and a heavy chain region having the amino acid sequence set forth in SEQ ID NO: 185. Conjugates A and B were prepared with chemical purities>99%, radiochemical purities>99%, and specific activities between 7-10 mCi/mg.
A 1.5 mL Eppendorf tube was charged with a SABST solution of Compound C-anti-EGFRvIII immunoconjugate (0.0492 mL of 3.05 mg/mL, i.e., 0.150 mg; prepared from EGFRvIII antibody hH3-hL1 following protocols similar to those described in Example 4 above), additional SABST to make a 2.0 mg/mL solution, and a 0.05±0.01 M HCl solution of [111In]Cl3 (2.0 mCi). After 60 minutes at 37° C., radio-TLC analysis of the reaction mixture (iTLC-SG plates, 5% methanol in 0.02 M citrate buffer as the mobile phase) indicated a radiochemical conversion (RCC) of 97%. Purification was carried out using a 1 mL column packed with Sephadex G50 resin (hydrated with SABST). The product fractions were eluted using SABST and combined. SABST solutions of sodium L-ascorbate and diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (DTPA) were added to give a final formulation of 10 mM ascorbate and 1 mM DTPA. Analysis of the resulting formulation by radio-TLC and SEC-HPLC at end-of-synthesis (EOS) indicated the formation of [111In]-Compound C-anti-EGFRvIII (364 μL, 0.299 mg/mL, 8.9 mCi/mg specific activity, 99% radiochemical purity, and >95% chemical purity).
A 1.5 mL Eppendorf tube was charged with a SABST solution of Compound C-IgG4 immunoconjugate (0.0318 mL of 4.72 mg/mL, i.e., 0.150 mg; prepared from isotype IgG4 antibody S228P (purchased from Bio X Cell, Cat #BE0349-5 MG-R) following similar protocols to those described in Example 4 above), additional SABST to make a 2.0 mg/mL solution and a 0.05±0.01 M HCl solution of [111In]C1; (2.25 mCi). After 60 minutes at room temperature, radio-TLC analysis of the reaction mixture (iTLC-SG plates, 5% methanol in 0.02 M citrate buffer as the mobile phase) indicated a radiochemical conversion (RCC) of 99%. Purification was carried out using a 1 mL column packed with Sephadex G50 resin (hydrated with SABST). The product fractions were eluted using SABST and combined. SABST solutions of sodium L-ascorbate and diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (DTPA) were added to give a final formulation of 10 mM ascorbate and 1 mM DTPA. Analysis of the resulting formulation by radio-TLC and SEC-HPLC at end-of-synthesis (EOS) indicated the formation of [111In]-Compound C-IgG4 (364 μL, 0.362 mg/mL, 9.3 mCi/mg specific activity, 99% radiochemical purity, and >95% chemical purity).
A 1.5 mL Eppendorf tube was charged with a SABST solution of Compound C-anti-EGFRvIII immunoconjugate (0.0656 mL of 3.05 mg/mL, i.e., 0.200 mg; prepared from anti-EGFRvIII antibody hH3-hL1 following the protocols in Example 4 above), additional SABST to make a 2.0 mg/mL solution and a 0.001 M HCl solution of [225Ac]Cl3 (10.9 μCi). After 2 hours at 37° C., radio-TLC analysis of the reaction mixture (iTLC-SG plates, 5% methanol in 0.02 M citrate buffer as the mobile phase) indicated a radiochemical conversion (RCC) of 99% (the plate was scanned on the radio-TLC scanner the following day). Purification was carried out using a 1 mL column packed with Sephadex G50 resin (hydrated with SABST). The product fractions were eluted using SABST and combined. SABST solutions of sodium L-ascorbate and diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (DTPA) were added to give a final formulation of 10 mM ascorbate and 1 mM DTPA. Analysis of the resulting formulation by radio-TLC and SEC-HPLC at end-of-synthesis (EOS) indicated the formation of [225Ac]-Compound C-anti-EGFRVIII (461 μL, 0.406 mg/mL, 0.0454 mCi/mg specific activity, 99% radiochemical purity, and >95% chemical purity).
A 1.5 mL Eppendorf tube was charged with a SABST solution of IgG4 immunoconjugate (0.0847 mL of 4.72 mg/mL, i.e., 0.400 mg; prepared from isotype IgG4 antibody S228P following protocols similar to those described in Example 4 above), additional SABST to make a 2.0 mg/mL solution and a 0.001 M HCl solution of [225Ac]Cl3 (21.1 μCi). After 2 hours at 37° C., radio-TLC analysis of the reaction mixture (iTLC-SG plates, 5% methanol in 0.02 M citrate buffer as the mobile phase) indicated a radiochemical conversion (RCC) of 68% (the plate was scanned on the radio-TLC scanner the following day). Purification was carried out using a 1 mL column packed with Sephadex G50 resin (hydrated with SABST). The product fractions were eluted using SABST and combined. SABST solutions of sodium L-ascorbate and diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (DTPA) were added to give a final formulation of 10 mM ascorbate and 1 mM DTPA. Analysis of the resulting formulation by radio-TLC and SEC-HPLC at end-of-synthesis (EOS) indicated the formation of [225Ac]-Compound C-IgG4 (463 μL, 0.773 mg/mL, 0.0427 mCi/mg specific activity, 99% radiochemical purity, and 95% chemical purity).
A study was conducted to evaluate the receptor binding affinity of [177Lu]-Compound C-anti-EGFRvIII conjugates, i.e., Conjugates A and B, with the U87-EGFRvIII glioblastoma cell line over-expressing EGFRvIII (obtained from National Research Council of Canada).
The materials used in this study are summarized in Table 3 below.
This study followed the procedures described below:
The results from the in vitro binding study are shown in
A study was conducted to evaluate the internalization of [177Lu]-Compound C-anti-EGFRvIII conjugates, i.e., Conjugates A and B, with the U87-EGFRvIII glioblastoma cell line over-expressing EGFRvIII (obtained from National Research Council of Canada) following the protocol described below.
The main purpose of this study is to: 1) quantitatively measure the amount of radiolabeled test article, i.e., EGFRvIII radioimmunoconjugate, on the cell surface and inside cells; and 2) determine the amount of internalized test article that is retrieved in the medium, retained in the cells, or recycled back on the cell surface.
The materials used in this study are summarized in Table 4 below.
This study followed the procedures below:
1. Cell preparation:
The results from the internalization study are shown in
As shown in
A U87-EGFRvIII cell line xenograft mouse model was used to assess the in vivo biodistribution of [177Lu]-DOTA-anti-EGFRvIII conjugates. Two [177Lu]-DOTA-anti-EGFRvIII conjugates, i.e., Conjugates A and B, were synthesized using a pure R enantiomer of Compound C (see Example 4), two humanized variants of anti-EGFRvIII antibody 4E11, and lutetium-177.
Groups of tumor-bearing animals were injected intravenously with [177Lu]-DOTA-anti-EGFRvIII conjugates. Doses contained about 9.6-9.8 microcuries (μCi)/μg of activity on 2 μg (0.1 mg/kg) of antibody. Animals were euthanized at 4 h, 24 h, 96 h, and 168 h after injection to determine levels of radioactivity in the blood, heart, intestines, kidneys, liver, lungs, spleen, tumor, urine, and tail (n=3 per time point).
Results were expressed as the percentage injected dose per gram of tissue (% ID/g) and are depicted in
A U87-EGFRvIII-GFP-Luc orthotopic model was used to assess the in vivo biodistribution of radiolabeled anti-EGFRvIII conjugates, i.e., Conjugates A and B.
Groups of tumor-bearing animals were injected intravenously with [177Lu]-DOTA-anti-EGFRvIII. Doses contained about 9.6-9.8 microcuries (μCi)/μg of activity on 2 μg (0.1 mg/kg) of antibody. Animals were euthanized at 96 h after injection to determine levels of radioactivity in the blood, tumor, normal brain, spleen, liver, kidneys, and tail (n=10 per time point for Conjugate A and n=10 per time point for Conjugate B).
Results were expressed as the percentage injected dose per gram of tissue (% ID/g) and are depicted in
Target specificity and tumor uptake of [111In]-Compound C-anti-EGFRvIII (see Example 5 above) was investigated in an imaging biodistribution study using orthotopic glioblastoma multiforme (GBM) PDX models with varying degrees of blood-brain barrier (BBB) permeability. Although all GBM tumors have some degree of BBB permeability as a characteristic of the disease, this can vary considerably between tumors and even within the same tumor. The large size of antibodies (about 150 kDa) prevents them from crossing an intact BBB, and intravenous administration of radiolabeled anti-EGFRvIII immunoconjugates may be dependent on BBB permeability for delivery to GBM tumors. To characterize the extent of tumor uptake after intravenous administration of [111In]-Compound C-anti-EGFRvIII, an imaging biodistribution study was performed in a GBM PDX model with a relatively intact BBB (G06-GFP-Luc) and another model with a completely disrupted BBB (G39-GFP-Luc). In addition, to validate the target specificity, the biodistribution of a non-targeted [111In]-Compound C-IgG4 (see Example 5 above) was also assessed for comparison.
Orthotopic GBM PDX tumors were established in 7-8 week old female Balb/c athymic nude mice by intracranial injection of 3×105 G06-GFP-Luc or 2.5×105 G39-GFP-Luc cells in 3 μL PBS. Cells were injected into the right cerebral hemisphere 1 mm posterior from bregma, 1.5 mm to the right of the sagittal suture, and at a depth of 2.5 mm. Bioluminescence 3D tomography (BLT) was performed 13 days after intracranial injections to confirm the presence of tumors and determine tumor volumes. 150 mg/kg D-luciferin in PBS was injected intraperitoneally into mice 15 min prior to imaging. Mice were anesthetized with isoflurane and placed on the imaging bed. Images were acquired using the optical imaging (OI) module of the VECTor6CT SPECT/PET/CT/OI imaging system and reconstructed using MiLabs BLT Recon 1.0 software. Tumor volume was quantified using Imalytics software and region of interest (ROI) analysis. Mice were triaged into groups based on tumor size such that each group had a relatively equal distribution of tumor volumes.
Tumor-bearing mice were injected intravenously via the lateral tail vein with 0.2 mL of [111In]-Compound C-anti-EGFRvIII or [111In]-Compound C-IgG4 containing 100 μCi of radioactivity (10.8-11.8 μg of antibody) formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, 0.01% Tween 80 (n=4 mice/compound). BLT and SPECT/CT imaging was performed 96 hours post-injection using the VECTor6CT SPECT/PET/CT/OI imaging system to determine the tissue biodistribution. Images were reconstructed using MiLabs BLT Recon 1.0 software and tumor/normal tissue uptake was determined using PMOD software. Uptake was calculated as percentage of injected dose per cubic centimeter of tissue (% ID/cc).
In the G39-GFP-Luc PDX model with a disrupted BBB, [111In]-Compound C-anti-EGFRvIII biodistribution data demonstrated high tumor uptake of 50.2±13.6% ID/cc at 96 h. In comparison, non-targeting [111In]-Compound C-IgG4 had a tumor uptake of 17.4±1.2% ID/cc at 96 h, confirming that the majority of [111In]-Compound C-anti-EGFRvIII tumor uptake is target specific. Importantly, normal brain showed very low uptake (1.6-2.1% ID/cc) providing further evidence of tumor-specific uptake of [111In]-Compound C-anti-EGFRVIII (
In the G06-GFP-Luc PDX model with a relatively intact BBB, [111In]-Compound C-anti-EGFRvIII biodistribution data demonstrated a tumor uptake of 10.4±1.6% ID/cc at 96 h while non-targeting [111In]-Compound C-IgG4 had a tumor uptake of 9.4±1.7% ID/cc at 96 h (
The results of the imaging biodistribution studies confirm that uptake of [111In]-Compound C-anti-EGFRvIII is highly tumor-specific with minimal off-target uptake in normal tissues. It was further demonstrated that high tumor uptake of [111In]-Compound C-anti-EGFRvIII can be achieved via intravenous administration in tumors with a highly disrupted BBB, but uptake is restricted by an intact BBB.
The single-dose radiotherapeutic efficacy of [225Ac]-Compound C-anti-EGFRvIII (see Example 5 above) was determined using orthotopic glioblastoma multiforme (GBM) models generated with the cell line U87-EGFRvIII-GFP-Luc or the patient-derived xenograft (PDX) model, G06-GFP-Luc. Both the GBM cell line and PDX cells were transduced with lentiviral particles expressing green fluorescent protein (GFP) and luciferase (Luc) for in vivo and ex vivo imaging purposes. GBM brain tumors were established in 7-8 week old female Balb/c athymic nude mice by intracranial injection of 2×104 U87-EGFRvIII-GFP-Luc cells or 3×105 G06-GFP-Luc in 3 μL PBS. Cells were injected into the right cerebral hemisphere 1 mm posterior from bregma, 1.5 mm to the right of the sagittal suture, and at a depth of 2.5 mm. Bioluminescence imaging (BLI) was performed 8-10 days after intracranial injections to confirm the presence of tumors and quantify tumor burden. 150 mg/kg D-luciferin in PBS was injected intraperitoneally into mice 15 min prior to imaging. BLI signal was quantified in a region of interest (ROI) drawn around the tumor and expressed as area averaged signal. The area averaged signal represents a measurement of tumor burden and mice were triaged into groups such that the average BLI tumor signal was equal across groups.
Therapy was initiated 10-13 days post-intracranial injection and after tumors were verified by imaging. Tumor-bearing mice were injected intravenously (IV) via the lateral tail vein with 0.2 mL [225Ac]-Compound C-anti-EGFRvIII formulated in 20 mM sodium citrate pH5.5, 0.82% NaCl, 0.01% Tween 80 or vehicle alone for control mice (n=5/group). Actinium-225 radiolabeled test articles contained 100-200 nCi of activity (up to 4 μg of antibody protein/dose). Mice were weighed three times per week and monitored daily for signs of brain tumor development such as hunched posture, lethargy, seizures, impaired mobility, paralysis, or enlarged skull. For the U87-EGFRVIII-GFP-Luc cell line model, BLI was used to monitor tumor burden and response to therapy over time. Mice were imaged once per week as described above, and BLI signals were plotted as a fold change from the pre-treatment area averaged tumor signal to determine the therapeutic response. Animals were euthanized when humane endpoints were reached (>20% weight loss, BC2) or signs of brain tumors developed (as described above). Overall survival was plotted on a Kaplan-Meier survival curve. The log rank (Mantel-Cox) test was used to statistically compare the survival between groups and determine p-values. To facilitate the interpretation of treatment response between models, the survival benefit for a given treatment was calculated by determining the ratio of the median survival for mice receiving the treatment versus vehicle control.
Radiotherapeutic studies of [225Ac]-Compound C-anti-EGFRvIII in the U87-EGFRvIII-GFP-Luc cell line model demonstrated significant therapeutic efficacy at both 100 nCi and 200 nCi doses. BLI analysis revealed that tumor growth was significantly suppressed in comparison to the vehicle control group (
The radiotherapeutic efficacy of [225Ac]-Compound C-anti-EGFRVIII was examined using an orthotopic GBM patient-derived xenograft (PDX) model. Multiple fractionated doses of [225Ac]-Compound C-anti-EGFRvIII or [225Ac]-Compound C-IgG4 (see Example 5 above) were compared to single dose administration to determine the optimal dosing regimen to maximize therapeutic efficacy. Aside from the dosing schedule, the study was carried out as described above for the single dose radiotherapeutic efficacy studies.
Mice bearing orthotopic G39-GFP-Luc tumors were administered [225Ac]-Compound C-anti-EGFRvIII at a single dose of 100, 200, or 400 nCi or a total cumulative radiochemical dose of 400 nCi fractionated as follows: 100 nCi dosed once weekly for 4 weeks (100 nCi×4) or 200 nCi dosed every 2 weeks for 2 doses (200 nCi×2). Each dose contained 8.6 μg of antibody protein. Control groups were administered a single dose of [225Ac]-Compound C-IgG4 containing either 200 nCi or 400 nCi of radioactivity (9.4 μg antibody protein/dose) or vehicle alone (n=5 mice/group).
Survival analysis showed a dose-dependent increase in survival in response to treatment with [225Ac]-Compound C-anti-EGFRvIII. The survival benefits of the 400 nCi [225Ac]-Compound C-anti-EGFRvIII single-dose or fractionated dose groups were comparable with survival benefit ratios of 3.1, 3.2, and 3.1 for the 400 nCi single dose, fractionated 200 nCi×2, and fractionated 100 nCi×4 dose groups, respectively. These results further demonstrated that efficacy was related to the total radioactivity dose, regardless of the dosing schedule. In addition, the survival benefit of 400 nCi single-dose [225Ac]-Compound C-IgG4 was significantly lower (1.9), confirming that at least some of the therapeutic efficacy of [225Ac]-Compound C-anti-EGFRvIII is target-mediated (
The standard of care (SoC) for GBM patients is maximal surgical resection followed by external beam radiation therapy (EBRT) in combination with the chemotherapeutic temozolomide (TMZ). The therapeutic efficacy of combined SoC and [225Ac]-Compound C-anti-EGFRvIII in comparison to single-agent was tested using the orthotopic G06-GFP-Luc GBM PDX model. Tumors were engrafted and verified by BLT imaging as described above. Tumor bearing mice were treated with a single intravenous dose (0.2 mL) of vehicle alone, 200 nCi dose [225Ac]-Compound C-anti-EGFRvIII, or 200 nCi dose [225Ac]-Compound C-anti-EGFRvIII in combination with TMZ (25 mg/kg administered daily for 5 days via oral gavage). Two additional treatment groups (SoC alone and combination group) received 2 Gy EBRT once daily for 5 consecutive days in combination with 25 mg/kg temozolomide (administered daily for 5 days via oral gavage 1 hr prior to EBRT). The combination group then subsequently received a single dose of 200 nCi [225Ac]-Compound C-anti-EGFRvIII 6 days after completion of EBRT/TMZ treatments (n=5 mice/group).
Survival analysis demonstrated that SoC alone (2 Gyx5 EBRT+25 mg/kg TMZ), 200 nCi [225Ac]-Compound C-anti-EGFRvIII, and 200 nCi [225Ac]-Compound C-anti-EGFRvIII in combination with TMZ all significantly extend survival in comparison to vehicle controls with a survival benefit of approximately 2 for all. However, combined treatment with SoC (2 Gyx5 EBRT+25 mg/kg TMZ) followed by 200 nCi [225Ac]-Compound C-anti-EGFRvIII resulted in a significant extension in survival (survival benefit of 3.6) compared to SoC alone, [225Ac]-Compound C-anti-EGFRvIII, or [225Ac]-Compound C-anti-EGFRvIII in combination with TMZ (survival benefit of approximately 2) (
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.
MRPSGTAGAALLALLAALCPASRA
LEEKKVCQGTSNKLTQLGTFEDH
HFPWT
FGGGTKLEIK
HYSTPLT
FGAGTKLELK
GTSNLAS
GVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT
HGTNLED
GVPSRFSGSGSGTDYSLTISSLESEDFADYYCVQYAQFPYTF
GTSNLAS
GVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLT
DTSNLAS
GVPIRFSGSGSGTSYSLTISSVEAEDAATYYCQQWSSYPLTF
HVPFT
FGSGTKLEIK
MHT
FGGGTKLEIK
HVPFT
FGSGTKLEIK
MHT
FGGGTKLEIK
HVPFT
FGSGTKLEIK
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEW
MGYIGYNGRTSYNPSLKSRISITRDTSKNQFFLQLNYVTTEDTATFYCA
RLGRGFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLV
DILMTQSPSSMSVSLGDTVSITCHASQGINSNIGWLLQKPGKSFKGLIYH
GTNLEDGVPSRFSGSGSGTDYSLTISSLESEDFADYYCVQYAQFPYTFG
GGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
EVQLQQSGAELARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLE
WIGAIYPGNSDISYNQKFKGKAKLTAVTSATTAYMELSSLTNEDSAVY
YCTLYDYDPDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALG
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSP
KRLIYLASKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQAT
HFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
QVOLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEW
VAVIWYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCARDGWQQLAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPR
LLIYKISNRFSGVPDRFSGSGAGTAFTLKISRVEAEDVGVYYCMQATQL
PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
T
FGAGTKLELX3a
SSYPLT
FGX6dGTKLEX7dX8d
A
CVRACGADSYEMEEDGVRKCKK
GASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEW
IGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ
LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV
SSVE
GGSGGSGGSGGSGG
VDDIQLTQSPAIMSASPG
EKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDT
SKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY
CQQWSSNPLTFGAGTKLELK
DIQMTQSPSSLSASVGDRVTITCHASQGINSNIGWYQQKP
GKAPKLLIYHGTNLEDGVPSRFSGSGSGTDYTLTISSLQP
EDFATYYCVQYAQFPYTFGQGTKLEIKRTVAAPSVFIFPP
DIQMTQSPSSLSASVGDRVTITCHASQGINSNIGWLQQKP
GKAPKGLIYHGTNLEDGVPSRFSGSGSGTDYTLTISSLQP
EDFATYYCVQYAQFPYTFGQGTKLEIKRTVAAPSVFIFPP
DIQMTQSPSSLSASVGDRVTITCHASQGINSNIGWLQQKP
GKAFKGLIYHGTNLEDGVPSRFSGSGSGTDYTLTISSLQP
EDFATYYCVQYAQFPYTFGQGTKLEIKRTVAAPSVFIFPP
QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIR
QPPGKGLEWIGYIGYNGRTSYNPSLKSRVTISVDTSKNQF
SLKLSSVTAADTAVYYCARLGRGFAYWGQGTLVTVSSA
QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIR
QPPGKGLEWIGYIGYNGRTSYNPSLKSRVTISRDTSKNQF
SLKLSSVTAADTAVYYCARLGRGFAYWGOGTLVTVSSA
QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIR
QPPGKGLEWMGYIGYNGRTSYNPSLKSRITISRDTSKNQF
SLKLSSVTAADTAVYYCARLGRGFAYWGQGTLVTVSSA
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051360 | Sep 2021 | WO | international |
The present application claims priority to International Patent Application No. PCT/CA2021/051360, filed Sep. 29, 2021, the entire contents of which are hereby incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051447 | 9/29/2022 | WO |
