Patent Application
20230107890
The present disclosure relates to the field of HER2-targeted therapy.
Engineered scaffold proteins (ESPs) attract an increasing interest as specific targeting vectors in radionuclide molecular imaging (Krasniqi 2018). ESPs have a number of attractive features such as efficient and inexpensive production in prokaryotic hosts or even by peptide synthesis. Moreover, they show high stability in a broad range of temperature and pH, facilitating the use of a large variety of labeling methods. In addition, their high stability and mutational tolerance promotes the ease of engineering multimeric, multispecific and multifunctional constructs. “ADAPT”is an ESP derived from the small (46 amino acids) albumin-binding domain (ABD) of protein G (Nilvebrant Hober S 2013). Feasibility of using ADAPTs as imaging probes has been demonstrated using ADAPT6, which shows high affinity to human epidermal growth factor receptor type 2 (HER2) (Garousi 2015). However, the use of ADAPT6-based radionuclide therapy has been precluded by high renal reabsorption after clearance via glomerular filtration. The re-absorption cannot be reduced by co-injection of L-lysine or Gelofusine (Lindbo 2016). Therefore, other strategies are needed to allow for therapeutic use.
The present inventors have realized that a non-covalent binding to serum albumin is a possible strategy to extend circulatory half-life, increase bioavailability and reduce renal uptake of small therapeutics undergoing renal clearance.
An objective of the present disclosure is to provide a HER2-targeting therapeutic agent that overcomes at least some of the shortcomings of the prior art.
To meet this objective, there is provided a therapeutic conjugate comprising a fusion protein and a cytotoxic radionuclide, which cytotoxic radionuclide is bound to the fusion protein, wherein:
other,
wherein, independently of each other,
In an embodiment, the cytotoxic radionuclide is coupled to a terminal end (preferably the C-terminal end) of the fusion protein, typically by chelator-based conjugation or covalent conjugation.
In an embodiment, the spacer region connects the C-terminal end of the HBR to the N-terminal end of the ABR.
In an embodiment, the ABR comprises the amino acid sequence
wherein, independently of each other,
In an embodiment, the ABR comprises the amino acid sequence
wherein, independently of each other,
In an embodiment, the ABR comprises or consists of an amino acid sequence selected from the group consisting of:
In an embodiment of amino acid sequence i):
In an embodiment, the HBR comprises or consists of an amino acid sequence selected from the group consisting of:
In an embodiment, the cytotoxic radionuclide is selected from the group consisting of 177Lu, 90Y, 188Re; 186Re;166Ho, 153Sm, 67Cu, 64Cu,149Tb, 161Tb, 47Sc; 225Ac; 212Pb; 213Bi, 212Bi, 227Th, 223Ra; 58mCo, 131I, 76As, 77As and 211At.
In an embodiment, the group of cytotoxic radionuclides is limited to 177Lu, 90Y, 131I, 153Sm, 225Ac;188Re and 186Re.
In an embodiment, the radionuclide is a radiometal and the fusion protein is conjugated to the radiometal by a chelator that is covalently bound to the fusion protein, preferably to a cysteine (C) residue of the fusion protein. Accordingly, an embodiment of the fusion protein further comprises a terminal cysteine (C) residue, preferably a C-terminal cysteine (C) residue. The terminal cysteine (C) residue is preferably the only cysteine (C) residue of the fusion protein.
In an embodiment, the number of amino acids of the spacer region is at least 8, such as 8-30, such as 8-20, preferably 8-14, more preferably 10-14.
In an embodiment, the spacer region comprises a repeat of the amino acid sequence SSSG.
In an embodiment, the therapeutic conjugate comprises no more than 150 amino acid residues, such as no more than 130 amino acid residues.
In an embodiment, the fusion protein further comprises an additional region comprising the amino acid sequence DEAVDANS (SEQ ID NO:25). In such an embodiment, the additional region may extend from the N-terminal end of the HBR.
The present disclosure further provides a pharmaceutical composition comprising the above-mentioned therapeutic conjugate and a pharmaceutically acceptable carrier.
The present disclosure also provides a therapeutic conjugate according to the above for use in a method of treating a cancer, typically a cancer overexpressing HER2. The cancer type may be a breast cancer or a gastric/gastroesophageal cancer.
The therapeutic conjugate of the present disclosure exhibits excellent biodistribution, which means that side effects are limited during treatment of patients having HER2-expressing tumors.
As explained above, the therapeutic conjugate of the present disclosure comprises a fusion protein having an albumin-binding region (ABR) and a HER2-binding region (HBR), which are linked to each other by a spacer region. The function of the spacer region is to enable free refolding and prevent steric hindrance of binding to albumin and HER2.
In
In
MG-DEAVDANS-ADATNeg-(SSSG)3-ABD035 -GSSC-DOTA-177Lu (single injection) is represented by a dotted line;
MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-DOTA (unlabelled) is represented by a dotted line; and
PBS is represented by a solid line.
ADAPT6 has shown great promise in both pre-clinical and clinical imaging applications mainly because of its small size and fast blood clearance. However, the small size and hence the substantial kidney uptake also prevents further therapeutic use. In the present disclosure, half-life extension strategy where the protein of interest is fused to a sequence based on the Albumin Binding Domain (ABD) of Protein G has been utilized, to take advantage of the long half-life of the patient's own serum albumin.
Our molecular design of HBR-ABR-based fusion proteins permits production of probes capable of correct folding and high-affinity binding to both albumin and HER2 (Table 1). The ability of the fusion proteins to refold after thermal denaturation has allowed the use of high temperatures for labeling, e.g. with1?7Lu, which should ensure high coupling stability (Price 2014). Indeed, the label was stable under challenge with a large molar excess of EDTA, and binding specificity and affinity of 177Lu-labeled fusion proteins was not compromised (Table 4). Cellular processing study with the use of residualizing177Lu label suggested that both MG-DEAVDANS-ABDon-(SSSG)3-ADAPT6-GSSC-DOTA-177Lu and MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-DOTA-177Lu internalized slowly, to the same extent as a non-fused ADAPT6 (
An in vivo targeting experiment (
Unexpectedly, the present disclosure shows that the relative positioning of ABDos (the ABR) and ADAPT6 (the HBR) had an effect, in particular in case of using 177Lu as the cytotoxic radionuclide. Connecting the ABR to the C-terminal end of the HBR resulted not only in significantly higher tumor uptake, but also significantly lower kidney uptake. The fact that absorbed dose to kidneys might be dose limiting underlines the importance of these results.
The data presented herein demonstrates that both the 177Lu-label and the 125I-label were delivered to the tumor and that the delivery was more efficient in case of the 177Lu-labelled fusion protein. Accumulation of both177Lu- and25I-labeled ADAPT6-ABD035 in tumors was apparently dependent on HER2 expression (
Results of an experimental therapy confirmed the dosimetry data. It is shown herein that control groups of mice treated with only PBS or unlabelled HBR-ABR (i.e. fusion protein lacking cytotoxic radionuclide) had a median survival of 25 days. Treatment with a radionuclide-labelled variant of the fusion protein that had been modified to remove the HER2-binding capacity caused slight but significant extension of median survival (31 d) (
Currently, there are several treatment options for patients with disseminated HER2-expressing breast cancers, e.g. treatment with monoclonal antibodies trastuzumab and pertuzumab or antibody-drug conjugated trastuzumab-DM1. These therapeutics significantly improved survival of breast cancer patients. Unfortunately, tumors eventually develop resistance to such therapies. Remarkably, the resistant tumors preserve a high level of HER2 expression. Therapy using the therapeutic conjugate of the present disclosure is an additional option for such patients.
The present disclosure provides a therapeutic conjugate comprising a fusion protein and a cytotoxic radionuclide. The cytotoxic radionuclide is bound to the fusion protein.
In a preferred embodiment, the cytotoxic radionuclide is coupled to a terminal end of the fusion protein. Such coupling is typically achieved by chelator-based conjugation or covalent conjugation (further discussed below). In a particularly preferred embodiment, the cytotoxic radionuclide is coupled to the C-terminal end of the fusion protein.
The fusion protein comprises and an HER2-binding region (HBR), an albumin-binding region (ABR) and a spacer region. Preferably, the spacer region connects the C-terminal end of the HBR to the N-terminal end of the ABR. Advantages thereof is described in the Examples section below.
The number of amino acids of the spacer region is at least 7, such as 7-30. A spacer region that is relatively short, but still enables free refolding and prevents steric hindrance to binding to albumin and HER2 is preferred. Accordingly, the number of amino acids of the spacer region maybe 8-30, such as 8-20, preferably 8-14, more preferably 10-14.
As an example, the spacer region may comprise or consist of a repeat of the amino acid sequence SSSG. Consequently, the spacer region may comprise or consist of an amino acid sequence selected from the group consisting of SSSGSSSG ((SSSG)2, SEQ ID NO:31), SSSGSSSGSSSG ((SSSG)3, SEQ ID NO:32), SSSGSSSGSSSGSSSG ((SSSG)4, SEQ ID NO:33), SSSGSSSGSSSGSSSGSSSG ((SSSG)5, SEQ ID NO:34) and SSSGSSSGSSSGSSSGSSSGSSSG ((SSSG)6, SEQ ID NO:35). Preferably, the group consists of (SSSG)2, (SSSG)3 and (SSSG)4.
The HBR comprises an amino acid sequence selected from i) LAX3AKX6TX8X9Y HLX13X14X15GVX18DX20 YKX23LIDKX28KT VEX33VX38 AX37YX39X40 ILX43ALP, wherein, independently of each other,
Data showing binding activity of i) and ii) to HER2 is presented in WO2014o76179, Nilvebrant 2014 and the Examples section below.
In preferred embodiment of the amino acid sequence i)
In another preferred embodiment, the HBR comprises an amino acid sequence selected from the group consisting of:
A particularly preferred group consists of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:13. SEQ ID NO:6 is used in the Examples section below. SEQ ID NO:9 and 13 were identified by both phage display and FACS in Nilvebrant 2014, which the inventors consider to be beneficial.
The ABR comprises an amino acid sequence selected from
The scaffold of sequence a) is based on the wild type of the albumin binding domain (ABDwt, SEQ ID NO:4) of protein G. Data showing binding activity of i) and ii) to serum albumin is presented in Jonsson 2008, WO2012004384 and the Examples section below.
In a preferred embodiment, the ABR comprises the amino acid sequence LAX3AKX6X7AX9X,o ELDX14YGVSDX2o YKX23LIX26X27AKTVEGVX35ALX38X3X4OILAALP, wherein, independently of each other,
In a particularly preferred embodiment, the ABR comprises the amino acid sequence LAF.AK6X7ANX,o ELDX14YGVSDF YKRLIX2eKAKT VEGVEALKX39X40 ILAALP, wherein, independently of each other,
This sequence covers ABD3s and PEPo7914 in WO2012004384. The reduced immune-stimulatory properties of the sequences disclosed in WO2012004384 are considered to be beneficial for the conjugate of the present disclosure. WO2012004384 has resulted in two granted European patents, EP2590993B1 and EP2933262B1. The ABR of the present disclosure maybe defined as in these patents.
Consequently, the ABR may, as in EP2933262B1, comprise an amino acid sequence selected from aa) LAX3AKX6X7ANX,o ELDX14YGVSDF YKRLIX26KAKT VEGVEALKX39X40 ILX43X44LP, wherein, independently of each other,
Further, the ABR may, as in EP2590993B1 comprise an amino acid sequence selected from aaa) LAX3AKEAANA ELDX14YGVSDF YKRLIDKAKT VEGVEALKDAILAALP, wherein, independently of each other,
In one embodiment, the ABR comprises an amino acid sequence selected from the group consisting of:
A particularly preferred group consists of SEQ ID NO:1 (ABD35), SEQ ID NO:2 and SEQ ID NO:3 (PEPo7914 in WO2012004384).
The cytotoxic radionuclide is preferably selected from the group consisting of 177Lu, 90Y, 188Re; 186Re;166Ho, 153Sm, 67Cu, 64CU,149Tb, 161Tb, 47Sc; 255Ac; 212Pb; 213Bi, 212Bi, 227Th, 223Ra; 58mCo, 131I, 76As, 77As and 211At.
A preferred radionuclide is177Lu.
77Lu, 90Y, 188Re; 186Re;166Ho, 153Sm, 67Cu, 64Cu,149Tb, 161Tb, 47Sc; 225Ac; 212Pb; 213Bi, 212Bi, 227Th, 223Ra and 58mCo are radiometals that maybe bound to the fusion protein by means of chelator-based conjugation. The chelator is preferably covalently bound to a cysteine residue of the fusion protein, optionally via a thiol-reactive linker. Binding to an amine of an amino acid residue of the fusion protein is also possible, but generally less preferred.
The chelator may be selected from the group consisting of DOTA and its derivatives (e.g. the maleimido-derivative of DOTA), cross-bridged macrocyclic chelators and sterically-restricted acyclic chelators.
Particularly suitable chelators for 177Lu, 90Y, 166Ho, 153Sm, 149Tb, 161Tb, 47Sc; 225Ac; 212Pb; 213Bi, 212Bi, 227Th and 58mCo are DOTA and its derivative DOTAGA.
For67Cu and 64Cu a cross-bridged chelator, such as CB-TE2A, is a better option.
For 188Re and 186Re a chelator based on a cysteine- or mercaptoacetyl-containing peptide is preferred.
131I, 76As, 77As and 211At are non-metal radionuclides that can be bound to the fusion protein by means of covalent conjugation.
The radioiodination may be achieved using ((4-hydroxyphenyl)ethyl) maleimide (HPEM), which can be bound to a cysteine (C) residue of the fusion protein. 76As and 77As (and 74As) in As (III) form may be coupled directly to a (freshly) reduced thiol group of a cysteine (C) residue of the fusion protein.
For coupling of 211At, N-[4-(tri-n-butylstannyl) phenethyl]-maleimide can be used as a linker. In such case, 211At is first coupled to the linker by astatodestannylation forming 4-astato-phenethyl-maleimide (AtPEM), which in turn can be coupled to a cysteine (C) residue of the fusion protein.
Consequently, the cytotoxic radionuclide is preferably linked to the fusion protein via a cysteine (C) residue of the fusion protein. To avoid cross/side reactions, the fusion protein in such case preferably comprises only one cysteine (C) residue.
The cysteine (C) residue that links the cytotoxic radionuclide to the fusion protein may be a placed in a terminal position. Preferably, the cysteine (C) residue is the C-terminal residue of the fusion protein.
In one embodiment, the cysteine (C) residue is the C-terminal residue of an amino acid sequence extending from the C-terminal end of the ABR. Such an amino acid sequence may for example be GSSC.
The fusion protein may further comprise an additional region, e.g. comprising or consisting of the amino acid sequence DEAVDANS (SEQ ID NO:25) or MGDEAVDANS (SEQ ID NO:18). This additional region is typically a terminal region of the fusion protein, preferably an N-terminal region of the fusion protein. In an embodiment, the additional region extends from the N-terminal end of the HBR. The additional region may result in improved biodistribution and facilitate production.
At the same time, it is beneficial to limit the size of the therapeutic conjugate of the present disclosure. Accordingly, it preferably comprises no more than 150 amino acid residues, such as no more than 130 amino acid residues. The total molecular weight of the therapeutic conjugate is preferably below 20 kDa, such as below 15 kDa.
In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:21(ADAPT6-(SSSG)3-ABD3s), SEQ ID NO:22 (ABD3s-(SSSG)3-ADAPT6), SEQ ID NO:23 (ADAPT6-(SSSG)5-ABD3s) or SEQ ID NO:24 (ABD3s-(SSSG)5-ADAPT6). Out of these four sequences, SEQ ID NO:21(ADAPT6-(SSSG)3-ABD3s) is considered to be the most preferred.
In an embodiment, the fusion protein consists of the amino acid sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30. Out of these four sequences, SEQ ID NO:27 is considered to be the most preferred.
The present disclosure further provides a pharmaceutical composition comprising the therapeutic conjugate discussed above and a pharmaceutically acceptable carrier.
The composition is preferably adapted for intravenous administration. Accordingly, the composition is typically water-based. The water-based composition is preferably buffered, such as phosphate-buffered. As an example, the composition may be based on phosphate-buffered saline. The water-based composition may comprise human serum albumin (HSA). HSA scavenges free radicals and prevents radiolytic damages to the therapeutic conjugate. The amount of HSA may be 10-150 mg/ml, such as 50-100 mg/ml, preferably 75 mg/ml.
In one embodiment, the composition comprises pentetic acid (DTPA). DTPA converts free nuclide into a form that is rapidly excreted via kidneys. The amount of DTPA may be o.1-1.o mg/ml, such as 0.2-0.8 mg/ml, preferably 0.4 mg/ml.
The present disclosure further provides the above-mentioned therapeutic conjugate for use in a method of treating a cancer, typically a cancer overexpressing HER2. Diagnosis of HER2 overexpression is known to the skilled person. In an embodiment, the cancer has developed resistance to a first line HER2-targeting treatment, such as an antibody-based treatment, e.g. trastuzumab, pertuzumab or antibody-drug conjugated trastuzumab-DM1. The cancer type is preferably breast cancer or gastric/gastroesophageal cancer.
The method may comprise intravenous administration of the therapeutic conjugate, e.g. in the pharmaceutical composition described above. The number of injections of the therapeutic conjugate maybe 1-5, such as 1-3. The injections are typically carried out several days apart. A low number of injections is preferred, not only from a cost perspective, but also to minimize the pain and possible side effects of the injections.
Statistical treatment was performed using GraphPad Prism software (version 4.00 for Microsoft Windows; GraphPad Software) to determine significant differences (P<0.05). Data on cellular uptake and processing and biodistribution were analyzed by unpaired 2-tailed t test when 2 groups were compared. Comparison of data for more than 2 groups was performed using ANOVA with a Bonferroni test for multiple comparison. Biodistribution data concerning dual-label studies were analyzed using paired t test.
An ABD variant was selected as the ABR. This variant, referred to as ABD3s (SEQ ID NO:1), is engineered to bind human serum albumin with subpicomolar affinity (Jonsson 2008). Further, ADAPT6 (SEQ ID NO:6) was selected as the HBR because it shows high affinity to HER2. SSSG repeats were selected for the spacer region.
Genes encoding ADAPT6-(SSSG)3-ABD3s (SEQ ID NO:21), ABD3s-(SSSG)3-ADAPT6 (SEQ ID NO:22), ADAPT6-(SSSG)5-ABD3s (SEQ ID NO:23) and ABD3s-(SSSG)5-ADAPT6 (SEQ ID NO:24) were synthesized by Thermo Fisher Scientific (Waltham, Mass., USA) and subcloned into expression vectors through amplification with primers containing the desired restriction sites as well as the N-terminal sequence MGDEAVDANS (SEQ ID NO:18) and the C-terminal sequence GSSC. Proteins were expressed in E. coli BL21*(DE3) cells and extracted by sonication. The proteins were purified by loading the lysates on an in-house produced affinity chromatography column coupled with Human Serum Albumin (HSA).
Proteins intended for177Lu labeling were conjugated with the chelator DOTA (Macrocyclics, TX, USA) through standard maleimide coupling at the free thiol group of the C-terminal cysteine. Both the DOTA-conjugated as well as the unconjugated protein intended for125I labeling, were further purified through semipreparative RP-HPLC (Zorbax, 300SB-C18, 9.4×250 mm, 5 μm, Agilent).
Non-fused ADAPT6 (MGDEAVDANS-ADAPT6-GSSC, SEQ ID NO:26) was used as a control in the biodistribution measurements and was produced and purified like previously described (Garousi 2019).
For the purpose of radionuclide therapy, a non-target binding version of ADAPT was designed, denoted ADAPTNeg (SEQ ID NO:19).
The gene of a non-target-binding fusion protein (MGDEAVDANS-ADAPTNeg-(SSSG)3-ABD035-GSSC (SEQ ID NO:20)) was synthesized by Thermo Fisher Scientific (Waltham, Mass., USA) and produced as described above.
The molecular weights of all proteins were confirmed by liquid chromatography-electro-spray ionization-mass spectrometry (LC-ESI-MS) using an Impact II UHR QqTOF MS (Bruker Daltonics, MA, USA) and the secondary structure and thermal stability was evaluated on a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK) like previously described (Garousi 2019).
Target binding analysis was performed on a Biacore T200 system (GE Healthcare, Stockholm, Sweden) by injecting analytes over a CM5 chip immobilized with Murine Serum Albumin (MSA), Human Serum Albumin (HSA) and Human Epidermal growth factor receptor 2 (HER2) to a response level of -10oo RU each. Experimental parameters have been described previously (Garousi 2019).
Yield of125I-HPEM was measured by radio-TLC using Silica gel 60 F254 TLC plates (20x100 mm, elution path 80 mm; E. Merck, Darmstadt, Germany) eluted with acetonitrile. Radiochemical yield and purities were measured using radio-ITLC (Varian Medical Systems, Palo Alto, Calif., USA). For analysis of radioiodinated proteins, a mixture of acetone:water (7:3) was used for development. In analysis of 177Lu-labeled proteins, 0.2 M citric acid, pH 2.0, was used for development. To cross-validate radio-ITLC data, radio-HPLC analysis was performed. Hitachi Chromaster HPLC systems with radioactivity detector and Vydac RP C18 column (300 A; 3X150 mm; 5-μm) at room temperature was used. Solvent A was o.i % trifluoroacetic acid (TFA) in H2O; solvent B was 0.1 % TFA in acetonitrile. The flow rate was 1 mL/min, with a 5% B to 80% B gradient over 20 min.
Labelling with 125I was performed according to the method described by (Tolmachev 2009). Briefly, 125I (14 MBq) was mixed with a solution of HPEM (10 pg, 31.5 nmol) in 5% acetic acid in methanol. Chloramine-T (10 μL, 8 mg/mL in MQ water) and 10 μL 5% acetic acid in methanol were added, and the mixture was incubated for 5 min at room temperature. The reaction was quenched by adding 10 μL sodium metabisulphite (12 mg/mL in water). ADAPT6 derivatives (500 mg, 38.9 nmol) were reduced by incubation in PBS with 20 mM dithiothreitol (DTT) for 60 min at 40° C. Reduced proteins were purified using size-exclusion NAP-5 column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with NH4OAc 0.2 M buffer, pH 6. Radioiodinated HPEM was added, and the mixture was incubated for 60 min at 40° C. Radiolabeled proteins were purified using NAP-5 columns.
Labeling with 177Lu was performed as previously described (Altai 2017). Briefly, a DOTA-conjugated protein (80 pg, 6.2 nmol) in 1 M ascorbate buffer, pH 5.5, was mixed with 345 MBq 177Lu in 0.1 M HC. The mixture was incubated for 45 min at 95° C. Thereafter, a 5000-fold molar excess of EDTA was added to scavenge a loosely bound nuclide, and the mixture was incubated for 15 min at 95° C. Radiolabeled proteins were purified using NAP-5 columns.
Stability of radioiodine labels was tested by 3-h incubation with 1000-fold molar excess of potassium iodide. Stability of labeling with177Lu was evaluated by 3-h challenge with 5000-fold molar excess of EDTA.
Cell-lines were purchased from American Type Culture Collection (ATCC, Manassas, Va.). The specificity of binding of the radiolabeled ADAPT6 derivatives to HER2-expressing cancer cells was evaluated using ovarian carcinoma SKOV-3 and breast carcinoma BT474 cell lines using a saturation method described earlier (Willberg 2008). The affinity of radiolabeled conjugates binding to living HER2-expressing SKOV-3 cells was determined using LigandTracer (Ridgeview Instruments AB) by an established method as described previously (Tolmachev 2014). Internalization of radiolabeled ADAPT6 derivatives by SKOV-3 and BT474 cells during continuous incubation was evaluated by a modified acid wash method as described earlier (Wllberg 2008).
Animal studies were planned in agreement with Swedish national legislation concerning protection of laboratory animals and were approved by the Ethics Committee for Animal Research in Uppsala.
Biodistribution and targeting properties were evaluated in BALB/C nu/nu mice bearing HER2-positive SKOV-3 xenografts. As a specificity control, HER2-negative Ramos xenografts were used. To establish xenografts, 107cells were implanted subcutaneously.
To reduce the number of tumor-bearing mice, a dual-label approach was used. The mice were injected into tail vein with a mixture of 12I- and177Lu-labeled ADAPT6 variants (15 kBq of12I and 90 kBq of177Lu per animal in 100 mL of PBS). The total injected protein dose was adjusted to 3 μg/mouse for un-fused ADAPT6 and 6 μg/mouse for ABD-fused variants using the corresponding unlabeled protein. After exsanguination under anesthesia, the organ and tissues of interest were excised and their activity was measured using an automated gamma-spectrometer (1480 WIZARD; Wallac Oy). Activity was measured in the energy window from 10 to 45 keV for 125I, and from 90 to 370 keV for177Lu
To evaluate influence of fusion with ABD035, effect of positioning of ABDo35 relative to ADAPT6 moiety and a chemical nature of a label, biodistribution of MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-HPEM-12I, MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-DOTA-177Lu, MG-DEAVDANS-ABD035 -(SSSG)3-ADAPT6-GSSC-HPEM-125I, MG-DEAVDANS-ABD035-(SSSG)3-ADAPT6-GSSC-DOTA-177Lu, MG-DEAVDANS-ADAPT6-GSSC-HPEM-1sI and MG-DEAVDANS-ADAPT6-GSSC-DOTA-177Lu was compared 48 h after injection. Five mice per group was used in this experiment. The average mouse weight was 16.9 * o.6 g, and the average tumor weight 0.42 ±0.6 g.
To evaluate dosimetry of the most promising variants, MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-HPEM-125I and MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-DOTA-177Lu, their biodistribution was measured 4, 24, 48, 72, 168 and 336 h after injection in mice bearing SKOV-3 xenografts (average mouse weight was 16.3 ±1.3 g, and average tumor weight o.11 * 0.06 g). To control that the tumor uptake is HER2-specific, the biodistribution of these conjugates in mice bearing HER2-negative Ramos xenografts (average tumor weight o.11 * 0.06 g) was measured 48 h after injection. Four mice per data point were used in this experiment. Dosimetry was evaluated as described earlier (Westerlund 2019).
To evaluate the therapeutic effect of the ADAPT6 fused to ABD, 107 SKOV-3 cells per mouse were implanted subcutaneously in the abdomen. At least twice a week, the mice were weighed and visually inspected, and tumors were measured using electronic calipers. Tumor volumes (mm3) were calculated as [length (mm)]·[width (mm)]2·0.5. The animals were euthanized when tumors reached a size of 1,000mm3 or became ulcerated, or if an animal's weight dropped by more than 10 % during 1 week or by more than 15% since the study began. After euthanasia, tumors and kidneys were excised for subsequent histologic evaluation.
Treatment started 7 d after tumor implantation, when the average tumor volume was 0.07 * 0.02 mm3 and the average mouse weight was 15.1 * o.4 g. The mice were randomly divided into 5 groups of 11 animals each. The first group of treated animals was injected with 6 μg (4.7nmol)/18 MBq of MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-DOTA-177Lu. Animals in the second treatment group received two treatments, the first at day 7 and the second three weeks after the first one. Control group 1 received vehicle alone, PBS. To evaluate the influence of non-labeled ABD-fused ADAPT6 on tumor growth, control group 2 was injected with 6 μg (4.7 nmol) of unlabeled MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-DOTA. To evaluate the effect of nontargeting radiolabeled protein, a control group 3 was injected with MG-DEAVDANS-ADAwiNthg-(SSSG)3-ABDo3-GSSC-DOTA-177Lu (6 μg/18 MBq) only.
To confirm tumor targeting, SPECT/CT imaging was performed. A mouse was imaged at 72 h as described in (Westerlund 2018).
All proteins used in this study was produced in E. coli and purified to homogeneity. To establish the optimal length of the SSSG repeat linker between the HBR and the ABR, an SPR binding analysis was performed to compare affinities between variants with the (SSSG)3spacer region and the longer (SSSG)5 spacer region. As can be seen in Table 1, the SPR data show no gain of affinity when the length of the spacer region was increased from 12 to 20 amino acid residues. Therefore, the rest of the study was focused on the shorter variants.
The purity of the variants intended for in vivo studies were determined by RP-HPLC to be above 95% (
SPR measurements of the ABD-fused constructs revealed similar affinities towards HER2 (around 5 nM), regardless of the placement of the ABD. This is comparable to the previously determined 4 nM affinity of the unfused control (Garousi 2016). The affinity towards the serum albumins were also similar between all constructs, with measured affinities around 1.8 nM towards MSA and around 50 μM for HSA (
As previously mentioned, a non-target binding fusion protein was designed to be used as a control in the therapy studies. SPR measurements confirm that the three-point mutations efficiently remove the binding to HER2 (
Data concerning radiochemical yields and radiochemical purity are provided in Table 3. All labeling protocols were efficient and purification using NAP-5 provided complete absence of non-conjugated radionuclide. Radiochemical yield of labelling of HPEM with 12I (covalent coupling) was 98%. For experimental therapy, the maximum molar activity up to 55.6 GBq/pmol was obtained for MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-DOTA-177Lu. Identity of radiolabeled ADAPT6 derivative was confirmed by radio-HPLC (
Pre-saturation of HER2 receptors in cells with unlabeled constructs resulted in a significant (p<5×10−5) reduction of binding of all radiolabeled ADAPT6 variants to both SKOV-3 and BT-474 cell lines. Data concerning binding and processing of radiolabeled ADAPT6 derivatives by HER2-expressing cells are presented in
Data concerning affinity of radiolabeled ADAPT6 derivatives to living HER2 expressing cells are presented in Table 4 and
Biodistribution Measurements
Effect of fusion with ABD035 is demonstrated in
Effect on biodistribution, caused by the positioning of ABD035 region relative to the ADAPT6 region as well as by the chemical nature of the label, is shown in
Data concerning biodistribution of MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-HPEM-125I and MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-DOTA-177Lu up to 48 hours after injection are presented in
The biodistribution data (Tables 5 and 6) demonstrate slow clearance of activity from blood. The biological half-lives in blood were 29.4 (95% CI 25.7 to 33.7) and 28.4 (95% CI 24.6 to 32.8) h for MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-HPEM-125I and MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035-GSSC-DOTA-177Lu, respectively. The level of the blood-borne activity was slightly but significantly (p<0.05 in a paired t-test) higher4for the radioodine label at all time points. Both conjugates showed efficient targeting, as already by 24 h, the uptake in HER2-positive tumors exceeded uptake in any other organ or tissues for both variants. Starting from 72 h after injection, the tumor uptake was higher for177Lu label. Overall, the use of177Lu provided significantly higher tumor-to-blood but lower tumor-to-kidney ratios compared to125I label.
The biodistribution data of Tables 5 and 6 can be compared to that presented in the prior art document Liu 2019, in which a cytotoxic polypeptide (PE38X8 or PE25) is coupled to the C terminal end of an ADAPT6-ABD035 fusion protein (see Tables 7 and 8). Such a comparison shows that the conjugates of the present disclosure result in a drastically improved biodistribution. Notably, the fusion toxins of Liu 2019 accumulate in the liver and/or the kidney, which means that a large proportion of the drug fails to target the tumor and that severe and probably unacceptable side effects are expected in the liver and/or the kidney. At 24 h post-injection, the blood to kidney ratio is very low (0.04 and 0.12, respectively) in Liu 2019, but much higher (0.6 and 3.5, respectively) for the inventive conjugates having cytotoxic radionuclides. Similarly, the blood to liver ratio is very low (0.3 and 0.04, respectively) in Liu 2019, but much higher for the inventive conjugates having cytotoxic radionuclides (3.5 and 5.9, respectively) at 24 h post-injection. Notably, at this point in time, Liu 2019 does not show a blood concentration above 1.8% despite having the albumin-binding region ABD035. In contrast, the blood concentration of the conjugates of the present disclosure is 17% and 19%, respectively.
Imaging performed during experimental therapy, 72 hours post injection (
The tumor growth data (
The therapy was well tolerated. The appearance of the skin, fat pads, and eyes did not differ between treated and untreated mice, and there was no behavior indicating pain or suffering. The average animal weight did not differ significantly between the treated groups and the control groups (data not shown). The average weight in the groups treated with MG-DEAVDANS-ADAPT6-(SSSG)3-ABD035 -GSSC-DOTA-177Lu increased between day 28 and day 90, which reflects recovery from the disease and treatment (data not shown).
| Number | Date | Country | Kind |
|---|---|---|---|
| 20161922.8 | Mar 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/055925 | 3/9/2021 | WO |
