Patent Application
20250215093
The present disclosure relates to selective targeting of EGFR expressed on tumor cells.
Overexpression of oncogenic receptors is common in cancer and presents an opportunity for designing tumor-selective drugs. However, in many cases, these receptors are also often abundantly expressed in healthy tissues thus limiting the potential of therapies in terms of safety and efficacy. The epidermal growth factor receptor (EGFR or ErbB-1) is a tyrosine kinase receptor involved in tumorigenesis for many cancers and is often due to overexpression. Therapeutic targeting of this receptor has been marked with challenges of systemic toxicity due to its abundant endogenous expression. Designing drugs that are more active in the tumor microenvironment and less active in circulation and healthy tissues would have the potential to significantly improve toxicity profiles and efficacies, allowing for more aggressive treatment options otherwise limited by systemic toxicity.
Affibody molecules are small (58 amino acid residues, 6.5 kDa) three-helical affinity proteins and promising alternatives to antibody-based drugs because of efficient tissue penetration, high stability, simple modularity of functional domains and ease of production in prokaryotic hosts. Novel binding specificities are generated from large combinatorial libraries by randomization of surface exposed residues on helices 1 and 2 of the affibody molecule (
The present disclosure is based on the development a conditionally activated affibody-based prodrug targeting EGFR. To achieve conditional activation, Staphylococcus carnosus cell-surface display was used to select for an anti-idiotypic affibody molecule masking the binding interface of a preexisting EGFR-targeting affibody molecule (ZEGFR:2377, see: Friedman et al., J. Mol. Biol. 2008, 376 (5), 1388-1402; Tolmachev et al. Eur. J. Nucl. Med. Mol. Imaging 2010, 37(3), 613-622 and WO 2007/065635). ZEGFR:2377 is hereinafter referred to as “ZEGFR”.
A masking domain (“ZB05”) showing high binding propensity in flow cytometry (FC) on the surface of S. carnosus has been isolated. The ZB05 affibody was produced as a soluble monomer and characterized in terms of binding to ZEGFR and thermostability. From kinetic evaluation using SPR, rapid association and dissociation rates were observed, which are favorable for the utility of a masking domain. Additionally, the protein demonstrated high thermostability (Tm 64.1° C.) and refolding capacity following heat denaturation, a common trait seen for monomeric affibody molecules.
The binding contribution of various residues of ZB05 was investigated by mutations. Results from the mutagenesis study revealed flexibility and interchangeability of residues in several randomized positions in terms of retained binding (see
To further investigate the potential of ZB05 as a masking domain, a proof-of-concept pro-affibody (POC-PA) construct was designed in which ZB05 was fused to ZEGFR-ABP using a TEV-cleavable linker (ABP means “albumin-binding protein”). The capacity to mask EGFR-binding was initially analyzed using FC as a displayed protein on staphylococcal cells in the presence of fluorescently labelled soluble EGFR. Binding to EGFR could not be observed for cells displaying the intact protein but after treating the cells with TEV-protease the binding to EGFR was restored.
The construct was produced as a soluble molecule and evaluated for cleavage by TEV protease. After 1 hour incubation with TEV protease, the protein was completely digested showing distinct bands of correct size on an SDS-page gel. Binding to recombinant immobilized EGFR on Surface Plasmon resonance (SPR) was similarly masked by ZB05 for intact POC-PA and restored for cleaved POC-PA. A separate surface immobilized with human serum albumin was used to confirm equal injection amounts for intact and cleaved POC-PA.
Intact and cleaved POC-PA were tested for binding to endogenously expressed EGFR on H292 human mucoepidermoid pulmonary carcinoma cells and A431 human squamous carcinoma cells expressing moderate to high levels of EGFR respectively. Binding relative to cells alone could be observed not only for cleaved POC-PA, but also for intact POC-PA. Nonetheless, cleaved POC-PA increased the signal for both cell lines and was comparable to the construct POC-PA-DM where the ZB05 had been exchanged for a dummy masking domain without specificity for ZEGFR.
The capacity of an [111In]In-labeled prodrug along with a variant thereof having a non-cleavable linker (“dummy-linker”) and a non-masked control to target EGFR-expressing matriptase-positive cancer cells in vivo without binding to EGFR-expressing hepatocytes was evaluated using a radioactive label. For labelling using 111In, a maleimido derivative of DOTA-chelator was conjugated to C-termini of all constructs. This site-specific approach ensures that the radioactive label reflects the distribution of ZEGFR-ABD035-fusion. Additionally, the site-specific labelling provides a homogenously labelled protein, not a mixture of proteins with different numbers of conjugated chelators in different positions. All radiolabeled constructs had a high radiochemical purity and demonstrated excellent stability. The in vitro tests demonstrated that the binding of [111In]In-labeled non-masked control to both cell lines is significantly reduced by saturation of receptors using both non-labelled ZEGFR-ABD035-fusion and cetuximab, which shows that the binding was specific. In vitro binding of [111In]In-labeled prodrug and dummy-linker was much lower and predominantly unspecific. This suggests that the incorporation of anti-idiotypic masking domain efficiently prevents binding of these constructs to EGFR in vitro.
Initial in vivo evaluation of the [111In]In-labeled prodrug, the dummy-linker and the non-masked control demonstrated that concentration of all these proteins in blood was much higher that the concentration of non-ABD035-fused 111In-ZEGFR and the renal uptake was much lower. This phenomenon demonstrates that there is a binding of the ABD035 to the murine albumin in vivo. This prevents glomerular filtration of the constructs and their reabsorption in proximal tubuli. Accordingly, the bioavailability of the constructs is higher and potential renal toxicity is lower compared to targeted constructs based on non-ABD035-fused ZEGFR. The most important observation from this experiment concerns the hepatic uptake. The uptake of non-masked control in liver was high, 17.2±1 and 12.7±1.8% ID/g at 4 and 24 h after injection, respectively. This is expected because of a noticeable expression of EGFR on hepatocytes and high affinity of ZEGFR to murine EGFR. The prodrug and the dummy-linker had much lower hepatic uptake, which shows that the incorporation of the masking domain served its purpose.
Data from the experiment in mice bearing EGFR-expressing H292 xenografts confirmed extended residence time in circulation of all constructs and low hepatic uptake of the [111In]In-labeled prodrug and the dummy-linker. Importantly, the uptake of the [111In]In-Prodrug in H292 xenografts was significantly higher than in EGFR-negative Ramos xenografts. This was also confirmed in the imaging experiment. This demonstrates that uptake in H292 xenografts was EGFR-specific. Another interesting finding is that the tumor uptake was equally high for the prodrug and the dummy-linker.
To elucidate the role of matriptases in the tumor uptake of the [111In]In-labeled prodrug, an additional experiment was performed. The tumor uptake was compared in mice bearing H292 and A431 xenografts simultaneously. The results of this experiment demonstrated that the uptake in H292 xenografts with high matriptase expression was two times higher than in A431 xenografts with low matriptase expression. At the same time, the EGFR expression per cell is three-fold higher in A431 cells than H292 cells. Taken together, these data suggests that matriptases play a role in the tumor uptake of the [111In]In-labeled prodrug. At the same time, the uptake in A431 xenograft was higher than in EGFR-negative Ramos xenografts, which suggests that a matriptase-independent mechanism is also in play.
In conclusion, the data show that the masked EGFR binders have a significantly lower liver uptake than a non-masked version and, interestingly, that tumor uptake of the masked EGFR binders was on the same level as that of the non-masked version independent of a protease-cleavable linker. The masking domain thus improves the tumor to liver ratio significantly.
Accordingly, the following itemized listing of embodiments of the present disclosure is provided.
1. A fusion protein comprising an EGFR-binding domain, a masking domain and a linker linking the masking domain to the EGFR-binding domain, wherein:
2. The fusion protein of item 1, wherein, in the masking domain, no more than four of X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 are substitutions and/or no more than one of X19-X22 is absent.
3. The fusion protein of item 2, wherein, in the masking domain, no more than two of X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 are substitutions and/or none of X19-X22 is absent.
4. The fusion protein of any one of the preceding items, wherein the masking domain comprises the amino acid sequence
5. The fusion protein of item 4, wherein the masking domain comprises the amino acid sequence
6. The fusion protein of item 5, wherein, in the masking domain, no more than two of X1-X8 are substitutions.
7. The fusion protein of any one of the preceding items, wherein the masking domain comprises the amino acid sequence
8. The fusion protein of any one of the preceding items, wherein, in the masking domain, no more than two of X36-X44 are substitutions.
9. The fusion protein item 7 or 8, wherein the masking domain comprises the amino acid sequence
10. The fusion protein of item 9, wherein, in the masking domain, no more than seven of X36-X54 are substitutions.
11. The fusion protein of item 10, wherein, in the masking domain, no more than five of X36-X54 are substitutions.
12. The fusion protein of any one of items 9-11, wherein the masking domain comprises the amino acid sequence
13. The fusion protein of item 12, wherein, in the masking domain, no more than seven of X36-X58 are substitutions.
14. The fusion protein of item 13, wherein, in the masking domain, no more than five of X36-X58 are substitutions.
15. The fusion protein of any one of the preceding items, wherein, in the masking domain, X10 is R.
16. The fusion protein of any one of the preceding items, wherein, in the masking domain, X13 is absent.
17. The fusion protein of any one of the preceding items, wherein, in the masking domain, X14 is T.
18. The fusion protein of any one of the preceding items, wherein, in the masking domain, X24 is A.
19. The fusion protein of any one of the preceding items, wherein, in the masking domain, X25 is D.
20. The fusion protein of any one of the preceding items, wherein, in the masking domain, X28 is W.
21. The fusion protein of any one of the preceding items, wherein, in the masking domain, X31 is I.
22. The fusion protein of any one of the preceding items, wherein the masking domain comprises the amino acid sequence
23. The fusion protein of any one of the preceding items, wherein the masking domain comprises the amino acid sequence IRSATEIWWLPNLTADQKWAFIYKLV (SE ID NO:1).
24. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X3 is W.
25. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X6 is V or W.
26. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X10 is R or G.
27. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X10 is W or G.
28. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X18 is W or G, preferably W.
29. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X21 is T or D, preferably T.
30. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises the amino acid sequence
31. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X2 is M.
32. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X4 is I, V, G or S.
33. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X11 is D, N or E.
34. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X20 is M.
35. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X25 is A or S.
36. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X28 is L.
37. The fusion protein of any one of the preceding items, wherein, in the EGFR-binding domain, X2 is M, X20 is M and X28 is L.
38. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises the amino acid sequence
39. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises the amino acid sequence
40. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises the amino acid sequence
41. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises the amino acid sequence
42. The fusion protein of any one of the preceding items further comprising a half-life-extending region, such as an Fc-binding region or an albumin-binding region (ABR).
43. The fusion protein of any one of the preceding items further comprising an albumin-binding region (ABR), which ABR comprises an amino acid sequence selected from
44. The fusion protein of item 43, wherein the ABR comprises the amino acid sequence
45. The therapeutic conjugate according to item 43, wherein the ABR comprises an amino acid sequence selected from the group consisting of:
46. The fusion protein of any one of items 42-45, wherein the half-life-extending region is located on the C-terminal side of the EGFR-binding domain.
47. The fusion protein of any one of the preceding items, wherein the linker is a protease-cleavable linker.
48. The fusion protein of item 47, wherein the protease-cleavable linker comprises a sequence selected from the group consisting of GFLG (SEQ ID NO:11), Glutamic acid-Valine-Citrulline, GILGVP (SEQ ID NO:13), GPLGIAGQ (SEQ ID NO:14), VHMPLGFLGP (SEQ ID NO:15), SGGPGPAGMKGLPGS (SEQ ID NO: 16), PLGLAG (SEQ ID NO:17), LALGPG (SEQ ID NO:18), KRALGLPG (SEQ ID NO: 19), GGGRR (SEQ ID NO:20), LSGRSDNH (SEQ ID NO:21), PMAKK (SEQ ID NO:22), RQARVVNG (SEQ ID NO:23), MSGRSANA (SEQ ID NO:38), HSSKLQL (SEQ ID NO:24) and RRSSYYSG (SEQ ID NO:25).
49. The fusion protein of any one of the preceding items, wherein the length of the linker is at least 12 amino acid residues, such as 12-60 amino acid residues, such as 20-50 amino acid residues.
50. The fusion protein of any one of the preceding items, wherein the linker comprises no cysteine (C) residue.
51. The fusion protein of any one of the preceding items, wherein the masking domain comprises no cysteine (C) residue.
52. The fusion protein of any one of the preceding items, wherein the EGFR-binding domain comprises no cysteine (C) residue.
53. The fusion protein of any one of the preceding items, wherein masking domain is located on the N-terminal side of the EGFR-binding domain.
54. A therapeutic conjugate comprising the fusion protein of any one of the preceding items and a cytotoxic agent, such as a cytotoxic molecule, peptide, protein or radionuclide.
55. The therapeutic conjugate according to item 54, wherein the cytotoxic agent is a cytotoxic radionuclide 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.
56. The therapeutic conjugate according to item 54, wherein the cytotoxic agent is a cytotoxic molecule selected from the group consisting of Doxorubicin (DOX), Duocarmycins (DUO), Docetaxel (DTX), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Paclitaxel (FTX), Mertansine (DM1), emtansine (DM1), Ravtansine (DM4), Soravtansine (DM4), Pyrrolobenzodiazepine (PBD) and Calicheamicin.
57. The therapeutic conjugate according to item 54, wherein the cytotoxic agent is a protein-based toxin selected from the group consisting of Pseudomonas exotoxin (PE), Diphtheria toxin (DT), Ricin toxin A-chain (RTA) and deBouganin.
58. A therapeutic conjugate according to any one of items 54-57 for use in a therapeutic method of treatment.
59. The therapeutic conjugate for use according to item 58, wherein the therapeutic method of treatment is a method of treatment of a subject suffering from a cancer, such as a cancer overexpressing EGFR.
60. The therapeutic conjugate for use according to item 59, wherein the cancer is selected from the group consisting of lung cancer, preferably non-small cell lung cancer, prostate cancer, breast cancer, colon and rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervix cancer, ovary cancer, bladder cancer, kidney cancer and pancreatic cancer.
As a first aspect of the present disclosure, there is provided a fusion protein comprising an EGFR-binding domain, a masking domain and a linker linking the masking domain to the EGFR-binding domain.
The masking domain binds to the EGFR-binding domain and thereby restricts binding to EGFR under certain conditions.
The masking domain comprises the amino acid sequence
The amino acid residues of positions X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 are not believed to form part of the binding site actively interacting with the EGFR-binding domain. Hence substitutions and in some cases (X19-X22) even deletions (as compared to ZB05) are allowed in these positions. However, the three-dimensional structure is believed to be tweaked to such a degree that the binding capacity is lost if all or most of them are substituted or (in case of X19-X22) deleted. Hence no more than six of X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 can be substitutions and no more than two of X19-X22can be absent in the fusion protein of the first aspect.
In one embodiment of the masking domain, no more than four of X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 are substitutions. Preferably, no more than two of X12, X15, X16, X19, X20, X21, X22, X23, X26, X29, X30, X33 and X34 are substitutions.
In an alternative or complementary embodiment, no more than one of X19-X22 is absent. Preferably, none of X19-X22 is absent.
In one embodiment, the masking domain comprises further amino acid residues (X5-X8) at the N-terminus. Hence the masking domain may comprise the amino acid sequence
In one embodiment, the masking domain comprises still further amino acid residues (X1-X4) at the N-terminus. Hence the masking domain may comprise the amino acid sequence
In one embodiment, the masking domain comprises further amino acid residues (X36-X44) at the C-terminus. Hence the masking domain may comprise the amino acid sequence
In one embodiment, the masking domain comprises still further amino acid residues (X45-X54) at the C-terminus. Hence the masking domain may comprise the amino acid sequence
In one embodiment of the masking domain, no more than seven of, such as no more than five of, X36-X54 are substitutions.
The masking domain may be further extended another four (X55-X58) amino acid residues at the C-terminus. Hence the masking domain may comprise the amino acid sequence
In one embodiment of the masking domain, no more than seven of, such as no more than five of, X36-X58 are substitutions.
In a preferred embodiment of the masking domain:
Accordingly, the masking domain may comprise the amino acid sequence
In one embodiment, the masking domain comprises no cysteine (C) residue.
In one embodiment, the masking domain, just like ZB05, comprises the amino acid sequence IRSATEIWWLPNLTADQKWAFIYKLV (SEQ ID NO:1).
In one embodiment, the masking domain comprises the amino acid sequence VDAKYAKEIRSATEIWWLPNLTADQKWAFIYKLVDDPSQSSELLSEAKKLNDSQAPK (SEQ ID NO:26), which is the sequence of ZB05.
The EGFR-binding domain of the fusion protein of the first aspect comprises the amino acid sequence
The rationale behind this amino acid sequence (and the embodiments thereof presented below) is explained in WO 2007/065635, which is incorporated herein by reference.
In a preferred embodiment of the EGFR-binding domain:
In one embodiment, the EGFR-binding domain comprises the amino acid sequence EX2WX4AWX7EIRX11LPNLNGWQX20TAFIX25SLX28D,
In a preferred embodiment of the EGFR-binding domain, X2 is M, X20 is M and X28 is L.
In a particularly preferred embodiment, the EGFR-binding domain comprises an amino acid sequence selected from:
Optionally there are additional N-terminal amino acid residues such that the EGFR-binding domain comprises an amino acid sequence selected from:
Optionally there are additional C-terminal amino acid residues such that the EGFR-binding domain comprises an amino acid sequence selected from:
In one embodiment, there are additional amino acid residues at the N-terminus and the C-terminus such that the EGFR-binding domain comprises an amino acid sequence selected from:
Sequence (vii) is the sequence of the EGFR-binding affibody used in the Examples section below (also referred to as ZEGFR).
In one embodiment, the EGFR-binding domain comprises no cysteine (C) residue.
In one embodiment of the fusion protein of the first aspect, the masking domain is located on the N-terminal side of the EGFR-binding domain. In this embodiment, the linker is thus linking the C-terminus of the masking domain to the N-terminus of the EGFR-binding domain.
In one embodiment of the fusion protein of the first aspect, the linker is a protease-cleavable linker. Preferably, such a linker is cleavable by one or more of the following proteases: Cathepsin B; MMP-2; MMP-9; MMP-7; Urokinase-type plasminogen activator; Matriptase; and Prostate-specific antigen (PSA). These proteases are found in tumor microenvironments.
The above-mentioned proteases recognize the cleaving sites listed below
Consequently, an embodiment of the protease-cleavable linker comprises at least one of these cleaving site sequences listed above.
To allow the masking sequence and the EGFR-binding sequence to bind to each other, the length of the linker is typically at least 12 amino acid residues, such as at least 20 amino acid residues. The maximum length may for example be 50 or 60 amino acid residues.
In one embodiment, the linker comprises no cysteine (C) residue.
The fusion protein of the first aspect may further comprise a half-life-extending region, such as an Fc-binding region or an albumin-binding region (ABR). The half-life-extending region may for example be located on the C-terminal side of the EGFR-binding domain.
Alternatively, a half-life-extending group is not part by the fusion protein, but connected to the fusion protein in another way. In this alternative embodiment, the fusion protein and the half-life-extending group together forms a construct that may comprise further parts or groups.
Various half-life-extending strategies for proteins are described in a review article by Kontermann (EXPERT OPINION ON BIOLOGICAL THERAPY, 2016 VOL. 16, NO. 7, 903-915).
In one embodiment, the fusion protein of the first aspect comprises an ABR comprising an amino acid sequence selected from
The rationale behind this ABR sequence (and the embodiments thereof described below) is explained in WO 2021/180727.
The ABR preferably comprises the amino acid sequence LAX3AKX6X7AX9X10 ELDX14YGVSDX20 YKX23LIX26X27AKTVEGVX35ALX38X39X40 ILAALP, wherein, independently of each other,
One embodiment of the ABR comprises no cysteine (C) residue.
In one embodiment, the ABR comprises an amino acid sequence selected from the group consisting of:
As a second aspect of the present disclosure, there is provided a therapeutic conjugate comprising the fusion protein of the first aspect and a cytotoxic agent, such as a cytotoxic molecule, peptide, protein or radionuclide. In case of a cytotoxic peptide or protein, the whole therapeutic conjugate may be a fusion protein.
The cytotoxic molecule may for example be Doxorubicin (DOX), Duocarmycins (DUO), Docetaxel (DTX), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Paclitaxel (FTX), Mertansine (DM1), emtansine (DM1), Ravtansine (DM4), Soravtansine (DM4), Pyrrolobenzodiazepine (PBD) or Calicheamicin.
In one embodiment, the cytotoxic molecule is DM1, MMAE, MMAF or DM4 (see Tarcsa et al. Drug Discovery Today: Technologies; Volume 37, December 2020, Pages 13-22 and the review by Khongorzul et al. Mol Cancer Res; 18(1) January 2020).
Examples of cytotoxic proteins are Pseudomonas exotoxin (PE) and engineered variants thereof, including PE38, diphtheria toxin (DT) and deBouganin (see Antignani et al, Biomolecules; 2020 Sep. 17; 10(9):1331).
Other examples of cytotoxic proteins are targeting domains against immunomodulatory targets such as CD3, CD47, PD-1, PD-Li, CTLA-4, 4-1BB and OX40 (see the review by Blanco et al., Clin Cancer Res 2021 Oct. 15; 27(20):5457-5464).
The cytotoxic radionuclide may for example be selected from the group consisting of 177Lu, 90Y, 188Re; 186Re; 166Ho, 153Sm, 67Cu, 64Cu, 149b, 161Tb, 47Sc; 25Ac; 212Pb; 213Bi, 212Bi, 227Th, 223Ra; 58mCo, 131I, 76As, 77As and 211At.
Preferred cytotoxic radionuclides are 177Lu 90Y and 188Re (see the review by Rondon et al., Cancers (Basel); 2021 Nov. 7; 13(21):5570).
177Lu, 90Y, 188Re; 186Re; 166Ho, 153Sm, 67Cu, 64Cu, 1497b, 161Tb, 47Sc; 225Ac; 212Pb; 213Bi, 212Bi, 227Th, 223Ra and 58mCo are radiometals that may be 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.
For 67Cu 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, this 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 EEEC (SEQ ID NO:33) or GSSC (SEQ ID NO:34).
Adoptive transfer of immune cells (e.g. T cells, NK cells and macrophages) expressing engineered chimeric antigen receptors (CARs), has recently shown enormous potential for cancer treatment in humans. Adoptive transfer of CD19-directed CAR T cells has generated complete and durable remissions in patients with refractory and relapsed B cell malignancies. The extracellular portion of a CAR comprises an affinity protein directed against a tumor antigen (TA). The affinity protein is typically fused to a transmembrane domain, and intracellular stimulatory domains. Upon binding of the cells to the TA on the cancer cells, endogenous downstream signaling molecules are recruited to transduce signaling, leading to T-cell activation and cancer cell killing. However, in spite of the extraordinary responses observed for B cell malignancies, the potent cell killing and the very long serum circulation time of engineered T cells in the patients (probably life-long), may result in challenges with controlling toxicity in healthy organs and long-term side effects. Accordingly, the treatment is predominantly used for cancers with favorable TA expression profiles. Substantial efforts are therefore focused on strategies for controlling the activity of CAR T cells (see also the review by Krug et al., Cancers (Basel) 2021 Dec. 30; 14(1):183).
The fusion protein of the first aspect may be used in protease-activated CAR immune cells. Such CAR immune cells have the potential to preferentially be active in the protease-containing tumors. Alternatively, the corresponding protease could be co-administered along with the transfusion of CAR immune cells for activation of the cells during a defined time window. After completed treatment, no protease will be available in the patient and the cells will remain inactive. Both strategies have potential to reduce toxicity and long term-side effects and make the treatment available for more cancer forms.
In a variant of the second aspect, a fusion protein of the first aspect is thus expressed on the surface of a cell for use in cell therapy. It follows from the discussion above that the cell may be a T cell, an NK cell or a macrophage, in particular a T cell or an NK cell.
As a third aspect of the present disclosure, there is provided the therapeutic conjugate of the second aspect for use in a therapeutic method of treatment.
The therapeutic method of treatment typically is a method of treatment of a subject suffering from a cancer, such as a cancer overexpressing EGFR. Examples of cancers overexpressing EGFR are lung cancer (especially non-small cell lung cancer), prostate cancer, breast cancer, colon and rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervix cancer, ovary cancer, bladder cancer, kidney cancer and pancreatic cancer.
In one embodiment, the method of treatment comprises a diagnostic step of quantifying the degree of EGFR expression in the tumor and administration of the therapeutic conjugate in case of overexpression of EGFR in the tumor.
The quantification of the degree of EGFR expression may for example by based on imaging, e.g. using an imaging agent comprising the fusion protein of the first aspect. In such an imaging agent, the fusion protein may be coupled to a radionuclide suitable for imaging. When used for imaging, the fusion protein typically comprises no half-life-extending region.
Subsequent to the administration of the imaging agent comprising the radionuclide, the patient is scanned to detect, visualize and/or quantify EGFR expression. The scanning is typically a tomography, preferably positron emission tomography (PET) or single-photon emission computed tomography (SPECT). For the latter, a CZT-based camera technology may be used.
In an embodiment, the radionuclide suitable for imaging is selected from the group consisting of 18F, 14I, 76Br, 68Ga, 44Sc, 61Cu, 64Cu, 89Zr, 55Co, 41Ti, 66Ga, 86Y, 110mIn, 123I, 131I, 99mTc, 111In and 67Ga. A preferred group consists of 18F, 68Ga, 99mTc and 111In. Another preferred group consists of 18F, 68Ga and 111In.
For radiolabelling with 18F, a prosthetic group (forming a covalent bond to 18F) may be coupled to the fusion protein. Examples of resulting structures are N-(2-(4-[18F]-fluorobenzamido) ethyl) maleimido ([18F]FBEM), 4-[18F]-fluorobenzaldehyde ([18F]-FBA) and [18F]-fluorophenyloxadiazole methylsulfone ([18F]-FPOS. Another option is [18F]aluminium monofluoride in combination with a triaza chelator.
Also in case of radiolabeling with 123I, 124I, 131I and 76Br, a prosthetic group may be used. Examples of resulting structures are iodo-/bromo-benzoate and iodo-/bromo-hydroxyphenylethyl mealeimide.
For radiolabeling with 68Ga, 67Ga, 66Ga, 44Sc, 55Co, 41Ti, 86Y, 110mIn and 111In, it is preferred to couple a chelator to the HBP. Examples of chelators are DOTA, NOTA, NODAGA and DOTAGA and their derivatives.
For 61Cu and 64Cu, a cross-bridged chelator, such as CB-TE2A, is a better option.
For radiolabelling with 99mTc, a variety of chelators can be used, such as hexahistidine (H6) and chelators based on a cysteine- or mercaptoacetyl-containing peptide.
In case of 18F, 124I, 76Br, 68Ga, 44Sc, 61Cu, 64Cu, 89Zr, 55Co, 41Ti, 66Ga, 86Y or 110mIn, the scanning technique is preferably PET.
In case of 123I, 131I, 99mTc, 111In or 67Ga, the scanning technique preferably comprises SPECT, e.g. using a CZT-based camera.
The radionuclide is preferably coupled to a terminal end of the fusion protein, such as the C-terminal end of the fusion protein.
In an embodiment, the fusion protein comprises an extension that forms a chelator for the radionuclide. As an example, the chelator-forming part may comprise the sequence HHHHHH (SEQ ID NO:35), which can bind 99mTc. An alternative to HHHHHH is HEHEHE (SEQ ID NO:30).
As a fourth aspect of the present disclosure, there is provided a method of treatment of a subject suffering from cancer comprising the step of administration of the therapeutic conjugate of the second aspect.
As a fifth aspect of the present disclosure, there is provided a pharmaceutical composition comprising the therapeutic conjugate of the second aspect and a pharmaceutically acceptable carrier.
The composition may for example be adapted for intravenous administration. Accordingly, the composition may be 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.
S. carnosus cells were grown O/N in B2 medium (i % casein hydrolysate, 2.5% yeast extract, 0.5% D-glucose, 2.5% NaCl, 0.08% K2HPO4, pH=7.4) supplemented with 10 μg/mL Chloramphenicol. A combinatorial S. carnosus library expressing 109 different Affibodies variants on the surface was used for the isolation of binders.
ZEGFR-His6-Cys was purified by IMAC and biotinylated using EZ-Link™ Maleimide-PEG2-Biotin (Thermo Scientific), according to manufacturer's recommendations. EGFR (His-tag) (Sino Biological Inc., China) was biotinylated using Biotin-XX Microscale Protein Labelling Kit, (Invitrogen, USA) according to manufacturer's recommendations. Absorbance at 280 nm was used to determine the protein concentrations.
Streptavidin-coated Dynabeads (Invitrogen, USA) (500 μL) were washed twice with 800 μL of PBS-P (phosphate-buffered saline supplemented with 0.1% Pluronic® F108 NF Surfactant, pH 7.4; BASF Corporation, USA) and incubated with 150 nM of biotinylated ZEGFR for 1 h at RT with gentle rotation. Afterwards, the ZEGFR-coated magnetic beads were washed with PBS-P supplemented with 2 mM EDTA (PBSP-E). A number of cells corresponding to a ten-fold library coverage (1010 cells) were washed with PBSP-E and the pellet resuspended in PBSP-E to a final concentration of 5·108 cells/mL. The cells were incubated with 1.25 mg of ZEGFR-coated magnetic beads for 1 h at RT with gentle rotation followed by 5 minutes on ice. The tubes were placed on a magnetic rack for 4 minutes to capture the beads and the supernatant was removed. The beads were then resuspended in 30 mL of ice-cold PBS-P. Bead capture and washing was repeated three times. Lastly, the cells were resuspended in 50 mL of B2 media supplemented with 10 μg/mL Chloramphenicol. The cultures were incubated O/N at 37° C. and 150 rpm. Serial dilutions of samples taken before and after the magnetic sorting were used to calculate the population size. Finally, the libraries were incubated with 225 nM Alexa Fluor 647-HSA conjugate and 33.3 nM streptavidin R-phycoerythrin conjugate (SAPE) in PBS-P. The cells were washed twice with ice-cold PBS-P and resuspended in 300 μL of ice-cold PBS-P. The cells were analyzed using a Gallios™ flow cytometer (Beckman Coulter, CA, USA) and the laser protocol used for the detection was FL6-660/20 nm for detection of Alexa Fluor 647-HSA, and FL2-575/20 nm for detection of SAPE.
The output cells from the MACS selection (library size≈105 cells) were subsequently sorted using Fluorescence-activated cell sorting (FACS). A 100-fold coverage of the new library size was used for the O/N culture. Cells were washed twice with ice-cold PBS-P and mixed with biotinylated ZEGFR suspended in PBS-P at a concentration of 150 nM. The samples were incubated for 1 h at RT with gentle rotation. Afterwards, the cells were washed twice using ice-cold PBS-P. In order to visualize and sort the cell library by FACS, the cells were incubated with 225 nM Alexa Fluor 647-HSA conjugate and 33.3 nM SAPE on PBS-P. Finally, the cells were washed twice with ice-cold PBS-P and resuspended in 300 μL of ice-cold PBS-P. The cells were subsequently sorted using an Astrios FC cell sorter (Beckman Coulter, USA). The laser protocol used for the detection was 561-585/40 height-log for detection of SAPE, and 640-671/30 height-log for detection of Alexa Fluor 647-HSA. A total of 106 cells were sorted into 1.5 mL B2 media and incubated for 1 h at 37° C. with gentle rotation. Afterwards, the cells were mixed with B2 media supplemented with 10 μg/mL Chloramphenicol to a final volume of 3 mL and incubated at 37° C. O/N with gentle rotation. Finally, the libraries were studied by FC and aliquoted as glycerol stocks to be stored at −80° C. for further experiments.
Individual affibody candidates were selected by plating the library on Agar plates supplemented with 10 μg/mL Chloramphenicol and growing single colonies O/N on TSB-Y media (Merck, Germany) supplemented with 10 μg/mL Chloramphenicol. The cells were analysed by FC and sent for sequencing (Microsynth Seqlab, Germany). The coding sequence for the selected candidate Affibody was cloned by restriction cloning into a pETb26+ bacterial expression vector (Addgene, USA). Escherichia coli BL21* cells were transformed with the previously cloned plasmid by standard heat-shock treatment. A single colony from the transformation was inoculated and grown O/N in 10 mL of TSB-Y media supplemented with 50 ng/mL of Kanamycin. After 16 h, the O/N culture was diluted 1:100 in TSB-Y supplemented with 50 ng/mL of Kanamycin to a final volume of 100 mL. The culture was induced with IPTG to a final concentration of 1 mM at OD600≈1 and incubated O/N at 27° C. and 200 rpm. After 16 h, the cells were harvested by centrifugation for 8 min at 5000×g and stored at −20° C. The cells were lysed by sonication for 1.5 min (1s ON/1s OFF) followed by centrifugation at 4° C. and 10000×g for 20 min. The supernatant was filtered (0.45 μm) and the protein of interest purified using immobilized metal ion affinity chromatography (IMAC). For this purpose, PD-10 columns packed with 3 ml TALON Metal Affinity Resin. Wash buffer (50 mM Na2HPO4, 500 mM NaCl, 15 mM imidazole, pH 8) and elution buffer (50 mM Na2HPO4, 500 mM NaCl, 300 mM imidazole, pH 8) were used according to manufacturer's recommendations (GE Healthcare, Sweden). Finally, the buffer was changed to PBS using PD-10 desalting columns according to manufacturer's recommendations (GE Healthcare, Sweden). Purified proteins were analyzed by mass spectrometry (MS) (4800 MALDI TOF/TOF, Applied Biosystems, USA) and SDS-page gels (NuPAGE, Invitrogen, USA).
Evaluation of Secondary Structure. Thermostability and Refolding Capacity of ZB05 Using Circular Dichroism
Circular dichroism spectroscopy was performed using a Chirascan spectropolarimeter (Applied Photophysics, UK) with an optical path length of 1 mm to analyse the alpha-helical content, thermal stability, and refolding capacity of the constructs at a concentration of 0.4 mg/mL. The thermal stability was evaluated by measuring the change in ellipticity at 221 nm during heating (5° C./min) from 20 to 95° C. The melting temperatures (Tm) were approximated from the data acquired from variable temperature measurements (VTM) by curve fitting using a Boltzmann Sigmoidal model (GraphPad Prism version 7, USA). The refolding capacity was assessed by comparing spectra obtained from measurements at wavelengths in the range of 195-260 nm at 20° C., before and after thermal denaturation.
Target binding was measured for soluble ZB05 Affibody masking candidate and POC-PA molecules using a Biacore 8K SPR instrument (GE Healthcare, Sweden). Approximately 676 RU of ZEGFR and 1000 RU HSA were immobilized by amine coupling on a dextran CM-5 sensor chip according to manufacturer's recommendations (GE Healthcare, Sweden). PBS-T (phosphate-buffered saline supplemented with 0.05% Tween20, pH 7.4) was used as running buffer. The analytes ZB05 and ZEGFR were injected at 5 different concentrations (200, 100, 50, 25 and 12.5 mM) for 150 s followed by dissociation for 150 sec. Intact and cleaved POC-PA were injected at a concentration of 100 nM for 150 s followed by dissociation for 150 sec. The experiments were performed at 25° C. with a flow rate of 100 μL/min. The chips were regenerated by injection of 10 mM HCl for 30 s. The binding kinetics were analysed by the Biacore evaluation software using a Langmuir 1:1 kinetic model.
Each randomized position in the ZB05 sequence were individually mutated to each amino acid except for cysteine, producing a total of 253 sequences. Oligos for each sequence were synthesised and pooled (Twist Bioscience, USA). The pooled oligos were cloned into the S. carnosus display vector pSC2 using NEB Builder (New England Biolabs, USA). The resulting plasmids were transformed into Stellar™ Electrocompetent Cells (Takara Bio, Japan) for amplification and extracted using Qiagen Maxi prep kit (Qiagen, USA). The amplified vectors were subsequently transformed into electrocompetent S. carnosus TM300 cells according to a standard electroporation protocol.
S. carnosus cells were grown O/N in B2 medium supplemented with 10 μg/mL Chloramphenicol. After 16 h, the cells were washed twice with PBS-P and mixed with biotinylated ZEGFR resuspended in PBS-P at a concentration of 150 nM. The samples were incubated for 1 h at RT with gentle rotation. Afterwards, the cells were washed twice using ice-cold PBS-P. To visualize and sort the library by FACS, the cells were incubated with 225 nM Alexa Fluor 647-HSA conjugate and 33.3 nM SAPE in PBS-P. Finally, the cells were washed twice with ice-cold PBS-P and resuspended in 300 μL of ice-cold PBS-P. The cells were subsequently sorted for binding and for non-binding using an Astrios FC cell sorter (Beckman Coulter, USA). The laser protocol used for the detection was 561-585/40 height-log for detection of SAPE, and 640-671/30 height-log for detection of Alexa Fluor 647-HSA. A total of 106 cells per population were sorted into 1.5 mL B2 medium and incubated for 1 h at 37° C. with gentle rotation. Afterwards, the cells were mixed with B2 medium supplemented with 10 μg/mL Chloramphenicol to a final volume of 3 mL and incubated at 37° C. O/N with gentle rotation. Finally, the sorted binding and non-binding populations as well as the naïve library were studied using FC and used for next-generation sequencing.
Deep Sequencing of Libraries from Mutagenesis Study
S. carnosus cells were grown O/N in B2 medium supplemented with 10 μg/mL Chloramphenicol. The Qiagen Miniprep kit was used to purify plasmids from each library populations according to manufacturer instructions (Qiagen, USA). The samples were prepared for deep sequencing by PCR amplifying the plasmids with primers containing the TrueSeq adapters and specific index (Illumina, USA). The sequencing was performed at Scilifelab (National Genomics Infrastructure, Sweden) using a MiSeq 300 cycle instrument (Illumina, USA). The output FASTQ files were analyzed by Geneious version 2020.1.1 (Geneious, New Zealand). Binding and non-binding populations of mutated ZB05 variants as well as the naïve mutagenesis library were sequenced using NGS. The data was normalized to the prevalence of amino acids in each position of the naïve library. Allowed substitutions that retained binding to ZEGFR was determined from a twofold enrichment in the binding population compared to the naïve library with at least a 50% depletion for the corresponding variant in the non-binding population.
POC-PA Sub-Cloning into the Staphylococcal Display Vector
The gene encoding for the EGFR-binding Affibody molecule ZEGFR and the anti-idiotypic Affibody molecule ZB05 were cloned by restriction cloning into a pSC2 vector separated by a TEV protease substrate sequence and linked to an albumin binding protein (ABP). The TEV protease substrate consisted of the sequence ENLYFQG (SEQ ID NO:36) flanked by G4S repeats in order to extend the length of the linker (the exact sequence of the linker including the TEV protease substrate is shown in
The full amino acid of the POC-PA prodrug is shown in
POC-PA Activation by TEV Protease on S. carnosus Cell Surface
S. carnosus cells displaying the different prodrug constructs were grown O/N following the standard protocol previously described. Approximately 107 bacterial cells (10 μL O/N culture) were washed twice with 800 μL 1×PBS-P and pelleted by centrifugation for 6 min at 4° C. and 6000 rpm. The cells were resuspended either in assay buffer (50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, pH 8) supplemented with 5 Units of TEV protease or in assay buffer only (controls) and incubated for 1 h at 30° C. Bacterial cells were washed three times with PBS-P and incubated in 100 μL of PBS-P supplemented with 50 nM biotinylated EGFR receptor or just PBS-P during 45 min at 37° C. with gentle rotation. Finally, the cells were washed twice with PBS-P and incubated with Alexa Fluor 647-HSA and streptavidin R-phycoerythrin conjugate to be analysed by FC.
The different prodrug constructs were cloned into pET26b+ expression vector and produced in E. coli BL21* cells. The proteins were purified using an automated purification system (ÄXKTA, GE Healthcare, Sweden). For this purpose, a PD-10 desalting column packed with 10 mL HSA-sepharose, 1×TST pH 8, 5 mM, NH4Ac pH 5.5 and 0.5 M HAc was used according to manufacturer's instructions. Proteins were freeze dried and stored at −20° C. The POC-PA (ZB05-TEVsubstrate−ZEGFR-ABP) protein was resuspended in Assay buffer (50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, pH 8) supplemented with 33 μg of TEV for 1 h at 30° C. The buffer was changed to PBS-0.1% BSA using PD-10 desalting columns according to manufacturer's recommendations (GE Healthcare, Sweden).
The H292 (human mucoepidermoid pulmonary carcinoma) and A431 (human Squamous cell skin cancer) (American Type Culture Collection, ATCC via LGC Promochem, Sweden) cell lines were used for cell cytotoxicity assays. The cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Flow laboratories, UK) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO., USA), and a mixture of penicillin 100 IU/mL and 100 μg/mL streptomycin (PEST, Biokrom Kg, Berlin, Germany) (for H292), and Dulbecco's Modified Eagle Medium (DMEM) medium (Flow, Irvine, UK) supplemented with 10% fetal bovine serum (for A431). The cells were grown at 37° C. in a 5% CO2 atmosphere.
A431 and H292 cells were cultured according to manufacturer's recommendations (ATCC, USA) and as described above. Trypsin-treated cells (5·105) were resuspended and washed in 500 μL of PBS-0.1% BSA. The cells were incubated in 100 μL of PBS-0.1% BSA supplemented with 100 nM of POC-PA (previously treated with or without TEV protease) for 1 h at RT. The cells were washed once more and incubated with 225 nM Alexa Fluor 647-HSA in PBS-0.1% BSA for 45 min on ice. After a final washing step, the cells were resuspended in 300 μL of PBS-0.1% BSA and analyzed by FC using a Gallios™ flow cytometer (Beckman Coulter, USA).
An anti-idiotypic affibody masking domain, denoted ZB05, with specificity for the binding surface of ZEGFR was successfully generated using staphylococcal display by randomization of 14 surface exposed residues (
A naïve combinatorial S. carnosus library with a theoretical size of 109 affibody variants was used to isolate an anti-idiotypic affibody with affinity for the binding surface of the EGFR-binding affibody molecule ZEGFR. Magnetic-assisted cell sorting (MACS) was initially used for two rounds to reduce the complexity of the library prior to several rounds of FACS. The estimated maximal cell diversity after the second MACS selection round was 4·8·105 variants. Three rounds of FACS selection were performed to further enrich the library for binders. In the first round of FACS a 0.42% gate was used to sort cells from the output of the second MACS, creating the library F1A. In the second round of FACS, F1A was sorted using a 3.02% gate resulting in F2A1. Finally, a third round of FACS was performed on the F2A library with a gate encompassing 12.77% of the population resulting in the library F3A1.2. The output from each selection round was analyzed by Flow Cytometry (FC) to confirm enrichment for binding to ZEGFR. Negative controls were used to eliminate the potential for streptavidin-binders (data not shown). The number of putative binders increased after each selection round as seen from flow cytometric analysis of the sorted outputs.
Binding to ZEGFR was analyzed for more than 30 sequenced candidates (both recurring and unique) by FC. One of these candidates (ZB05) was found to exhibit high binding propensity to ZEGFR and was chosen as an anti-idiotypic masking domain candidate for the construction of a pro-affibody targeting EGFR. The sequence of ZB05 is shown in
The mutagenesis study of the ZB05 masking domain was performed to evaluate the binding contribution of amino acids in each randomized position and how substituting to any other amino acid affects binding to ZEGFR. The original sequence and the randomized positions of ZB05 are shown in
The ZB05 masking candidate was cloned into a pET26b+ vector and produced in E. coli BL21* cells as a soluble monomer containing a C-terminal His-tag, with an expected molecular weight of 7.6 kDa. The protein was purified by IMAC and subsequently analyzed with mass spectrometry (MS) and SDS-page to confirm the mass identity and to evaluate purity, respectively (data not shown). Circular dichroism spectroscopy was used to investigate the secondary structure and determine the thermal stability and refolding capacity. The results from variable temperature measurements (VTM) are shown in
Analysis of POC-PA Activity on S. carnosus Cell Surface
To produce a proof-of-concept pro-affibody (POC-PA), both ZB05 and ZEGFR sequences were subcloned into a staphylococcal display vector containing a linker with the TEV substrate coding sequence and a C-terminal albumin binding protein (ABP). The TEV-protease was chosen to facilitate the initial analysis of the interaction between the EGFR-binding ZEGFR and masking ZB05 affibody molecules. In later experiments, the protease substrate sequence could easily be exchanged to accommodate any protease specificity. The POC-PA (ZB05-TEVSubstrate−ZEGFR-ABP) was displayed on staphylococci and the binding to recombinant human EGFR was analyzed using FC before and after treatment with TEV-protease. The results demonstrated the masking capacity of ZB05, impeding the binding interaction of ZEGFR with its target, since no binding to EGFR could be detected for the intact POC-PA in this particular experiment (data not shown). However, following treatment with TEV-protease, the binding of ZEGFR to EGFR was restored (data not shown). Overall, the results demonstrated that protease cleavage was a requirement for the POC-PA to bind to EGFR in solution. Furthermore, another construct consisting of an anti-ZHER2 Affibody masking domain and the ZEGFR binding domain (POC-PA-DM) was evaluated in the same conditions to verify that the masking by ZB05 was conferred by the specificity for ZEGFR and not due to steric hindrance or unspecific interactions (data not shown). This result shows that the interaction between the two domains of POC-PA is due to the specific anti-idiotypic binding of ZB05 to ZEGFR.
The purified proteins were analyzed both by MS (data not shown) and SDS-page gel to confirm the size and purity of the sample, respectively (
The binding of POC-PA to native EGFR on the two human cancer cell lines H292 (human mucoepidermoid pulmonary carcinoma) and A431 (human Squamous carcinoma), with moderate and high EGFR-expression respectively, was analyzed for both intact POC-PA and pre-cleaved with TEV-protease (
There are several differences between the prodrug used in this in vivo biodistribution study and the POC-PA prodrug used in Example 1:
The full amino acid sequence (SEQ ID NO:12) of the prodrug of this Example 2 is shown in
Human epidermoid carcinoma cell line A431 (EGFR positive, matriptase low expression), lung mucoepidermoid carcinoma cell line H292 (EGFR positive, matriptase high expression) and lymphoma cell line Ramos (EGFR negative) were obtained from American Type Culture Collection (ATCC, via LGC Promochem, Boris, Sweden). They were maintained in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich), 2 mM L-glutamine, and a mixture of penicillin 100 IU/mL and 100 μg/mL streptomycin. Cells were grown in a humidified incubator at 37° C. in 5% CO2 atmosphere.
Indium chloride [111In]InCl3 was purchased from Curium Pharma (Curium Netherlands B.V., Petten, The Netherlands). Buffer were prepared in high quality Mil-Q water, purified from metal contamination using Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) overnight and filtered 0.2 μm. Three compounds were stored at −20° C. in 0.2 M ammonium acetate, pH 6.0.
Radiolabeling was performed by mixing the compounds (21-25 μg in 88-97 μL 0.2M ammonium acetate, pH 6.0) with [111In]InCl3 (30 MBq in 40 μL 0.05 M HCl). The mixture was thoroughly vortexed and incubated at 80° C. for 60 min. After incubation, 500-fold molar excess of EDTA (160 μg in 16 μL 0.2 M ammonium acetate, pH 6.0) was added and the mixture was incubated at 80° C. for 10 min. The reaction mixture was then purified using NAP-5 size exclusion column pre-equilibrated and eluted with phosphate-buffered saline (PBS). Radiochemical yield and purity of the compounds were determined with iTLC eluted with 0.2 M citric acid, pH 2.0. In this system, the [111In]In-labeled compounds stay in application point while free [111In]In moves with the solvent front. Distribution of the activity among the strips was measured using a Storage Phosphor System (CR-35 BIO Plus, Elysia-Raytest, Bietigheim-Bissingen, Germany) and analyzed using AIDA Image Analysis software (Elysia-Raytest, Bietigheim-Bissingen, Germany).
In vitro stability was evaluated in PBS and in the presence of 1000-fold molar excess of ethylenediaminetetraacetic acid (EDTA). After purification, samples of labeled compounds (1-1.6 μg, 50 μL in 1% BSA in PBS) were mixed with EDTA (20 μg, 2 μL in PBS) and incubated at room temperature for up to 48 h. Samples were taken for iTLC analysis at i h, 4 h, 24 h and 48 h.
An in vitro binding specificity test was performed according to previously described method (Wällberg et al., Cancer Biother Radiopharm. 2008; 23(4):435-42). In brief, EGFR-positive A431 and H292 cells were seeded in 6-well plates with a density of 106 cells/well one day before the experiment. During the experiment day, a solution of radiolabeled compounds (10 nM) was added to the cell plates. For blocking, 1000 nM of non-labeled ZEGFR-ABD035-DOTA was added 15 min before the radiolabeled compounds to saturate the receptors in a set of (n=3) wells at room temperature. Same volume of culture medium was added to another set of dishes to compensate the volume. The cells were incubated at 37° C. for 60 min. Thereafter, the medium was removed, and the cells were washed with 1 mL PBS wash. The cells were then detached by a trypsin-EDTA solution and collected. The cell-associated activity was measured using an automated γ-spectrometer (2480 Wizard; Wallac, Finland).
An additional in vitro binding experiment was performed to compare EGFR expression levels in H292 and A431 in vitro. Cells were incubated with the radiolabeled ZEGFR-ABD035-DOTA (10 nM), without blocking, and with blocking with cetuximab (1000 nM) or non-labelled ZEGFR-ABD035-DOTA (1000 nM) as described above. The same amount of radioactivity (1.64×106 counts per minute (CPM)) was added to each cell culture dish. Additionally, the cells were counted in additional culture dishes (n=3) for each cell line. The specific cell bound activity was calculated as CPM/106 cells for non-blocked cells minus CPM/106 cells blocked with cetuximab.
The animal experiments were approved by the Local Ethics Committee for Animal Research in Uppsala.
Female NMRI mice and Balb/c nu/nu mice were supplied from Scanbur A/S (Karlslunde, Denmark) and had an adaptation period of one week before the start of experimental procedures.
To test if masking of the binding site prevents hepatic uptake, the biodistribution study was performed at 4 h and 24 h post-injection in 24 female NMRI mice. For each time point, a group of four mice was intravenously (i.v.) injected with equimolar amount (133 μmol) of respective compounds: 3 μg of [111In]In-labeled prodrug, 3 μg of [111In]In-labeled dummy-linker or 1.8 μg of [111In]In-labeled non-masked control (40 kBq in 100 μL 1% BSA in PBS per mouse). In the prodrug, the linker (linking ZB05 to ZEGFR) was based on the sequence LSGRSDNH (SEQ ID NO:21), which was extended on both sides by repeats of G4S (the exact sequence of this linker is shown in
The mice were euthanized by an overdose of anesthesia solution (30 μl of solution per gram body weight, ketamine 10 mg/mL and xylazine 1 mg/mL) and sacrificed by heart puncture. Blood, salivary gland, lung, liver, spleen, pancreas, small intestine, large intestine, kidney, muscle, bone were collected and weighed. Gastrointestinal tract (with its content) and remaining carcass were also collected. The activity of the organs and standards of injected solution was measured using an automated γ-spectrometer (2480 Wizard; Wallac, Finland). The uptake values were calculated as percentage of injected dose per gram sample weight (% ID/g) except for the gastrointestinal tract (with its content) and remaining carcass, which was calculated as percentage injected dose (% ID) per whole sample.
For H292 tumor implantation, female Balb/c nu/nu mice were subcutaneously injected with 10×106 H292 cells in 100 μL of culture medium 1:1 mixed with Matrigel in the right hind leg. The experiments were performed 13-16 days after the implantation. For Ramos implantation, female Balb/c nu/nu mice were subcutaneously injected with 5×106 Ramos in 100 μL of culture medium in the left hind leg. The experiments were performed 13-15 days after the implantation. For A431 tumor implantation, female Balb/c nu/nu mice were subcutaneously injected with 10×106 A431 cells in 100 μL of culture medium in the left hind leg. The experiments were performed 16 days after the implantation. The average animal weight was 17.6±1.3 g in the H292 groups, 18.6±1.3 g in the Ramos groups and 17.3±1.8 g in the A431 groups. The average tumor weight was 0.38±0.23 g for H292 xenografts, 0.19±0.11 g for Ramos xenografts and 0.11±0.06 g for A431 xenografts.
To evaluate the biodistribution of 3 compounds over time, 24 mice bearing H292 xenografts were randomized into 6 group (n=4). Each two groups were i.v. injected with 133 μmol of [111In]In-labeled Prodrug, [111In]In-labeled dummy-linker or [111In]In-labeled non-masked control (40 kBq in 100 μL 1% BSA in PBS per mouse), respectively. Organs and tumors were collected at 4 h and 48 h post-injection, weighed and measured for activity as described above.
In Vivo Specificity of EGFR Binding (H292 vs. Ramos 4 h and 48 h)
To evaluate the in vivo specificity, 8 mice bearing EGFR-positive H292 xenografts and 8 mice bearing EGFR-negative Ramos xenografts were injected with 133 μmol of [111In]In-labeled Prodrug. Organs and tumors were collected at 4 h and 48 h p.i., weighed and measured for activity as described above.
To study the relationship between tumor uptake of Prodrug and matriptase level, 5 mice bearing H292 xenografts (EGFR positive, matriptase high expression) in the right hind leg and A431 xenografts (EGFR positive, matriptase low expression) in the left hind legs were i.v. injected with 133 μmol of [111In]In-labeled Prodrug (40 kBq in 100 μL 1% BSA in PBS per mouse). Mice were euthanized at 48 h p.i., organs, half of the liver and partially of the A431 and H292 xenografts were collected, weighted and measured for activity as described above. Another half of the liver and partially A431 and H292 xenografts were collected, formalin fixed and embedded in paraffin for histopathologic exam.
To image the EGFR expression, whole-body SPECT/CT scans were performed using a nanoScan SPEC/C (Mediso Medical Imaging Systems, Budapest, Hungary). One mouse bearing H292 xenograft was i.v. injected with [111In]In-labeled Prodrug (14.7 μg, 1.9 MBq). As a control, one mouse bearing H292 xenograft was i.v. injected with [111In]In-labeled non-masked control (8.8 μg, 1.1 MBq). To clarify the in vivo specificity, one mouse bearing Ramos xenograft was i.v. injected with [111In]In-labeled Prodrug (11.2 μg, 2.9 MBq). The mice were imaged at 4 h and 48 h p.i. Imaging of [111In]In-labeled labeled compounds and image reconstruction were performed as described earlier (Rinne et al. J. Mol Sci. 2020 Feb. 15; 21(4):1312).
Statistical analysis was performed using GraphPad Prism (version 9.0.0; GraphPad Software, Inc., La Jolla, CA, USA). The in vitro and in vivo data were analyzed using an unpaired two-tailed t-test. A p value <0.05 was considered a statistically significant difference.
Radiolabeling of three compounds with indium-111 was performed in radiochemical yield ranged from 10% to 21%. Purification after size exclusion column provided over 97% purity (Table 1). No release of activity during incubation with an excess of EDTA was observed (Table 2).
111In-Prodrug
111In-Dummy-linker
111In-Non-masked control
111In-
111In-
111In-non-
Binding specificity test was performed with A431 and H292 cell lines. Both cell lines over-express EGFR. A significant (p<0.05, t-test) reduction of activity was observed for Non-masked control in the blocked group for both cell lines. This confirmed EGFR-mediated binding of Non-masked control to A431 and H292 cells. No significant difference was observed for prodrug or dummy-linker between blocked and non-blocked groups in A431 cell line (
Data concerning biodistribution of the three compounds in NMRI mice are presented in Table 3. The activity concentration in blood was over 15% ID/g at 4 h p.i. and over 6% ID/g at 24 h p.i for all three compounds. For comparison, the blood concentration of non-ABD035-fused 111In-ZEGFR was 0.34*0.02 and 0.12*0.03% ID/g, at 4 and 24 h, respectively (Tolmachev et al. Eur J Nucl Med Mol Imaging. 2010 March; 37(3):613-22). This indicates that fusion to ABD035 results in prolongation of circulation time. The biodistribution of [111In]In-labeled Prodrug and Dummy-linker was very similar, except a small but significant (P<0.05, t-test) different kidney uptake at 4 h p.i., as well as significant different of salivary gland uptake and liver uptake at 24 h p.i. Both [111In]In-labeled prodrug and dummy-linker had significantly lower liver uptake than Non-masked control at 4 h and 24 h p.i.
To evaluate the tumor uptake over time, mice bearing H292 xenografts were injected with [111In]In-labeled compounds. Data concerning biodistribution at 4 h and 48 h p.i. are presented in Table 4. Equal levels (p>0.3, t-test) of tumor uptake at both time points for three compounds was observed. [111In]In-labeled prodrug and dummy-linker had significantly (p<0.05, t-test) higher blood activity, higher uptake in organs such as: lung, kidney and muscle and significantly (p<0.05, t-test) lower liver uptake than [111In]In-labeled non-masked control at both 4 h and 48 h p.i.
To determine the in vivo EGFR binding specificity, biodistribution was compared in mice bearing H292 xenografts and Ramos xenografts at 4 h and 48 h p.i. At both time points, EGFR-positive H292 xenograft has significantly (p<0.05, unpaired t-test) higher Prodrug uptake than EGFR-negative Ramos xenograft (
To evaluate the uptake of Prodrug in the presence of matriptase, biodistribution was compared in mice bearing both H292 and A431 xenografts at 48 h p.i. The uptake in matriptase-positive H292 xenograft (11±2% ID/g) was significantly (p<0.05, paired t-test) higher than in matriptase-negative A431xenografts (6±1% ID/g) (
The biodistribution data were confirmed by experimental microSPECT/CT imaging for [111In]In-labeled Prodrug and Non-masked control in H292 xenografts (
Four new pro-affibody (PA) variants were designed to improve the cleavage efficiency by matriptase. A new protease recognition sequence (MSGRSANA, SEQ ID NO:38, “ZW”) developed by the company Zymeworks was used for PA-ZW-(G4S)1, PA-ZW-(G4S)2, PA-ZW-(G4S)3 and PA-ZW3, replacing the original LSGRSDNH sequence used above:
In the new PAs, the linker interconnecting ZB05 and ZEGFR thus contains a single ZW sequence flanked by 1-3 G4S sequences or a concatemer of three ZW substrate sequences.
The new variants were produced and purified using affinity chromatography, freeze-dried and dissolved in PBS. Four identical samples were prepared for each variant. Each sample contained 10 uM of PA protein and 20 nM of recombinant human matriptase (cat. Nr. 3946-SEB) in a total volume of 50 uL PBS. The samples were incubated at 37 degrees Celsius for 1, 3, 5 or 24 hours followed by freezing at −20 degrees Celsius. The frozen samples were thawed and analyzed with SDS-PAGE, which included the intact (int.) non-cleaved protein from the same stock used for sample preparation.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 2250114-2 | Feb 2022 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/052882 | 2/6/2023 | WO |
