SERUM ALBUMIN BINDERS

Abstract

The present invention relates to amino acid sequences that can bind to serum albumin. In particular, the present invention relates to immunoglobulin single variable domains, and in particular heavy-chain immunoglobulin single variable domains, that can bind to serum albumin. The invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the immunoglobulin single variable domains binding to serum albumin that are described herein.

Claims

1. A method for preventing or treating a disease or condition in a subject, comprising administering to the subject an immunoglobulin single variable domain (ISVD), or a polypeptide comprising said ISVD, wherein the ISVD is capable of binding to serum albumin and comprises: a CDR1 (according to Abm) that is the amino acid sequence GSNISSYVMG (SEQ ID NO:11) or GSTISSYVMG (SEQ ID NO:12) or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a CDR2 (according to Abm) that is the amino acid sequence AISRSGGYTY (SEQ ID NO: 13) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13; and a CDR3 (according to Abm) that is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, optionally wherein the serum albumin is human serum albumin.

2. The method according to claim 1, wherein the ISVD comprises: a CDR1 (according to Abm) that is the amino acid sequence GSNISSYVMG (SEQ ID NO:11) or GSTISSYVMG (SEQ ID NO:12); and a CDR2 (according to Abm) that is the amino acid sequence AISRSGGYTY (SEQ ID NO: 13); and a CDR3 (according to Abm) that is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14).

3. The method according to claim 1, wherein the ISVD can bind to human serum albumin with an affinity better than 100 nM, optionally better than 50nM, as determined using surface plasmon resonance.

4. The method according to claim 1, wherein the ISVD is a heavy-chain immunoglobulin single variable domain.

5. The method according to claim 1, wherein the ISVD is a VHH, a humanized VHH or a camelized VH, optionally wherein the camelized VH is a camelized human VH.

6. The method according to claim 1, wherein the ISVD, or the polypeptide comprising the ISVD has a serum half-life in human (expressed as t1/2 beta) that is more than 6 hours, 12 hours, 24 hours, or 72 hours; and/or.

7. The method according to claim 1, wherein the ISVD has: a degree of sequence identity with the sequence of SEQ ID NO: 18 and/or 19 of at least 85%, at least 90%, or at least 95%; and/or: -no more than 7, no more than 5, no more than 3, no more than 2, or no more than 1 amino acid differences with the sequence of SEQ ID NO: 18 and/or 19; wherein the CDRs and any C-terminal extension that may be present are not taken into account for determining the degree of sequence identity or the amino acid differences.

8. The method according to claim 1, wherein the ISVD has: a degree of sequence identity with the sequence of SEQ ID NO: 18 of at least 85%, at least 90%, or at least 95%; and/or: no more than 7, no more than 5, no more than 3, no more than 2, or no more than 1 amino acid differences with the sequence of SEQ ID NO: 18; wherein the CDRs and any C-terminal extension that may be present are not taken into account for determining the degree of sequence identity or the amino acid differences.

9. The method according to claim 1, wherein the ISVD has: a degree of sequence identity with the sequence of SEQ ID NO: 19 of at least 85%, at least 90%, or at least 95%; and/or: no more than 7, no more than 5, no more than 3, no more than 2, or no more than 1 amino acid differences with the sequence of SEQ ID NO: 19; wherein the CDRs and any C-terminal extension that may be present are not taken into account for determining the degree of sequence identity or the amino acid differences.

10. The method according to claim 1, wherein the ISVD contains, compared to the sequence of SEQ ID NO: 18, one or more mutations that reduce the binding by pre-existing antibodies.

11. The method according to claim 1, wherein the ISVD is a VHH and that contains, compared to the sequence of SEQ ID NO:18, one or more humanizing substitutions.

12. The method according to claim 1, wherein the ISVD comprises the amino acid sequence of any one of SEQ ID NOs: 18-59.

13. The method according to claim 1, wherein a polypeptides the ISVD.

14. The method according to claim 13, wherein the polypeptide comprises at least one therapeutic moiety or entity.

15. The method according to claim 13, wherein the polypeptide is a fusion protein.

16. The method according to claim 13, wherein the polypeptide has a serum half-life in human (expressed as t1/2 beta) that is more than 6 hours, more than 12 hours, more than 24 hours, or more than 72 hours.

17. The method according to claim 13 wherein the polypeptide is such that: when the polypeptide comprises a C-terminal ISVD, it further comprises a C-terminal extension (X).sub.n; and/or when the polypeptide comprises a C-terminal ISVD, said C-terminal ISVD contains one or more mutations that reduce the binding of pre-existing antibodies; and/or when the polypeptide comprises a N-terminal ISVD, said N-terminal ISVD contains a D at position 1; and/or when the polypeptide comprises a N-terminal ISVD, said N-terminal ISVD contains a D or an EID at position 1; and/or essentially all of the ISVDs present in said polypeptide, contain one or more mutations that reduce the binding of pre-existing antibodies.

18. The method according to claim 13, comprising administering to the subject a pharmaceutical composition comprising the ISVD or the polypeptide comprising said ISVD.

19. A nucleic acid that encodes an immunoglobulin single variable domain (ISVD), or a polypeptide comprising said ISVD, wherein the ISVD is capable of binding to serum albumin and comprises: a CDR1 (according to Abm) that is the amino acid sequence GSNISSYVMG (SEQ ID NO:11) or GSTISSYVMG (SEQ ID NO:12) or an amino acid sequence that has 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and a CDR2 (according to Abm) that is the amino acid sequence AISRSGGYTY (SEQ ID NO: 13) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 13; and a CDR3 (according to Abm) that is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) with the amino acid sequence of SEQ ID NO: 14, optionally wherein the serum albumin is human serum albumin, optionally wherein the nucleic acid is a genetic construct.

20. A host or host cell that comprises the nucleic acid according to claim 19.

21. A method for preparing a polypeptide comprising cultivating or maintaining a host cell according to claim 20 under conditions such that said host cell produces or expresses the ISVD or polypeptide, and optionally further comprising isolating the ISVD or polypeptide.

Description

EXPERIMENTAL PART

Example 1

Affinity for Serum Albumin

[0179] The affinity of the serum albumin binder of SEQ ID NO:19 (invention) for human (Sigma-Aldrich A3782), cynomolgus monkey (generated in-house), mouse (Albumin Bioscience 2601), rat (Sigma-Aldrich A4538), rabbit (Sigma-Aldrich A0764), guinea pig (Gentaur GPSA62), pig (Sigma-Aldrich A4414), sheep (Sigma-Aldrich A3264), dog (Abcam 119814) and bovine (Sigma-Aldrich A3059) serum albumin (SA) was measured via Surface Plasmon Resonance (SPR) on a ProteOn XPR36 (BioRad) instrument. Serum albumin was immobilized via amine coupling on GLC ProteOn chip using ProteOn Amine Coupling Kit (BioRad). Different concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 1.23 nM) of The serum albumin binder of SEQ ID NO:19 (invention) was injected in HBS-P+ pH 7.4 buffer (GE Healthcare) at 45 ?L/min for 120 s, followed by dissociation for 900 s. There was no or very low binding observed for SEQ ID NO: 19 on sheep and bovine SA. The affinity of SEQ ID NO: 19 for human, cynomolgus monkey, rat, mouse and guinea pig SA was in the pM range (Table 1). In addition, SEQ ID NO:19 is cross-reactive with dog SA and also binds with weak affinity to pig and rabbit SA.

TABLE-US-00003 TABLE 1 Kinetic parameters for binding of The serum albumin binder of SEQ ID NO: 19 (invention) on SA from different species. SEQ ID NO: 19 (invention) SEQ ID NO: 1 (reference) ka ka (s-1 Kd KD (s-1 Kd KD SA M-1) (s-1) (M) M-1) (s-1) (M) human 7.9E+05 3.7E?05 4.6E?11 4.9E+05 1.6E?03 3.3E?09 cyno 8.3E+05 1.4E?05 1.6E?11 4.6E+05 1.4E?03 3.1E?09 rat 1.2E+06 1.5E?05 1.2E?11 3.9E+05 2.6E?01 6.7E?07 mouse 9.4E+05 1.5E?05 1.6E?11 6.6E+05 3.0E?02 3.9E?08 guinea pig 9.4E+05 5.6E?05 5.9E?11 9.4E+05 1.9E?02 2.0E?08 dog 9.0E+05 2.5E?03 2.8E?09 no binding pig 1.2E+05 3.6E?02 2.9E?07 no binding The serum albumin binder of SEQ ID NO: 19 (invention) was injected at different concentrations on immobilized SA on a ProteOn instrument. Binding and dissociation kinetics were analysed at pH 7.4. Kinetic parameters for SEQ ID NO: 1 are listed as a reference and were determined in separate experiments.

[0180] The long half-life of albumin in blood is mainly driven by two characteristics: (i) the large size (65 kDa) of albumin limits its glomerular filtration and (ii) albumin binds to FcRn at low pH (pH 6), which protects albumin from degradation in the lysosomes after passive endocytosis in endothelial and epithelial cells, by recycling from early endosome back to the extracellular environment. For albumin-binding Nanobodies to result in long serum half-life through albumin binding and subsequent recycling, these should stay bound to albumin in the pH range from 5.0 to 7.4. The dissociation rate of the serum albumin binder of SEQ ID NO: 19 (invention) from HSA at pH 5, pH 6 and pH 7.4 was measured on a ProteOn instrument as described above, including SEQ ID NO: 1 as a reference. The serum albumin binders of SEQ ID NO:19 (invention) and SEQ ID NO:1 were injected at 500 nM and 300 nM respectively in HBS-P+ pH 7.4 buffer. Dissociation buffers were 50 mM NaOAc/HOAc+150 mM NaCl+0.05% Tween-20 pH 5.0, 50 mM NaOAc/HOAc+150 mM NaCl+0.05% Tween-20 pH 6.0 and HBS-P+pH 7.4 respectively. Dissociation was analysed for 2700 s. Dissociation rates for SEQ ID NO:19 do not differ significantly across the pH range from 5.0 to 7.4 (Table 2).

TABLE-US-00004 TABLE 2 Dissociation rate of the serum albumin binder of SEQ ID NO: 19 (invention) from HSA at different pH. kd (s?1) pH SEQ ID NO: 19 (invention) SEQ ID NO: 1 (reference) pH 7.4 6.0E?05 1.3E?03 pH 6.0 6.7E?05 9.2E?04 pH 5.0 7.5E?05 1.1E?03 SEQ ID NO: 19 (invention) and SEQ ID NO: 1 (reference) were injected on immobilized HSA. Dissociation was monitored at pH 5.0, 6.0 and 7.4 on a ProteOn instrument.

Example 2

Epitope

[0181] Epitope binning was analysed in a competition ELISA. Human serum albumin was coated at 125 ng/ml in PBS at 4? C. overnight. After blocking with PBS +1% casein, 1.5 nM SEQ ID NO:1-cMycHis6 and a concentration series of competitors (His6Flag3-SEQ ID NO:20, His6Flag3-SEQ ID NO:1 as positive control or hen egg lysozyme binding single domain antibody cAblys3-Flag3His6 as negative control) were added. Bound SEQ ID NO:1-cMycHis6 was detected with goat anti-cMyc (Abcam ab19234) and HRP-labelled rabbit anti-goat (Genway 18-511-244226) antibodies. The albumin binder of SEQ ID NO:20 and SEQ ID NO:1 do not bind identical epitopes on HSA (FIG. 6).

Example 3

Interference with Interaction Between SA and FcRn

[0182] For the serum albumin binder of SEQ ID NO:19 (invention) to provide long half-life via albumin binding and subsequent recycling, it should not interfere with the binding of albumin to FcRn. This was analysed in SPR on a Biacore T100 (GE Healthcare) instrument. Human FcRn-human ?2 microglobulin heterodimer (Sino Biological CT009-H08H) was immobilized on CM5 chip via standard amine coupling (Biacore amine coupling kit). A mixture of 1 ?M HSA and 2 ?M Nanobody (His6Flag3-SEQ ID NO:20, His6Flag3-SEQ ID NO: 1 or cAblys3-Flag3His6) in 50 mM NaPO4 +150 mM NaCl +0.05% Tween-20 pH 6.0 was injected at 10 ?l/min for 120 s, followed by dissociation for 600 s. Binding curves were qualitatively compared with binding curve of 1 ?M HSA in absence of Nanobody. The albumin binder of SEQ ID NO:20 (invention) did not interfere with the binding of HSA to FcRn (FIG. 7).

Example 4

PK Profile in Dog

[0183] Pharmacokinetics of a trivalent Nanobody construct (SEQ ID NO:68) after single i.v. dose are studied in dog. The trivalent construct was produced in Pichia pastoris and comprises two Nanobodies against respiratory syncytial virus (RSV) with the albumin binding Nanobody of SEQ ID NO: 19 at the C-terminal end (in which the three Nanobodies are linked to each other using (Gly4Ser)7 linkers).

[0184] The pharmacokinetic profile of the construct is evaluated in a pharmacokinetic study (non-crossover, single dose) in twelve fasted healthy male Beagle dogs (healthy as determined by a pre-study health examination). The construct is administered intravenously.

[0185] The animals are fasted (with uninterrupted access to water) overnight prior to dosing, and prior to collection of blood samples at pre-dose, 24, 168, and 336 hours after dosing (total fasting time no longer than 24 hours). Food is returned 4 hours post-dose, or immediately after blood collection at 24, 168, and 336 hours. Body weight is also recorded and tracked during the study.

[0186] 1 ml of whole blood (or 2-3 ml pre-dose and at 24, 168 and 336 hours) for plasma is collected from a peripheral vessel pre-dose and at 5, 15, 30 min and 1, 4, 8, 12, 24, 72, 168, 240, 336, 504, and 672 hours post initial dose. Collected samples for serum processing are kept at room temperature and are allowed to clot. Blood is be centrifuged at 10,000 rpm for 2 minutes using a refrigerated centrifuge (set to maintain 4?? C.). Samples are frozen within 30 minutes from collection until they are submitted for analysis (including measurement of concentration of the relevant Nanobody construct).