BIOLOGICAL MATERIALS AND USES THEREOF
20220233706 · 2022-07-28
Inventors
- Mahendra Persaud Deonarain (Surrey, GB)
- Gokhan Yahioglu (London, GB)
- Ioanna Stamati (London, GB)
- Savvas Saouros (London, GB)
- Prashant Bhimrao Kapadnis (Harlow Essex, GB)
Cpc classification
A61K47/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K47/6835
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
A61K47/6809
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
A61K47/6855
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
The invention provides compounds comprising a therapeutic agent coupled to a carrier molecule, with a minimum coupling ratio of 5:1; wherein the carrier molecule is (i) an antibody fragment or derivative thereof or (ii) an antibody mimetic or derivative thereof; and wherein the therapeutic agents are coupled onto a lysine amino acid residue; and further wherein the therapeutic agent is not a photosensitising agent. There is also provided uses, methods relating to such compounds, as well as processes for their manufacture.
Claims
1. A compound comprising a therapeutic agent coupled to a carrier molecule, with a minimum coupling ratio of 5:1; wherein the carrier molecule is (i) an antibody fragment or derivative thereof or (ii) an antibody mimetic or derivative thereof; and wherein the therapeutic agents are coupled onto a lysine amino acid residue; and further wherein the therapeutic agent is not a photosensitising agent.
2. The compound of claim 1 wherein the functional and physical properties of the therapeutic agent and the carrier molecule are qualitatively substantially unaltered in the coupled form in comparison to the properties when in an uncoupled form.
3. A compound according to claim 1 wherein the compound has (a) an IC50 of 100 nM or lower; and/or (b) an IC50 of at least 10-fold lower than the therapeutic agent when unconjugated; and or (c) a serum half-life of at least 2 hours, optionally the serum half-life of at least 2 hours is measured in mice or in humans; and/or (d) a serum half-life of at least 50% of that of the free antibody when unconjugated; and/or (e) a solubility of at least 1 mg/ml in phosphate-buffered saline at room temperature; and or (f) a solubility of at least 1 mg/ml in phosphate-buffered saline at room temperature in the presence of an excipient a concentration and type approved by the FDA, wherein the excipient is up to 0.5% polysorbate, 1% glycerol, 0.5% glycine, 0.1% histidine, 0.5% chlorobutanol, 5% propylene glycol, 2% benzyl alcohol, 0.05% octanoic acid and/or 0.1% N-acetyl tryptophan; and/or (g) an aggregation level of up to 5% in phosphate-buffered saline at room temperature; and/or
4.-10. (canceled)
11. A compound according to claim 1, wherein the therapeutic agents, when coupled to the carrier molecule, are (h) separated by a distance of at least two amino acids (3.5 to 7.5 angstroms), and/or (i) separated by a distance of two amino acids (3.5 to 7.5 angstroms), three amino acids (9 to 12 angstroms), four amino acids (10 to 15 angstroms), five amino acids (15 to 20 angstroms) or six amino acids (20 to 25 angstroms).
12. (canceled)
13. A compound according to claim 1, wherein the therapeutic agents are directly coupled to the carrier molecule at the amino acid, optionally wherein the direct coupling to the amino acid is via an N-hydroxy-succinamide ester.
14. (canceled)
15. A compound according to claim 1 wherein the therapeutic agents are indirectly coupled to the carrier molecule at the amino acid, optionally wherein the indirect coupling to the amino acid is via a thiol or maleimide.
16. (canceled)
17. The compound of claim 1, wherein the carrier molecule binds selectively to a target, optionally wherein the target is a target cell or an extracellular target molecule.
18. (canceled)
19. The compound according to claim 17 wherein the carrier molecule, on binding a target cell, is internalised into the cell and following binding of the target, is decoupled from the therapeutic agent.
20.-21. (canceled)
22. The compound of claim 1 wherein the carrier molecule is an antibody fragment that does not include the CH2 and CH3 antibody regions of a whole antibody.
23. The compound of claim 1 wherein the carrier molecule is an antibody fragment selected from scFv, Fv, Fab, F(ab′)2, Fab-SH, dsFv, be-scFv, sdAb, di-scFvs (also known as bi-scFvs), Fcabs, domain antibodies, nanobodies, VHH domains, bispecific formats such as bispecific T-cell engagers, diabodies, and tandabs.
24. The compound of claim 1 wherein the carrier molecule is an antibody mimetic selected from DARPins, affibodies, affitins, anticalins, avimers, kunitz domain peptides, adnectins, centyrins, Fynomers, IgNARs and monobodies.
25. The compound of claim 1 wherein the carrier molecule is humanised or human.
26. The compound of claim 1 wherein the carrier molecule binds specifically to HER2, EGFR, HER3, MUC1, EpCAM, CEA, Fibronectin-EDB, CD19, CD20, CD22, LeY, CD30, CD33, CD79b, GPNMB, PSMA, CD56, CD37, Folate receptor, CA6, CD27L, MUC16, CD66e, CD74, Trop-2 or guanylate cyclase.
27. The compound of claim 1 wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.
28. The compound of claim 1 wherein the therapeutic agent is selected from cell cycle progression inhibitors, angiogenesis inhibitors, MAPK signaling pathway inhibitors, PI3K/m-TOR/AKT pathway inhibitors, kinase inhibitors, HDAC inhibitors, protein chaperone inhibitors, PARP inhibitors, Wnt/Hedgehog/Notch signalling pathway inhibitors, RNA polymerase inhibitors. DNA-binding drugs, DNA damaging drugs, DNA alkylating drugs, microtubule stabilizing agents, microtubule destabilizing agents, platinum compounds, kinase inhibitors, pyridocarbazole and its derivatives, and topoisomerase I inhibitors, cemadotin, P5, P5-C5, doxorubicin, ellipticine, MMAE, paclitaxel, auristatins, maytansines, dolostatins, camptothecin, SN-38 and pyrrolobenzodiazepine dimers (PBDs), PNU-159862, indolino-benzodiazepine dimers (IGNs) and MMAF.
29. (canceled)
30. The compound according to claim 1 wherein the carrier molecule is an scFv and the therapeutic agent is selected from the group consisting of cemadotin, doxorubicin, ellipticine, MMAE, P5-C5, maytansine, pyrrolobenzodiazepine dimer (PBD), and MMAF.
31-37. (canceled)
38. The compound of claim 30 wherein the scFv binds specifically to HER2, optionally wherein the scFv has the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 5.
39. (canceled)
40. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically-acceptable carrier, excipient or diluent.
41. A compound as defined in claim 1 for use in the diagnosis, treatment and/or prevention of disease.
42.-44. (canceled)
45. A process of making a compound as defined in claim 1 comprising the steps of: providing a therapeutic agent; providing a carrier molecule; conjugating the therapeutic agent and the carrier molecule in the presence of at least one polar aprotic solvent and an aqueous buffer.
46.-54. (canceled)
Description
EXAMPLES
[0156] Examples embodying an aspect of the invention will now be described with reference to the following figures in which:
[0157]
[0158] Modified from Knappik et al (2000) J. Mol. Biol 296, 57-86. The grey-scale indicate the percentage Surface/solvent accessible, from dark (=high) to light (=low).
[0159] Numbering scheme according to Honegger A & Pluckthun A [J. Mol. Biol. 2001, 309:657-70]. Around 70% is considered to be predominantly solvent exposed.
[0160]
[0161] Lane 1: Molecular Markers
[0162] Lane 2: Clarified lysate after 2.sup.nd pass through Ni-NTA resin
[0163] Lane 3: Final wash with Wash Buffer 1
[0164] Lane 4: 1.sup.st wash with Wash Buffer 2
[0165] Lane 5: Elution fraction
[0166] Lane 6: Pooled elution fraction after dialysis in TEV cleavage buffer. The rectangle denotes the fusion-TCT.
[0167] Lane 7: 16 hours after TEV cleavage initiation. The upper square denotes the cleaved scFv (TCT). The lower square denotes the cleaved thioredoxin fusion partner.
[0168] Lane 8: Molecular Markers
[0169] Lane 9: Cleaved TCT
[0170] Lane 10: Proteins remaining bound to Ni-NTA
[0171] Lanes 11-18: Fractions from size exclusion column (35 kDa), of
[0172]
[0173] Peak 1—too dilute to appear on coomassie-stained PAGE gel, high molecular weight contaminants
[0174] Peak 2—High molecular weight aggregates of scFv TCT.
[0175] Peak 3—Pure monomeric scFv TCT, corresponding to Lanes 11-18 on the gel (
[0176]
[0177] Ellipticine (compound 21) conjugates. 1=32 equivalents, 14% DMSO; 2=16 equivalents, 14% DMSO; 3=16 equivalent, 6% DMSO; D=dialysed, Z=Zeba column desalted; CS=soluble crude reaction; P=insoluble/precipitated crude reaction. Sample loading=2.4 μg (ADCs), 1.9 μg (scFv).
[0178] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0179]
[0180]
[0181] Ellipticine (compound 21) and PEG-Ellipticine (compound 23) conjugates. 1=scFv (TCT)-PEG-Ellipticine, 20 equivalents; 2=scFv (TCT)-Ellipticine, 20 equivalents; 3=scFv (TCT)-PEG-Ellipticine, 32 equivalents; 4=scFv (TCT)-PEG-Ellipticine, 64 equivalents; Z=Zeba column desalted; CS=soluble crude reaction; P=insoluble/precipitated crude reaction. Sample loading=1.8 μg.
[0182] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0183]
[0184] D=dialysed; Z=Zeba column desalted; C=soluble crude reaction; P=insoluble/precipitated crude reaction. Ell=Ellipticine Sample loading=2.5 μg.
[0185] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0186]
[0187] D=dialysed; Z=Zeba column desalted; C=soluble crude reaction; P=insoluble/precipitated crude reaction, Ell=Ellipticine. Sample loading=2.5 μg. MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0188]
[0189] Upper panel, HMFG1 IgG conjugates, Lower panel C6.5 scFv conjugates.
[0190] HMFG1/C6=free antibody, S=soluble fraction, P=precipitate, A=Doxorubicin-maleimide (compound 12) conjugates, B=Doxorubicin-PEG-maleimide (compound 48) conjugates.
[0191] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0192]
[0193]
[0194] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10. Cemadotin (compound 2) conjugates (compound 69). 1=16 drug equivalents; 2=48 drug equivalents; 3=112 drug equivalents; S=HPLC-SEC purification; Z=further Zeba buffer exchange.
[0195]
TABLE-US-00003 RT Molecular (min) Weight (kDa) Sample 6.43 80 Alcohol dehydrogenase 8.48 30 Carbonic anhydrase 11.25 13 Lysozyme 10.9 <1 Cemadotin [0197] b) Upper trace, scFv (TCT), lower trace, scFv (TCT)-Cemadotin ADC sample-1 analyses by SEC-HPLC, The column was run at 0.5 ml/min in PBS/20% isopropanol. [0198] c) Upper trace, scFv (TCT)-Cemadotin ADC sample-2, middle trace, scFv (TCT)-Cemadotin ADC sample-3 analyses by SEC-HPLC, lower trace-UV-Vis spectrum confirming protein/peptide content. The column was run at 0.5 ml/min in PBS/20% isopropanol.
[0199]
[0200]
[0201]
[0202]
[0203]
[0204]
[0205]
[0206]
[0207]
[0208]
[0209] P5C5 drug (compound 6) and conjugates (compound 71). 1=scFv (TCT)-P5C5, 30 equivalents; 2=scFv (TCT)-P5C5, 112 equivalents; C=crude reaction mix; S=sample after SEC purification and concentrating; F=final sample after SEC purification, concentrating, and buffer exchange. Sample loading=2 μg.
[0210] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10
[0211]
[0214]
[0215] P5C5 drug (compound 6), CemadotinC5 drug (compound 4) and conjugates (compound 71). 1=scFv (TCT)-P5C5, 16 equivalents; 2=scFv (TCT)-P5C5, 30 equivalents; 3=scFv (TCT)-P5C5, 112 equivalents; 4=Trastuzumab-P5C5, 16 equivalents; 5=Trastuzumab-P5C5, 32 equivalents; 6-7=scFv (TCT)-Cemadotin-C5; 8=Trastuzumab-Cemadotin-C5; Z=final Zeba desalted sample; Sc=sample after HPLC-SEC and concentration; C=crude reaction mix. Sample loading=1.9 μg.
[0216] MW markers (M), kDa top to bottom: 250, 130, 100, 70, 55, 35, 25, 15, 10.
[0217]
[0218] Upper trace shows the free trastuzumab IgG, the lower 3 traces show various ADCs at different conjugation reaction equivalents. Samples were run on a G3000SWxl column calibrated with markers. The column was run at 0.5 ml/min in PBS/20% isopropanol. The markers eluted as follows:
TABLE-US-00004 Molecular Retention weight (kDa) time (mins) Sample 223.8 18.1 beta amylase 146.8 18.9 alcohol dehydrogenase 66.5 21.2 BSA 29 23.7 carbonic anhydrase 14.5 29.7 lysozyme
[0219] The IgG (retention time=11.7 min) and the IgG-ADCs (retention time=10.9-11.4 min) all elute at around 150 kDa indicating little/no aggregation.
[0220]
[0221] Peak 1=Trastuzumab, 2=Trastuzumab-P5C5 (16 equivalents conjugation reaction), 3=2=Trastuzumab-cemadotin (16 equivalents conjugation reaction), 2=Trastuzumab-P5C5 (32 equivalents conjugation reaction)
[0222]
[0223] The scFv (TCT)-P5-C5 ADCs had faster retention times (15.7 min, 15.6 min and 15.4 with increasing conjugation equivalents and hence DAR) than the free scFv (retention time=16.9 min). The scFv (TCT)-Cemadotin retention times were 16.1 min and 15.7 with increasing DAR). All still remained in the range of monomeric scFv with little or no aggregation. The column was run at 0.5 ml/min in PBS/20% isopropanol.
[0224]
[0225]
[0226]
[0227]
[0228]
[0229]
[0230]
[0231]
[0232]
[0233]
[0234] Blots 1-3 and 5-7, 10, and 11 are loaded as follows: Marker; ADC; TCT; and free Cemadotin.
[0235] Blots 4 and 8 are loaded as follows: Marker; ADC; and free Cemadotin.
[0236]
[0237] Cemadotin conjugate (compound 69).
[0238]
[0239] Cemadotin conjugate (compound 69).
[0240]
[0241] Cemadotin conjugate (compound 69).
[0242]
[0243] Cemadotin conjugate (compound 69).
[0244]
[0245]
[0246] P5C5 conjugate (compound 71).
[0247]
[0248]
[0249] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 10 mins.
[0250]
[0251] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 10.3 mins.
[0252]
[0253]
[0254] Size markers used are shown.
[0255]
[0256] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 10.2 mins.
[0257]
[0258] (A) is the LCMS trace (UV and TIC) and (B-E) show the deconvoluted masses for the main peaks 10-12 mins.
[0259]
[0260]
[0261] (A) and (B) are the LCMS data for scFv (TCT1067)-MMAF-C5 ADC1 where (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peaks between 9.3-10.6 mins. (C) and (D) are the LCMS data for scFv (TCT1067)-MMAF-C5 ADC2 where (C) is the LCMS trace (UV and TIC) and (D) is the deconvoluted mass for the main peaks between 9.9-12 mins.
[0262]
[0263] Size markers as shown in
[0264]
[0265]
[0266] Size markers as shown in
[0267]
[0268] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 8.2 mins.
[0269]
[0270]
[0271] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 9.5 mins.
[0272]
[0273]
[0274] (A) and (B) are the LCMS data for scFv (TCT) ADC 1 where (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 8.1 mins. (C) and (D) are the LCMS data for scFv (TCT)-AF-C5 ADC 2 where (C) is the LCMS trace (UV and TIC) and (D) is the deconvoluted mass for the main peak at 9.4 mins. (E) and (F) are the LCMS data for scFv (TCT)-AF-C5 ADC 3 where (E) is the LCMS trace (UV and TIC) and (F) is the deconvoluted mass for the main peak at 9.9 mins.
[0275]
[0276] Size markers as shown in
[0277]
[0278]
[0279] (A) is the LCMS trace (UV and TIC) and (B-E) show the deconvoluted masses for the main peaks.
[0280]
[0281] Size markers as shown in
[0282]
[0283]
[0284] (A) is the LCMS trace (UV and TIC) and (B-F) show the deconvoluted masses for the main peaks.
[0285]
[0286]
[0287] (A) and (B) are the LCMS data for scFv (TCT1067)-Maytansine-(PEG12) DM1 ADC 1 where (A) is the LCMS trace (UV and TIC), and (B) shows the deconvoluted masses for the main peak. (C) and (D) are the LCMS data for scFv (TCT1067-Maytansine-PEG(12) DM1 ADC 2 where (C) is the UV LCMS trace and (D) is the TIC LCMS trace indicating the DAR of the species present in the sample.
[0288]
[0289] Size markers are as shown in (
[0290]
[0291] M=molecular weight markers.
[0292] P=unconjugated scFv (panitumumab),
[0293] T=unconjugated scFv (TCT1067).
[0294] The higher DAR species migrate more slowly due to increased molecular weight, with the scFv (TCT1067) conjugates demonstrating an increasing DAR. Size markers as shown in
[0295]
[0296]
[0297] (A) and (B) is the LCMS data for scFv (Pani-AF-C5) ADC 1, where (A) is the LCMS trace (UV and TIC) and (B) shows the deconvoluted masses for the main peaks of sample 1. (C) is the LCMS trace (UV and TIC) and (D) shows the deconvoluted masses for the main peaks of sample 2. (E)-(H) is the LCMS data for scFv (TCT1067-AF-C5) ADCs 4-6 where (E) is the LCMS trace (UV and TIC) for sample 4, (F) shows the deconvoluted masses for the main peak of sample 4, (G) shows the deconvoluted masses for sample 5 and (H) shows the deconvoluted masses for sample 6.
[0298]
[0299] Cell killing dose-response profiles of free MMAF cytotoxin on (A) U87 cells (B) SKBr3 cells (C) BT474 cells.
[0300]
[0301] Cell killing dose-response profiles of unconjugated scFv(TCT1067) and trastuzumab on (A) U87 cells (B) BT474 cells (C) SKBr3 cells
[0302]
[0303] Cell killing dose-response profiles of unconjugated scFv(TCT) on BT474 cells.
[0304]
[0305] Cell killing dose-response profiles of antibody fragment ADCs scFv (TCT)-MMAF-C5 DAR 6.6, scFv (TCT0167)-MMAF-C5 DAR 6.4, Unconjugated trastuzumab and trastuzumab-MMAF-C5 on (A) U87 cells (B) BT474 cells (C) SKBr3 cells.
[0306]
[0307] Cell killing dose-response profiles of antibody fragment conjugates scFv (TCT)-MMAF-C5 DAR 8, scFv (TCT0167)-MMAF-C5 DAR 8.7 and trastuzumab-MMAF-C5 on (A) U87 cells (B) BT474 cells (C) SKBr3 cells.
[0308]
[0309] Cell killing dose-response profiles of (A) Free P5-C5 drug and (B) antibody fragment conjugates scFv (TCT1067)-P5-C5 DAR 10.6 (H1) and DAR 12.5 (H2) on U87 cells.
[0310]
[0311] Cell killing dose-response profiles of (A) Free P5-C5 drug and (B) antibody fragment conjugates scFv (TCT1067)-P5-C5 DAR 10.6 (H1) and DAR 12.5 (H2) on SKBr3 cells.
[0312]
[0313] Cell killing dose-response profiles of (A) Free P5-C5 drug and (B) antibody conjugates scFv (TCT1067)-P5-C5 DAR 10.6 (H1) and trastuzumab DAR6 on BT474 cells.
[0314]
[0315] Cell killing dose-response profiles of free Auristatin cytotoxin on (A) U87 cells (B) SKBr3 cells (C) BT474 cells.
[0316]
[0317] Cell killing dose-response profile of antibody fragment ADCs scFv (TCT1067)-AF-C5, DAR 2.7 (L), scFv (TCT1067)-AF-C5, DAR 6.2 (M), scFv (TCT1067)-AF-C5, DAR 11.8 (H) and trastuzumab-AF-C5, DAR 4.8 on (A) U87 cells (B) BT474 cells (C) SKBr3 cells (D) NCI-N87 cells.
[0318]
[0319] Cell killing dose-response profiles of free DM1-PEG9 cytotoxin on (A) U87 cells (B) SKBr3 cells
[0320]
[0321] Cell-killing dose-response profiles of antibody fragment ADCs scFv (TCT1067)-DM1-(dPEG12), DAR 3.5 (L), scFv (TCT1067)-DM1-(dPEG12) DAR 5.5 (M), scFv (TCT1067)-DM1-(dPEG12), DAR 8 (H) on (A) U87 cells (B) SKBr3 cells.
[0322]
[0323] Cell killing dose-response profiles of free MMAE cytotoxin on (A) U87 cells (B) SKBr3 cells
[0324]
[0325] Cell killing dose-response profiles of antibody fragment ADCs scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG9, DAR9 and Trastuzumab-MMAE-PAB-Cit-Val-dPEG9, DAR 4 on (A) U87 cells (B) BT474 cells (C) SKBr3 cells
[0326]
[0327] Cell killing dose-response profiles of free Auristatin cytotoxin on SKBr3 cells for (A) 4 and (B) 96 hours incubation
[0328]
[0329] Cell killing dose-response profiles of trastuzumab-Auristatin-C5 and scFv (1067)-Auristatin-C5 on SKBr3 cells for (A) 4 and (B) 96 hours incubation.
[0330]
[0331] (A) High affinity scFv (TCT1067)-P5C5 conjugate, (B) Medium affinity scFv (TCT)-P5C5 conjugate, (C) Trastuzumab-P5C5 conjugate (D) Saline administered control.
[0332]
[0333] A single i.v. dose was injected into female BALB/c mice at 5 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT)-MMAF-C5 (circles) (n=3), trastuzumab-MMAF-C5 (triangles) (n=3) and scFv (TCT) (squares) (n=4). Control scFv (TCT) values supplied from a separate PK study.
[0334]
[0335] A single i.v. dose was injected into female BALB/c mice at 5 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT1067)-MMAF-C5 (circles) (n=3), trastuzumab-MMAF-C5 (triangles) (n=3) and scFv (TCT1067) (squares) (n=3).
[0336]
[0337] A single i.v. dose was injected into female BALB/c mice at 5 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT)-P5C5 (circles) (n=3), trastuzumab-P5C5 (triangles) (n=3) and scFv (TCT) (squares) (n=4). Control scFv (TCT) and trastuzumab-P5C5 values supplied from a separate PK study.
[0338]
[0339] A single i.v. dose was injected into female BALB/c mice at 2 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT1067)-AF-C5 (circles) (n=3) and scFv (TCT1067) (squares) (n=3).
[0340]
[0341] A single i.v. dose was injected into female BALB/c mice at 2 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT1067)-DM1 (dPEG.sub.12) (circles) (n=3) and scFv (TCT1067) (squares) (n=3).
[0342]
[0343] (A) A single i.v. dose was injected into male Sprague-Dawley rats at 4 mg/kg. Plasma samples were taken at time points indicated and analysed by ELISA using anti-protein detection (total protein, indicated by solid lines, closed symbols) and where relevant anti-drug detection (total ADC, indicated by dashed lines, open symbols). The SE of the mean of each group and experimental triplicates are shown. ADC scFv (TCT1067)-P5C5 (circles) (n=3) and scFv (TCT1067) (squares) (n=3). (B) 10-fold concentrated urine collected over 24 hours analysed on a HER2-Biacore SPR chip for the scFv (TCT1067)-injected rats, 3 animal samples. The scFv reference is shown. The bulk shifts in the urine samples are due to the concentration of urine components. (C) 10-fold concentrated urine collected over 24 hours analysed on a HER2-Biacore SPR chip for the scFv (TCT1067)-P5C5 conjugate-injected rats, 3 animal samples. The scFv (TCT1067)-P5C5 reference is shown. The bulk shifts in the urine samples are due to the concentration of urine components.
[0344]
[0345] (A) Tumour volume against time (days) is plotted with 3 doses of scFv (TCT1067)-MMAF-C5 ADC (circles), 2 doses of trastuzumab-MMAF-C5 conjugate (triangles) and controls (squares). Each group consists of 6 animals and the SE of the mean is shown. Inset is a zoomed-in view of the first 30 days). The second plot is an enlargement of a portion of the first plot (shown by the boxed region). (B) The percentage change in body weight from the start of the treatment of the same groups in (A).
[0346]
[0347] (A) Tumour volume against time (days) is plotted with 2 doses of scFv (TCT1067)-MMAF-C5 ADC (circles), 2 doses of scFv (TCT)-MMAF-C5 ADC (crosses), 2 doses of trastuzumab-MMAF-C5 conjugate (triangles) and controls (squares). Each group consists of 6 animals and the SE of the mean is shown. (B) The percentage change in body weight from the start of the treatment of the same groups in (A).
[0348]
[0349] (A) Tumour volume against time (days) is plotted with one dosing regimen of scFv (TCT1067)-P5-C5 ADC (diamonds), one dosing regimen of scFv (TCT1067)-MMAF-C5 ADC (circle), one dosing regimen of trastuzumab-MMAF conjugate (triangles), one dosing regimen of trastuzumab-P5-C5 conjugate (diamonds) and controls (squares). Each group consists of 6 animals and the SE of the mean is shown. (B) The percentage change in body weight from the start of the treatment of the same groups in (A).
[0350]
[0351] (A) Tumour volume against time (days) is plotted for two therapeutic agents, scFv (TCT1067)-AuristatinF (L) Low DAR, 2.7 and. scFv (TCT1067)-AuristatinF (M) medium DAR, 5.7 and vehicle control. Each group consists of 6 animals and the SE of the mean is shown. (B) The percentage change in body weight from the start of the treatment of the same groups in (A).
[0352]
[0353] Tumour volume against time (days) is plotted for two therapeutic agents, scFv (TCT1067)-AuristatinF (L) Low DAR, 2.7 and. scFv (TCT1067)-AuristatinF (M) medium DAR, 5.7, scFv (TCT1067)-AuristatinF (H) High DAR, 11 and vehicle control. Each group consists of 6 animals and the SE of the mean is shown.
[0354]
[0355] Lanes 2-4 are purified antibody fragment (scFv) conjugate.
[0356] 1=unmodified scFv (TCT) stock;
[0357] 2=scFv (TCT) conjugate with 4 drug equivalent;
[0358] 3=TCT conjugate with 6 drug equivalent;
[0359] 4=scFv (TCT) conjugate with 16 drug equivalent.
[0360] Lanes 6-8 are antibody fragment conjugates before purification.
[0361] 6=scFv (TCT) conjugate with 4 drug;
[0362] 7=scFv (TCT) conjugate with 6 drug equivalent;
[0363] 8=scFv (TCT) conjugate with 16 drug equivalent.
[0364] Size markers used are shown.
[0365]
[0366] (A) is the LCMS trace (UV and TIC) and (B) is the deconvoluted mass for the main peak at 10.76 mins.
[0367]
[0368]
[0369]
COMMON FACTORS TO ALL EXAMPLES
[0370] All SDS-PAGE gels are reducing.
TABLE-US-00005 TABLE 3 Molar extinction coefficients used in examples Molar extinction Item coefficient ε280 nm for C6.5 andscFv (TCT) 65422 ε280 nm for HMFG1 IgG 210000 ε343 nm thione 8080 ε280 nm thione 5100 εDoxorubicin 488 nm 11294 εDoxorubicin 280 nm 8487 εDoxorubicin-PEG 488 nm 10218 εDoxorubicin-PEG 280 nm 14021 εEllipticine 429 nm 3603 εEllipticine 280 nm 22990 εEllipticine-PEG 429 nm 1810 εEllipticine-PEG 280 nm 8838
[0371] Synthetic Experimental Procedures
[0372] Experiments were generally carried out under inert atmosphere (nitrogen) especially in cases where oxygen- or moisture sensitive reagents or intermediates were employed unless otherwise stated. Commercial solvents and reagents were the best grade available and used without further purification. Anhydrous solvents were obtained from either Acros or Sigma-Aldrich. Reactions were followed by thin-layer chromatography (tic), LCMS or HPLC and purifications carried out by either Biotage automated chromatography using normal or reverse phase supports or by reverse phase HPLC. Reverse phase fractions from either the Biotage or HPLC were concentrated via lyophilisation/freeze-drying. Mass spectrometry data is reported from LCMS or by direct injection using electro-spray (ES) as ionisation mode unless otherwise stated. Chemical shifts for both proton and carbon nuclear magnetic resonance (NMR) are expressed as part per million (ppm) with the deuterated solvent as internal reference.
Example 1—Preparation of Cemadotin-NHS (2)
[0373] ##STR00001##
[0374] To a stirred solution of P5 (100 mg, 0.18 mmol) in DMF (5 mL), HATU (62 mg, 0.16 mmol) was added, followed by N,N-diisopropylethylamine (DIPEA) (63 μL, 0.36 mmol), and the resultant mixture was stirred at room temperature for 30 min. The reaction mixture was then added dropwise over 10 min to a slurry of 4-(aminomethyl)benzoic acid (30 mg, 0.20 mmol) in DMF (5 mL) and stirred at room temperature under nitrogen for 30 min, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 4 mL/min; 4 min 20% MeCN, 20-23% over 2 min, 23-25% over 14 min, 25-30% over 2 min, 30-80% over 3 min, 5 min 80% MeCN) collecting t.sub.R=9.96 min to give the title product 1 85 mg, (69%) as a white solid. HRMS (m/z) calculated for C.sub.36H.sub.57N.sub.6O 685.4289 [M+H] found 685.4307; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.86 (br. s, 1H), 9.62 (br. s, 1H), 8.92 (br. s, 1H), 8.40 (t, J=6.0 Hz, 1H), 7.87 (d, J=8.0 Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 6.55 (br. s, 1H), 4.99 (d, J=11.0 Hz, 1H), 4.61-4.51 (m, 2H), 4.42-4.24 (m, 3H), 3.77-3.63 (m, 3H), 3.60-3.51 (m, 2H), 3.09 (s, 3H), 2.78 (s, 3H), 2.75 (s, 3H), 2.32-2.22 (m, 1H), 2.20-1.87 (m, 8H), 1.86-1.69 (m, 3H), 1.00-0.93 (m, 6H), 0.88 (dd, J=11.8, 6.6 Hz, 6H), 0.71 (d, J=6.7 Hz, 3H) ppm.
[0375] To a stirred solution of cemadotin acid 1 (15 mg, 0.02 mmol) and DIPEA (16 μL 0.09 mmol) in DMF (2 mL) TSTU (12 mg, 0.04 mmol) was added and stirred at room temperature under nitrogen for 1 h, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 4 mL/min; 25-35% MeCN over 20 min, 35-80% over 5 min, 2 min at 80% MeCN) collecting t.sub.R=12.29 min to give the title product 2 13 mg, (76%) as a white solid; HRMS (ES) (m/z) calculated for C.sub.40H.sub.60N.sub.7O.sub.9 [M+H] 782.4453 found: 784.4449 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.64 (br. s, 1H), 9.18-9.13 (m, 1H), 8.93 (br. s, 1H), 8.39 (t, J=6.3 Hz, 1H), 8.07 (d, J=8.2 Hz, 2H), 7.85 (d, J=8.1 Hz, 2H), 7.58 (d, J=8.1 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 6.55 (br. s, 4H), 4.99 (d, J=10.9 Hz, 1H), 4.61 (d, J=5.9 Hz, 2H), 4.58-4.52 (m, 2H), 4.42-4.22 (m, 3H), 3.77-3.63 (m, 3H), 3.60-3.51 (m, 2H), 3.08 (s, 3H), 2.89 (s, 3H), 2.81-2.72 (br. d, 5H), 2.69-2.66 (m, 1H), 2.32-2.22 (m, 1H), 2.20-2.05 (m, 3H), 2.00-1.87 (m, 3H), 1.85-1.69 (m, 3H), 1.00-0.93 (m, 6H), 0.91-0.81 (m, 6H), 0.71 (d, J=6.7 Hz, 3H) ppm.
Example 2—Preparation of Cemadotin C.SUB.5.-NHS (4)
[0376] ##STR00002##
[0377] To a stirred solution of cemadotin acid 1 (20 mg, 0.03 mmol) in dry DMF (1.5 mL), HATU (10 mg, 0.03 mmol) was added, followed by DIPEA (10 μL, 0.06 mmol), and the resultant mixture was stirred at room temperature under nitrogen for 30 min. The reaction mixture was then added dropwise over 10 min to a slurry of 5-aminovaleric acid (3.8 mg, 0.03 mmol) in dry DMF (1.5 mL) and stirred at room temperature for 30 min, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 4 mL/min; 4 min 20% MeCN, 20-23% over 2 min, 23-25% over 14 min, 25-30% over 2 min, 30-80% over 3 min, 5 min 80% MeCN) collecting t.sub.R=12.18 min to give the title product 3 20 mg, (88%) as a white solid; HRMS (m/z) calculated for C.sub.41H.sub.66N.sub.7O.sub.8 [M+H] 784.4973 found: 784.4921; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.64 (br. s, 1H), 8.93 (br. s, 1H), 8.45 (t, J=5.8 Hz, 1H), 8.36 (t, J=6.1 Hz, 1H), 7.77 (d, J=7.8 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 6.56 (br. s, 2H), 4.99 (d, J=10.9 Hz, 1H), 4.60-4.51 (m, 2H), 4.42-4.30 (m, 2H), 4.28-4.20 (m, 1H), 3.77-3.62 (m, 3H), 3.60-3.52 (m, 2H), 3.28 (q, J=6.4 Hz, 3H), 3.09 (s, 3H), 2.81 (s, 3H), 2.80-2.70 (m, 3H), 2.35-2.22 (m, 2H), 2.18-1.88 (m, 7H), 1.85-1.58 (m, 7H), 1.29-1.22 (m, 2H), 0.99-0.93 (m, 5H), 0.90-0.81 (m, 8H), 0.71 (d, J=6.9 Hz, 2H) ppm.
[0378] To a stirred solution of cemadotin C5 3 (20 mg, 0.03 mmol) and DIPEA (10 μL 0.06 mmol) in dry DMF (2 mL), TSTU (14 mg, 0.05 mmol) was added, and the resultant mixture was stirred at room temperature under nitrogen for 1 h, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 3 mL/min; 25-35% MeCN over 20 min, 35-80% over 5 min, 8 min at 80% MeCN) collecting t.sub.R=12.13 min to give the title product 4 15 mg, (65%) as a white solid; MS (m/z) 881.5 [M+H]; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.64 (br. s, 1H), 8.93 (br. s, 1H), 8.45 (t, J=5.8 Hz, 1H), 8.36 (t, J=6.1 Hz, 1H), 7.77 (d, J=7.8 Hz, 2H), 7.30 (d, J=8.0 Hz, 2H), 6.56 (br. s, 2H), 4.99 (d, J=10.9 Hz, 1H), 4.60-4.51 (m, 2H), 4.42-4.30 (m, 2H), 4.28-4.20 (m, 1H), 3.77-3.62 (m, 3H), 3.60-3.52 (m, 2H), 3.28 (q, J=6.4 Hz, 3H), 3.09 (s, 3H), 2.81 (s, 3H), 2.80-2.70 (m, 7H), 2.35-2.22 (m, 2H), 2.18-1.88 (m, 7H), 1.85-1.58 (m, 7H), 1.29-1.22 (m, 2H), 0.99-0.93 (m, 5H), 0.90-0.81 (m, 8H), 0.71 (d, J=6.9 Hz, 2H) ppm.
Example 3—Preparation of P5-C.SUB.5.-NHS (6)
[0379] ##STR00003##
[0380] To a stirred solution of P5 (100 mg, 0.18 mmol) in dry DMF (5 mL), HATU (62 mg, 0.16 mmol) was added, followed by DIPEA (63 μL, 0.36 mmol), and the resultant mixture was stirred at room temperature under nitrogen for 30 min. The reaction mixture was then added dropwise over 10 min to a slurry of 5-aminovaleric acid (23 mg, 0.20 mmol) in dry DMF (5 mL) and stirred at room temperature for 30 min, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 4 mL/min; 4 min 10% MeCN, 10-20% over 4 min, 20-30% over 8 min, 2 min 30% MeCN) collecting t.sub.R=13.58 min to give the title product 5 93 mg, (79%) as a white solid; MS (m/z) 651.4 [M+H]; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.59 (br. s, 1H), 8.92 (d, J=7.7 Hz, 1H), 7.78 (t, J=5.8 HZ, 0.6H), 7.73 (t, J=5.8 Hz, 0.4H), 4.97 (d, J=11.0 Hz, 1H), 4.57 (t, J=8.2 Hz, 1H), 4.51 (dd, J=8.4, 5.2 Hz, 1H), 4.23 (dd, J=8.4, 3.7 Hz, 1H), 3.75-3.68 (m, 3H), 3.66-3.59 (m, 1H), 3.57-3.50 (m, 2H), 3.25-3.11 (m, 1H), 3.08 (s, 3H), 3.01-2.89 (m, 1H), 2.77 (dd, J=14.2, 4.2 Hz, 6H), 2.67 (t, J=7.3 Hz, 1H), 2.60 (s, 1H), 2.32-2.24 (m, 1H), 2.22-2.09 (m, 3H), 2.06-1.99 (m, 2H), 1.96-1.86 (m, 3H), 1.84-1.68 (m, 3H), 1.64-1.56 (m, 2H), 1.50-1.43 (m, 2H), 0.96 (dd, J=9.4, 6.6 Hz, 6H), 0.88-0.82 (m, 9H), 0.71 (d, J=6.6 Hz, 3H) ppm.
[0381] To a stirred solution of P5C5 5 (93 mg, 0.14 mmol) and DIPEA (58 μL 0.33 mmol) in dry DMF (10 mL), TSTU (76 mg, 0.25 mmol) was added, and the resultant mixture was stirred at room temperature under nitrogen for 1 h, concentrated under reduced pressure, and purified by preparative HPLC (MeCN in H.sub.2O [0.1% TFA]; 4 mL/min; 4 min 15% MeCN, 15-30% over 8 min, 5 min at 30%, 30-40% over 2 min, 3 min at 40% MeCN) collecting t.sub.R=13.29 min to give the title product 6 78 mg, (73%) as a white solid; MS (m/z) 748.4 [M+H]; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.59 (br. s, 1H), 8.92 (d, J=7.7 Hz, 1H), 7.78 (t, J=5.8 HZ, 0.6H), 7.73 (t, J=5.8 Hz, 0.4H), 4.97 (d, J=11.0 Hz, 1H), 4.57 (t, J=8.2 Hz, 1H), 4.51 (dd, J=8.4, 5.2 Hz, 1H), 4.23 (dd, J=8.4, 3.7 Hz, 1H), 3.75-3.68 (m, 3H), 3.66-3.59 (m, 1H), 3.57-3.50 (m, 2H), 3.25-3.11 (m, 1H), 3.08 (s, 3H), 3.01-2.89 (m, 1H), 2.82 (s, 3H), 2.77 (dd, J=14.2, 4.2 Hz, 6H), 2.67 (t, J=7.3 Hz, 1H), 2.60 (s, 1H), 2.32-2.24 (m, 1H), 2.22-2.09 (m, 3H), 2.06-1.99 (m, 2H), 1.96-1.86 (m, 3H), 1.84-1.68 (m, 3H), 1.64-1.56 (m, 2H), 1.50-1.43 (m, 2H), 0.96 (dd, J=9.4, 6.6 Hz, 6H), 0.88-0.82 (m, 9H), 0.71 (d, J=6.6 Hz, 3H) ppm.
Example 4—Preparation of Doxorubicin-dPEG.SUB.(7).-NHS: (7)
[0382] ##STR00004##
[0383] To a stirred suspension of doxorubicin.HCl (15 mg, 0.026 mmol) in dry DMF (2 ml) DIPEA (22.5 μl, 0.013 mmol) was added and stirred under nitrogen for 30 min. resulting in a clear dark-red solution. This was taken up in a 5 ml syringe and added dropwise over 20 min. to a stirred solution of the bis-dPEG.sub.7-NHS (24.1 mg, 0.039 mmol) and DIPEA (22.5 μl, 0.13 mmol) in dry DMF (2 ml). The resulting solution was then stirred at room temperature under nitrogen for 3 h and evaporated under high vacuum to give a dark red-orange oil. This was purified by flash chromatography [silica gel: 10% MeOH/DCM] and the appropriate fractions (R.sub.f 0.38) collected, combined and evaporated to give the title product 7 10.4 mg (39%) as a red-orange viscous oil; MS (m/z) calculated for C.sub.49H.sub.64N.sub.2O.sub.23Na 1071.3798 (M+Na) found 1071.3805.
Example 5—Preparation of Doxorubicin-dPEG.SUB.8.-NHS Ester (10)
[0384] ##STR00005##
[0385] Doxorubicin hydrochloride (94 mg, 0.161 mmol) was dissolved in anhydrous DMF (10 mL) and DIPEA (89 μl, 0.483 mmol) was added. The mixture was stirred for 10 min, after which NHS-PEG.sub.8-N.sub.3 (100 mg, 0.177 mmol) was added followed by stirring for 18 h at room temperature under nitrogen in the dark. The reaction mixture was evaporated under vacuum and purified by flash chromatography [silica gel: 5% MeOH/DCM] To give the desired Dox-dPEG.sub.8-azide 8 as a red oil 121 mg, (76%). (R.sub.f 0.395, 5% MeOH/DCM); MS (m/z): 1010.44 [M.sup.++NH4], 1015.39 [M.sup.++Na], 1031.37 [M.sup.++K], .sup.1H NMR (CDCl.sub.3): δ 14.00 (1H, s, 6-OH), 13.31 (1H, s, 11-OH), 8.08 (1H, d, J=8 Hz, 3-H), 7.84 (1H, t, J=8 Hz, 2-H), 7.43 (1H, d, J=8 Hz, 1-H), 5.53 (1H, d, J=4 Hz, c-OH), 5.33 (1H, s, 1′-OH), 4.79 (2H, s, 14-H), 4.19-4.11 (5H, m, CH3-O—, 5′-H, 7-H), 3.71-3.64 (33H, m, 3′-H, —CH.sub.2—O—(CH.sub.2—CH.sub.2—O).sub.7—CH.sub.2—), 3.42 (2H, t, J=8 Hz, —CH.sub.2—N.sub.3), 3.34-3.05 (2H, q, J=20 Hz, 10-H), 2.46-2.16 (3H, m, 4′-H, b-H, d-H), 1.94-1.77 (4H, m, 2′-H, 8′-H), 1.31 (3H, d, J=8 Hz, 6′-H).
[0386] To a solution of Dox-dPEG.sub.8-azide 8 (120 mg, 0.121 mmol) in 2.5 mL of tert-butanol/water (1:1 v:v) a solution of 5-hexynoic acid (14 mg, 0.121 mmol) in 2.5 mL of tert-butanol/water (1:1) was added. The reaction was stirred at room temperature for 30 min, followed by addition of copper (II) sulfate (2 mg, 0.012 mmol) and (+)-sodium L-ascorbate (5 mg, 0.024 mmol). The reaction was warmed to 40° C. and stirred for 24 h. The reaction mixture was then diluted with DCM (15 mL) and a solution of citric acid added until pH 4 was reached. The organic layer was then washed with brine (2×10 mL) and the aqueous layers combined and back-extracted with DCM (4×10 mL). The organic fractions were combined, dried over sodium sulfate, filtered, and concentrated to give a dark red residue. This was purified flash chromatography [silica gel: DCM increasing upto 20% MeOH/DCM) to give the product 9 as a red solid 53.4 mg, (40%). (R.sub.f0.20, 10% MeOH/DCM); MS (m/z): 1106.05 [M.sup.++H], 1128.00 [M.sup.++Na .sup.1H NMR (CDCl.sub.3): δ 14.01 (1H, s, 6-0H), 13.33 (1H, s, 11-OH), 8.08 (1H, d, J=8 Hz, 3-H), 7.82 (1H, t, J=8 Hz, 2-H), 7.64 (1H, s, —N—CH═CN—), 7.44 (1H, d, J=8 Hz, 1-H), 5.55 (1H, d, J=4 Hz, c-OH), 5.33 (1H, s, 1′-OH), 4.81 (2H, s, 14-H), 4.55 (2H, t, J=4 Hz, —CH═CN—CH.sub.2—), 4.16-4.11 (5H, m, CH.sub.3—O—, 5′-H, 7-H), 3.86 (2H, t, J=8 Hz, ˜O—CH.sub.2—CH.sub.2—CN—), 3.70-3.62 (33H, m, 3′-H, —(CH.sub.2—CH.sub.2—O).sub.5—CH.sub.2—), 3.35-3.06 (2H, q, J=20 Hz, 10-H), 2.83 (2H, t, J=4 Hz, —CH.sub.2—COOH), 2.47-2.16 (2H, m, b-H, d-H), 2.07-2.02 (3H, m, 2′-H, 4′-H), 1.83-1.79 (2H, m, 8-H), 1.36-1.28 (5H, m, 6′-H, —CH.sub.2—CH.sub.2—CH.sub.2-000H).]. A solution of the Dox-dPEG.sub.8 acid is stirred in dry DMF with TSTU and DIPEA for 1 h. The solvent is taken off using gigh vacuum and the residue purified by reverse phase HPLC to give the NHS ester derivative 10.
Example 6—Preparation of Doxorubicin-dPEG.SUB.12.-SPDP (11)
[0387] ##STR00006##
[0388] To a stirred suspension of Dox.HCl (10 mg, 0.0172 mmol) in dry DMF (2 ml) DIPEA (7.7 μl) was added and the reaction mixture stirred for 10 min. under nitrogen to give a clear red solution. To this, SPDP-dPEG.sub.12-NHS ester (17.3 mg, 0.044 mmol) dissolved in dry DMF (1 ml) was added and the reaction stirred at room temperature, under nitrogen and protected from light overnight. The DMF was removed by high vacuum and the dark red oil purified by flash chromatography [silica gel: 10% MeOH/DCM R.sub.f 0.36] to give the desired product 11 16.2 mg (70%) as a red viscous oil; HRMS (m/z) calculated for C.sub.62H.sub.85N.sub.8O.sub.21S.sub.2 [M+H] 1341.5271 found: 1341.5380.
Example 7—Preparation of Doxorubicin-SMCC (12)
[0389] ##STR00007##
[0390] To a suspension of Dox.HCl (0.05 g, 0.086 mmol) in dry DMF (10 ml) SMCC cross-linker (0.0346 g, 0.104 mmol) and DIPEA (22.5 μl, 0.129 mmol) were added and the reaction stirred at room temperature for 12 h under nitrogen shielded from light. The suspension goes into solution within 1 h. The solvent was taken off under high vacuum at 35° C. to give a dark-red residue. This was taken up in DCM (50 ml), washed with brine, dried over MgSO.sub.4, filtered and evaporated to give a dark-red solid. This was purified by flash column chromatography [silica gel: 1-5% MeOH/DCM, R.sub.f 0.25] to give 12 as a orange-red solid 0.053 g (78%). MS (m/z) found 785.25 (M+Na) calculated for C.sub.39H.sub.42N.sub.2O.sub.14Na
Example 8—Preparation of Doxorubicin-PAB-Cit-Val-dPEG.SUB.7.-NHS Ester (16)
[0391] ##STR00008##
[0392] To a suspension of Dox.HCl (25 mg, 0.043 mmol) and Fmoc-Val-Cit-PNP 13 (30 mg, 0.039 mmol) in dry DMF (1 ml) DIPEA (7.5 μl, 0.043 mmol) was added, resulting in a dark-red solution. This was stirred at room temperature under nitrogen for 24 h after which the solvent was evaporated under high vacuum and the residue triturated with dry diethyl ether to give a red solid R.sub.f 0.22 [silica gel: 10% MeOH/DCM]. Purification by flash chromatography [silica gel: 5% MeOH/DCM] gave the desired product 14 as a red solid 25.2 mg (55%); HRMS (m/z) calculated for C.sub.61H.sub.67N.sub.6O.sub.18 [M+H] 1171.4512 found: 1171.4534.
[0393] To a stirred solution of 14 (20 mg, 0.017 mmol) in dry DCM (5 ml) piperidine (10 mol %) was added. The bright red predominantly in solution mixture immediately became a dark brown clear solution and was stirred for 10 min. after which all the solvent was taken off to give the desired compound 15 a red sticky solid. HRMS (m/z) calculated for C.sub.46H.sub.57N.sub.6O.sub.16 [M+H] 949.3831 found: 949.3874. This was used without further purification in the preparation of 16.
[0394] A solution of compound 15 in dry DMF is added dropwise over 20 min. to a stirred solution of bis-dPEG.sub.7-NHS and DIPEA (22.5 μl, 0.13 mmol) in dry DMF. The resulting solution is then stirred at room temperature under nitrogen for 3 h and evaporated under high vacuum to give a dark red-orange oil. This is purified by flash chromatography [silica gel: 10% MeOH/DCM] and the appropriate fractions collected, combined and evaporated to give the title product 16 as a red-orange viscous oil.
Example 9—Preparation of Camptothecin-dPEG.SUB.3.-NHS Ester (19)
[0395] ##STR00009##
[0396] To a stirred solution of camptothecin (400.0 mg, 1.1 mmol) in dry DCM (100 ml) were subsequently added 5-hexynoic acid (319.8 mg, 2.9 mmol), EDC (437.1 mg, 2.28 mmol) and DMAP (139.4 mg, 1.14 mmol). The yellow suspension was left stirring at RT under N.sub.2 and in the dark for 16 hours. The resulting light brown solution was washed with H.sub.2O (120 ml) and extracted with DCM (100 mL). Organic phases were combined, washed with brine (100 mL), dried over MgSO.sub.4 and concentrated in vacuo. The crude was purified by flash chromatography [silica gel: with a 1-3% MeOH/DCM gradient] to give the camptothecin alkyne 17 as an off-white/yellow powder 471.1 mg, (91.6%); HRMS (m/z): calculated for C.sub.26H.sub.22N.sub.2O.sub.5 443.1623 [M+H], found 443.1607. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=8.43 (s, 1H), 8.25 (d, J=8.5 Hz, 1H), 7.97 (d, J=8.3 Hz, 1H), 7.86 (ddd, J=8.5, 6.8, 1.5 Hz, 1H), 7.70 (t, J=7.8 Hz, 1H), 7.28 (s, 1H), 5.71 (d, J=17.3 Hz, 1H), 5.43 (d, J=17.2 Hz, 1H), 5.32 (s, 2H), 2.78-2.59 (m, 2H), 2.35-2.28 (m, 3H), 2.18 (dq, J=13.6, 7.5 Hz, 1H), 2.05 (t, J=2.6 Hz, 1H), 1.90 (p, 7.2 Hz, 2H), 1.01 (t, J=7.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=172.1, 167.5, 157.4, 152.4, 148.9, 146.2, 146.0, 131.2, 130.70, 129.6, 128.5, 128.2, 128.1, 120.3, 96.0, 83.0, 75.9, 69.5, 67.1, 49.9, 32.4, 31.9, 23.2, 17.7, 7.6; IR λ.sub.max: 3302.5, 2984.3, 2943.6, 1753.6, 1737.3, 1669.3, 1624.3, 1564.1, 1446.8, 1405.6, 1365.6, 1351.3, 1296.6, 1234.1, 1205.3, 1166.5, 1131.8, 1088.4, 1045.3, 1011.2, 976.5, 946.6, 909.3, 825.4, 786.2, 762.2, 722.4, 652.0;
[0397] To a stirred solution of the camptothecin alkyne 17 (60 mg, 0.136 mmol) in 15 ml 1:2 H.sub.2O: tert-butanol were subsequently added 63.2 mg azido-PEG.sub.3-acid (0.271 mmol, 2 eq), 2.7 mg Na ascorbate (0.0136 mmol, 0.1 eq) and 2.2 mg CuSO.sub.4 (0.0136 mmol, 0.1 eq). The white suspension was stirred at 80° C. under N.sub.2 and in the dark for 5 h. After the clear solution was cooled down to RT, 15 ml DCM and 15 ml distilled H.sub.2O were added and the organic layer was separated. The obtained organics were washed with 25 ml 0.5M HCl and 25 ml 1:1 1M HCl:brine (organic layer becomes a fluorescent yellow), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude was purified by flash chromatography [silica ge:l 10-15% MeOH/DCM gradient, followed by 0.1% formic acid 10% MeOH/DCM. The appropriate fractions were combined, concentrated in vacuo and washed with hot ether under reflux for 1 hour. A yellow, sticky solid was obtained as the desired product 18 71 mg (78.4%); HRMS (m/z): 676.2644 [M+H], calculated mass 676.2669. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=8.44 (s, 1H, 5-CH-aromatic), 8.28 (d, J=8.6 Hz, 1H, 4-CH-aromatic), 7.97 (d, J=8.2 Hz, 1H, 1-CH-aromatic), 7.87 (t, J=7.7 Hz, 1H, 3-CH-aromatic), 7.70 (t, J=7.6 Hz, 1H, 2-CH-aromatic), 7.30 (s, 1H, 7-CH-aromatic), 5.71 (d, J=17.2 Hz, 1H, 8-CH2-O), 5.44 (d, J=17.2 Hz, 1H, 8-CH2-O), 5.33 (s, 2H, 6-CH2-N), 4.52 (td, J=4.8, 1.9 Hz, 2H), 4.18 (s, 2H), 3.86 (t, J=5.2 Hz, 2H), 3.75 (dd, J=5.8, 3.1 Hz, 2H), 3.68-3.57 (m, 6H), 2.82 (t, J=7.4 Hz, 3H), 2.68-2.49 (m, 3H), 2.31 (dq, J=14.8, 7.4 Hz, 1H), 2.18 (dq, J=14.7, 7.4 Hz, 1H), 2.06 (p, J=7.4 Hz, 2H), 1.01 (t, J=7.5 Hz, 3H, 10-CH3); .sup.13C NMR (100 MHz, CDCl.sub.3): δ=172.39, 167.73, 157.40, 152.23, 146.08, 131.52, 130.86, 129.36, 128.56, 128.23, 122.57, 120.29, 96.37, 75.82, 70.39, 69.50, 67.13, 50.12, 49.99, 32.84, 31.81, 24.43, 24.24, 7.59; IR cm.sup.−1: 3422.20, 2913.21, 1745.90, 1664.85, 1616.45, 1562.85, 1501.82, 1457.03, 1404.63, 1352.03, 1299.22, 1231.80, 1132.95, 1088.07, 1048.25, 994.41, 947.36, 815.25, 787.33, 763.06, 724.93.
[0398] To a stirred solution of the camptothecin acid 18 acid (10 mg) were added 160.3 mg disuccinimidyl carbonate (DSC) (0.64 mmols) and 24 mg triethylamine (0.24 mmols) in dry DMF (3 ml). The yellow solution was left stirring at RT, under N.sub.2 and in the dark. Further addition of 160.4 mg DSC (43 eq) and 24.1 mg triethylamine was carried out after 16 hours. After 6 more hours, the reaction was stopped and concentrated in vacuo to give an orange oil. This was redissolved in 15 ml DCM, washed with 15 ml 0.5M HCl and 15 ml brine and dried over Na.sub.2SO.sub.4. This work-up procedure was repeated twice, and one last wash was performed with 2×5 ml H.sub.2O and 5 ml brine. The dried organics were filtered and concentrated in vacuo and lyophilized to give the desired compound 19 as a white hygroscopic powder 9 mg. MS (m/z) 773.2832 (M+1), 796.2639 (M+Na)
Example 10—Preparation of Ellipticine-C.SUB.6.-NHS Ester (21)
[0399] ##STR00010##
[0400] To a solution of ellipticine (35 mg, 0.14 mmol) in dry DMF (5 ml), 6-bromohexanoic (55.4 mg, 0.284 mmol) acid was added and the reaction mixture stirred at 120° C. for 4 hr and then at room temperature for a further 12 hr to give a mustards-yellow precipitate. This was filtered and washed with cold anhydrous ether. Some precipitation was also observed in the filtrate which was also collected. The total combined yield obtained of compound 20 was 49.1 mg (78%). Analysis by TLC [silica gel: MeCN:Water: KNO3 (satd.)] showed the product to be a single yellow spot (Rf 0.55, Ellipticine Rf 0.67). HRMS (m/z) calculated for C.sub.23H.sub.25N.sub.2O.sub.2 361.1916 (M+1) found 361.1924.
[0401] To a partial suspension of the acid 20 (10 mg, 0.023 mmol) in dry DMF (1.5 ml), TSTU (12 mg, 0.04 mmol) followed by DIPEA (16.2 μl, 0.093 mmol) were added and the reaction mixture stirred for 1 h at room temperature under nitrogen. Over the course of the reaction the suspension slowly gave way to a clear mustard-yellow coloured solution. The reaction was followed by TLC [silica gel: MeCN:Water:KNO3 (satd.)] and once complete, the DMF was taken off using high vacuum keeping the temperature below 30° C. The residue was triturated with anhydrous ether and air dried to give the ester 21 as a mustard-yellow coloured solid; HRMS (m/z) calculated for C.sub.27H.sub.28BrN.sub.3O.sub.4
Example 11—Preparation of Ellipticine-PEG.SUB.4.-NHS Ester (23)
[0402] ##STR00011##
[0403] To a stirred solution of ellipticine (0.035 g, 0.142 mmol) in dry DMF (4 ml) under nitrogen, Br-PEG.sub.4-acid (0.0936 g, 0.0284 mmol) dissolved in dry DMF (1 ml) was added. The reaction was stirred at 120° C. for 4 h, allowed to cool and stirred at room temperature for a further 12 h. The DMF was taken off using high vacuum and the residue purified by preparative HPLC [Chromolith HighResolution RP-18e 100×4.6 mm] 100% 10 mM Na.sub.3PO.sub.4/pH7 to 100% MeCN over 27 mins step gradient at 20° C., detecting at 280 and 435 nm collecting t.sub.R 7.9 min to give the acid 22 as a yellow hygroscopic solid 40.6 mg (50%); HRMS (m/z) calculated for C.sub.25H.sub.35N.sub.2O.sub.6 M-Br) 495.2495 found 495.2498.
[0404] The ellipticine-PEG.sub.4-acid 22 (0.0143 g, 0.00256 mmol) was dissolved in dry DMSO (1 ml) and stirred under nitrogen. To this, TSTU (0.0136 g, 0.00451 mmol) was added followed by DIPEA (18.5 μl, 0.105 mmol) and the bright yellow solution was stirred at room temperature under nitrogen for 1 h. The solvent was taken under high vacuum and the sticky residue triturated with dry ether and after decanting the ether, dried under high vacuum to give 23 as a yellow sticky solid 11.4 mg (66%); HRMS (m/z) calculated for C.sub.32H.sub.38N.sub.3O (M-Br) 592.2659 found 592.2643.
Example 12—Preparation of Ellipticine-PAB-Cit-Val-dPEG.SUB.3.-NHS Ester (29)
[0405] ##STR00012## ##STR00013##
[0406] To a stirred solution of Cit-Val-PAB-OH (24) (0.10 g, 0.43 mmol) in dry DMF (8 mL), 11-Azido-3,6,9-trioxaundecanoic acid (0.16 g, 0.43 mmol) in dry DMF (1 mL) was added. EEDQ (2-Ethoxy-1-Ethoxycarbonyl-1,2-dihydro quinoline (100 mg, 0.5 mmol) was then added and the solution was stirred at room temperature under nitrogen overnight. The solvent was removed in vacuo and purified by flash chromatography [silica gel:10% MeOH/DCM] to yield the product 25 (0.21 g (82%) as a white solid. mp 139° C.; HRMS (m/z) calculated for C.sub.26H.sub.42N.sub.8O.sub.8 617.3023 [M+Na]. Found 617.2999, IR 3270, 2925, 2103, 1629, 1538, 1272, 1094, 799 cm.sup.−1; .sup.1H NMR (400 MHz, MeOD) δ 7.54 (m, 2H), 7.29 (d, J=8.7 Hz, 2H), 4.43-4.52 (m, 2H), 4.30 (d, J=7.2 Hz, 2H), 4.05 (s, 2H), 3.59-3.77 (m, 10H), 3.30 (m, 2H), 3.04-3.25 (m, 2H), 2.04-2.17 (m, 1H), 1.67-1.95 (m, 2H), 1.57 (m, 2H), 0.97 (m, 6H); NMR (100 MHz, DMSO-d6) δ 17.9, 19.2, 26.8, 29.3, 31.1, 39.6, 49.9, 53.2, 56.5, 62.6, 69.2, 69.6, 69.6, 69.7, 69.8, 70.3, 118.8, 126.9, 17.5, 158.9, 170.3, 170.7;
[0407] To a stirred solution of the PEG.sub.3 azide linker 25 in dry CH.sub.2Cl.sub.2 (60 mg in 2 mL), HBr (33% in AcOH, 1M, 0.04 mL) was added in a dropwise. After 10 min, the flask was put on ice, NaHCO.sub.3 was than added slowly, and the solution was stirred for 30 min. After stirring, the solution was filtered, washed with water and diethyl ether and dried in vacuo to yield the benzyl bromide derivative 26 (20 mg (33%) as a cream solid; HRMS (m/z) calculated for C.sub.26H.sub.42N.sub.8O.sub.7Br 657.2360 (M+1). Found 657.2357. .sup.1H NMR (400 MHz, MeOD) δ 7.63-7.50 (m, 2H), 7.43-7.21 (m, 2H), 4.60-4.47 (m, 2H), 4.33-4.24 (m, 1H), 4.06 (s, 2H), 3.88-3.60 (m, 10H), 3.52 (s, 2H), 3.30-3.1 (m, 2H), 2.20-2.07 (m, 1H), 2.00-1.72 (m, 2H), 1.72-1.54 (m, 2H), 1.09-0.90 (m, 6H);
[0408] 9-Hydroxyellipticine (10 mg, 0.04 mmol) and K.sub.2CO.sub.3 (0.12 g, 0.08 mmol) were dissolved in dry DMF (4 mL) and stirred for 5 min. The brominated linker 26 (30 mg, 0.04 mmol) was added as a solution in dry DMF and the mixture was stirred at room temperature for 17 hours. A black solid was obtained after concentration in vacuo, which was then dissolved in CHCl.sub.3:MeOH 9:1, washed with water, dried, and concentrated to give the alkylated ellipticine derivative 27 24 mg (76%) as a dark brown solid; MS (m/z) 840 [M].sup.+; HRMS (m/z) calculated for C.sub.43H.sub.55N.sub.10O.sub.8 839.4204. Found 839.4202. IR 3272, 2937, 2107, 1646, 1526, 1462, 1415, 1254, 1103, 807 cm-1; .sup.1H NMR (400 MHz, MeOD) δ 8.38-8.10 (m, 1H), 8.01 (s, 1H), 7.99-7.81 (m, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.68-7.48 (m, 2H), 7.48-7.32 (m, 1H), 7.30-6.98 (m, 1H), 5.84 (s, OH), 4.54 (dd, J=1.5, 8.9 Hz, 1H), 4.31 (q, J=7.8 Hz, 1H), 4.08 (d, J=9.3 Hz, 2H), 3.92-3.46 (m, 9H), 3.35 (d, J=17.3 Hz, 12H), 3.17 (d, J=18.8 Hz, 3H), 3.02 (s, 3H), 2.89 (s, 3H), 2.70 (d, J=7.6 Hz, 2H), 1.90 (s, 1H), 1.78 (s, 1H), 1.60 (s, 2H), 1.06-0.86 (m, 6H);
[0409] The azide ellipticine derivative 28 undergoes 1,3 cycloaddition with hexynoic-acid under ‘click’ conditions using Cu(II)SO.sub.4 and ascorbic acid to give the derivative with a carboxylic acid 28. Activating this terminal carboxylic acid of derivative 28 with TSTU and DIPEA in dry DMF will give the activated succinimidyl ester derivative 29.
Example 13—Preparation of Ellipticine-N-PAB-Cit-Val-dPEG.SUB.3.-NHS Ester (33)
[0410] ##STR00014## ##STR00015##
Example 14—Preparation of N-Ellipticinepentyl Amine (36)
[0411] ##STR00016##
[0412] Sodium azide (0.6 g, 9.6 mmol) was dissolved in DMF (20 mL) and 1,5-dibromopentane (1.2 mL, 8.7 mmol) was added. The mixture was heated to 50° C. overnight with a blast shield in place. The solution was cooled to 0° C. and water (20 mL) was added. The mixture was then extracted with EtOAc (3×20 mL), washed with water (20 mL) and brine (20 mL), dried over Na.sub.2SO.sub.4, and concentrated to form an oil that was purified by column chromatography with n-hexane to yield 34 (1.5 g, 90%) as a clear oil. Azide staining reagent was used to follow the azide, and iodine visualization was used to stain for the starting dibromopentane, the first compound off the column. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.44 (t, J=6.7 Hz, 2H), 3.32 (t, J=6.7 Hz, 2H), 1.92 (p, J=7.0 Hz, 2H), 1.69-1.61 (m, 2H), 1.61-1.51 (m, 2H).
[0413] Ellipticine (50 mg g, 0.2 mmol) was added to 1-azido-5-bromopentane 34 (80 mg, 0.4 mmol) in DMF (10 mL) and heated to 120° C. for 4 hours, followed by stirring at room temperature for three days. The orange suspension was treated with ether (10 mL) and filtered to give the quaternised ellipticine derivative 35 64 mg (90%) as a yellow solid. m.p. decomposed without melting >150° C. IR 3065, 2088, 1598, 1578, 1463, 1420, 1401, 1154, 744, 716, 606 cm.sup.−1; .sup.1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 10.09 (s, 1H), 8.62-8.52 (m, 1H), 8.44 (dd, J=11.7, 7.6 Hz, 2H), 7.71-7.57 (m, 2H), 7.37 (s, 1H), 4.72 (t, J=7.5 Hz, 2H), 3.37 (m, 4H), 3.30 (s, 3H), 2.84 (s, 3H), 1.62 (p, J=7.0 Hz, 2H), 1.51-1.28 (m, 2H); .sup.1C NMR (100 MHz, DMSO-d6) δ 206.5, 146.5, 144.3, 142.6, 13.3, 12.5, 10.8, 128.7, 125.6, 124.4, 122.1, 120.8, 120.2, 111.6, 110.45, 59.2, 50.4, 30.4, 27.7, 22.9, 15.1, 12.1; MS (ES+) m/z 358 [M].sup.+; HRMS (m/z) mass calculated for C.sub.22H.sub.24N.sub.5 358.2032. Found 358.2036.
[0414] The Ellipticine azide 35 (10 mg, 0.036 mmol) was dissolved in methanol (2 mL). Pd/C was added and a hydrogen balloon was attached to the stirring solution. After 6.5 hours the reaction mixture was filtered through celite and concentrated in vacuo to give the ellipticine amine 36 (7.0 mg, 58%) as bright orange crystals. Importantly, reduction of the pyridine ring can occur when left under hydrogen overnight so careful monitoring by TLC (MeCN:H.sub.2O:KNO.sub.3 8:1:1) is required. m.p. decomposed without melting >150° C. IR 2934, 1598, 1578, 1419, 1244, 1176, 747, 628 cm.sup.−1; .sup.1H NMR (400 MHz, MeOD) δ 9.86 (d, J=1.1 Hz, 1H), 8.38-8.27 (m, 3H), 7.64-7.54 (m, 2H), 7.36 (ddd, J=8.0, 6.5, 1.7 Hz, 1H), 4.81-4.71 (m, 2H), 3.25 (s, 3H), 3.00-2.92 (m, 2H), 2.78 (s, 3H), 2.18 (ddd, J=12.2, 10.2, 6.8 Hz, 2H), 1.83-1.72 (m, 2H), 1.59 (m, 2H); .sup.13C NMR (100 MHz, MeOD) δ 146.5, 144.3, 142.6, 133.3, 132.5, 130.8, 128.7, 125.9, 124.4, 122.1, 120.8, 120.5, 120.1, 111.6, 110.4, 59.2, 48.5, 40.1, 39.9, 39.7, 39.4, 39.2, 39.0, 38.8, 30.3, 26.9, 22.6, 15.2, 15.1, 12.0; MS (m/z) 332 [M].sup.+; HRMS (m/z) calculated for C.sub.22H.sub.26N.sub.3 322.2127. Found 332.2123.
[0415] The PEG.sub.3 azide linker 25 (20 mg, 0.03 mmol) and bisnitrophenyl carbonate (30 mg, 0.10 mmol) were dissolved in DMF (2 mL). DIPEA (0.1 mL, 0.07 mmol) was added and the solution was heated to 50° C. for 3 hours. The DMF was removed in vacuo, water was added, and the product was extracted with CH.sub.2Cl.sub.2:MeOH 9:1, before being dried and concentrated to yield activated p-nitrophenyl derivative 30 20 mg (74%) as a dark yellow oil. IR 1652, 1590, 1516, 1498, 134, 1288, 1216, 1109, 850, 753, 629 cm.sup.−1; .sup.1H NMR (400 MHz, MeOD) 8.30-8.39 (m, 2H), 7.61-7.71 (m, 2H), 7.40-7.53 (m, 4H), 5.28 (s, 2H), 4.50-4.61 (m, 1H), 4.33 (d, J=7.1 Hz, 1H), 4.09 (s, 2H), 3.64-3.79 (m, 10H), 3.22-3.14 (m, 1H), 2.16 (h, J=6.9 Hz, 1H), 1.79-1.92 (m, 2H), 8.6 Hz, 2H), 1.60 (m, 6H); MS (ES+) 782 [M+Na]; .sup.13C NMR (100 MHz, MeOD) δ 172.0, 171.3, 171.0, 163.8, 163.8, 161.1, 155.8, 152.6, 145.3, 140.4, 130.6, 129.2, 125.7, 121.9, 119.8, 115.1, 70.8, 70.3, 70.2, 70.1, 69.7, 50.4, 48.3, 48.1, 47.8, 47.6, 47.4, 47.2, 47.1, 30.9, 18.4, 17.4; HRMS (m/z) calculated for C.sub.33H.sub.45N.sub.9O.sub.12 782.3170 [M+Na].sup.+. Found 782.3173.
[0416] The ellipticine amine 36 (11 mg, 0.03 mmol) and the activated linker 30 (24 mg, 0.03 mmol) were dissolved in dry DMF. DIPEA (6 μL, added via Gilson pipette, 0.035 mmol) was added and the reaction mixture stirred in the dark at room temperature for 24 hours. The product was precipitated by addition of diethyl ether and centrifuged. The supernatant was removed and the resulting solid was washed with diethyl ether and dried to give the ellipticine linker derivative 31 10 mg, (36%) as a yellow solid. MS (ES+) m/z 952 [M].sup.+; HRMS calculated for C.sub.49H.sub.66N.sub.11 952.5045. Found 952.4993.
[0417] The azide ellipticine derivative 31 undergoes 1,3 cycloaddition with hexynoic-acid under ‘click’ conditions using Cu(II)SO.sub.4 and ascorbic acid to give the derivative 32. Activating the terminal carboxylic acid of derivative 32 with TSTU and DIPEA in dry DMF will give the succinimidyl ester derivative 33.
Example 15—Preparation of 6-Maleimidocaproyl-MMAE (37)
[0418] ##STR00017##
[0419] To a suspension of MMAE (0.05 g, 0.0694 mmol) in freshly distilled dry DCM (2 ml), 6-maleimidocaproic acid (0.0221 g, 0.104 mmol) was added followed by diethylcyanophosphonate (21 μl, 0.139 mmol) and DIPEA (37 μl, 0.208 mmol). On addition of DIPEA, the reaction mixture became clear and was stirred at room temperature under nitrogen for 12 h, TLC [silica gel: 5% MeOH/DCM, Rf 0.31]. The reaction mixture was diluted with DCM (30 ml) and washed with 10% citric acid (2×20 ml), water (20 ml), brine (20 ml) and concentrated to dryness. The crude was purified by flash column chromatography [silica gel: 5% MeOH/DCM] to give 6-Maleimidocaproyl-MMAE 37 as a white solid 0.023 g (36%). MS (m/z) found 911.58 (M+1) calculated for C.sub.49H.sub.79N.sub.6O.sub.10
Example 16—Preparation of 6-Maleimidocaproyl-Val-Cit-PAB-MMAE (40)
[0420] ##STR00018##
[0421] To a stirred solution of val-cit-PAB 24 (0.11 g, 0.29 mmol) in dry N-methylpyrrolidinone, NMP (5 ml) under nitrogen, N-succinimidyl-6-maleimidohexanoate (0.0983 g, 0.318 mmol) was added and the resulting light-brown solution stirred at room temperature for 16 h. The NMP was removed by high vacuum at <40° C. The resulting thick oily residue was triturated with dry ether (20 ml) the solid collected by filtration and washed several times with dry ether and air dried to give the desired product 38 an off-white powder 0.16 g (98%). TLC [silica gel: 10% MeOH/DCM R.sub.f 0.21. MS (m/z) 572.653 (M+1) calculated for C.sub.28H.sub.41N.sub.6O.sub.7
[0422] To stirred solution of 6-maleimidocaproyl-val-cit-PAB 38 in dry DMF under nitrogen, bis-(p-nitrophenyl)carbonate was added followed by DIPEA, resulting in a colour change from colourless to bright yellow. The solution was stirred at room temperature under nitrogen for 1 h after which the DMF was removed by high vacuum to give an oily residue. This was triturated with ethyl acetate for 15 min resulting in precipitation which was completed by the addition of ether. The solid was collected and washed well with ether and air dried to give an off-white solid. TLC [silica gel: 10% MeOH/DCM R.sub.f 0.46]. This was purified by chromatography [silica gel: 5-10% MeOH/DCM gradient elution] to give the activated linker 39 as a white solid 0.006 g, (46%). MS (m/z) 738.3091 (M+H), HRMS (m/z) calculated for C.sub.35H.sub.43N.sub.7O.sub.11Na M+Na 760.2918 found 760.2922.
[0423] The activated linker 39 (50 mg, 0.068 mmol), MMAE (32.6 mg, 0.045 mmol) and N-hydroxybenzotriazole (1.4 mg, 0.0091 mmol) are stirred in dry DMF (1 ml) for 2 min. after which a drop of pyridine is added and the reaction stirred for 24 h. The solvent is then removed by high vacuum and the residue purified by reverse-phase preparative HPLC to give the desired product 40 after lyophilisation as a white powder; MS (m/z) 1316.7 (M+H).
Example 17—Preparation of Paclitaxel-dPEG.SUB.6.-NHS Ester (44)
[0424] ##STR00019## ##STR00020##
[0425] Paclitaxel (100 mg, 0.12 mmol) and glutaric anhydride (17 mg, 0.14 mmol) were dissolved in dry DCM (10 ml) and stirred for 10 min, followed by addition of dry pyridine (100 μl, 0.0013 mmol). The reaction mixture was stirred for 3 days at room temperature and evaporated under vacuum. The residue obtained was recrystallized from DCM to afford the paclitaxel acid 41 as a white solid 60.7 mg (52.3%). (R.sub.f0.26, 3% MeOH/DCM). .sup.1H NMR (CDCl.sub.3): δ 8.16 (2H, d, J=4 Hz, 23-H, 27-H), 7.78 (2H, d, J=4 Hz, 39-H, 43-H), 7.66-7.36 (11H, CH, Ar), 6.28 (2H, m, 10-H, 13-H), 6.01 (1H, q, J=4 Hz, 3′-H), 5.71 (1H, d, J=7.2 Hz, 2-H), 5.52 (1H, d, J=3.2 Hz, 2′-H), 5.00 (1H, d, J=8 Hz, 5-H), 4.47 (1H, q, J=6.4 Hz, 7-H), 4.29 (2H, d, J=8.4 Hz, 20-H), 3.83 (1H, d, J=6.8 Hz, 3-H), 2.53-2.16 (15H, m, 7-OH, 6-H, 14-H, g2-H, g4-H, 29-H, 31-H), 2.06-1.70 (7H, m, 1-OH, g3-H, 6-H, 18-H, 19-H), 1.28-1.16 (6H, m, 16-H, 17-H). MS (m/z): 968.36 [M.sup.+], 985.39 [M.sup.++NH.sub.4], 990.35 [M.sup.++Na]. (Theoretical: C.sub.52H.sub.57NO.sub.17 968.01).
[0426] To a stirred solution of paclitaxel acid 41 (26 mg, 0.027 mmol) and SDPP (20 mg, 0.058 mmol) in dry acetonitrile (5 ml), TEA (20 μl, 0.143 mmol) was added. The reaction mixture was stirred overnight at room temperature under nitrogen, followed by evaporation and purification by silica gel chromatography (MeOH/DCM=3:97) to give paclitaxel NHS ester 42 as a white solid 38 mg (76%). (R.sub.f 0.48). .sup.1H NMR (CDCl.sub.3): δ 8.15 (2H, d, J=7.6 Hz, 23-H, 27-H), 7.72 (2H, d, J=7.6 Hz, 39-H, 43-H), 7.64-7.37 (11H, CH, Ar), 6.28 (2H, m, 10-H, 13-H), 6.01 (1H, q, J=4 Hz, 3′-H), 5.71 (1H, d, J=7.2 Hz, 2-H), 5.52 (1H, d, J=3.2 Hz, 2′-H), 5.00 (1H, d, J=8 Hz, 5-H), 4.47 (1H, q, J=6.4 Hz, 7-H), 4.29 (2H, d, J=8.4 Hz, 20-H), 3.83 (1H, d, J=6.8 Hz, 3-H), 2.99-2.36 (15H, m, 7-OH, 6-H, 14-H, g2-H, g4-H, 29-H, 31-H), 2.29-1.82 (15H, m, 1-OH, g3-H, 6-H, 18-H, 19-H, n3-H, n4-H), 1.28-1.16 (6H, m, 16-H, 17-H). MS (m/z): 1065.38 [M.sup.+], 1087.36 [M.sup.++Na]. (Theoretical: C.sub.66H.sub.60N.sub.2O.sub.19 1065.08).
[0427] To a solution of the paclitaxel NHS ester 42 (32 mg, 0.03 mmol) in dry DCM (5 mL), H.sub.2N-PEG.sub.6-COOH (10.6 mg, 0.03 mmol) and TEA (5 μl, 0.03 mmol) were added. The reaction mixture was stirred overnight under nitrogen, followed by washing with HCl (2×10 mL, 0.1 M) and brine (2×10 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated to give 43 as a clear oil 25 mg, (64%). [Silica gel: 5% MeOH/DCM R.sub.f 0.16]. .sup.1H NMR (CDCl.sub.3): δ 8.16 (2H, d, J=7.6 Hz, 23-H, 27-H), 7.86 (2H, d, J=7.6 Hz, 39-H, 43-H), 7.65-7.30 (11H, CH, Ar), 6.28 (2H, m, 10-H, 13-H), 6.01 (1H, q, J=4 Hz, 3′-H), 5.71 (1H, d, J=7.2 Hz, 2-H), 5.50 (1H, d, J=3.2 Hz, 2′-H), 5.00 (1H, d, J=8 Hz, 5-H), 4.47 (1H, q, J=6.4 Hz, 7-H), 4.29 (2H, d, J=8.4 Hz, 20-H), 3.83 (1H, d, J=6.8 Hz, 3-H), 3.73-3.47 (24H, m, —CO—NH—(CH.sub.2—CH.sub.2—O).sub.6—CH.sub.2—), 2.62-1.87 (24H, m, 7-OH, 6-H, 14-H, 18-H, g2-H, g4-H, 29-H, 31-H, 1-OH, g3-H, 6-H, 19-H), 1.28-1.16 (6H, m, 16-H, 17-H). MS (m/z): 1303.56 [M.sup.+], 1325.56 [M.sup.++Na], 1341.55 [M.sup.++K]. (Theoretical: C.sub.67H.sub.86N.sub.2O.sub.23 1303.40).
[0428] To a stirred solution of paclitaxel-PEG.sub.6-acid 43 (22 mg, 0.017 mmol) in anhydrous DMF (2 mL), TSTU (11 mg, 0.034 mmol) and DIPEA (15 μl, 0.085 mmol) were added. The reaction mixture was stirred for 2 h at room temperature under nitrogen, followed by concentration to afford the crude product as a yellow oil. This was purified by flash chromatography [silica gel, 3-5% MeOH/DCM] to afford the NHS ester 44 15.2 mg (64%). [silica gel 3% MeOH/DCM R.sub.f 0.18]. .sup.1H NMR (CDCl.sub.3): δ 8.16 (2H, d, J=7.6 Hz, 23-H, 27-H), 7.86 (2H, d, J=7.6 Hz, 39-H, 43-H), 7.65-7.30 (11H, CH, Ar), 6.28 (2H, m, 10-H, 13-H), 6.01 (1H, q, J=4 Hz, 3′-H), 5.71 (1H, d, J=7.2 Hz, 2-H), 5.50 (1H, d, J=3.2 Hz, 2′-H), 5.00 (1H, d, J=8 Hz, 5-H), 4.47 (1H, q, J=6.4 Hz, 7-H), 4.29 (2H, d, J=8.4 Hz, 20-H), 3.83 (1H, d, J=6.8 Hz, 3-H), 3.73-3.47 (24H, m, —CO—NH—(CH.sub.2—CH.sub.2—O).sub.6—CH.sub.2—), 2.62-1.87 (28H, m, 7-OH, 6-H, 14-H, 18-H, g2-H, g4-H, 29-H, 31-H, 1-OH, g3-H, 6-H, 19-H, n3-H, n4-H), 1.28-1.16 (6H, m, 16-H, 17-H). MS (m/z): 1400.60 [M.sup.+], 1417.62 [M.sup.++NH4], 1422.58 [M.sup.++Na], 1338.60 [M.sup.++K]. (Theoretical: C.sub.71H.sub.89N.sub.3O.sub.26 1400.47).
Example 18—Preparation of Paclitaxel-PAB-Val-Cit-dPEG.SUB.7 .NHS Ester (47)
[0429] ##STR00021##
[0430] To a stirred mixture of paclitaxel (100 mg, 0.117 mmol) and Fmoc-Val-Cit-PAB (74.8 mg, 0.00976 mmol) in dry DCM (10 ml) DMAP (14.3 mg, 0.117 mmol) is added and stirred at room temperature under nitrogen for 48 h. The solvent is evaporated to give a light-yellow crystalline solid which is purified by flash column chromatography [silica gel: 3-5% MeOH/Chloroform] giving the desired compound 45.
[0431] To a stirred solution of 45 in dry THF, DBU is added and stirred for 10 min. after which the solvent is removed to give the deprotected derivative 46 which is used without further purification.
[0432] A solution of 46 in dry DCM is added dropwise over 20-30 min. to a stirred solution of the bis-dPEG.sub.7 NHS ester in dry DCM under nitrogen after which it is stirred for 2 h, quenched by the addition of water, back-extracted with DCM and the combined organic extracts dried and evaporated to give crude 47.
Example 19—Preparation of Doxorubicin-dPEG.SUB.12.-Maleimide (48)
[0433] ##STR00022##
[0434] To a stirred suspension of Dox.HCl (10 mg, 0.017 mmol) in dry DMF (2 ml) DIPEA (7.7 μl) was added and the reaction mixture stirred for 10 min. under nitrogen to give a clear red solution. To this, Maleimide-dPEG.sub.12 NHS ester (16.4 mg, 0.019 mmol) dissolved in dry DMF (1 ml) was added and the reaction stirred at room temperature, under nitrogen and protected from light overnight. The DMF was removed by high vacuum and the dark red oil purified by flash chromatography [silica gel: 10% MeOH/DCM R.sub.f 0.5] to give the desired product 48 17.8 mg (80%) as a red viscous oil; HRMS (m/z) calculated for C.sub.61H.sub.87N.sub.3O.sub.27Na [M Na] 1316.5424 found: 1316.5601.
Example 20—Preparation of Cemadotin-SH (53)
[0435] ##STR00023##
Example 21—Preparation of Cemadotin-OH (54)
[0436] ##STR00024##
Example 22—Preparation of Cemadotin-O-PAB-Cit-Val-PEG.SUB.3.-NHS Ester (57)
[0437] ##STR00025##
Example 23—Preparation of Seco CBI-β-Glucuronide-NHS Ester (65)
[0438] ##STR00026## ##STR00027##
Example 24—Preparation of 6-Maleimidocaproyl-SGD-1910 (67)
[0439] ##STR00028##
Example 25—Preparation of Maytansinol DM4 Mal-PEG.SUB.4.-NHS Ester (68)
[0440] ##STR00029##
Example 26—Scheme for the Synthesis of Conjugates
[0441] ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
Example 27—Expression and Purification of a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0442] Construction of the Anti-HER2 Cytoplasmic-Expression scFv Clone, TCT
[0443] The open reading frame (ORF) of the scFv C6.5 [Adams G P et al. Cancer Res, 2001, 61:4750-55], which is known to have multiple, well-spaced, surface lysine residues, was cloned into the expression vector pET32 Xa/LIC (Novagen) carrying the ORF of thioredoxin as a fusion tag to enable the cytoplasmic expression of the protein. To facilitate the cleavage of the fusion tag at low cost and effective detection and monitoring of the resulting scFv, the following features were engineered into the vector: [0444] a) TEV protease cleavage site, downstream of the Factor Xa cleavage site [0445] b) Linker region between the TEV protease cleavage site and the C6.5 ORF. Without this the TEV protease fails to cleave, probably due to the fact that the structure of scFv C6.5 sterically hinders access to its cleavage site. [0446] c) T7 tag sequence at the C-terminus of the C6.5. This Tag was chosen because it lacks lysine residues.
[0447] The resulting protein was called scFv (TCT) (Tev cleavage site, C6.5, T7 tag). The DNA sequence can be found below:
TABLE-US-00006 DNA sequence of cleaved TCT [SEQ ID NO: 1] AGCGGTAGCGGAGGTAGCGGACAGGTGCAGCTGGTGCAGTCTGGGGCAGA GGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGAT ACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAA GGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATA CAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCA GCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTG TATTTTTGTGCGAGACATGACGTGGGATATTGCAGTAGTTCCAACTGCGC AGCGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCG TCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGA TCGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACA GAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATT ATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATC TATGGTCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTC CAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGG ATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGG GTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
Amino Acid sequence of cleaved TCT [SEQ ID NO: 2] SGSGGSGQVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVT ISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSS NCAAWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGG SQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWY QQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAI SGFRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
Number of Amino acids: 272 Molecular weight: 28,160 Da Theoretical PI: 7.54 Extinction coefficient: 65 235 KEY: Bold = Residual Ser left after TEV cleavage Underlined = Linker region (GSGGSG (SEQ ID NO: 8)) Unformatted = C6 sequence Bold italics = T7 tag sequence
[0448] Bacterial Expression in 15 L Bioreactor of the Anti-HER2 Cytoplasmic-Expression scFv Clone, scFv (TCT)
[0449] TCT was produced in SHUFFLE® T7 Competent E. coli (NEB). Four to five single colonies of transformed cells grown overnight on selective agar plate were first inoculated in 5 ml of selective 2TY medium+ 1% glucose. 1 μl from the culture that was observed to be growing faster was transferred to fresh selective 5 ml cultures of 2YT+ 1% glucose and allowed to grow at 30° C. for about 10 hours. These steps were taken to ensure that the cell growth does enter the lag phase for too long and hence ensure the plasmid stability within growing cells.
[0450] The next day, a selective 0.5 L+1% glucose preculture was inoculated with one of two 5 ml cultures. Medium used, Supercharged Terrific Broth [12 g/l tryptone, 24 g/l yeast extract, 9 g/l Na.sub.2HPO.sub.4, 2.2 g/l KH.sub.2PO.sub.4, 2.6 g/l NH.sub.4Cl, 0.7 g/l Na2SO.sub.4 1 g/l NaCl, 5 g/l glycerol]. Adjust pH to 7.4, autoclave and add 2 mM MgSO.sub.4
[0451] After 3.5 hrs, the preculture (OD.sub.600 0.8-1.2) was transferred to a 15 L Fermenter (Applikon P1000) containing 14.5 L of selective (carbenicillin 100 ug/L) Supercharged Terrific Broth+0.5% glucose and 0.05 ml/L antifoam PPG 2025. The stirrer blade speed was adjusted to between 200-500 RPM to ensure adequate dissolving of oxygen in the medium. Typically 200 RPM initially and 400-500 post-induction. The initial temperature was either 37° C. or 30° C. depending on the doubling time of the culture (typical culture doubling times (Td) 35-55 minutes).
[0452] When the culture OD.sub.600˜1.0 the culture temperature control was adjusted to 26° C. and allowed about 30 minutes to stabilise. Induction was carried out typically 3.5-5 hours after inoculation with 15 ml of 50 mM IPTG. Final IPTG culture concentration 50 uM. It is very important that the cells are well adjusted to 26° C. before induction with a low concentration of IPTG otherwise the amount of soluble protein produced decreases significantly. The fermenter was coupled with an automatic antifoam dispenser which is triggered when foam builds up.
[0453] The culture was allowed to grow for about 16 hours and harvested using Beckman JLA8.1000 for 15′ @ SKRPM. The final OD.sub.600=35.7.
[0454] 3) Protein Purification
[0455] Cells were resuspended in Lysis buffer (40 mM Tris-HCl pH 8, 750 mM NaCl, 2 mM Imidazole) and frozen in liquid nitrogen. On lysis day, the frozen cells were thoroughly thawed and the lysis buffer was adjusted to have a final concentration of 2M Urea. Urea and a high concentration of NaCl were employed to ensure better IMAC purification. The 2M Urea-treated scFv was probed with 1D NMR to ensure that the structure of scFv (TCT) was not affected.
[0456] Complete EDTA free tablets (Roche Diagnostics, 1/100 ml lysis solution) and Benzonase (Novagen >99 purity, 5 ul/100 ml lysis solution) were added. Lysis was performed with a Constant Cell Disruption Systems (model TS5) coupled to a chiller keeping the cell disruption chamber at 4° C. Cell disruption was achieved three times over at a pressure of 27 kpsi. Total volume of the lysate amounted at 2 L.
[0457] The Lysate was initially spun using an Eppendorf centrifuge 5810 R at 4000 rpm for 40 minutes to remove the bulk of cell debris and then twice using a Sorvall RC 6+, rotor F21-8×50 at 17000 rpm for 40 minutes. The clarified supernatant was then filtered through 0.22 um PES filter (Corning) under vacuum.
[0458] IMAC was then performed using the HisPur Ni-NTA resin from Thermo scientific under gravity flow in columns. The column was equilibrated with lysis buffer containing 2M urea. The clarified supernatant was passed through the column twice, followed by 10 bed volumes wash with the lysis buffer. The resin was then further washed with 10 bed volumes of Wash buffer 1 (40 mM Tris-HCl pH 8, 750 mM NaCl, 2M Urea, 10 mM Imidazole) and then Wash buffer 2 (40 mM Tris-HCl pH 8, 750 mM NaCl, 2M Urea, 30 mM Imidazole) until there was no significant absorbance at OD 280 nm.
[0459] The protein was then eluted ((40 mM Tris-HCl pH 8, 750 mM NaCl, 250 mM Imidazole) until there was no reading at OD 280 nm. The eluate was then dialysed extensively in TEV cleavage buffer (50 mM tris-HCl pH8, 150 mM NaCl). The protein solution was then adjusted to a concentration of about 2 mg/ml and reduced glutathione was added to a final concentration of 3 mM. In-house produced TEV protease, fused with a polyhistidine tag was added at 0.15 mg/100 mg of fusionscFv (TCT)(fTCT) and allowed the cleavage to proceed for 14-18 hours on a rolling incubator at 4° C.
[0460] The cleaved protein solution was allow to pass 3 times through Ni-NTA resin. The cleaved scFv (TCT) flowed through while the thioredoxin fusion tag, TEV protease and other proteins remain bound to the resin. A summary SDS-PAGE of the purification is shown in
[0461] TCT scFv was dialysed into Storage buffer (20 mM Sodium Acetate pH5, 150 mM NaCl) and then SEC was carried out to eliminate high molecular weight contaminants and scFv (TCT) soluble aggregates (
Example 28—Protein Engineering, Expression and Purification of a Single-Chain Fv Antibody Fragment in Order to Bear Multiple, Well-Dispersed, Surface Lysine Residues
[0462] An antibody fragment that does not possess sufficient well-spaced lysine residues and demonstrates poor conjugation properties (typical DARs<5) can be modified by directed mutagenesis to bear a configuration similar to the scFv (TCT). Using general and accepted antibody and protein structural concepts from the literature [Alzari P M et al Annual Rev. Immunol. 1988. 6:555-80; Davies D R & Metzger H. Annual Rev Immuno. 1983. 1:87-117; Mariuzza R A et al. Annual Review Biophys. & Biophysical Chem, 1987, 16:139-59] in combination with 3-dimensional molecular modelling software (e.g. PyMOL, http://www.pymol.org Schrodinger K K, Japan) and alignment tools such as Clustal, positions within the protein primary sequence can be identified that can be mutated to lysine residues, where lysine residues are known to be well-tolerated at that position (using databases such as IMGT or Kabat) or are known (from a solved 3D structure) or predicted (using software such as Phyre) to be at the protein surface (
Example 29—Bioconjugation of Ellipticine Derivatives onto a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0463] Ellipticine
[0464] Ellipticine-C.sub.6-NHS (compound 21) was conjugated to scFv-TCT in PBS at pH 8.0 in 6% MeCN with varying amounts of DMSO (either 14% or 6%) and two different sets of excess drug equivalents. The NHS was added in 5 equivalent portions for reaction 1 and in 2.7 equivalent portions for reactions 2 and 3. In more detail, Ellipticine-NHS was dissolved in anhydrous DMSO to obtain a clear yellow/orange 50 mM stock solution. A scFv (TCT) stock solution in PBS pH 8.0, stored at 4° C., was diluted in degassed PBS pH 8.0 pre-equilibrated with 6% MeCN and either 14% or 6% DMSO. The NHS was added in portions of either 5 or 2.7 equivalents every 75 min whilst mixing on a vortex at room temperature. 4 hr from completion of addition, the samples were recovered by centrifugation (2.5 min, 11 krpm). The supernatant was recovered and purified by zeba columns (Pierce) pre-equilibrated with the same buffer as the reaction mixture of each sample. The samples were then dialysed over 4000× in 6% MeCN/PBS pH 7.3 overnight at 4° C. then 8000×.
[0465] The samples were recovered and analysed by SDS-PAGE (
[0466] Conjugates became insoluble and precipitated out of solution once a certain DAR was obtained. As an example, sample 1 above contained small amounts of protein/conjugate following centrifugation and even less once purification was attempted via zeba columns, indicating that the residual soluble conjugate was very hydrophobic and adhered to the column. There was a significant amount of protein/conjugate in the pellet sample of this reaction, as seen in mainly the fluorescent gel, i.e. recovery of soluble conjugate was low. This is also supported by the UV/Vis data. Precipitation was far less pronounced for samples 2 and 3 which had 16 equivalents of drug compared to the 32 of reaction 1. The pellet samples were less intense and the soluble material more prominent both on Coomassie and fluorescence detection. There is an indication that sample 2 migrated less far on the gel than sample 3 supporting the rationale that increased amount of DMSO can lead to increased solubility of the drug, thereby increasing the efficiency of the reaction and leading to higher DARs. Overall, reaction 2 had less NHS equivalents than 1, leading to lower DARs which appear to be more soluble, but at the same time having the same number of equivalents as 3, thereby supporting the organic solvent argument.
[0467] DARs were calculated for these reactions using their UV/vis absorption spectra in buffer (
[0468] Quantification of Drug to Antibody Loading of an scFv-Ellipticine Conjugate
TABLE-US-00007 TABLE 4 Final DARs for scFv (TCT)-Ellipticine ADCs. No Reaction Final DAR 1 32 equivalents, 14% DMSO 5.4 2 16 equivalents, 14% DMSO 5.1 3 16 equivalents, 6% DMSO 3.9
[0469] PEG-Ellipticine
[0470] ScFv-TCT was conjugated to another Ellipticine-NHS derivative with a short PEG chain to increase water solubility (compound 23). The conjugation was carried out in parallel with Ellipticine-NHS as a control, using the best conditions for Ellipticine in order to obtain a DAR 5, which was the maximum obtained in the soluble phase. The reactions were set up as described previously, using 99% pure scFv. ScFv in PBS pH 8.0 was diluted in PBS pH 8.0 pre-equilibrated with DMSO (14%) and MeCN (6%), and then incubated for 5 min on a vortex, shaking gently at RT. The crude NHS drugs were dissolved in anhydrous DMSO to a 50 mM stock solution and were added in two portions over 15 min and incubated for a further 2 hrs at RT. The samples were recovered by centrifugation and stored at 4° C. before being purified using zeba columns pre-equilibrated with 14% DMSO/6% MeCN/PBS pH 7.3. The pellets were resuspended in buffer and gel loading buffer and all samples were analysed by SDS-PAGE (Coomassie and fluorescence,
[0471] Comparing Ellipticine with PEG-Ellipticine, it is clear that under the same reaction conditions (1 and 2), the PEG derivative leads to higher recovery of soluble conjugate/protein (compound 73). The bands for 2 are very faint in comparison to 1 both in the Coomassie and the fluorescence detection. Comparing the three reaction conditions where the number of equivalents was investigated to raise the DAR, there was a shift on the gel indicating that perhaps 4 has a higher DAR than 3 and 1. Protein recovery is less for 4Z than the other two indicating that again, the maximum loading has been reached, at which point the higher DAR conjugates precipitate out of solution.
[0472] Using the UV/Vis in combination with the densitometry data (to calculate % non-covalent binding) DAR values were calculated as follows: (1): 4.1 (2): 2.0 (3): 5.1 and (4): 4.3 (Table 5). This confirmed that the PEG Ellipticine resulted in two-fold higher protein recovery and up to two-fold higher DAR compared to Ellipticine. Conjugate precipitation seems to have improved.
TABLE-US-00008 TABLE 5 Final DARs for scFv (TCT)-PEG-Ellipticine ADCs (compound 73) No Reaction Final DAR 1 20 equivalents, 14% DMSO, 6% MeCN 4.1 2 20 equivalents, 14% DMSO, 6% MeCN 2.0 3 32 equivalents, 14% DMSO, 6% MeCN 5.1 4 64 equivalents, 14% DMSO, 6% MeCN 4.3
[0473] The conjugation to Ellipticine was carried out on a whole IgG as a comparison to the scFv (TCT) under identical conditions. The SDS-PAGE gels indicate at least equivalent conjugation fluorescence (
[0474] Lysosomally-Releasable Ellipticine
[0475] A cleavable dipeptide Ellipticine-NHS drug (compound 29) was conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction was controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated cleavable dipeptide Ellipticine-NHS was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0476] Buffer—bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol; [0477] Temperature—25° C.; [0478] Mixing conditions—Thermomixer 1000 rpm; [0479] Antibody at 1 mg/ml, [0480] Cleavable dipeptide Ellipticine-NHS—8 equivalent addition portions; and, [0481] NHS-drug addition rate (every 70-90 minutes).
[0482] Typically, scFv (TCT) was defrosted on the thermomixer at 4° C., then the temperature of the aliquot was slowly raised to 20° C. Any precipitate was spun down before using.
[0483] A cleavable dipeptide Ellipticine-NHS (compound 29) 100 mM stock solution was made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol in eppendorf microtubes and the buffer was equilibrated on the thermomixer at 4° C., then the temperature of the aliquot was raised to 20° C. whilst mixing at 1000 rpm. The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the cleavable dipeptide Ellipticine-NHS was started. This was carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 mins, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation was in the sample with the highest number of drug equivalents and that was very low.
[0484] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0485] The unconjugated and conjugated scFv (TCT) were analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0486] Mass spectrometric analysis was performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), were analysed by both MALDI-MS and then further analysed by LC-MS. All samples gave well resolved peaks.
Example 30—Bioconjugation of Doxorubicin Derivatives onto a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0487] (a) One-Step Conjugation with Doxorubicin-NHS Derivative
[0488] Doxorubicin derivatives (compounds 7, 10, 16) with an NHS reactive group were conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction was controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated Doxorubicin-NHS derivatives was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0489] Buffer—bicarbonate buffer with NaCl at pH7.8 with 20% DMSO, 30% glycerol and 1% Tween; [0490] Temperature—25° C.; [0491] Mixing conditions—Thermomixer 1000 rpm; [0492] Antibody at 1 mg/ml; [0493] Doxorubicin-NHS derivatives—2 equivalent addition portions; and, [0494] NHS-drug addition rate—every 70-90 minutes.
[0495] Typically, scFv (TCT) was defrosted on the thermomixer at 4° C., then the temperature of the antibody aliquot was slowly raised to 20° C. The aliquots were spun down to collect any precipitate before using.
[0496] Doxorubicin-NHS derivatives 100 mMstock solution were made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol in eppendorf microtubes and the buffer was equilibrated on the thermomixer at 4° C., then the temperature of the aliquot was raised to 20° C. whilst mixing at 1000 rpm. The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the Doxorubicin-NHS derivatives was started.
[0497] This was carried out by adding 4 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 mins, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation was in the sample with the highest number of drug equivalents and that was very low.
[0498] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and then analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0499] The unconjugated and conjugated scFv (TCT) were analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0500] Mass spectrometric analysis was performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), were analysed by both MALDI-MS and then further analysed by LC-MS. All samples gave well resolved peaks. The DAR was determined using the extinction coefficient for the doxorubicin drug and antibody
[0501] (b) Two-Step Conjugation to a Doxorubicin-Maleimide Derivative
[0502] To conjugate Doxorubicin maleimide derivatives (compounds 48, 12) onto the antibody, the antibody's native lysines were chemically converted to firstly a protected thiol which was subsequently reduced to obtain the free thiol. The free thiols could then be reacted with maleimide derivatives of Doxorubicin to obtain conjugates containing thioether bonds.
[0503] The first step of introducing the thiols onto the antibody was optimised. This involved the conjugation of the SPDP linker onto the antibody to form an amide bond between the linking group and the antibody. A lysine-optimised scFv facilitated the production of high SPDP-substituted conjugates for subsequence conjugation of a maleimide-derived drug.
[0504] Overall, SPDP conjugated well even at lower pH (7 or 8). When reduced with sufficient TCEP (115 molar excess) it gave a SH:scFv ratio of up to 12. The SPDP conjugation was carried out to introduce various ratios of SPDP linker per antibody.
[0505] The antibodies, both at 1 mg/ml, C6.5 and HMFG1 were diluted into degassed PBS pH8 containing 1 mM EDTA, 3% DMSO and 6% MeCN. A fresh colourless solution of SPDP was prepared in anhydrous DMSO and the required amount was added to the antibody solution. The samples were incubated on a roller for 3 hrs at RT and at 4° C. overnight. The samples were collected by centrifugation when minimal precipitation was observed. The excess/unconjugated SPDP linker was removed using Zeba spin columns (ThermoScientific) and buffer exchanging into degassed PBS pH8 with 1 mM EDTA. The UV/vis spectra of the samples were recorded.
[0506] For the reduction of the linker to release the free thiol on the antibody and at the same time the pyridine-2-thione, the following was carried out. TCEP was first dissolved (fresh) in water to make up a 500 mM stock solution. The SPDP linked samples were incubated with 115 equivalents of TCEP for 20 mins at 37° C. The samples were collected by centrifugation and immediately chilled on ice. The UV/vis spectra of the crude samples were recorded before removing the excess TCEP and pyridine-2-thione using zeba desalting columns using 3% DMSO/6% MeCN in degassed PBS pH7 with 1 mM EDTA as the eluent.
[0507] At this point, the efficiency of the SPDP conjugation was determined. The quantity of the released pyridine-2-thione in the crude reduced sample was determined using the spectrophotometric data. The Δmax for pyridine-2-thione is 343 nm and the extinction coefficient 8080M.sup.−1cm.sup.−1. The extinction coefficient at 280 nm is 5100M.sup.−1 cm.sup.−1 was used to correct the absorption at 280 nm. The concentration of the thione in the crude reduced solution was calculated using the A343 nm and using this concentration corrected the absorption at 280 nm to account for the thione absorption. The antibody concentration was calculated and the ratio of the SPDP:Ab was determined. The same process was repeated for the pre-reduction sample and this DAR was subtracted from the reduced sample DAR to obtain the actual SPDP:Ab ratio.
[0508] After the purification of the reduced sample, the following conjugates were obtained (Table 6) showing that up to 9 linkers could be conjugated to the scFv (TCT):
TABLE-US-00009 TABLE 6 Ratio of SPDP linker conjugated to a lysine-optimised scFv and a control IgG C6.5 scFv HMFG1 IgG SPDP linker Linker to Ab Linker to Ab reaction ratio Ab Ratio Recovery Ab Ratio Recovery 5 0.9 71% 4.4 93% 8 1.8 71% 6.3 81% 16 4 65% 8.2 80% 32 9 38% 13 44%
[0509] In another example, the above procedure was carried out similarly using 32 equivalents of SPDP and subsequently reducing the samples with 115 equivalents of TCEP. The antibody recovery in this case was much higher (92%). The reduced, purified and quantified samples were then conjugated to Doxorubicin. Doxorubicin maleimide and doxorubicin-PEG-maleimide were added to the antibody samples (in degassed PBS pH7/1 mM EDTA/3% DMSO/6% MeCN) at 2 equivalents each. The samples were incubated on a roller at RT for 3 hrs followed by 4° C. overnight. Samples were recovered by centrifugation and analysed by SDS-PAGE gel (
[0510] The DAR for the Dox conjugates was calculated from the crude samples using UV/Vis spectroscopy and gel densitometry. From the spectroscopic data, the DAR was calculated using the Doxorubicin c at 488 nm and 280 nm and the antibody's c at 280 nm (Table 7).
TABLE-US-00010 TABLE 7 Ratio of SPDP linker conjugated to a lysine-optimised scFv and a control IgG, followed by doxorubicine derivative conjugations SPDP C6.5 scFv HMFG1 IgG linker Linker Dox: Linker Dox: reaction to Ab Ab Ab Dox-PEG: to Ab Ab Ab Dox-PEG: ratio Ratio Recovery DAR Ab DAR Ratio Recovery DAR Ab DAR 32 7.5 92% 3.5 1.6 6.5 50% N/A 2.5
[0511] The DAR was determined using the experimentally-determined molar extinction coefficient for the doxorubicin drugs (Table 7) and antibody and confirmed by mass spectrometry as described above.
[0512] Binding of High Ratio SPDP scFv Conjugates
[0513] C6.5 scFv was conjugated to SPDP as in example 30(b) with 16 equivalent excess reagent followed by reduction with 115 molar equivalents of TCEP to obtain a linker to antibody ratio of 5.4 (SPDP:scFv). This sample, as well as an unmodified control and a non-SPDP modified but reduced controls were used.
[0514] Ninety-six-well Immunosorb ELISA plates were coated with 10 μg/ml HER2-Fc in PBS, followed by the test samples, anti-myc IgG (Sigma) and anti-mouse peroxidase conjugate (Sigma). Extensive PBS washes were in between each layer and detection was with BM-Blue substrate. The plot (
Example 31—Bioconjugation of P5 and Cemadotin Derivatives onto a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0515] (A). ScFv (TCT)-Cemadotin
[0516] Cemadotin-NHS (compound 2) was conjugated to scFv (TCT) to obtain conjugates (compound 69) with various DARs. The reaction was controlled to obtain products with low, medium, and high DARs. Initially, the hydrolysis rate of the pure, isolated Cemadotin-NHS was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug, and the concentration of the drug in the buffer. The latter is a crucial parameter: the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0517] Buffer—bicarbonate buffer with NaCl at pH 8.8 with 20% DMSO; [0518] Temperature: 20° C.; [0519] Mixing conditions—Thermomixer 1000 rpm; [0520] Antibody at 1 mg/ml; [0521] Cemadotin/Cemadotin-C5 and P5C5, all NHS (16 equivalent addition portions); and, [0522] NHS-drug addition rate (every 70-90 min).
[0523] Typically, scFv (TCT) was defrosted on the Thermomixer at 4° C., then the temperature of the aliquot was slowly raised to 20° C. Aliquots were spun down to collect any precipitate before using.
[0524] A Cemadotin-NHS 100 mM stock solution was made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO in eppendorf microtubes and the buffer was equilibrated on a Thermomixer (with the temperature raised from 4° C. to 20° C., whilst mixing at 1000 rpm). The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 min before the addition of the Cemadotin-NHS. This was carried out by adding 16 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 min, before replacing on the Thermomixer and mixing at 20° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the Thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 min, 11 krpm). The only visible precipitation was in the sample with the highest number of drug equivalents and was very low.
[0525] All samples were purified from crude on the HPLC-SEC with 10% IPA/PBS pH 7, 20° C. and analysed by SDS-PAGE (
[0526] Reaction 1—scFv-TCT-Cemadotin 16 equivalents;
[0527] Reaction 2—scFv-TCT-Cemadotin 48 equivalents; and
[0528] Reaction 3—scFv-TCT-Cemadotin 112 equivalents.
[0529] The unconjugated and conjugated scFv (TCT) were analysed by HPLCsize-exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time of 15.5-16 min correlating to a MW of around 30 kDa. The three conjugates all eluted slightly and progressively earlier indicating a larger molecular weight (due to varying drug loads), but as single, sharp, monomeric peak, indicating no aggregation (
[0530] The DAR was accurately determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility. From the AAA (Table 7A-C), the amount in mol of both the protein and the drug (due to the drug's fingerprint-release of 4-aminomethylbenzoic acid) can be derived and the DAR calculated (No mol drug/No mol protein). The concentration of the protein in the solution can be calculated by first calculating the conjugates molecular weight based on the DAR, and then subsequently converting the concentration obtained from AAA to mg/ml of protein. For example, in sample 1: scFv (TCT) is 28162 (MS), DAR is 3.9, and each Cemadotin molecule adds 667 onto the antibody. Therefore conjugate MW=28162+(3.9×667)=30763. The concentration is 9.02 nmol/ml which is equal to 277 μg/ml of protein.
TABLE-US-00011 TABLE 8A Sample: 1 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 excluded — Asp 15 15.35 better than 5% Thr 14 13.67 better than 5% Ser 46 44.48 better than 5% Glu 22 21.72 better than 5% Gly 44 43.97 better than 5% Ala 17 17.21 better than 5% Val 18 19.74 within 5-10% Met 5 excluded — Ile 8 7.87 better than 5% Leu 15 14.84 better than 5% Norleu std — Tyr 14 13.42 better than 5% Phe 7 6.86 better than 5% His 3 2.98 better than 5% Lys 12 12.72 within 5-10% Arg 5 5.17 better than 5% Pro 0 excluded — Trp 8 8.00 (not determined) Total 259 residues Average of 2 runs Total sample 0.726 nmoles 0.72 20.43 ug 20.32 Concentration 9.07 nmoles/ml 9.02 255.42 ug/ml 254.00 Cemadotin 2.82 2.81 2.81 nmoles DAR = 3.90
TABLE-US-00012 TABLE 8B Sample: 2 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 excluded — Asp 15 15.38 better than 5% Thr 14 13.69 better than 5% Ser 46 44.17 better than 5% Glu 22 21.61 better than 5% Gly 44 44.25 better than 5% Ala 17 16.91 better than 5% Val 18 20.00 within 5-10% Met 5 excluded — Ile 8 8.04 better than 5% Leu 15 14.93 better than 5% Norleu std — Tyr 14 13.24 within 5-10% Phe 7 7.07 better than 5% His 3 3.03 better than 5% Lys 12 12.62 better than 5% Arg 5 5.05 better than 5% Pro 0 0.00 — Trp 8 8.00 (not determined) Total 259 residues Average of 2 runs Total sample 0.806 nmoles 0.80 22.69 ug 22.64 Concentration 6.20 nmoles/ml 6.18 174.54 ug/ml 174.19 Cemadotin 6.66 6.54 6.60 nmoles DAR = 8.21
TABLE-US-00013 TABLE 8C Sample: 3 Integer fit of a measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 excluded — Asp 15 15.76 better than 5% Thr 14 13.78 better than 5% Ser 46 44.47 better than 5% Glu 22 21.92 better than 5% Gly 44 44.59 better than 5% Ala 17 17.20 better than 5% Val 18 excluded — Met 5 excluded — Ile 8 7.66 better than 5% Leu 15 15.09 better than 5% Norleu std — Tyr 14 13.71 better than 5% Phe 7 6.81 better than 5% His 3 3.06 better than 5% Lys 12 12.81 within 5-10% Arg 5 5.14 better than 5% Pro 0 0.00 — Trp 8 8.00 (not determined) Total 259 residues Average of 2 runs Total sample 0.796 nmoles 0.79 22.42 ug 22.21 Concentration 7.24 nmoles/ml 7.17 203.78 ug/ml 201.88 Cemadotin 8.83 8.46 8.65 nmoles DAR = 10.96
[0531] Tables 8A-C. Summary of AAA results showing DARs of 3.9, 8.2 and 11 from the three conjugation reactions 1-3
[0532] Mass spectrometric analysis was performed by SGS M-Scan. Samples 1-3, as well as ScFv-TCT (control), were analysed by MALDI-MS and then further analysed by LC-MS. All samples gave well resolved peaks and these are summed up below.
[0533] Electrospray Ionisation, Mass Spectrometry (ESI-MS)
[0534] Equipment: Analyses were performed using a Waters Xevo Q-TOF (Quadrupole-Time of Flight (Q-TOF)) mass spectrometer coupled with a Dionex Ultimate 3000 MDLC system (SOPs MS900 to MS905 and HPLC012 and HPLC019).
[0535] Buffer exchange: The samples were buffer exchanged and concentrated, using Millipore Amicon Centrifugal filter units (10 kDa MWCO), into 0.05% (v/v) Formic acid.
[0536] Online ESI-MS analysis: aliquots of the TCT-Cemadotin samples were analysed using online HPLC/ES-MS analysis to provide data relating to the intact mass of the constituents as follows:
[0537] Instrument: Waters Xevo Q-ToF (Quadrupole-Time of Flight) G1 mass spectrometer equipped with a Dionex Ultimate 3000 MDLC system. Column: PLRP-S Column, Temperature: 60° C., Flow rate: 0.2 mL/minute, UV detection: 214 nm and 280 nm, Solvent A: 0.05% (v/v) Formic acid, Solvent B: 90% (aq) Acetonitrile containing 0.05% (v/v) Formic acid.
[0538] Gradient:
TABLE-US-00014 Gradient: Time (mins) % B 0 20 5 55 65 75 73 75 73.1 98 83 98 83.1 20 111 20
[0539] The mass spectrometer was calibrated externally using Glu-Fibrinopeptide B, which was also utilised as a lockspray internal calibrant. The mass spectrometer was scanned from m/z 200 to 4000.
[0540] ESI-MS of TCT-Cemadotin 2 Samples
[0541] Aliquots of TCT-Cemadotin 2 samples were analysed using online HPLC/ES-MS analysis to provide data relating to the intact mass of the constituents. The Total Ion Current (TIC) chromatograms, spectra and transformed data samples TCT-Cemadotin 2 are shown below (
[0542] A major peak was observed in the TIC of the TCT-Cemadotin 2 sample eluting at 35.9 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 32,164, 32,831 and 33,498, which was consistent with the supplied theoretical mass of the scFv (TCT) molecule, together with 6-8 additions of the Cemadotin molecule. This correlated well with the AAA determination of the DAR of 8.21
[0543] ESI-MS of Samples 1, 3 and TCT
[0544] Aliquots of samples TCT-Cemadotin 1, TCT-Cemadotin 3 and scFv (TCT) control were analysed using on-line HPLC/ES-MS analysis to provide data relating to the intact mass of the constituents. The Total Ion Current (TIC) chromatograms, spectra and transformed data samples are shown below (
[0545] A major peak was observed in the TIC of the scFv (TCT) control sample eluting at 33.2 min. The zero-charge deconvoluted mass spectrum for this peak produces a single major component at m/z 28162 which was consistent with the theoretical mass of the scFv (TCT) molecule (
[0546] A major peak was observed in the TIC of the TCT-Cemadotin 1 eluting at 33.5 min. The zero-charge deconvoluted mass spectrum for this peak produces a series of major peaks at m/z 29495, 30162 and 30829, which was consistent with the supplied theoretical mass of the scFv (TCT) molecule, together with 2-4 additions of the Cemadotin molecule (
[0547] A major peak was observed in the TIC of the TCT-Cemadotin 3 sample eluting at 37.1 min. The zero-charge deconvoluted mass spectrum for this peak produces a series of major peaks at m/z 33496, 34163 and 34830, which was consistent with the supplied theoretical mass of the scFv (TCT) molecule, together with 8-10 additions of the Cemadotin molecule (
TABLE-US-00015 TABLE 9 Summary of the ESI-MS analyses of the ScFv (TCT)-Cemadotin ADCs Observed Added peak mass Mass Calculated Sample (m/z) (m/z) DAR ScFv (TCT) 28162 0 0 TCT-Cem ADC1 29495 1335 2 30162 2002 3 30829 2669 4 TCT-Cem ADC2 32164 4004 6 32831 4671 7 33498 5338 8 TCT-Cem ADC3 33496 5336 8 34163 6303 9 34830 6670 10 scFv (TCT) molecular weight = 28160 (protein sequence) Da Cemadotin drug molecular weight = 667 Da
[0548] MALDI-Mass Spectrometry
[0549] Equipment: Analyses were performed using the following equipment: Shimadzu Scientific Instruments AXIMA Performance MALDI TOF-TOF mass spectrometer.
[0550] Linear MALDI MS Analysis: a sample of myoglobin was used to calibrate the instrument externally in both positive and negative ion high mass linear mode.
[0551] Samples of TCT-Cemadotin conjugate 3, TCT-Cemadotin conjugate 1, TCT-Cemadotin conjugate 2, TCT-Control were diluted 1:1 (v/v) in 50% (aq.) acetonitrile and spotted in 1 μl aliquots onto a steel 384 spot non-coated MALDI plate. Replicate spots were made for each MALDI matrix: Norharmane, 2′,4′,6′-Trihydroxyacetophenone monohydrate (THAP), Norharmane:THAP (4:1, v/v), and sinapinic acid matrix solutions. Spots were also made using undiluted samples, for sinapinic acid. Each spot was overlaid with 1 μL aliquots of corresponding MALDI matrix, and allowed to co-crystallise and dry under a gentle stream of air. Sinapinic acid was prepared as a saturated solution in 1:1 (v/v) 0.1% aq. Trifluoroacetic acid (TFA):acetonitrile. Norharmane was prepared as a 10 mg/mL solution in 1:1 (v/v) 0.1% aq. Trifluoroacetic acid (TFA):acetonitrile.
[0552] 2′,4′,6′-Trihydroxyacetophenone monohydrate (THAP) was prepared as a saturated solution in 1:1 (v/v) 0.1% aq. Trifluoroacetic acid (TFA):acetonitrile. Spots containing Norharmane, 2′,4′,6′-Trihydroxyacetophenone monohydrate (THAP) and Norharmane:THAP (4:1, v/v) were analysed in high-mass linear mode negative ion; and spots containing sinapinic acid were analysed in high-mass linear mode positive ion. Mass spectra were collected over an appropriate mass range and the laser power was varied to achieve optimal results.
[0553] MALDI-MS of scFv (TCT) (Control Sample) in Sinapinic Acid
[0554] The MALDI-MS data obtained from the linear mode positive ion analysis of undiluted scFv (TCT). Control in sinapinic acid matrix is shown in
[0555] MALDI-MS of TCT-Cemadotin Conjugate 1 in Sinapinic Acid
[0556] The MALDI-MS data obtained from the linear mode positive ion analysis of TCT-Cemadotin conjugate 1 in sinapinic acid matrix is shown in
[0557] MALDI-MS of TCT-Cemadotin Conjugate 2 in Sinapinic Acid
[0558] The MALDI-MS data obtained from the linear mode positive ion analysis of TCT-cemadotin conjugate 2 in sinapinic acid matrix is shown in
[0559] MALDI-MS of TCT-Cemadotin Conjugate 3 in Sinapinic Acid
[0560] The MALDI-MS data obtained from the linear mode positive ion analysis of TCT-Cemadotin conjugate 3 in sinapinic acid matrix is shown in
TABLE-US-00016 TABLE 10 Summary of the MALDI analyses of the ScFv (TCT)-Cemadotin ADCs Observed Added peak mass Mass Calculated Sample (m/z) (m/z) DAR ScFv (TCT) 28251 0 0 TCT-Cem ADC1 29559 1308 2 30223 1972 3 30884 2633 3.9 31531 3280 4.9 TCT-Cem ADC2 32293 4042 6.1 32948 4697 7 33588 5337 8 TCT-Cem ADC3 33809 5558 8.3 34415 6164 9.2 35057 6806 10.2 Cemadotin drug molecular weight = 667 Da
[0561] Binding ELISA of scFv (TCT)-Cemadotin Conjugates
[0562] ScFv (TCT)-Cemadotin ADCs (compound 69) were made and characterised as described above. Their binding affinity against immobilised HER2 target antigen was determined by ELISA compared to the unmodified scFv (
[0563] Overall TCT-Cemadotin Conclusions, Biophysical Data
[0564] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a very high yield of low, medium and high DAR conjugates. There was no precipitation of antibody/conjugate observed in any of the conjugates, therefore recovery was very high. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜500 μg/ml and were stable in the buffer for several weeks. Prior to using them for in vitro or in vivo testing, these conjugates were buffer exchanged into PBS and sterile filtered. Again, recovery was very high.
[0565] The products were analysed extensively by reducing SDS-PAGE, SEC-HPLC, AAA, MS and ELISA.
[0566] The techniques used for analysis are in agreement and support the argument that an optimised scFv structure, exemplified by TCT, can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding affinity. Purified conjugates with low DAR (sample 1) run closer to the control scFv (TCT) on the gel and were less polydispersed than the medium DAR (sample 2) which run slightly higher and was more polydispersed, whereas for the high DAR (sample 3) there was a clear migration shift on the gel where the sample was clearly bigger in size than the control, unmodified TCT.
[0567] These observations were further supported by the HPLC where the samples had progressively shorter retention times than TCT, eluting faster from the SEC column due to their increasing size. Amino acid analysis was an extremely useful tool for further quantitative analysis and complemented the MS data. The mass spectrometry identified both high and low DAR within the same sample whereas AAA gave an average.
[0568] For sample 1, DAR was 3.9 by AAA and 3.4 and 3 by MS (ES and MALDI)
[0569] For sample 2, DAR was 8.2 by AAA and 7 and 7 by MS (ES and MALDI)
[0570] For sample 3, DAR was 10.9 by AAA and 9.2 and 9 by MS (ES and MALDI)
[0571] (B) ScFv (TCT)-P5C5
[0572] ScFv-TCT was conjugated to P5C5-NHS (compound 6) using the same method employed for Cemadotin-NHS. The HPLC purified P5C5-NHS was dissolved in filtered anhydrous DMSO to make up a 100 mM stock solution and spun down. This was stored at −20° C. when not in use. In this example, the set up was: [0573] Reaction 1—scFv-TCT-P5C5 30 equivalents [0574] Reaction 2—scFv-TCT-P5C5 112 equivalents
[0575] The antibody was defrosted at 4° C. and the temperature of the antibody was slowly raised to 20° C. on the Thermomixer. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8, was combined with anhydrous, filtered DMSO in a 1 or 5 ml eppendorf and equilibrated on the Thermomixer 20° C., 10 min, 1000 rpm before adding the antibody and equilibrating for a further 10 mins. P5C5-NHS was added in portions of 16 equivalents for reaction 2 and 10 equivalents for reaction 1 by adding the solution, inverting to mix, and replacing on the Thermomixer.
[0576] Additions were carried out every 90 min, after which point the samples were left overnight at 4° C. at 1000 rpm on the Thermomixer. Samples were recovered by centrifugation (2.5 min, 10 krpm) to obtain clear solutions. Minimal precipitation was observed for sample 2.
[0577] The samples were purified from crude on the HPLC by SEC using the Tosoh TSKGel G2000, eluting with 10% IPA/PBS pH 7.3 20° C. (same method as previously, loading ˜300 μg per injection run) and analysed by SDS-PAGE (
[0580] The ADCs (compound 71) eluted with a faster retention time than the unmodified antibody indicating a higher molecular weight, but as soluble monomeric conjugates with no visible aggregation.
[0581] (C) scFv (TCT)-P5C5, scFv (TCT)-Cemadotin-C5, Trastuzumab-P5C5 and Trastuzumab-Cemadotin-C5
[0582] The following reactions were carried out following the same process as previously described for Cemadotin (4) and P5C5 (6) drugs. In short, the antibodies were equilibrated in buffer/DMSO through incubation at 20° C./1000 rpm, and the drug was added in 16 equivalent portions every 90 min. Samples were recovered by centrifugation and purified by SEC-HPLC (G2000SWxl for scFv (TCT) and G3000SWxl for Trastuzumab) (10% IPA/PBS isocratic). Purified fractions were then concentrated using vivaspin 20 spin concentrators 5-fold and buffer exchanged into PBS using zeba spin columns (Pierce). Samples were analysed by SDS-PAGE (
[0583] After synthesis of these conjugates, it was clear that the three P5 based derivatives behave very similarly to the cemadotin-NHS derivatives (70), leading to very soluble, monomeric, highly loaded conjugates (compound 71). These have not been quantified for DAR but when compared to previous samples (that were quantified by AAA and MS) on SDS-PAGE, and compared to the scFv (TCT) control, it is clear that low, medium and high DARs can be formed with scFv (TCT) and Cemadotin, P5C5 and Cemadotin-C5. Trastuzumab IgG was also conjugated to P5C5 and Cemadotin-C5 with shifts observed on the gel (albeit smaller than TCT). These observations were supported by the HPLC-SEC traces where the samples gave similar retention times for low, medium and high DAR conjugates with cemadotin, P5C5 and cemadotin-C5.
[0584] (D) Binding Affinity of scFv (TCT)-P5C5 ADCs
[0585] ScFv (TCT)-P5C5 ADCs were made and characterised as described in examples above. The DAR was determined by AAA as before (Table 11A & B), this time following the release of the di-proline fragment to identify and quantify the P5-based drug. Their binding affinity against immobilised HER2 target antigen was determined by ELISA compared to the unmodified scFv. All proteins were detected using the C-terminal T7 Tag which was not expected to be chemically modified (no lysines present). 96-well Immunosorb ELISA plates were coated with 10 μg/ml HER2-Fc in PBS, followed by the test samples, anti-T7-peroxidase conjugate. Extensive PBS/tween-20 and PBS washes were in between each layer and detection was with BM-Blue substrate. The plot (
TABLE-US-00017 TABLE 11A Sample: TCT-P5C5(1) Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 excluded — Asp 15 15.76 better than 5% Thr 14 13.90 better than 5% Ser 46 43.51 within 5-10% Glu 22 21.87 better than 5% Gly 44 45.32 better than 5% Ala 17 16.46 better than 5% Val 18 excluded — Met 5 4.69 within 5-10% Ile 8 8.22 better than 5% Leu 15 15.79 within 5-10% Norleu std 0 — Tyr 14 14.64 better than 5% Phe 7 6.89 better than 5% His 3 3.07 better than 5% Lys 12 12.00 better than 5% Arg 5 4.89 better than 5% Pro 0 excluded — Trp 8 8 (not determined) Total 259 residues Average of 2 runs Total sample 0.271 nmoles 0.27 7.65 ug 7.62 Concentration 18.10 nmoles/ml 18.03 509.69 ug/ml 507.70 P5C5 nmoles 2.1980 2.2211 2.21 DAR 8.17
TABLE-US-00018 TABLE 11B Sample: TCT-P5C5(2) Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 excluded — Asp 15 15.99 within 5-10% Thr 14 14.20 better than 5% Ser 46 43.55 within 5-10% Glu 22 21.79 better than 5% Gly 44 45.11 better than 5% Ala 17 16.58 better than 5% Val 18 excluded — Met 5 excluded — Ile 8 8.15 better than 5% Leu 15 15.74 better than 5% Norleu std 0 — Tyr 14 14.16 better than 5% Phe 7 6.74 better than 5% His 3 3.20 within 5-10% Lys 12 11.91 better than 5% Arg 5 4.88 better than 5% Pro 0 0.00 — Trp 8 8 (not determined) Total 259 residues Average of 2 runs Total sample 0.225 nmoles 0.23 6.33 ug 6.54 Concentration 14.98 nmoles/ml 15.49 421.82 ug/ml 436.14 P5C5 nmoles 2.7742 2.6534 2.71 DAR 11.68
[0586] Table 11A & B. DAR by AAA of scFv (TCT)-P5C5 (compound 71) used in example 31D.
[0587] (E) Cell Killing Potency of scFv (TCT)-P5C5 ADCs Compared to IgG-Based ADCs
[0588] ScFv (TCT)-P5C5 and Trastuzumab-P5C5 ADCs were made and characterised as described above (examples 31B & 31C), which had similar DARs as before. SKBr3, human breast cancer cell line, high HER2 expression levels, up to 1,000,000 receptors per cell [Lazar G A, et al Proc Natl Acad Sci USA. 2006, 103:4005-10] were grown in DMEM, at 37° C., 5% CO.sub.2 in a humidified atmosphere. When confluency was 70-80%, cells were washed with PBS (2×10 ml) and incubated with trypsin for 5-7 min. Complete media was added and the cells were resuspended by pipetting. The cells were recovered by centrifugation (2 min, 2000 rpm), the supernatant was discarded, and the cells were resuspended in complete DMEM (5 ml). The cells were then counted using a haemocytometer and diluted accordingly. They were plated at 4500 cells/well (200μl) using attachment factor and incubated overnight at 37° C., 5% CO.sub.2 in a humidified atmosphere. U87 is a non-HER2 expressing glioblastoma cell line and was grown in a similar way, plated at 1000 cells/well.
[0589] The cells were exposed to the various ADCs diluted in complete media for 96 hours at 37° C., 5% CO.sub.2 in a humidified atmosphere. Cell viability was measured using the Promega Aqueous Celltitre96™ aqueous one solution cell proliferation kit according to manufacturer's instructions. Briefly, the media was removed and 100 μl of complete phenol red free media, pre-combined with MTS reagent, was added to the cells (20 μl of reagent per 100 μl of media). The plates were read on an ELISA plate reader at 490 nm after a 2 hr incubation in the dark (5% CO.sub.2, 37° C.).
[0590] The data (absorption units) were converted to % cell survival by using the untreated controls as the 100% cell survival and the Triton X-100 controls as the 100% cell death. The average absorption value for the latter was subtracted from all the rest of the data in order to get a suitable baseline. The averages were converted to survival and standard error values were obtained for each n value (as a % cell survival). The data were plotted and fitted to a dose-response sigmoidal logistic 3-parameter curve using the equation y=y.sub.0+a/(1+(x/x.sub.0)b) where, x.sub.0=IC50 and x.sub.0>0 and a=100 using SigmaPlot 11.0. Experiments were repeated at least 3 times for each compound tested and a set or an average of the data was plotted and fitted to obtain a dose response curve.
[0591] The data (
TABLE-US-00019 TABLE 12 Summary of Cell killing potency of scFv (TCT) & Trastuzumab P5C5 ADCs (compounds 5 and 71) SKBr3 Cells U87 Cells (HER2+++) (HER2−) IC50 IC50 IC50 IC50 Sample μg/ml nM μg/ml nM scFv (TCT)- 1.33 ± 0.1 47.5 ± 0.36 — — P5C5, DAR = 5 scFv (TCT)- 0.8 ± 0.1 .sup. 28 ± 0.36 — — P5C5, DAR = 8 scFv (TCT)- 5.01 ± 1.9 178.6 ± 68 100 3570 P5C5, DAR = 12 Trastuzumab- 0.07 ± 0.01 2.5 ± 0.4 — — P5C5, DAR = 5 Trastuzumab- 0.02 ± 0.004 0.7 ± 0.14 — — P5C5, DAR = 6 Free P5C5 (acid) N/A 2200 ± 400 N/A 7300 ± 900 drug
Example 32—Bioconjugation of Other Payloads (Camptothecin, Paclitaxel, MMAE, Maytansine) Derivatives onto a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0592] Camptothecin
[0593] A water-soluble derivative of camptothecin-NHS ester (compound 19) was conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction was controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated camptothecin-NHS was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0594] Buffer—bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol; [0595] Temperature—25° C.) [0596] Mixing conditions—Thermomixer 1000 rpm; [0597] Antibody at 1 mg/ml; [0598] Camptothecin-NHS—8 equivalent addition portions; and, [0599] NHS-drug addition rate—every 70-90 minutes.
[0600] Typically, scFv (TCT) was defrosted on the thermomixer at 4° C., then the temperature of the antibody aliquot was slowly raised to 20° C. Aliquots were spun down to collect any precipitate before using.
[0601] A camptothecin-NHS (compound 19) 100 mM stock solution was made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol in eppendorf microtubes and the buffer was equilibrated on the thermomixer at 4° C., and then the temperature was raised to 20° C. whilst mixing at 1000 rpm. The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the camptothecin-NHS was started.
[0602] This was carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 min, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation was in the sample with the highest number of drug equivalents and was very low.
[0603] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C., and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0604] The unconjugated and conjugated scFv (TCT) were analysed by HPLC size-exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0605] Mass spectrometric analysis was performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), were analysed by both MALDI-MS, and then further analysed by LC-MS. All samples gave well resolved peaks.
[0606] Paclitaxel
[0607] A water-soluble derivative of paclitaxel-NHS ester (compound 44) was conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction was controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated paclitaxel-NHS was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0608] Buffer—bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol; [0609] Temperature—25° C.; [0610] Mixing conditions—Thermomixer 1000 rpm; [0611] Antibody at 1 mg/ml; [0612] Paclitaxel-NHS—8 equivalent addition portions; and, [0613] NHS-drug addition rate—every 70-90 minutes.
[0614] Typically, scFv (TCT) was defrosted on the thermomixer at 4° C., then the temperature of the antibody aliquot was slowly raised to 20° C. Aliquots were spun down to collect any precipitate before using.
[0615] A paclitaxel-NHS 100 mM stock solution was made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol in eppendorf microtubes and the buffer was equilibrated on the thermomixer at 4° C., and then the temperature was raised to 20° C. whilst mixing at 1000 rpm. The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the paclitaxel-NHS was started.
[0616] This was carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 min, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation was in the sample with the highest number of drug equivalents and was very low.
[0617] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0618] The unconjugated and conjugated scFv (TCT) were analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0619] Mass spectrometric analysis was performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), were analysed by both MALDI-MS and then further analysed by LC-MS. All samples gave well resolved peaks.
[0620] MMAE
[0621] A water-soluble derivative of MMAE-NHS ester is conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction is controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated MMAE-NHS is determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were: [0622] Buffer—bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol; [0623] Temperature—25° C.; [0624] Mixing conditions—Thermomixer 1000 rpm; [0625] Antibody at 1 mg/ml; [0626] MMAE-NHS—8 equivalent addition portions; and, [0627] NHS-drug addition rate—every 70-90 minutes.
[0628] Typically, scFv (TCT) is defrosted on the thermomixer at 4° C., then the temperature of the antibody aliquot was slowly raised to 20° C. Aliquots are spun down to collect any precipitate before using.
[0629] An MMAE-NHS 100 mM stock solution is made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 is combined with filtered DMSO and glycerol in eppendorf microtubes and the buffer is equilibrated on the thermomixer at 4° C., and then the temperature is raised to 20° C. whilst mixing at 1000 rpm). The antibody is added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the MMAE-NHS is started.
[0630] This is carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 min, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples are left on the thermomixer for a further 2 hrs after the last addition. The samples are then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation is in the sample with the highest number of drug equivalents and that was very low.
[0631] All samples are initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0632] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all elute earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0633] Mass spectrometric analysis are performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), are analysed by both MALDI-MS and then further analysed by LC-MS. All samples give well resolved peaks.
[0634] Maytansine (DM4)
[0635] A water-soluble derivative of MaytansineDM4-NHS ester (compound 68) is conjugated to scFv (TCT) to obtain conjugates with various DARs (compound 74). The reaction is controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated MaytansineDM4-NHS is determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease. Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were:
[0636] Buffer (bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol), Temperature (25° C.), Mixing conditions (Thermomixer 1000 rpm), Antibody at 1 mg/ml, MaytansineDM4-NHS (8 equivalent addition portions), NHS-drug addition rate (every 70-90 minutes).
[0637] Typically, scFv (TCT) is defrosted on the thermomixer at 4° C., then slowly raising the temperature of the antibody aliquot to 20° C. Spun down to collect any precipitate before using.
[0638] A MaytansineDM4-NHS 100 mM stock solution is made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol into eppendorf microtubes and the buffer is equilibrated on the thermomixer at 4° C., and then the temperature is raised to 20° C. whilst mixing at 1000 rpm. The antibody is added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the MaytansineDM4-NHS was started.
[0639] This is carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 min, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples are left on the thermomixer for a further 2 hrs after the last addition. The samples are then collected by centrifugation (2.5 mins, 11 krpm). The only visible precipitation is in the sample with the highest number of drug equivalents and that was very low.
[0640] All samples are initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0641] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all elute earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0642] Mass spectrometric analysis are performed by SGS M-Scan. Conjugates, as well as ScFv-TCT (control), are analysed by both MALDI-MS and then further analysed by LC-MS. All samples give well resolved peaks.
[0643] Maytansine (DM1), 2-Step Method
[0644] DM1 drug is conjugated to scFv (TCT) to obtain conjugates with various DARs (compound 75). The reaction was controlled to obtain products with low, medium and high DARs. The procedure is carried out treating the scFv (TCT) with 32 equivalents of SPDP and subsequently reducing the samples with 115 equivalents of TCEP. The reduced, purified and quantified samples are then conjugated to DM1. DM1 was added to the antibody samples (in degassed PBS pH7/1 mM EDTA/20% DMSO/10% propylene glycol) at 2 equivalents each. The samples are incubated on the thermomixer (25° C., 1000 rpm) followed by 4° C. (1000 rpm) overnight. Recovered the samples by centrifugation and analysed by SDS-PAGE gel and UV/Vis spectroscopy.
[0645] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0646] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all elute earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0647] Pyrrolobenzodiazepine Conjugation, 2-Step Method
[0648] A PBD derivative, 6-maleimidocaproyl-SGD-1910 (compound 67) is conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction is controlled to obtain products with low, medium and high DARs. The procedure is carried out treating the scFv (TCT) with 32 equivalents of SPDP and subsequently reducing the samples with 115 equivalents of TCEP. The reduced, purified and quantified samples are then conjugated to 6-maleimidocaproyl-SGD-1910. 6-maleimidocaproyl-SGD-1910 was added to the antibody samples (in degassed PBS pH7/1 mM EDTA/20% DMSO/20% propylene glycol) at 2 equivalents each. The samples are incubated on the thermomixer for 3 hrs (25° C., 1000 rpm) followed by 4° C. (1000 rpm) overnight. Recovered the samples by centrifugation and analysed by SDS-PAGE gel and UV/Vis spectroscopy.
[0649] All samples were initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0650] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all elute earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0651] MMAE Conjugation, 2-Step Method
[0652] An MMAE derivative, 6-maleimidocaproyl-MMAE (compound 37) is conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction is controlled to obtain products with low, medium and high DARs. The procedure was carried out treating the scFv (TCT) with 32 equivalents of SPDP and subsequently reducing the samples with 115 equivalents of TCEP. The reduced, purified and quantified samples were then conjugated to 6-maleimidocaproyl-MMAE. 6-maleimidocaproyl-MMAE is added to the antibody samples (in degassed PBS pH7/1 mM EDTA/20% DMSO) at 2 equivalents each. The samples are incubated on the thermomixer for 3 hrs (25° C., 1000 rpm) followed by 4° C. (1000 rpm) overnight. Recovered the samples by centrifugation and analysed by SDS-PAGE gel and UV/Vis spectroscopy.
[0653] All samples are initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0654] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
[0655] Duocarmycin Conjugates
[0656] A water-soluble derivative of seco CBI-β-Glucuronide-NHS ester (compound 65) is conjugated to scFv (TCT) to obtain conjugates with various DARs. The reaction is controlled to obtain products with low, medium and high DARs. Initially, the hydrolysis rate of the pure isolated seco CBI-β-Glucuronide-NHS ester was determined in various buffer conditions. The conditions that gave a reasonable hydrolysis rate, i.e. not too fast so that the NHS would hydrolyse to the acid before it reacted with the lysines and not too slow so that the reaction would take too long to complete. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug and the concentration of the drug in the buffer. The latter is a crucial parameter; the more concentrated the drug is in the solution, the more the hydrolysis rate will decrease.
[0657] Therefore, the concentration needs to be controlled to allow for an efficient rate of hydrolysis. The conditions identified and carried forward were:
[0658] Buffer (bicarbonate buffer with NaCl at pH8.8 with 20% DMSO and 30% glycerol), Temperature (25° C.), Mixing conditions (Thermomixer 1000 rpm), Antibody at 1 mg/ml, seco CBI-β-Glucuronide-NHS ester (8 equivalent addition portions), NHS-drug addition rate (every 70-90 minutes).
[0659] Typically, scFv (TCT) is defrosted on the thermomixer at 4° C., then slowly raising the temperature of the antibody aliquot to 20° C. Spun down to collect any precipitate before using.
[0660] A seco CBI-β-Glucuronide-NHS ester 100 mM stock solution is made up in anhydrous filtered DMSO. Any precipitate was collected by centrifugation. Bicarbonate buffer pH 8.8 was combined with filtered DMSO and glycerol into eppendorf microtubes and the buffer is equilibrated on the thermomixer at 4° C., and then the temperature was raised to 20° C. whilst mixing at 1000 rpm. The antibody is added and equilibrated further (20° C., 1000 rpm) for 10 mins before the addition of the seco CBI-β-Glucuronide-NHS ester is started.
[0661] This is carried out by adding 8 equivalents of the NHS-drug DMSO stock and inverting to mix every 70 mins, before replacing on the thermomixer and mixing at 25° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples are left on the thermomixer for a further 2 hrs after the last addition. The samples were then collected by centrifugation (2.5 mins, 11 krpm).
[0662] The only visible precipitation was in the sample with the highest number of drug equivalents and was very low.
[0663] All samples are initially passed through a Zeba column (Pierce) pre-equilibrated with 10% IPA/PBS before being further purified on the HPLC-SEC with 20% IPA/PBS pH7, 25° C. and analysed by SDS-PAGE, HPLC-SEC, UV/Vis spectroscopy and mass spectrometry as described above.
[0664] The unconjugated and conjugated scFv (TCT) are analysed by HPLC-size exclusion chromatography using a Tosoh TSKGel G2000Wxl column. The ScFv has a retention time correlating to a MW of around 30 KDa. The conjugates all eluted earlier, indicating a larger molecular weight (due to varying drug loads), but as primarily monomeric peaks, indicating little or no aggregation.
Example 33—Raising Anti-Cemadotin Drug Monoclonal Antibodies and Use in Detecting scFv-Cemadotin ADCs
[0665] Keyhole Limpet Haemocyanin (KLH)-Cemadotin conjugate was produced by conjugating Cemadotin-NHS (compound 2) to 1 mg/ml KLH at a molar excess and purified by desalting.
[0666] Four mice were immunized using a standard schedule [Ref: Lane Immunology book] by contract research organisation Generon Ltd. The anti-sera was tested by ELISA on scFv (TCT)-Cemadotin and unconjugated scFv (TCT) and all four responded similarly (
[0667] Mouse-4 was used to create a panel of hybridomas of which, 11 clones were identified as strong binders. These 11 clones were ranked by ELISA and also tested by Western Blot for ability to bind to scFv (TCT)-Cemadotin conjugates and not the free components.
[0668] By ELISA, Clone-11 (GA6) appeared to be the best binder, while clone-9 (1E11) also performed strongly. Clones 5, 3, and 6 also showed detectable but weaker activity. Clones-7 and 8 were very weak and clones 1, 2, 4, and 10 appeared to be non-reactive (
[0669] The conditioned media of the hybridomas was tested for expression levels (
[0670] Strong binding was seen in clones 5, 9 and 11, picking up higher MW species not visible by eye. Clones 3 and 6 were next strongest, picking up a possible degradation product and clones 7 & 8 were weaker. Very weak/no binding was observed for clones 1, 2, 4 and 10
[0671] Clone-9 (1E11) was selected. The hybridoma was expanded, cultured and pure Mab was prepared by protein A chromatography.
Example 34—Measurement of the Pharmacokinetic Profile and Blood Clearance of an scFv-Cemadotin Conjugate
[0672] (A) Radioactive Assay
[0673] An scFv, optimised for lysine conjugation was prepared as described in Example 27 or 28 and conjugated (as described in Example 31) to an NHS-derived Cemadotin drug (compound 2). The average DAR was 5 for scFv-TCT-Cem-1 and 4 for scFv-TCT-Cem-2 as determined by SDS-PAGE and IEF gels. This was radiolabelled with Iodine-125 using sodium-125-Iodide (MP Biologicals) and Iodogen™ tubes (Thermo) according to the manufacturer's instructions.
[0674] Ten micrograms of radiolabelled scFv or conjugate were injected intravenously into the tail vein of a group of 4 BALB/c female mice (Harlan UK) per time point. At each time point, blood was collected by cardiac puncture under terminal anaesthesia from 4 mice and the spleens were removed by dissection. The radioactivity from the blood and spleen tissues was measured using a gamma counter and compared to the injected dose, correcting for tissue weight. The percentage-injected dose per gram of tissue was plotted over time and pharmacokinetic parameters determined by fitting to a bi-exponential clearance model. For studying in vivo blood clearance pharmacokinetics, data values were fitted using SigmaPlot to equations that conform to the two-compartmental intravenous model of clearance, which takes into account the biexponential clearance phases, distribution phase, and elimination phase, of single intravenous doses. This is described by the exponential decay, double, four-parameter equation y=ae.sup.−bx+ce.sup.−dx, where the distribution phase clearance rate (t.sub.1/2 alpha) can be determined by ln 2/b, and the elimination clearance rate (t.sub.1/2 beta) can be determined by ln 2/d.
[0675] The scFv-drug conjugates blood clearance (
TABLE-US-00020 TABLE 13 Pharmacokinetic parameters, blood clearance in mice of cemadotin-based conjugates (compound 69). Total Protein Level by 125-I labelling Table 13 t.sub.1/2 (alpha) hrs t.sub.1/2 (beta) hrs ScFv (TCT) 0.4 4.3 ScFv (TCT)-Cem 1 (High 0.3 6.6 DAR) ScFv (TCT)-Cem 2 (Medium 0.3 5.5 DAR)
[0676] (B) Direct ADC/scFv Detection Assay
[0677] An anti-Cemadotin MAb was raised (Example 33) and used to follow the ADC in mouse blood, to determine the pharmacokinetic properties. Normal BALB/c mice, 6-8 weeks old were maintained in filtered cages until used. A single batch of material (scFv-TCT, scFv (TCT)-Cemadotin, DAR=5 or 8) were prepared as above (Example 31) and 0.1 mg was injected into groups of 20 mice that were sacrificed at 4 time points (5 mice analysed per time point). Around 0.5 ml of whole blood was removed by cardiac puncture into EDTA-tubes and the serum collected by centrifugation. The serum was diluted appropriately into PBS and analysed by ELISA on HER2 coated micro-titre plates (Example 11D), detecting either by anti-T7 Tag (total antibody) or anti-cemadotin (total ADC). Non-injected reference samples were used to create a calibration curve for a direct read-out of ADC/ScFv blood concentration.
[0678] The results are shown for total functional scFv (
[0679] When measured directly, the free scFv demonstrated typical alpha (distribution) and beta (elimination) phases of bi-exponential blood clearance (Table 14; Constantinou A, et al (2009) Bioconjugate Chem 20:924-31), which was a rapid blood clearance due to the small size. The medium DAR ADC (5 drug payloads attached) had a slower blood clearance both in terms of tissue distribution and elimination. This suggested that the slightly larger molecular mass led to slower clearance rather than any aggregation leading to rapid clearance via the reticulo-endothelial system. This effect was even more pronounced with the higher DAR conjugate (8 drug payloads).
[0680] When the total ADC was measured, a similar pattern was seen with similar pharmacokinetic parameters (Table 14). A comparative plot illustrates this (
TABLE-US-00021 TABLE 14 Summary of pharmacokinetic data Total Antibody ADC Levels Measured by Anti-HER2 Measured by Anti-Cemadotin binding drug ELISA t.sub.1/2 (alpha) t.sub.1/2 (beta) t.sub.1/2 (alpha) t.sub.1/2 (beta) hrs hrs hrs hrs ScFv (TCT) 0.4 5.25 N/A N/A ScFv (TCT)- 0.96 6.87 0.53 5.43 CEM (High DAR = 8) ScFv (TCT)- 0.55 6.65 0.51 3.91 CEM (Medium DAR = 5)
Example 35—Measurement of the Pharmacokinetic Profile and Blood Clearance of an scFv-Ellipticine Conjugate
[0681] An anti-ellipticine MAb is raised as above (e.g. example 33) and used to follow the ADC in mouse blood, to determine the pharmacokinetic properties. Normal BALB/c mice, 6-8 weeks old were maintained in filtered cages until used. A single batch of material (scFv-TCT, scFv (TCT)-ellipticine, DAR=5 or 8) is prepared as above (compound 21, Example 29) and 0.1 mg is injected into groups of 20 mice that are sacrificed at 4 time points (5 mice analysed per time point). Around 0.5 ml of whole blood is removed by cardiac puncture into EDTA-tubes and the serum collected by centrifugation. The serum is diluted appropriately into PBS and analysed by ELISA on HER2 coated micro-titre plates (Example 31D), detecting either by anti-T7 Tag (total antibody) or anti-ellipticine (total ADC). Non-injected reference samples were used to create a calibration curve for a direct read-out of ADC/ScFv blood concentration.
[0682] When measured directly, the free scFv demonstrates typical alpha (distribution) and beta (elimination) phases of bi-exponential blood clearance (Constantinou A, et al (2009) Bioconjugate Chem 20:924-31), which is a rapid blood clearance due to the small size. The medium DAR ADC (5 drug payloads attached) has a slower blood clearance both in terms of tissue distribution and elimination. This suggested that the slightly larger molecular mass led to slower clearance rather than any aggregation leading to rapid clearance via the reticulo-endothelial system. This effect is even more pronounced with the higher DAR conjugate (8 drug payloads).
Example 36—Measurement of the Pharmacokinetic Profile and Blood Clearance of an scFv-Doxorubicin Conjugate
[0683] An anti-doxorubicin MAb is commercially available and used to follow the ADC in mouse blood, to determine the pharmacokinetic properties. Normal BALB/c mice, 6-8 weeks old were maintained in filtered cages until used. A single batch of material (scFv-TCT, scFv (TCT)-doxorubicin, DAR=5 or 8) is prepared as above (compound 72) and 0.1 mg is injected into groups of 20 mice that are sacrificed at 4 time points (5 mice analysed per time point). Around 0.5 ml of whole blood is removed by cardiac puncture into EDTA-tubes and the serum collected by centrifugation. The serum is diluted appropriately into PBS and analysed by ELISA on HER2 coated micro-titre plates (Example 31D), detecting either by anti-T7 Tag (total antibody) or anti-doxorubicin (total ADC). Non-injected reference samples were used to create a calibration curve for a direct read-out of ADC/ScFv blood concentration.
[0684] When measured directly, the free scFv demonstrates typical alpha (distribution) and beta (elimination) phases of bi-exponential blood clearance (Constantinou A, et al (2009) Bioconjugate Chem 20:924-31), which is a rapid blood clearance due to the small size. The medium DAR ADC (5 drug payloads attached) has a slower blood clearance both in terms of tissue distribution and elimination. This suggested that the slightly larger molecular mass led to slower clearance rather than any aggregation leading to rapid clearance via the reticulo-endothelial system. This effect is even more pronounced with the higher DAR conjugate (8 drug payloads).
Example 37—Measurement of the Pharmacokinetic Profile and Blood Clearance of an scFv-MMAE Conjugate
[0685] An anti-MMAE MAb is commercially available and used to follow the ADC in mouse blood, to determine the pharmacokinetic properties. Normal BALB/c mice, 6-8 weeks old were maintained in filtered cages until used. A single batch of material (scFv-TCT, scFv (TCT)-MMAE, DAR=5 or 8) is prepared as above (Example 32) and 0.1 mg is injected into groups of 20 mice that are sacrificed at 4 time points (5 mice analysed per time point). Around 0.5 ml of whole blood is removed by cardiac puncture into EDTA-tubes and the serum collected by centrifugation. The serum is diluted appropriately into PBS and analysed by ELISA on HER2 coated micro-titre plates (Example 31D), detecting either by anti-T7 Tag (total antibody) or anti-MMAE (total ADC). Non-injected reference samples were used to create a calibration curve for a direct read-out of ADC/ScFv blood concentration.
[0686] When measured directly, the free scFv demonstrates typical alpha (distribution) and beta (elimination) phases of bi-exponential blood clearance (Constantinou A, et al (2009) Bioconjugate Chem 20:924-31), which is a rapid blood clearance due to the small size. The medium DAR ADC (5 drug payloads attached) has a slower blood clearance both in terms of tissue distribution and elimination. This suggested that the slightly larger molecular mass led to slower clearance rather than any aggregation leading to rapid clearance via the reticulo-endothelial system. This effect is even more pronounced with the higher DAR conjugate (8 drug payloads).
Example 38—Measurement of the Tumour Uptake and Tumour to Normal Tissue Ratios of an scFv-Cemadotin Conjugate
[0687] An scFv, optimised for lysine conjugation is prepared as described in Example 27 or 28 and is conjugated (as described in Example 31) to an NHS-derived Cemadotin drug (compounds 2, 69). The average DAR is between 6-10. This was radiolabelled with Iodine-125 using sodium-125-Iodide (MP Biologicals) and Iodogen™ tubes (Thermo) according to the manufacturer's instructions.
[0688] Ten micrograms of radiolabelled scFv or conjugate were injected intravenously into the tail vein of a group of 4 BALB/c nude female mice (Harlan UK) growing subcutaneous tumours of the appropriate target expression (e.g. SKOV3 for HER2 expression) per time point. At each time point, blood is collected by cardiac puncture under terminal anaesthesia from 4 mice and the tumours and normal organs are removed by dissection. The radioactivity from the blood and tissues are measured using a gamma counter and compared to the injected dose, correcting for tissue weight. The percentage-injected dose per gram of tissue is plotted over time.
Example 39—Measurement of the Tumour Uptake and Tumour to Normal Tissue Ratios of an scFv-Ellipticine Conjugate
[0689] An scFv, optimised for lysine conjugation is prepared as described in Example 27 or 28 and is conjugated (as described in Example 29) to an NHS-derived ellipticine drug (compounds 23, 73). The average DAR is between 6-10. This was radiolabelled with Iodine-125 using sodium-125-Iodide (MP Biologicals) and Iodogen™ tubes (Thermo) according to the manufacturer's instructions.
[0690] Ten micrograms of radiolabelled scFv or conjugate were injected intravenously into the tail vein of a group of 4 BALB/c nude female mice (Harlan UK) growing subcutaneous tumours of the appropriate target expression (e.g. SKOV3 for HER2 expression) per time point. At each time point, blood is collected by cardiac puncture under terminal anaesthesia from 4 mice and the tumours and normal organs are removed by dissection. The radioactivity from the blood and tissues are measured using a gamma counter and compared to the injected dose, correcting for tissue weight. The percentage-injected dose per gram of tissue is plotted over time.
Example 40—Measurement of the Tumour Uptake and Tumour to Normal Tissue Ratios of an scFv-Doxorubicin Conjugate
[0691] An scFv, optimised for lysine conjugation is prepared as described in Example 27 or 28 and is conjugated (as described in Example 30) to an NHS-derived doxorubicin drug (compounds 7, 72). The average DAR is between 6-8. This was radiolabelled with Iodine-125 using sodium-125-Iodide (MP Biologicals) and Iodogen™ tubes (Thermo) according to the manufacturer's instructions.
[0692] Ten micrograms of radiolabelled scFv or conjugate were injected intravenously into the tail vein of a group of 4 BALB/c nude female mice (Harlan UK) growing subcutaneous tumours of the appropriate target expression (e.g. SKOV3 for HER2 expression) per time point. At each time point, blood is collected by cardiac puncture under terminal anaesthesia from 4 mice and the tumours and normal organs are removed by dissection. The radioactivity from the blood and tissues are measured using a gamma counter and compared to the injected dose, correcting for tissue weight. The percentage-injected dose per gram of tissue is plotted over time.
Example 41—Measurement of the Tumour Uptake and Tumour to Normal Tissue Ratios of an scFv-MMAE Conjugate
[0693] An scFv, optimised for lysine conjugation is prepared as described in Example 27 or 28 and is conjugated (as described in Example 32) to an NHS-derived MMAE drug. The average DAR is between 6-10. This was radiolabelled with Iodine-125 using sodium-125-Iodide (MP Biologicals) and Iodogen™ tubes (Thermo) according to the manufacturer's instructions.
[0694] Ten micrograms of radiolabelled scFv or conjugate were injected intravenously into the tail vein of a group of 4 BALB/c nude female mice (Harlan UK) growing subcutaneous tumours of the appropriate target expression (e.g. SKOV3 for HER2 expression) per time point. At each time point, blood is collected by cardiac puncture under terminal anaesthesia from 4 mice and the tumours and normal organs are removed by dissection. The radioactivity from the blood and tissues are measured using a gamma counter and compared to the injected dose, correcting for tissue weight. The percentage-injected dose per gram of tissue is plotted over time.
Example 42—Tumour Regression Studies in Nude Mice Bearing SKBr3 Tumour Xenografts Treated with Two scFv (TCT)-P5C5 ADC DARs
[0695] Female nude BALB/c mice, 6-8 weeks old (Harlan UK), were used for in vivo studies. All in vivo research was carried out under a UK Home Office project license PPL 70/5833. Human tumor xenografts were set up by injecting mice subcutaneously into the left flank with 0.1 mL containing up to 5 million SKBr3 cells in 50% matrigel. Tumour growth was monitored and took 2-3 weeks to reach the required 3-5 mm diameter for subsequent testing. When tumours were about 100 mm.sup.3, 10 mg/kg (0.25 mg total dose) was injected IV with a scFv (TCT)-P5C5 conjugate (as prepared in example 31, compound 71) on 5 sequential days (total ADC dose=1.25 mg per animal and the tumour sizes were monitored. The tumour volume is plotted compared to the starting volume (
Example 43—Tumour Therapy of an scFv-MMAE Conjugate
[0696] Female nude BALB/c mice, 6-8 weeks old (Harlan UK), are used for in vivo studies. All in vivo research is carried out under a UK Home Office project license. Human tumor xenografts are set up by injecting mice subcutaneously into the left flank with 0.1 mL containing up to 5 million SKOV3 cells in 50% matrigel. Tumour growth is monitored and takes 2-3 weeks to reach the required 3-5 mm diameter for subsequent testing. When tumours are about 100 mm.sup.3, 10 mg/kg (0.25 mg total dose) is injected IV with a scFv (TCT)-MMAE conjugate (as prepared in example 32) on 5 sequential days (total ADC dose=1.25 mg per animal and the tumour sizes were monitored. The tumour volume is plotted compared to the starting volume.
Example 44—Pharmaceutical Formulations and Administration
[0697] A further aspect of the invention provides a pharmaceutical formulation comprising a compound according to the first aspect of the invention in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent or carrier.
[0698] Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
[0699] The compounds of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.
[0700] In human therapy, the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
[0701] For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds of the invention may also be administered via intracavernosal injection.
[0702] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
[0703] Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
[0704] The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
[0705] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
[0706] For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be from 1 mg/kg to 30 mg/kg. Thus, for example, the tablets or capsules of the compound of the invention may contain a dose of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
[0707] The compounds of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
[0708] Aerosol or dry powder formulations are preferably arranged so that each metered dose or “puff” delivers an appropriate dose of a compound of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
[0709] Alternatively, the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.
[0710] For ophthalmic use, the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
[0711] For application topically to the skin, the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
[0712] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
[0713] Generally, in humans, oral or topical administration of the compounds of the invention is the preferred route, as they are the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the drug may be administered parenterally, e.g. sublingually or buccally.
[0714] For veterinary use, a compound of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
Example 45—Preparation of MMAF-C.SUB.5.-NHS Ester (78)
[0715] ##STR00036##
[0716] To a solution of MMAF (0.1 g, 0.177 mmol) in DMF (4 ml) was added DIPEA (0.12 ml) and glutaric anhydride (50.5 mg, 0.44 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 16 h. The solvents were evaporated in vacuo and the obtained crude compound was purified on Prep HPLC using Phenomenex Synergi Polar-RP column (Eluents: A=0.1% TFA in Water, B=MeCN) gradient—0 to 11 Min: 15 to 40% B, 11 to 24 min: 40 to 55% B, 24 to 35 min: 55 to 85% B. compound collected at tR 16.4 min and lyophilised to give a white solid (81%). HRMS: ESI m/z Found 846.5200 [M+H].sup.+ calculated 846.5228 for C.sub.44H.sub.72N.sub.5O.sub.11
[0717] .sup.1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.46-6.98 (m, 5H), 4.93-4.38 (m, 3H), 4.04 (dd, J=13.7, 9.9 Hz, 2H), 3.66 (dq, J=12.8, 6.4 Hz, 4H), 3.50-3.37 (m, 6H), 3.29-3.15 (m, 9H), 3.15-3.00 (m, 4H), 2.94-2.65 (m, 12H), 2.30 (t, J=7.4 Hz, 12H), 1.94 (p, J=6.6 Hz, 3H), 1.76 (p, J=7.4 Hz, 9H), 1.51-1.22 (m, 28H), 1.13 (dt, J=19.1, 6.8 Hz, 6H), 1.04-0.69 (m, 20H).
[0718] To a solution of MMAF-C.sub.5 (50 mg, 0.05 mmol) in DMF (1 ml) was added DIPEA (15 μl) and TSTU (18 mg, 0.05 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 4 h. The solvents were evaporated in vacuo and the obtained crude compound was purified on Prep HPLC using Phenomenex Synergi Polar-RP column (Eluents: A=0.1% TFA in Water, B=MeCN) gradient—0 to 11 Min: 15 to 40% B, 11 to 24 min: 40 to 55% B, 24 to 35 min: 55 to 85% B. The desired compound was collected at tR 20.4 min and lyophilised to give a white solid (22 mg, 40%). HRMS: ESI m/z Found 943.5416 [M+H].sup.+ calculated 943.5392 for C.sub.48H.sub.75N.sub.6O.sub.13
Example 46—Preparation of MMAF-C.SUB.5.-NHS Ester by Direct Activation
[0719] ##STR00037##
[0720] To a solution of MMAF (50 mg, 0.06 mmol) in acetonitrile (5 ml) was added DIPEA (0.1 ml) and Di(N-succinimidyl) glutarate (193 mg, 0.6 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 72 h. The solvents were evaporated in vacuo and the obtained crude compound was purified on Prep HPLC using Phenomenex Synergi Polar-RP column (Eluents: A=0.1% TFA in Water, B=MeCN) gradient—0 to 11 Min: 15 to 40% B, 11 to 24 min: 40 to 55% B, 24 to 35 min: 55 to 85% B. The desired compound was collected at tR 20.4 min and lyophilised to give a white solid (10 mg, 18%).
[0721] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.34 (t, J=7.6 Hz, 1H), 7.14-6.90 (m, 5H), 4.41 (dt, J=11.3, 4.8 Hz, 1H), 4.22 (t, J=8.1 Hz, 1H), 3.74 (dd, J=10.0, 4.4 Hz, 1H), 3.10-2.90 (m, 8H), 2.88-2.66 (m, 6H), 2.54-2.41 (m, 2H), 2.28 (p, J=1.8 Hz, 38H), 2.07-1.90 (m, 3H), 1.74-1.31 (m, 5H), 1.14 (d, J=48.5 Hz, 3H), 0.88-0.77 (m, 3H), 0.77-0.47 (m, 20H).
Example 47—Preparation of MMAE-PAB-Cit-Val-C.SUB.5.-NHS (82)
[0722] ##STR00038##
[0723] To a solution of MMAE (0.15 g, 0.18 mmol) and Fmoc-Val-Cit-PAB-PNP 13 (0.152 g, 0.19 mmol) in DMF (1.5 ml), was added HOBt (58 mg, 0.36 mmol), pyridine (0.12 ml) and DIPEA (31 μl). The reaction mixture was stirred under N.sub.2 atmosphere at room temperature for 24 h. Solvents were evaporated in vacuo and the residue triturated with ethyl acetate. The resulting solid was filtered, washed with ethyl acetate, dried to give 79. LC-MS ESI m/z 1367.7 [M+Na].sup.+ This was used directly without further purification.
[0724] A solution Fmoc-Val-Cit-PAB-MMAE 79 (0.3 g, 0.22 mmol) in DMF (1.5 ml) and diethylamine (1.12 ml) was stirred for 3 hours at room temperature. The reaction mixture was then concentrated in vacuo. The product was precipitated in diethyl ether and filtered affording 0.15 g of 80 as an off white powder which was used without further purification. LC-MS ESI m/z 1145.6 [M+Na].sup.+
[0725] .sup.1H NMR (400 MHz, DMSO-d6) δ 10.20 (d, J=6.5 Hz, 1H), 8.70 (d, J=7.6 Hz, 1H), 8.07 (d, J=4.9 Hz, 4H), 7.61 (dd, J=26.0, 8.3 Hz, 2H), 7.45-7.21 (m, 7H), 7.18 (dd, J=8.9, 6.3 Hz, 1H), 6.04 (s, 1H), 5.48 (s, 2H), 5.21-4.88 (m, 2H), 4.63-4.37 (m, 4H), 4.26 (t, J=11.7 Hz, 1H), 3.98 (m, 3H), 3.33-3.08 (m, 12H), 3.10-2.69 (m, 8H), 2.27-1.88 (m, 5H), 1.90-1.66 (m, 5H), 1.64-1.19 (m, 7H), 1.15-0.52 (m, 36H).
[0726] To a solution of compound H-Val-Cit-PAB-MMAE 80 (0.1 g, 0.177 mmol) in DMF (4 ml) was added DIPEA (0.12 ml) and glutaric anhydride (50.5 mg, 0.44 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 16 h. The solvents were evaporated in vacuo, the obtained crude compound 81 was washed with diethyl ether and used directly in the next step. HRMS: ESI m/z Found 1237.7526 [M+Na].sup.+ calculated 1237.7448 for C.sub.63H.sub.11N.sub.10O.sub.15
[0727] To a solution of MMAE-PAB-Cit-Val-C.sub.5 81 (0.1 g, 0.08 mmol) in DMF (2 ml) was added DIPEA (29 μl) and TSTU (38 mg, 0.12 mmol) at room temperature and the reaction mixture was stirred under N.sub.2 atmosphere for 3 h. The solvents were evaporated in vacuo and the obtained product was purified on Prep HPLC using Phenomenex Synergi Polar-RP column (Eluents: A=0.1% TFA in Water, B=MeCN) gradient—0 to 14 Min: 15 to 85% B, the desired compound was collected at tR 10.1 min and lyophilised to give a white powder (40%). HRMS: ESI m/z Found 1356.7623 [M+Na].sup.+ calculated 1356.7431 for C.sub.67H.sub.103N.sub.11O.sub.17Na
[0728] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.05-7.79 (m, 2H), 7.68 (d, J=8.6 Hz, 2H), 7.37 (d, J=8.0 Hz, 3H), 7.25-7.00 (m, 7H), 6.96 (t, J=7.2 Hz, 1H), 4.85 (d, J=15.3 Hz, 4H), 4.55-4.09 (m, 10H), 4.00-3.56 (m, 12H), 3.13-2.85 (m, 12H), 2.75 (s, 5H), 2.67-2.58 (m, 7H), 2.54-2.36 (m, 4H), 2.28 (p, J=1.8 Hz, 88H), 2.14-1.99 (m, 4H), 1.95-1.67 (m, 5H), 1.68-1.40 (m, 8H), 1.39-0.93 (m, 7H), 0.90-0.31 (m, 40H).
Example 48—Preparation of MMAE-PAB-Cit-Val-dPEG.SUB.5.-NHS Ester (84)
[0729] ##STR00039##
[0730] To a solution of compound H-Val-Cit-PAB-MMAE 80 (0.2 g, 0.177 mmol) in DMF (7 ml) was added DIPEA (0.1 ml) and Acid-dPEG.sub.5-NHS (50.5 mg, 0.21 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 16 h. The solvents were evaporated in vacuo, the obtained crude compound 83 was washed with diethyl ether and directly used for the next step. LC-MS ESI m/z 1466.6 [M+Na].sup.+
[0731] To a solution of Acid-dPEG.sub.5-Val-Cit-PAB-MMAE 83 (0.25 g, 0.17 mmol) in DMF (7 ml) was added DIPEA (0.15 ml) and TSTU (120 mg, 0.39 mmol) at room temperature and the reaction mixture was stirred under N.sub.2 atmosphere for 3 h. The solvents were evaporated in vacuo and the crude product was purified on Biotage flash purification system using C18 column to yield the compound NHS-PEG.sub.3-Val-Cit-PAB-MMAE 84 as a white solid after lyophilisation, MS: ESI m/z 1562.9 [M+Na].sup.+
[0732] .sup.1H NMR (400 MHz, DMSO-d6) δ 10.03-9.96 (m, 1H), 8.12 (dd, J=19.3, 7.8 Hz, 1H), 7.90 (t, J=8.9 Hz, 1H), 7.69-7.55 (m, 2H), 7.38-7.23 (m, 5H), 7.23-7.13 (m, 1H), 5.11-4.93 (m, 2H), 4.52-4.33 (m, 3H), 4.32-4.19 (m, 2H), 3.99 (m, 2H), 3.83-3.68 (m, 2H), 3.65-3.41 (m, 17H), 3.22 (dd, J=19.9, 8.6 Hz, 6H), 3.12 (s, 1H), 3.10-2.74 (m, 12H), 2.42 (m, 12H), 2.26 (dd, J=15.9, 9.2 Hz, 1H), 2.19-1.90 (m, 4H), 1.79-1.65 (m, 3H), 1.65-1.52 (m, 2H), 1.52-1.31 (m, 3H), 1.08-0.96 (m, 6H), 0.96-0.71 (m, 23H).
Example 49—Preparation of MMAE-PAB-Cit-Val-dPEG.SUB.9.-NHS Ester (86)
[0733] ##STR00040##
[0734] MMAE-PAB-Cit-Val-dPEG.sub.9-NHS ester 86 was prepared as example 47 by reacting H-Val-Cit-PAB-MMAE 80 with Acid-dPEG.sub.9-NHS followed by activation with TSTU. The crude, obtained after evaporation was purified on Biotage flash purification system using C18 column to yield the compound MMAE-PAB-Cit-Val-dPEG.sub.9-NHS 86 as a white solid after lyophilisation, MS: ESI m/z 1718.3093 [M+H].sup.+
Example 50—Preparation of Auristatin F-C.SUB.5.-NHS Ester (88)
[0735] ##STR00041##
[0736] Auristatin F (0.1 g, 0.116 mmol) and HATU (40 mg, 0.104 mmol) was dissolved in DMF (3 ml) and DIPEA (40 μl) was added to it. The reaction mixture was stirred at room temperature for 40 min and then added dropwise to a solution of 5-Aminovaleric acid (15 mg, 0.127 mmol) in DMF (2 ml). The reaction mixture was stirred at room temperature for 4 h and evaporated in vacuo. The crude product was purified on Biotage flash purification system using C18 column to yield the compound Auristatin F-C.sub.5 acid 87 as a white solid (85 mg, 84%). LC-MS ESI m/z 867.5 [M+Na].sup.+
[0737] To a solution of the Auristatin F-C.sub.5 acid 87 (70 mg, 0.08 mmol) in DIPEA (72 μl) and DMF (3 ml) was added TSTU (57 mg 0.19 mmol) and the reaction mixture was stirred at room temperature for 1 h. The solvents were evaporated in vacuo and the crude compound was purified on Biotage flash purification system using C18 column to yield the compound Auristatin F-C.sub.5-NHS ester 88 as a white solid after lyophilisation (46 mg, 59%). LC-MS ESI m/z 964.5 [M+Na].sup.+
[0738] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.94 (q, J=7.9, 7.2 Hz, 1H), 8.05-7.84 (m, 1H), 7.31-7.10 (m, 5H), 4.60 (m, 2H), 3.99 (d, J=7.5 Hz, 1H), 3.84-3.73 (m, 2H), 3.70 (d, J=7.6 Hz, 1H), 3.50 (td, J=14.0, 7.5 Hz, 1H), 3.32-3.10 (m, 9H), 2.99 (m, 6H), 2.84-2.58 (m, 14H), 2.45 (dd, J=14.9, 5.0 Hz, 4H), 2.37-2.22 (m, 3H), 2.01 (dd, J=12.8, 5.7 Hz, 1H), 1.94-1.73 (m, 3H), 1.59-1.36 (m, 5H), 1.08-0.71 (m, 25H).
Example 51—Preparation of Maytansinol DM1-dPEG.SUB.4.-NHS Ester (89)
[0739] ##STR00042##
[0740] To a solution of DM1 (0.1 g, 0.135 mmol) in THF was added Et3N (18.9 μl) followed by addition of the Mal-dPEG.sub.4-NHS (77 mg, 0.15 mmol). The reaction mixture was stirred at room temperature under N.sub.2 atmosphere for 1 h and the solvents were removed in vacuo. The crude product was purified on Biotage flash purification system using C18 column to yield the compound DM1-dPEG.sub.5-NHS as a white solid after lyophilisation (105 mg, 75%). LC-MS ESI m/z 1274.60 [M+Na].sup.+
[0741] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.01 (q, J=5.6 Hz, 1H), 7.17 (dd, J=8.1, 1.8 Hz, 1H), 6.90 (s, 1H), 6.68-6.46 (m, 3H), 5.55 (dd, J=12.8, 8.9 Hz, 1H), 5.31 (q, J=6.7 Hz, 1H), 4.52 (dd, J=12.1, 2.9 Hz, 1H), 4.07 (t, J=10.8 Hz, 2H), 3.93 (d, J=1.5 Hz, 4H), 3.85 (dd, J=9.0, 4.0 Hz, 1H), 3.71 (t, J=6.0 Hz, 2H), 3.63-3.41 (m, 18H), 3.37 (t, J=5.9 Hz, 3H), 3.32-3.07 (m, 11H), 3.07-2.86 (m, 6H), 2.79 (d, J=11.6 Hz, 7H), 2.71 (s, 4H), 2.25 (m, 4H), 2.04 (d, J=14.4 Hz, 1H), 1.59 (s, 4H), 1.55-1.34 (m, 3H), 1.33-1.03 (m, 8H), 0.78 (d, J=2.1 Hz, 3H).
Example 52—Preparation of Maytansinol DM1-dPEG.SUB.12.-NHS Ester (90)
[0742] ##STR00043##
[0743] To a stirred and degassed solution of DM1 (0.05 g, 0.1678 mmol) in THF (3 ml) was added Et.sub.3N (9.45 μl) followed by addition of the Mal-dPEG.sub.9-NHS (58.7 mg, 0.1678 mmol) dissolved in THF (4 ml). The reaction mixture was stirred at room temperature under N.sub.2 atmosphere for 3 h and the solvents were removed in vacuo. The crude product was purified on Biotage flash purification system using C18 column to yield the compound DM1-dPEG.sub.12-NHS 90 as a white solid after lyophilisation (31 mg, 28%). LC-MS ESI m/z 1625.9 [M+Na].sup.+
Example 53—Preparation of Ellipticine-(DNMEA)-PAB-Cit-Val-dPEG.SUB.3.-NHS Ester (95)
[0744] ##STR00044## ##STR00045##
[0745] To a stirred solution of N,N′-dimethylethylene diamine (3.66 mL, 34 mmol) in dichloromethane (40 mL) at 0° C. was added dropwise a solution of di-tert-butyl dicarbonate (2.4 g, 11 mmol) in dichloromethane (20 mL) and allowed to warm to room temperature overnight, concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2×100 mL), brine (100 mL), dried and concentrated under reduced pressure to give the title product 91 as a colourless oil (1.54 g, 74% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.26 (t, J=6.15 Hz, 2H), 2.81 (s, 3H), 2.66 (t, J=6.57 Hz, 2H), 2.38 (s, 3H), 9.28 (s, 9H) ppm.
[0746] To a stirred solution of 9-hydroxyellipticine (80 mg, 0.27 mmol), DMAP (32 mg, 0.27 mmol) and triethylamine (261 μL, 1.88 mmol) in THF (2 mL) at 0° C., was added a solution of 4-nitrophenylchloroformate (81 mg, 0.40 mmol) in THF (1.5 mL), warmed to room temperature over 2 h, to which was added a solution of BOC-diamine 91 (151 mg, 0.80 mmol) in THF (0.5 mL), stirred overnight at room temperature, concentrated under reduced pressure and chromatographed (0-20% MeOH in CH.sub.2Cl.sub.2) to give BOC-amine-ellipticine 92 as a yellow solid (92 mg, 72% yield). R.sub.f=0.47 (10% MeOH in CH.sub.2Cl.sub.2), IR v.sub.max 3377, 2976, 2088, 1701, 1674, 1601, 1462, 1397, 1191, 1144, 1030, 816, 790, 721 cm.sup.−1; .sup.1H NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 9.88-9.75 (d, J=5.4 Hz, 1H), 8.48-8.35 (d, J=6.4 Hz, 1H), 8.13 (s, 1H), 8.10 (s, 1H), 7.64-7.54 (dd, J=8.7, 3.9 Hz, 1H), 7.42-7.25 (m, 1H), 3.45 (s, 6H), 2.85-2.82 (m, 4H), 2.84 (s, 6H), 1.41 (s, 9H) ppm; .sup.13C NMR (101 MHz, DMSO-d6) δ 226.28, 224.53, 209.31, 162.06, 142.18, 133.59, 121.52, 117.90, 116.45, 111.46, 109.63, 79.16, 55.12, 46.90, 46.13, 46.13, 28.56, 14.98, 12.47, 9.03 ppm; MS (EI.sup.+) m/z 477 [M+H].sup.+; HRMS (EI.sup.+) m/z calc'd for C.sub.27H.sub.33N.sub.4O.sub.4 [M+H].sup.+ 477.2502, found 477.2503.
[0747] A solution of BOC-amine-ellipticine 92 (93 mg, 0.19 mmol) in trifluoroacetic acid (2.5 mL) was stirred at room temperature for 3 h and concentrated under reduced pressure to give desired deprotected product 93 as a yellow solid (105 mg, 90% yield). R.sub.f=0.14 (20% MeOH in CH.sub.2Cl.sub.2); IR v.sub.max 2995, 2821, 1670, 1473, 1397, 1174, 1127, 1021, 813, 795, 721 cm.sup.−1; .sup.1H NMR (400 MHz, DMSO-d6) δ 9.03-8.72 (d, J=48.5 Hz, 2H), 8.58-8.36 (m, 2H), 8.86-8.69 (m, 1H), 12.57-11.95 (d, J=2.7 Hz, 1H), 10.18-9.85 (d, J=2.2 Hz, 1H), 8.31-8.08 (dd, J=11.3, 2.2 Hz, 1H), 7.87-7.58 (d, J=8.6 Hz, 1H), 7.54-7.37 (dt, J=8.8, 2.3 Hz, 1H), 3.35-3.26 (d, J=5.1 Hz, 3H), 3.17 (s, 6H), 3.15-3.06 (qd, J=7.3, 4.7 Hz, 4H), 2.87 (s, 3H) ppm; .sup.13C NMR (101 MHz, DMSO-d6) δ 159.01, 158.68, 157.26, 155.79, 144.77, 134.42, 134.14, 128.46, 125.98, 122.68, 120.29, 120.07, 112.01, 110.91, 49.06, 35.16, 34.96, 33.16, 15.35, 12.48 ppm; MS (EI.sup.+) m/z 377 [M].sup.+; HRMS (EI.sup.+) m/z calc'd for C.sub.22H.sub.25N.sub.4O.sub.2 [M].sup.+377.1978, found 377.1974.
[0748] To a stirred solution of the activated linker 30 (60.7 mg, 0.08 mmol) in DMF (3 ml) was added at room temperature a solution of the ellipticine amine 93 (48 mg, 0.10 mmol) and DIPEA (40 μL, 0.23 mmol) in DMF (1 mL), stirred overnight, concentrated under reduced pressure and chromatographed (0-20% MeOH in CH.sub.2Cl.sub.2) to give the title product 94 (19 mg, 26% yield). R.sub.f=0.30 (10% MeOH in CH.sub.2Cl.sub.2); IR v.sub.max 3310, 2935, 2103, 1650, 1541, 1466, 1402, 1202, 1130, 1027, 823, 800, 756, 720 cm.sup.−1; .sup.1H NMR (400 MHz, DMSO-d6) δ 11.79 (s, 1H), 10.01 (s, 1H), 9.82 (s, 1H), 8.44 (s, 2H) 8.39-8.30 (d, J=7.2 Hz, 1H), 8.23-8.16 (d, J=6.3 Hz, 1H), 8.15-8.11 (d, J=6.8 Hz, 1H), 7.63-7.54 (d, J=6.8 Hz, 2H), 2.05-1.96 (m, 1H), 7.48-7.42 (d, J=8.9 Hz, 1H), 7.32-7.27 (d, J=8.1 Hz, 3H), 5.42 (s, 2H), 5.10-5.00 (d, J=9.0 Hz, 2H), 4.46-4.28 (q, J=8.0, 7.3 Hz, 1H), 3.95 (s, 2H), 3.64-3.55 (m, 14H), 6.03 (s, 1H), 3.18-3.11 (ddd, J=10.8, 7.3, 3.7 Hz, 4H), 3.05-2.88 (m, 2H), 2.85 (s, 2H), 2.10-1.91 (dt, J=13.5, 7.2 Hz, 1H), 1.75-1.53 (m, 2H), 1.48-1.32 (m, 2H), 1.27 (s, 6H), 0.96-0.72 (ddd, J=22.7, 6.3, 3.2 Hz, 6H) ppm; .sup.13C NMR (126 MHz, DMSO-d6) δ 171.21, 170.95, 169.44, 162.80, 159.37, 158.36, 158.12, 155.46, 147.76, 145.15, 143.25, 140.41, 139.01, 133.58, 129.03, 128.77, 124.65, 123.33, 122.68, 121.45, 119.41, 117.59, 111.50, 109.74, 70.87, 70.07, 69.90, 69.69, 57.03, 53.98, 50.51, 49.13, 47.01, 42.24, 35.24, 31.60, 29.65, 27.39, 19.64, 18.51, 17.18, 15.01, 12.88 ppm; MS (EI.sup.+) m/z 1019 [M+Na]; HRMS (EI.sup.+) m/z calc'd for C49H641\11201 Na [M+Na]′ 1019.4715, found 1019.4722.
[0749] To a stirred solution of the azide 94 (2.5 mg, 0.003 mmol) in DMF (1 mL) was added (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (0.7 mg, 0.003 mmol), stirred at room temperature for 3 h and concentrated under reduced pressure to give the title product as a yellow solid (3 mg, 93% yield). IR v.sub.max 3323, 2924, 1811, 1786, 1740, 1664, 1604, 1537, 1402, 1199, 1126, 828, 799, 718 cm.sup.−1; .sup.1H NMR (500 MHz, DMSO-d6) δ 12.02 (s, 1H), 10.10-9.97 (m, 1H), 9.94-9.84 (d, J=8.6 Hz, 1H), 8.49-8.41 (d, J=6.7 Hz, 2H), 8.36-8.30 (dd, J=7.2, 3.4 Hz, 1H), 7.64-7.51 (td, J=12.0, 9.0, 4.7 Hz, 1H), 7.56-7.51 (d, J=8.4 Hz, 2H), 7.45-7.40 (d, J=8.6 Hz, 1H), 7.38-7.21 (m, 3H), 6.09-5.89 (q, J=5.5, 5.0 Hz, 1H), 5.42 (s, 2H), 5.11-4.97 (m, 2H), 4.53-4.45 (t, J=8.0 Hz, 2H), 4.39-4.32 (t, J=5.4 Hz, 2H), 3.96-3.91 (d, J=3.8 Hz, 2H), 3.71-3.66 (q, J=5.7, 4.2 Hz, 2H), 3.65-3.55 (dtt, J=20.5, 9.9, 4.7 Hz, 10H), 3.51-3.43 (m, 4H), 3.18-3.08 (qd, J=7.3, 4.1 Hz, 4H), 2.91-2.87 (s, 4H), 2.86-2.85 (s, 2H), 2.292.18 (m, 2H), 2.19-2.11 (m, 2H), 2.00-1.93 (d, J=6.7 Hz, 2H), 0.90-0.70 (ddd, J=29.9, 6.9, 4.3 Hz, 6H), 2.32-1.89 (m, 6H), 2.10-2.02 (m, 2H) ppm; .sup.13C NMR (126 MHz, DMSO-d6) δ 172.75, 170.73, 170.48, 169.94, 168.96, 162.29, 158.90, 154.87, 151.30, 144.85, 143.09, 139.99, 138.62, 133.72, 133.47, 128.53, 128.30, 122.61, 118.91, 117.16, 111.25, 109.72, 99.50, 98.90, 70.33, 70.29, 69.69, 69.65, 69.53, 69.31, 57.37, 56.55, 53.50, 53.20, 47.13, 46.54, 41.76, 40.15, 40.09, 40.00, 39.93, 39.84, 39.76, 39.67, 39.60, 39.50, 39.34, 39.17, 39.00, 38.51, 35.77, 34.82, 34.51, 31.05, 30.76, 29.17, 28.41, 26.85, 25.49, 25.34, 25.29, 25.21, 22.33, 22.04, 21.80, 21.22, 20.74, 20.69, 20.19, 19.89, 19.54, 19.15, 18.85, 18.04, 17.90, 16.92, 16.71, 16.67, 14.68, 12.41, 12.03 ppm; MS (EI.sup.+) m/z 1288 [M+H].sup.+; HRMS (EI.sup.+) m/z calc'd for C.sub.64H.sub.82N.sub.13O.sub.16 [M+H]′ 1288.5995, found 1288.5625.
Example 54—Preparation of Ellipticine-(DNMEA)-PAB-Cit-Val-BCN-dPEG.SUB.4.-NHS Ester (96)
[0750] ##STR00046##
[0751] To a stirred solution of the azide 94 (2.5 mg, 0.003 mmol) in DMF (1 mL) was added BCN-PEG.sub.4-NHS (ConjuProbe) (0.7 mg, 0.003 mmol), stirred at room temperature for 3 h and concentrated under reduced pressure to give the desired product 96 as a yellow solid (3 mg, 93% yield).
Example 55—Preparation of SN38-(DNMEA)-PAB-Cit-Val-BCN-dPEG.SUB.4.-NHS Ester (100)
[0752] ##STR00047## ##STR00048##
[0753] To a stirred solution of phosgene in toluene (15 wt %, 0.36 mL, 0.55 mmol) at 0° C. was added dropwise a solution of BOC-diamine 91 (94 mg, 0.50 mmol) and Et.sub.3N (77 μL, 0.55 mmol) in toluene (1.32 mL), stirred at 0° C. for 2 h, warmed to room temperature overnight, filtered, washed with toluene (5 mL), concentrated under reduced pressure and dissolved in pyridine (2.23 mL, 27.6 mmol), to which was added SN38 (150 mg, 0.39 mmol), stirred at room temperature overnight, diluted with EtOAc (100 mL), washed with water (3×100 mL), dried, concentrated under reduced pressure and chromatographed (0-10% MeOH in CH.sub.2Cl.sub.2) to give the title product as a yellow solid (72 mg, 31%). R.sub.f=0.57 (10% MeOH in CH.sub.2Cl.sub.2); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.15 (dd, J=9.3, 5.7 Hz, 1H), 7.85-7.73 (m, 1H), 7.64-7.61 (m, 1H, 7.53-7.48 (m, 1H), 5.64 (d, J=16.3 Hz, 1H), 5.22 (d, J=16.3 Hz, 1H), 5.18 (s, 2H), 3.65-2.85 (m, 6H), 2.82 (s, 6H), 1.85 (p, J=7.1 Hz, 2H), 1.40 (s, 9H), 1.34 (t, J=7.8 Hz, 3H), 0.94 (t, J=7.3 Hz, 3H) ppm; LCMS t.sub.R=2.10 min m/z 607.3 [M+H].sup.+.
Example 56—Preparation of DM1-Mal-SO.SUB.3.H-NHS (102)
[0754] ##STR00049##
Example 57—Preparation of Maytansinol-PEG.SUB.2.-Glu-BCN-NHS Ester (108)
[0755] ##STR00050## ##STR00051##
Example 58—Preparation of Maytansinol-SO.SUB.3.H-BCN-dPEG.SUB.4.-NHS Ester (110)
[0756] ##STR00052##
Example 59—Preparation of MMAE-β-Glucuronide-C.SUB.5.-NHS Ester (113)
[0757] ##STR00053##
Example 60—Preparation of PBD-MMAF-Dimer-C.SUB.5.-NHS Ester (116)
[0758] ##STR00054##
Example 61—Preparation of MMAF-Tetrazine (117)
[0759] ##STR00055##
Example 62—Scheme for Conjugates
[0760] ##STR00056##
##STR00057##
##STR00058##
##STR00059##
##STR00060##
##STR00061##
##STR00062##
##STR00063##
##STR00064##
##STR00065##
##STR00066##
##STR00067##
##STR00068##
##STR00069##
##STR00070##
##STR00071##
##STR00072##
##STR00073##
Example 63. Expression and Purification of a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0761] Construction of the High Affinity, Anti-HER2 Cytoplasmic-Expression scFv Clone, TCT1067
[0762] The anti-HER2 scFv in Example 27 was modified by mutagenesis to increase its binding affinity by over 1000-fold [Schier R et al (1996) J. Mol. Biol 263, 551-67].
[0763] This retained the multiple, well-spaced, optimally positioned, surface lysine residues configuration and was expressed and purified as described in Example 27.
[0764] The resulting protein was called scFv (TCT1067). The DNA and protein sequences are below:
TABLE-US-00022 DNA sequence of cleaved TCT1067 [SEQ ID NO: 3] GCGGTAGCGGAGGTAGCGGACAGGTGCAGCTGGTGCAGTCTGGGGCAGAG GTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATA CAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAG GCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATAC AGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAG CACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGT ATTTTTGTGCGAGACATGACGTGGGATATTGCACCGATCGTACCTGCGCA GCGTGGCCTGAATGGCTGGGCGTGTGGGGCCAGGGCACCCTGGTCACCGT CTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGAT CGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAG AAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTA TGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCT ATGGTCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCC AAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGA TGAGGCTGATTATTACTGTGCAAGCTGGGATTATACCCTGAGTGGTTGGG TGTTCGGCGGAGGGACCAAGCTGACCGTCCTAATGGCTAGCATGACTGGT GGACAGCAAATGGGTTGATGAGGCTCTAACTCTCCTCT Amino Acid sequence of cleaved scFv (TCT1067) [SEQ ID NO: 4] SGSGGSGQVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQVT ISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDR TCAAWPEWLGVWGQGTLVTVSSGGGGSGGGGSGGGG SQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWY QQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAI SGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVL
Number of Amino acids: 272, Molecular weight: 28,219 Da Theoretical PI: 8.14, Extinction coefficient: 70 735 KEY: Bold = Residual Ser left after TEV cleavage, Underlined = Linker region (GSGGSG(SEQ ID NO: 8)) Unformatted = scFv sequence, Bold italics = T7 tag sequence
Example 64A. Bioconjugation of a Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues to an NHS-Bearing Moiety
[0765] Generic Method for Conjugating an scFv to a Payload-NHS
[0766] Initially, the hydrolysis rate of the pure, payload-NHS was examined in selected buffer conditions in order to optimise its availability. Other factors that were taken into account were the stability of the antibody in the buffer/pH/organic solvent, the stability of the drug, and the amount and rate of the drug addition to the reaction. The conditions identified and used throughout unless otherwise specified are:
TABLE-US-00023 TABLE 15 Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 1 mg/ml concentration Payload NHS dissolved in 100% anhydrous filtered DMSO at a handling concentration of either 50 mM or 100 mM Payload addition every 70-120 mins rate Payload addition 10 to 16 equivalents equivalents
[0767] The scFv was thawed at 4° C. and any precipitate formed was collected by centrifugation (10 mins, 10 krpm, 4° C.).
[0768] The stock solution of payload-NHS was made up in anhydrous filtered DMSO and any precipitate was collected by centrifugation. Bicarbonate buffer pH8.8 was combined with filtered anhydrous DMSO in protein low-bind eppendorf microtubes and the mixture was equilibrated on a Thermomixer (with the temperature raised from 4° C. to 20° C., whilst mixing at 1000 rpm). The antibody was added and equilibrated further (20° C., 1000 rpm) for 10 min before the addition of the payload-NHS. This was carried out by adding the x number of equivalents (dependent on the payload) of the NHS-drug DMSO stock and inverting to mix every 70-120 min, before replacing on the Thermomixer and mixing at 20° C., 1000 rpm. The total number of equivalents used depended on the required DAR. The samples were left on the Thermomixer for a further 2-18 hrs after the last addition. The samples were then collected by centrifugation (2.5 min, 11 krpm, 4° C.).
[0769] All samples were pH neutralised (by the addition of 0.1M NaH.sub.2PO.sub.4) and purified by HPLC-SEC on a Tosoh TSKGel G2000 SW.sub.XL column eluting with 10% IPA/PBS at pH 7, 20° C. The sample and fractions were kept cold throughout (4° C.), the relevant fractions were combined and concentrated on Vivaspin columns (HY or PES membrane) (10,000 MWCO). The IPA was removed by spin concentrating several times (at least 500 fold) using PBS, filtered through a 0.2 μm supor membrane and quantified using UV/Vis spectroscopy and amino acid analysis.
[0770] The Final Product was Analysed by:
[0771] 1. Reducing SDS-PAGE
[0772] Precast reducing SDS-PAGE gels (12%) were routinely used for analysis of samples and stained with Coomassie Blue.
[0773] 2. Amino Acid Analysis
[0774] The DAR and sample concentration was accurately determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility. From the AAA, the amount in mol of both the protein and the drug (due to the drug's fingerprint-release, see Table 16A for each drug) can be derived and the DAR calculated (No mol drug/No mol protein). The concentration of the protein in the solution can be calculated by first calculating the conjugate's molecular weight based on the DAR, and then subsequently converting the concentration obtained from AAA to mg/ml of protein. For example: scFv (TCT) is 28162 (MS), DAR is ‘X’, and each drug molecule adds ‘XX’ onto the antibody. Therefore conjugate MW=28162+(X×X)=X. The concentration is X nmol/ml which is equal to X μg/ml of protein.
TABLE-US-00024 TABLE 16A Quantification using amino acid analyses Amino acid liberated and used for Payoad quantification P5C5 5-aminovaleric acid Cemadotin 4-aminomethylbenzoic acid MMAF no atypical ones, used calcn of excess Phenyl alanine AF 5-aminovaleric acid MMAE ornithine (from citrulline) DM1 no identifiable fragments
[0775] 3. HPLC-SEC
[0776] Samples were analysed and/or purified on a Tosoh TSKGel G2000SW.sub.XL or on a Sepax Zenix-C SEC-150, 3 μm, 7.8×300 mm, monitoring at 214 and 280 nm, flow rate 0.5 ml/min for 30 mins, 20° C., samples at 4° C. Typically, 20 μg of protein was injected for analytical runs and 300 μg for semi-preparative runs.
[0777] 4. Mass Spectrometry
[0778] Sample preparation: free antibody and conjugates (payload MMAF, P5C5, AuF and MMAE) were desalted on a COSTAR® SPIN-X® (Sigma Aldrich) by centrifuging for 2 min at 10.5 g.
[0779] Typically, for free antibody, P5, AF and MMAE conjugates, 20-30 μg of protein was used. For MMAF 80 μg of protein was used.
[0780] DM1 conjugates were desalted using a 0.5 mL zeba column (thermofisher), injecting 30 μg of protein.
[0781] Liquid chromatography experiments were performed on an Agilent 1100 system connected to MSQ Plus Single Quad Detector (SQD). An XBridge column, BEH300 C4 3.5 μm 2.1×100 mm was used at 0.5 ml/min, 10 min gradient from 95% A to 50% B then holding for 5 mins at 50% B where
[0782] A: H.sub.2O/0.1% FA
[0783] B: 90% CH.sub.3CN/H.sub.2O (0.1% FA)
[0784] Wavelength: 254 nm.
[0785] The parameters of the instruments were optimized to allow the stabilization and transmission of high molecular weight species. Scan Range: m/z=500-2000. Scan time: 1.5 s. Data obtained in continuum mode. The electrospray source of the MS was operated with a capillary voltage of 4.2 kV and a cone voltage of 50 V. Nitrogen was used as the nebulizer and desolvation gas at a total flow of 600 L/h. Ion series were generated by integration of the total ion chromatogram (TIC) over the 3.5-5.0 min range. Total mass spectra for protein samples were reconstructed from the ion series using the pre-installed ProMass software.
[0786] 5. Binding Analyses by Biacore Surface Plasmon Resonance (SPR)
[0787] The binding affinity against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv on a Biacore T200. A standard amine-coupling method was used to prepare all Biacore surfaces (CM5 or CM3), as follows. Flow cells were activated individually by injecting a freshly mixed solution of 0.2M EDC in 0.05 M NHS at 30 μl/min. 12.5 μg/ml of Her2 was injected until the desired level of immobilization was reached (typically ˜1500 RU). Excess NHS ester was deactivated using a 1 M ethanolamine solution. To measure the kinetics of the antibody and conjugate on the HER2 chip, a threefold serial dilution (typically at 5, 2.5, 1.25, 0.6 μg/ml or lower for free antibody and 20, 10, 5, 2.5 μg/ml for the conjugates), was injected for 160 s at 30 μl/min, allowing a 900-s dissociation phase. The dissociation time was extended to 3600 s for the highest concentration. Surfaces were regenerated with 45 s injection of MgCl.sub.2 8 M.
[0788] To evaluate binding ability, manual runs were performed at 30 μl/min with a 60 s injection of the desired compound followed by 60 s waiting time and a 45 s of regeneration at 10 μl/min using MgCl.sub.2 8 M. All data was analysed using the software, BIAEvaluation.
[0789] Method for Scaling Up Conjugations for In Vivo Sample Preparation
[0790] Freshly filtered bioconjugation buffer was combined with freshly filtered anhydrous DMSO in a 50 ml falcon tube and equilibrated on a Thermomixer (12 mins at 4° C. at 800 rpm, 6 mins, 20° C., 500 rpm). The scFv was thawed at 4° C. and any precipitate formed was collected by centrifugation (10 mins, 10 krpm, 4° C.). A stock solution of the payload-NHS was dissolved in anhydrous filtered DMSO and any precipitate formed was collected by centrifugation. The antibody was added to the buffer mixture and allowed to equilibrate on the thermomixer for 10 mins at 20° C., 350 rpm before the addition of the payload-NHS. This was carried out by adding the required equivalents of the payload-NHS DMSO stock, before replacing on the Thermomixer and mixing at 20° C., 350 rpm. The samples were left on the Thermomixer for a further 2 hrs/18 hrs after the last addition. The samples were then collected by centrifugation (20 min, 4 krpm, 4° C.) and purified by SEC on an AKTA Avant system using a Superdex 75, 26/600 column eluting with 10% IPA/PBS unless otherwise stated. A maximum flow rate for the column was used at 2.6 ml/min, detecting wavelengths 214 and 280 nm. The crude sample and fractions were kept cold throughout the purification process. Fractions were combined and concentrated using Vivacell 100 10kMWCO (PES membrane) (Sartorius) before buffer exchanging into PBS using the same process. The concentrated and buffer exchanged samples were quantified by UV/Vis spectroscopy filtered through a sterile 0.2 μm supor membrane, re-quantified, diluted accordingly and analysed as before by SDS-PAGE, HPLC-SEC, Amino acid analysis, mass spectrometry and binding analyses by Biacore SPR.
[0791] Trastuzumab-payload conjugates were synthesised as control samples. These reactions were carried out as described above for the scFv, noting the changes in the protein concentration and the payload NHS addition:
TABLE-US-00025 TABLE 16B Reaction conditions for trastuzumab bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM Nacl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 5 mg/ml concentration Payload-NHS 100 mM or 50 mM (as per the scFv) solution in 100% handling anhydrous filtered DMSO Payload-NHS N/A addition rate Payload-NHS all added in one portion addition portions
[0792] The following trastuzumab conjugates were made using the following reaction conditions:
[0793] Trastuzumab-P5-C5; 6 equivalents, Trastuzumab-MMAF-C5; 7 equivalents,
[0794] Trastuzumab-AF-C5; 5.5 equivalents, Trastuzumab-MMAE-PAB-Cit-Val-dPEG9; 6 equivalents.
[0795] The conjugates were processed as per the scFv using the appropriate HPLC-SEC columns, Tosoh TSKGel G3000SW.sub.XL and on the AKTA Avant a Superdex 200, 26/600.
Example 64B. Bioconjugation of Single-Chain Fv Antibody Fragments Bearing Multiple, Well-Dispersed, Surface Lysine Residues to Small NHS-Bearing Moieties Demonstrates Full OptiLinked Conjugation Capacity
[0796] A small molecule Acetate-NHS (CH3-CO-NHS) was conjugated to scFv (TCT and TCT1067) to obtain conjugates (compound 133) with a high DAR (where “drug”=small molecule acetate payload). The conditions identified and carried forward for the conjugations were:
TABLE-US-00026 TABLE 17 Reaction conditions for acetate-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration Acetate-NHS handling 100 mM solution in 100% anhydrous filtered DMSO Acetate-NHS addition Every 90 min rate Acetate-NHS addition 16 equivalents portions
[0797] The reactions were carried out as per Example 64A.
[0798] In this example, the set up was: [0799] Reaction 1—scFv (TCT): Ac-NHS, 110 equivalents; [0800] Reaction 2—scFv (TCT1067): Ac-NHS, 110 equivalents
[0801] The unconjugated and conjugated scFvs (TCT and TCT1067) were analysed by HPLC size-exclusion chromatography. The scFv (TCT1067) has a retention time of 7.53 min correlating to a MW of around 30 kDa. Its conjugate eluted earlier at 7.23 min indicating a larger molecular weight, but as a single, sharp, monomeric peak, indicating no aggregation (
[0802] LC-MS Method and Data Acquisition (Denaturing, Non-Covalent Conditions)
[0803] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT and TCT1067)-Acetate are shown in
[0804] A single major peak was observed in the TIC of the scFv (TCT and TCT1067)-Acetate samples. TCT-acetate eluting at 10.1 min and TCT1067-acetate eluting at 10.3 min. The zero-charge deconvoluted mass spectrum for this peak produced a major peaks at m/z 28792 for TCT-acetate and 28891 for TCT1067-acetate, which was consistent with the supplied theoretical mass of the scFv (TCT and TCT1067) molecule, together with 15 and 16 additions of the small molecule respectively.
TABLE-US-00027 TABLE 18 Summary of the LC-MS analyses of the scFv-Acetate conjugates (133) Observed Retention peak Added time mass mass Calculated Sample (min) (m/z) (m/z) DAR scFv(TCT) 8.27 28160 0 0 TCT-acetate 12.1 28792 632 15 conjugate 1 scFv(TCT1067) 8.35 28215 0 0 TCT1067- 10.72 28891 672 16 acetate conjugate 2
[0805] Binding Activity of scFv (TCT and TCT1067)-Acetate Conjugates
[0806] ScFv (TCT and TCT1067)-Acetate conjugates were made and characterised as described above. Their binding affinities against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv.
[0807] The scFv (TCT)-Acetate DAR 15 had an association rate of 5.93×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 1.71×10.sup.−2 s.sup.−1, giving an overall binding Kd of 2.9 nM. This was very similar to the unmodified scFv (TCT) which had an association rate of 2.8×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 4.17×10.sup.−3 s.sup.−1, with an overall binding Kd of 1.49 nM, indicating no loss of binding function.
[0808] The scFv (TCT1067)-Acetate DAR 16 had an association rate of 3.63×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 7.64×10.sup.−5 s.sup.−1, giving an overall binding Kd of 21 pM. This was very similar to the unmodified scFv (TCT1067) which had an association rate of 3.9×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.7×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM, indicating no loss of binding.
[0809] Overall Small Molecule (Acetate) Conclusions, Biophysical Data
[0810] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a very high yield of low, medium and high DAR conjugates. There was no precipitation of antibody/conjugate observed in any of the conjugates and recovery was overall very high. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜1 mg/ml.
[0811] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT or TCT1067), can be fully loaded with multiple molecules using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding function and affinity. The LCMS data supported by the HPLC SEC traces indicate that both antibodies can be effectively conjugated to full lysine occupancy to obtain monomeric conjugates. The conjugates showed shorter SEC retention times than the respective antibody controls in both cases. The LCMS indicates that the conjugates in both cases have a higher DAR than the total number of lysines of each antibody. When carrying out reactions at a highly alkaline pH with a large excess of a small NHS activated molecule it is possible that secondary amino acids will start to conjugate. In this case, we speculate that 12 Lysines, 1 terminal amine are predominantly modified.
Example 65. Bioconjugation of MMAF Derivatives with a Short Linker onto Two Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
Example 65A. ScFv (TCT)-MMAF with a C5 Linker
[0812] MMAF-C5-NHS (compound 78) was conjugated to scFv (TCT) to obtain conjugates (compound 118) with various DARs. The reaction was controlled to obtain products with medium and high DARs. The conditions identified and carried forward for the conjugations were:
TABLE-US-00028 TABLE 19 Reaction conditions for MMAFC5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration MMAF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling MMAF-C5 Every 90 min addition rate MMAF-C5-NHS 10 equivalents addition portions
[0813] The reactions were carried out as per Example 64A noting that some minor aggregation was observed in the crude sample which resolved once the sample was purified. Recovery was ˜50%. This reaction was scalable.
[0814] In this example, the set up was: [0815] Reaction 1—scFv-TCT: MMAF-C5-NHS, 60 equivalents and [0816] Reaction 2—scFv-TCT: MMAF-C5-NHS, 100 equivalents
[0817] The unconjugated and conjugated scFv (TCT) were analysed by HPLC size-exclusion chromatography. The scFv has a retention time of 17.9 min correlating to a MW of around 30 kDa. The two conjugates eluted progressively earlier at 16.4 mins for ADC 1 and 16.1 mins for ADC 2 indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0818] The DAR was determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility to obtain the results shown in Tables 20-21.
TABLE-US-00029 TABLE 20 Summary of AAA results showing DARs of 6.6 for reaction 1 Reaction: 1 TCT-MMAF-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not — determined Asp 15 16.20 within 5-10% Thr 14 14.12 better than 5% Ser 46 43.97 better than 5% Glu 22 21.57 better than 5% Gly 44 43.19 better than 5% Ala 17 16.46 better than 5% Val 0 excluded — Met 5 5.15 better than 5% Ile 8 8.27 better than 5% Leu 15 15.75 better than 5% Norleu std Tyr 14 14.62 better than 5% Phe 0 excluded — His 3 3.22 within 5-10% Lys 12 12.69 within 5-10% Arg 5 4.79 better than 5% Pro 0 excluded — Trp 8 (not determined) Total (used) 220 residues Average Total sample 0.169 nmoles 0.166 4.76 ug 4.68 Concentration 27.28 nmoles/ml 26.80 768.28 ug/ml 754.91 MMAF Total 1.13 nmoles 6.64 sample DAR
TABLE-US-00030 TABLE 21 Summary of AAA results showing DARs of 8 for reactions 2 Reaction: 2 TCT-MMAF-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not — determined Asp 15 15.39 better than 5% Thr 14 13.53 better than 5% Ser 46 42.72 within 5-10% Glu 22 22.56 better than 5% Gly 44 47.69 within 5-10% Ala 17 16.62 better than 5% Val 0 excluded — Met 5 4.69 within 5-10% Ile 8 7.89 better than 5% Leu 15 14.96 better than 5% Norleu std Tyr 14 13.87 better than 5% Phe 0 excluded — His 3 3.10 better than 5% Lys 12 12.20 better than 5% Arg 5 4.78 better than 5% Pro 0 excluded — Trp 8 (not determined) Total (used) 220 residues Average Total sample 0.058 nmoles 0.050 1.64 ug 1.40 Concentration 5.25 nmoles/ml 4.49 147.91 ug/ml 126.42 MMAF Total 0.476 7.96 sample DAR
[0819] LC-MS analysis was performed as described in Example 64A.
[0820] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT)-C5-MMAF 1 and 2 are shown in
[0821] A major peak was observed in the TIC of the scFv (TCT)-C5-MMAF 1 sample eluting at 10.2 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 30645, 31473, 32301 and 33129 which was consistent with the supplied theoretical mass of the scFv (TCT) molecule, together with 3-6 additions of the MMAF-C5 molecule. For sample 2, a major peak was observed in the TIC at 9.9-11.4 min corresponding to the zero-charge deconvoluted mass at m/z 31474, 32302, 33130 and 33958 which was consistent with the supplied theoretical mass of the scFv (TCT) together with 4-7 additions of MMAF-C5.
TABLE-US-00031 TABLE 22 Summary of the LC-MS analyses of the scFv (TCT)-MMAF C5 conjugates (118) Retention Observed Added time peak mass mass Calculated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT) 8.27 28160 0 0 0 TCT-MMAF- 10.2 30645 2485 3 4.5 C5 ADC 1 31473 3313 4 32301 4141 5 33129 4969 6 TCT-MMAF- 9.9 31474 3314 4 5.5 C5 ADC 2 10.4 32302 4142 5 10.9 33130 4970 6 11.4 33958 5798 7
[0822] Therefore, for:
[0823] Reaction 1, DAR was 6.64 by AAA and 4.5 by MS, with an average of 5.6
[0824] Reaction 2, DAR was 8.0 by AAA and 5.5 by MS, with an average of 6.8.
[0825] Binding Activity of scFv (TCT)-MMAF-C5 Conjugates
[0826] ScFv (TCT)-MMAF-C5 (compound 118) were made and characterised as described above. Their binding affinities against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as in Example 64A.
[0827] The scFv (TCT)-MMAF-C5 1 DAR 5.6 had an association rate of 7.7×10.sup.5 M.sup.−1 s.sup.−1 and a dissociation rate of 4.2×10.sup.−3 s.sup.−1, giving an overall binding Kd of 5.4 nM. The scFv (TCT)-MMAF-C5 DAR 6.8 had an association rate of 1.2×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 4.2×10.sup.−3 s.sup.−1, giving an overall binding Kd of 3.6 nM. This was essentially unchanged compared to the unmodified scFv (TCT) which had an association rate of 2.8×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 4.17×10.sup.−3 s.sup.−1, with an overall binding Kd of 1.49 nM, indicating no loss of binding function.
[0828] Overall TCT-MMAF-C5 Conclusions, Biophysical Data
[0829] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions to obtain medium and high DAR conjugates. There was minimal precipitation of antibody/conjugate observed during the synthesis. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜1.5 mg/ml and were stable in the buffer for several months.
[0830] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding function and affinity. On the HPLC the samples had progressively shorter retention times than the scFv (TCT), eluting faster from the SEC column due to their increasing size. Amino acid analysis was an extremely useful tool for further quantitative analysis and complemented the MS data.
Example 65B. ScFv (TCT1067)-MMAF with a C5 Linker
[0831] MMAF-C5-NHS (compound 78) was conjugated to scFv (TCT) to obtain conjugates (compound 118) with various DARs. The conditions identified and carried forward were:
TABLE-US-00032 TABLE 23 Reaction conditions for MMAF-C5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration MMAF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling MMAF-C5 Every 90 min addition rate MMAF-C5-NHS 10 equivalents addition portions
[0832] The reactions were carried out as per Example 64A noting that some minor aggregation was observed in the crude sample which resolved once the sample was purified. Total recovery was ˜50-60%. These reactions were scalable. In this example, the set up was: [0833] Reaction 1—scFv-TCT1067: MMAF-C5-NHS, 60 equivalents and [0834] Reaction 2—scFv-TCT1067: MMAF-C5-NHS, 100 equivalents.
[0835] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size-exclusion chromatography. The ScFv has a retention time of 18.1 min correlating to a MW of around 30 kDa. The two conjugates all eluted slightly and progressively earlier indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0836] The DAR was accurately determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility as shown in Table 24 and 25.
TABLE-US-00033 TABLE 24 Summary of AAA results showing DARs of 6.4 for reaction 1 Reaction: 1 TCT1067-MMAF-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 15.11 within 5-10% Thr 17 16.77 better than 5% Ser 44 41.02 within 5-10% Glu 21 20.99 better than 5% Gly 45 44.44 better than 5% Ala 16 15.65 better than 5% Val 0 excluded — Met 5 4.84 better than 5% Ile 8 8.22 better than 5% Leu 16 17.15 within 5-10% Norleu std Tyr 14 14.49 better than 5% Phe 0 excluded — His 2 2.11 within 5-10% Lys 12 12.93 within 5-10% Arg 6 6.30 better than 5% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.17 nmoles 0.16 4.83 ug 4.49 Concentration 38.80 nmoles/ml 36.01 1098.2738 ug/ml 1019.373151 MMAF Total 1.109 6.36 sample DAR
TABLE-US-00034 TABLE 25 Summary of AAA results showing DARs of 8.6 for reaction 2 Reaction: 2 TCT1067-MMAF-05 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.83 within 5-10% Thr 17 17.62 better than 5% Ser 44 41.77 within 5-10% Glu 21 20.66 better than 5% Gly 45 48.17 within 5-10% Ala 16 15.11 within 5-10% Val 0 excluded — Met 5 4.92 better than 5% Ile 8 8.07 better than 5% Leu 16 14.99 within 5-10% Norleu std Tyr 14 13.72 better than 5% Phe 0 excluded — His 2 2.04 better than 5% Lys 12 12.05 better than 5% Arg 6 6.06 better than 5% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.071 nmoles 0.078 2.00 ug 2.20 Concentration 6.47 nmoles/ml 7.12 183.28 ug/ml 201.43 MMAF Total 0.635 8.61 sample DAR
[0837] LC-MS analysis was performed as described in Example 64A.
[0838] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-C5-MMAF 1 and 2 are shown in
[0839] A major peak was observed in the TIC of the scFv (TCT1067)-C5-MMAF 1 sample eluting at 10.1 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 29874, 30702, 31530, 32358 and 33186, which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 2-6 additions of the MMAF molecule. For sample 2, the TIC had the main peaks eluting at 9.3-12 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 30703, 31531, 32358, 33186, 34015 and 34843 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 3-8 additions of the MMAF molecule.
TABLE-US-00035 TABLE 26 Summary of the LC-MS analyses of the scFV(TCT1067)- MMAF-C5 conjugates (118) Observed Retention peak Added time mass mass Calculated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT1067) 8.35 28215 0 0 0 TCT1067- 10.1 29874 1659 2 4.0 MMAF-C5 30702 2487 3 ADC 1 31530 3315 4 32358 4143 5 33186 4971 6 TCT1067- 9.9-12.0 30703 2484 3 5.5 MMAF-C5 31531 3312 4 ADC 2 32358 4139 5 33186 4967 6 34015 5796 7 34843 6624 8
[0840] Therefore,
[0841] For sample 1, DAR was 6.4 by AAA and 4.0 by MS with an average of 5.2
[0842] For sample 2, DAR was 8.6 by AAA and 5.5 by MS with an average of 7.1.
[0843] Binding Activity of scFv (TCT1067)-MMAF-C5 Conjugates
[0844] ScFv (TCT1067)-MMAF-C5 (compound 118) were made and characterised as described above. Their binding affinities against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as described in Example 64A.
[0845] The scFv (TCT1067)-MMAF-C5 DAR 5.2 had an association rate of 1.8×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.4×10.sup.−5 s.sup.−1, giving an overall binding Kd of 19.6 pM. The scFv (TCT)-MMAF-C5 DAR 7.1 had an association rate of 4.6×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 1.7×10.sup.−5 s.sup.−1, giving an overall binding Kd of 3.8 μM. This was essentially unchanged compared to the unmodified scFv (TCT1067) which had an association rate of 3.9×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.7×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM, indicating no loss of binding function.
[0846] Overall scFv (TCT1067)-MMAF-C5 Conclusions, Biophysical Data
[0847] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions to obtain medium and high DAR conjugates. There was very little precipitation of antibody/conjugate observed in the high DAR conjugates (none observed for the medium DAR) which resolved upon purification. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜1-3 mg/ml and were stable in buffer for several months.
[0848] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT1067), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding function and affinity. On the SDS gel (
Example 66. Bioconjugation of P5-C5 Derivatives onto a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0849] P5-C5-NHS (compound 6) was conjugated to scFv (TCT1067) to obtain conjugates (compound 71) with various DARs. The reaction was controlled to obtain products with high DARs. The conditions identified and carried forward for the conjugations were:
TABLE-US-00036 TABLE 27 Reaction conditions for P5-C5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration P5C5-NHS 100 mM solution in 100% anhydrous filtered DMSO handling P5C5-NHS Every 80 min addition rate P5C5-NHS 10 equivalents addition portions
[0850] The reaction was carried out as detailed in Example 64A.
[0851] In this example, the set up was: [0852] Reaction 1—scFv (TCT1067): P5-C5-NHS, 60 equivalents;
[0853] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size-exclusion chromatography. The scFv has a retention time of 18.1 min correlating to a MW of around 30 kDa. The conjugate eluted slightly and earlier at 16.5 mins indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0854] The DAR was also determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility and the results are shown in Table 28.
TABLE-US-00037 TABLE 28 Summary of AAA results showing DAR of 10.4 Reaction: 1 scFv (TCT1067)-P5C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.62 better than 5% Thr 17 18.20 within 5-10% Ser 44 43.22 better than 5% Glu 21 19.72 within 5-10% Gly 45 43.95 better than 5% Ala 16 15.25 better than 5% Val 19 20.20 within 5-10% Met 0 excluded — Ile 8 8.63 within 5-10% Leu 16 16.17 better than 5% Norleu std Tyr 0 excluded — Phe 0 excluded — His 2 1.87 within 5-10% Lys 12 12.63 within 5-10% Arg 6 5.53 within 5-10% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.274 nmoles 0.299 7.77 ug 8.47 Concentration 68.62 nmoles/ml 74.85 1942.45 ug/ml 2118.70 DAR 10.4
[0855] Mass spectrometric analysis was performed as described in Example 64A.
[0856] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-P5-C5 are shown in
[0857] A major peak was observed in the TIC of the scFv (TCT1067)-P5-C5 sample eluting at 7.8 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks as shown in Table 29 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 10-14 additions of the P5-C5 molecule giving an average DAR 11.7. This correlated well with the AAA determination of the DAR of 10.4.
TABLE-US-00038 TABLE 29 Summary of the LC-MS of scFv (TCT1067)- P5-C5 (compounds 71) Observed Retention peak Added Calcu- Weighed time mass mass lated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT1067) 8.35 28215 0 0 0 TCT1067- 8.2 34553 6334 10 11.7 P5C5 35186 6967 11 ADC 1 35819 7600 12 36452 8233 13 37085 8866 14
[0858] Overall, for scFv (TCT1067)-P5-C5, DAR was 10.4 by AAA and 11.7 by MS with an overall average 10.9.
[0859] Binding Activity of scFv (TCT1067)-P5-C5 Conjugates
[0860] ScFv (TCT1067)-P5-C5 (compound 71) was made and characterised as described above. Its binding affinity against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as described in Example 64A. The scFv (TCT1067)-P5-C5 DAR 10.9 had an association rate of 2.36×10.sup.5 M.sup.−1 s.sup.−1 and a dissociation rate of 7.13.×10.sup.−5 s.sup.−1, giving an overall binding Kd of 302 pM. There was a moderate reduction in the association rate (due to reversible steric hindrance of the antibody binding site by the high number of attached payloads), but once bound, there was insignificant effect on the dissociation rate compared to the unmodified scFv (TCT1067) which had an association rate of 3.9×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.7×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM.
[0861] Overall scFv (TCT1067)-P5-C5 Conclusions, Biophysical Data
[0862] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a high yield of high DAR conjugates. There was no precipitation of antibody/conjugate observed in any of the conjugates and overall recovery was very high ˜60%. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜3 mg/ml and were stable in the buffer for several months.
[0863] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT1067), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding affinity. The LCMS, SEC and AAA data support that a monomeric conjugate with a high DAR was made. In conjunction with the Biacore data, this conjugate retained binding to HER2. The mass spectrometry identified both high and lower DAR within the same sample whereas AAA gave an average.
Example 67. Bioconjugation of Auristatin F Derivatives with a Short Linker onto Two Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
Example 67A. ScFv (TCT)-Auristatin F with a C5 Linker
[0864] Auristatin-F-C5-NHS (compound 88) was conjugated to scFv (TCT) to obtain conjugates (compound 122) with various DARs. The reaction was controlled to obtain products with a high DAR. The conditions identified and carried forward were:
TABLE-US-00039 TABLE 30 Reaction conditions for Auristatin F-C5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration AF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling AF-C5-NHS Every 120 mins addition rate AF-C5-NHS 10 equivalents addition portions
[0865] The reaction was carried out as detailed in Example 64A. In this example, the set up was: [0866] Reaction 1—scFv-TCT-Auristatin-F-C5-NHS, 30 equivalents;
[0867] The unconjugated and conjugated scFv (TCT) were analysed by HPLC size-exclusion chromatography. The scFv has a retention time of 7.4 min correlating to a MW of around 30 kDa. The conjugate eluted slightly earlier at 7.2 min indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0868] LC-MS analysis was performed as described in Example 64A.
[0869] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT)-Auristatin F-C5 are shown in
[0870] A major peak was observed in the TIC of the scFv (TCT)-Auristatin F-C5 sample eluting at 9.5 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 33125, 33953, 34780, 35607, 36435 and 37261 which was consistent with the supplied theoretical mass of the scFv (TCT) molecule, together with 6-11 additions of the MMAF molecule.
TABLE-US-00040 TABLE 31 Summary of the LC-MS of scFv (TCT)- Auristatin-F-C5 conjugates (122) Retention Observed Added time peak mass mass Calculated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT) 28160 0 0 0 TCT-AF- 9.51 33125 4965 6 8.5 C5 ADC 1 33953 5793 7 34780 6620 8 35607 7447 9 36435 8275 10 37261 9101 11
[0871] Binding Activity of scFv (TCT)-C5-Auristatin-F Conjugates
[0872] ScFv (TCT)-Auristatin F-C5 (compound 122) were made and characterised as described above. Their binding function against immobilised HER2 target antigen was verified compared to the unmodified scFv as described in Example 64A.
[0873] Overall scFv (TCT)-Auristatin F-C5 Conclusions, Biophysical Data
[0874] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a high yield of high DAR conjugates. Following SEC HPLC purification, the resulting conjugates were concentrated to ˜600 μg/ml and were stable in the buffer for several weeks. Again, recovery was high at ˜50%.
[0875] The techniques used for analysis support that an optimised scFv structure, exemplified by TCT, can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding affinity. The LCMS data was further supported by the HPLC-SEC where the sample had a shorter retention time than TCT, eluting faster from the SEC column due to its increasing size.
Example 67B. ScFv (TCT1067)-Auristatin with a C5 Linker
[0876] Auristatin F-C5-NHS (compound 88) was conjugated to scFv (TCT1067) to obtain conjugates (compound 122) with various DARs. The reaction was controlled to obtain products with medium and high DARs. The conditions identified and carried forward were:
TABLE-US-00041 TABLE 32 Reaction conditions for Auristatin F-C5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 1 mg/ml concentration AF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling AF-C5-NHS every 120 mins addition rate AF-C5-NHS 1 × 5 equivalents (reaction 1); 2 × 10 equivalents addition (reaction 2); 3 × 8.3 equivalents (reaction 3) portions
[0877] The reactions were carried out as per Example 64A noting that the only minor visible precipitation was in the sample with the highest number of drug equivalents. This was resolved with centrifugation and subsequent purification.
[0878] In this example, the set up was: [0879] Reaction 1—scFv (TCT1067)-Auristatin-F-C5-NHS, 5 equivalents; [0880] Reaction 2—scFv (TCT1067)-Auristatin-F-C5-NHS, 10 equivalents; [0881] Reaction 3—scFv (TCT1067)-Auristatin-F-C5-NHS, 25 equivalents
[0882] The DAR was also determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility and the results are shown in Tables 33-35.
TABLE-US-00042 TABLE 33 Summary of AAA results showing DARs of 3.64 Reaction: 1 scFv (TCT1067)-Auristatin-F-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.7 better than 5% Thr 17 16.64 better than 5% Ser 44 40.77 within 5-10% Glu 21 20.87 better than 5% Gly 45 46.35 better than 5% Ala 16 16.19 better than 5% Val 0 excluded — Met 5 4.79 better than 5% Ile 8 8.08 better than 5% Leu 16 16.58 better than 5% Norleu std Tyr 14 14.47 better than 5% Phe 0 excluded — His 2 2.02 better than 5% Lys 12 12.49 better than 5% Arg 6 6.07 better than 5% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.159 nmoles 0.16 4.49 ug 4.54 Concentration 22.66 nmoles/ml 22.92 641.34 ug/ml 648.88 corrected 705.16 ug/ml DAR AAA 3.64
TABLE-US-00043 TABLE 34 Summary of AAA results showing DARs of 6.31 Reaction: 2 scFv (TCT1067)-Auristatin-F-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.46 better than 5% Thr 17 16.46 better than 5% Ser 44 41.20 within 5-10% Glu 21 20.84 better than 5% Gly 45 46.24 better than 5% Ala 16 15.89 better than 5% Val 0 excluded — Met 5 4.58 within 5-10% Ile 8 7.88 better than 5% Leu 16 17.15 within 5-10% Norleu std Tyr 14 14.32 better than 5% Phe 0 excluded — His 2 2.17 within 5-10% Lys 12 12.63 within 5-10% Arg 6 6.19 better than 5% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.163 nmoles 0.168 4.60 ug 4.74 Concentration 20.84 nmoles/ml 21.49 589.78 ug/ml 608.28 corrected ug/ml 661.03 DAR AAA 6.31
TABLE-US-00044 TABLE 35 Summary of AAA results showing DARs of 13.4 Sample: 3 scFv (TCT1067)-Auristatin-F-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.60 better than 5% Thr 17 16.66 better than 5% Ser 44 40.55 within 5-10% Glu 21 21.23 better than 5% Gly 45 46.74 better than 5% Ala 16 16.06 better than 5% Val 0 excluded — Met 0 excluded — Ile 8 7.74 better than 5% Leu 16 16.23 better than 5% Norleu std Tyr 14 14.31 better than 5% Phe 0 excluded — His 2 2.17 within 5-10% Lys 12 12.68 within 5-10% Arg 6 6.04 better than 5% Pro 0 excluded — Trp 9 (not determined) Total (used) 215 residues Average Total sample 0.169 nmoles 0.170 4.80 ug 4.80 Concentration 18.83 nmoles/ml 18.84 532.94 ug/ml 533.27 corrected 579.52 ug/ml DAR AAA 13.40
[0883] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size-exclusion chromatography. The scFv has a retention time of 18.1 min correlating to a MW of around 30 kDa. The three conjugates all eluted slightly and progressively earlier (at 17.9 min, 17.92 min and 17.87 min) indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0884] LC-MS analysis was performed as described in Example 64A.
[0885] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-C5-Auristatin-F are shown in
[0886] A major peak was observed in the TIC of the scFv (TCT1067)-C5-Auristatin-F reaction 1 eluting at 8.19 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 29037, 29865, 30692, 31519 and 32345 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 1-5 additions of the Auristatin F molecule with an average DAR of 2.9.
[0887] A major peak was observed in the TIC of the scFv (TCT1067)-C5-Auristatin-F reaction 2 eluting at 9.47 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 29866, 30692, 31519, 32347, 33174 and 34001 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 2-7 additions of the Auristatin F molecule with an average DAR of 4.98.
[0888] A major peak was observed in the TIC of the scFv (TCT1067)-C5-Auristatin-F reaction 3 eluting at 9.97 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 34826, 35653, 36480, 37307, 38134 and 38960 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 8-13 additions of the Auristatin F molecule with an average DAR of 10.3.
TABLE-US-00045 TABLE 36 Summary of the LC-MS of scFv (TCT1067)-Auristatin F-C5 (122) Observed Retention peak Added Calcu- Weighed time mass mass lated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT1067) 8.35 28215 0 0 0.00 TCT1067-AF- 8.11 29037 818 1 2.90 C5 ADC 1 29865 1646 2 30692 2473 3 31519 3300 4 32345 4126 5 TCT1067-AF- 9.47 29866 1647 2 4.98 C5 ADC2 30692 2473 3 31519 3300 4 32347 4128 5 33174 4955 6 34001 5782 7 TCT1067-AF- 9.97 34826 6607 8 10.30 C5 ADC 3 35653 7434 9 36480 8261 10 37307 9088 11 38134 9915 12 38960 10741 13
[0889] Therefore, overall,
[0890] For reaction 1, DAR was 3.64 by AAA and 2.9 by MS with an average DAR of 3.3.
[0891] For reaction 2, DAR was 6.31 by AAA and 4.98 by MS with an average DAR of 5.65.
[0892] For reaction 3, DAR was 13.4 by AAA and 10.4 by MS with an average DAR of 11.9.
[0893] An SDS-PAGE was run (
[0894] Binding Activity of scFv (TCT1067)-AF-C5 Conjugates
[0895] ScFv (TCT1067)-Auristatin-F-C5 conjugates (compound 122) were made and characterised as described above. Their binding affinities against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as described in Example 64A.
[0896] The scFv (TCT1067)-Auristatin-F-C5 DAR 3.3 had an association rate of 5.56×10.sup.5 M.sup.−1 s.sup.−1 and a dissociation rate of 1.82×10.sup.−5 s.sup.−1, giving an overall binding Kd of 32.8 pM.
[0897] The scFv (TCT1067)-Auristatin-F-C5 DAR 5.65 had an association rate of 3.36×10.sup.5 M.sup.−1 s.sup.−1 and a dissociation rate of 1.35×10.sup.−5 s.sup.−1, giving an overall binding Kd of 40.3 pM.
[0898] The scFv (TCT1067)-Auristatin-F-C5 DAR 11.9 had an association rate of 2.17×10.sup.4 M.sup.−1 s.sup.−1 and a dissociation rate of 1.76×10.sup.−5 s.sup.−1, giving an overall binding Kd of 810 pM.
[0899] The low and medium DAR samples had affinities that were very similar to the unmodified scFv (TCT1067) which had an association rate of 3.9×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.7×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM, indicating no loss of binding function. The high DAR sample had a moderately reduced association rate (due to reversible steric hindrance of the antibody binding site by the high number of attached payloads), but once bound, there was insignificant effect on the dissociation rate compared to the unmodified scFv.
[0900] Overall scFv (TCT1067)-Auristatin F-C5 Conclusions, Biophysical Data
[0901] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a high yield of low, medium and high DAR conjugates. Following purification and processing, the conjugates were concentrated to ˜9 mg/ml and were stable in the buffer for several months.
[0902] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT1067), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining function and binding affinity. Purified conjugates with low DAR (reaction 1) run closer to the control scFv (TCT) on the gel and were less polydispersed than the medium DAR (reaction 2) which run slightly higher and was more polydispersed, whereas for the high DAR (reaction 3) there was a clear migration shift on the gel where the sample was clearly bigger in size than the control, unmodified scFv (TCT1067). These observations were further supported by the HPLC SEC where the samples had progressively shorter retention times than TCT, eluting faster due to their increasing size. Amino acid analysis was an extremely useful tool for further quantitative analysis and supported the LC-MS data.
Example 68. Bioconjugation of a MMAE Derivative with a Protease Cleavable Linker onto a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0903] The cytotoxic drug MMAE-PAB-Cit-Val-dPEG.sub.9 NHS (compound 86) was conjugated to scFv (TCT1067) to obtain conjugates (compound 121) with a high DAR. The conditions identified and carried forward were:
TABLE-US-00046 TABLE 37 Reaction conditions for MMAE-PAB- Cit-Val-dPEG.sub.9 NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 20 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration MMAE-PABA-vc- 50 mM solution in 100% anhydrous filtered DMSO PEG9-NHS handling MMAE-PABA-vc- Every 120 mins PEG9-NHS addition rate MMAE-PABA-vc- 10 equivalents PEG9-NHS addition equivalents
[0904] The reaction was carried out as detailed in Example 64A noting that the MMAE-PABA-vc-PEG9-NHS required repeated vortexing to fully solubilise in DMSO. The reaction was carried out in a low salt buffer. The crude conjugate had no visible precipitation and was purified by SEC on an AKTA Avant system using a Superdex 75, 26/600 column eluting with 10% IPA/20 mM NaCl phosphate buffer pH7. Fractions were combined and concentrated using Vivacell 100 10kMWCO (PES membrane) (Sartorius) before buffer exchanging into 20 mM NaCl phosphate buffer pH7 using the same process. An HPLC-SEC run (
[0905] Reaction 1—scFv-TCT1067: MMAE-PAB-Cit-Val-dPEG.sub.9 30 equivalents;
[0906] The DAR was also determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility and the results are shown in Table 38.
TABLE-US-00047 TABLE 38 Summary of AAA results showing DAR of 9.6 for reaction 1 Reaction: 1 scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 13.96 better than 5% Thr 17 17.56 better than 5% Ser 44 40.04 within 5-10% Glu 0 excluded — Gly 45 45.75 better than 5% Ala 16 17.43 within 5-10% Val 0 excluded — Met 0 excluded — Ile 8 8.34 better than 5% Leu 16 16.28 better than 5% Norleu std Tyr 0 excluded — Phe 0 excluded — His 2 2.05 better than 5% Lys 12 12.39 better than 5% Arg 6 5.86 better than 5% Pro 13 13.35 better than 5% Trp 9 (not determined) Total (used) 193 residues Average Total sample 0.142 nmoles 0.151 4.01 ug 4.28 Concentration 2.18 nmoles/ml 2.32 61.69 ug/ml 65.77 Total sample Ornithine i 1.540 ii 1.370 average 1.46 DAR 9.6
[0907] Mass spectrometric analysis was performed as described in Example 64A. The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 are shown in
[0908] Several peaks corresponding to the various DAR species were observed in the UV/TIC of the sample eluting at between 10 and 12 min. The zero-charge deconvoluted mass spectrum for each peak corresponded to a series of major peaks at m/z 37837, 39439, 41042 and 42644 which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule with 6-9 additions of the MMAE moiety.
TABLE-US-00048 TABLE 39 Summary of the LC-MS of scFv (TCT1067)- MMAE-PAB-Cit-Val-dPEG.sub.9 (121) Observed Retention peak Added time mass mass Calculated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT1067) 8.35 28215 0 0 0 TCT1067- 10.7 37837 9618 6 7.5 MMAE ADC1 11.1 39439 11220 7 11.4 41042 12823 8 11.9 42644 14425 9
[0909] Therefore, overall,
[0910] For scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 reaction 1, DAR was 9.6 by AAA and 7.5 by LC-MS with an average DAR of 8.6.
[0911] An SDS-PAGE was run (
[0912] Binding Activity of scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 Conjugates
[0913] TCT(1067)-MMAE-PABA-vc-PEG9 (compound 121) were made and characterised as described above. Its binding affinity against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as described in Example 64A.
[0914] The scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 conjugate, DAR 8.6 had an association rate of 2.21×10.sup.4M.sup.−1 s.sup.−1 and a dissociation rate of 2.23×10.sup.−5 s.sup.−1, giving an overall binding Kd of 1 nM. There was a reduction in the association rate (due to reversible steric hindrance of the antibody binding site by the high number of attached payloads), but once bound, there was insignificant effect on the dissociation rate compared to the unmodified scFv (TCT1067) which had an association rate of 3.88×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.69×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM.
[0915] Overall scFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.sub.9 Conclusions
[0916] The conjugation conditions were optimised as detailed above obtaining a conjugate with a high DAR. Following purification, concentration and filtration, the resulting conjugates appeared stable in the buffer for several weeks.
[0917] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by TCT(1067), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding affinity. The purified conjugate with high DAR (reaction 1) showed a clear migration shift on the gel where the sample was bigger in size than the control, unmodified TCT1067. These observations were further supported by the HPLC SEC where the sample had a significantly shorter retention time eluting at 15.1 mins than the control eluting at 18.1 mins, eluting faster from the SEC column due to its increasing size. Amino acid analysis was an extremely useful tool for further quantitative analysis and supported the LC-MS data.
Example 69. Bioconjugation of Two Different Payload Types onto a scFv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0918] The cytotoxic drugs P5-C5-NHS (compound 6) and MMAF-C5-NHS (compound 78) were conjugated to scFv (TCT1067) to obtain conjugates (compound 135) with a high DAR. The conditions identified and carried forward for the conjugations were:
TABLE-US-00049 TABLE 40 Reaction conditions for MMAF/P5C5 dual payload bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody concentration 1 mg/ml MMAF-C5-NHS and P5-C5- 100 mM solution in 100% anhydrous NHS handling filtered DMSO MMAF-C5-NHS and P5-C5- Every 90 min NHS addition rate MMAF-C5-NHS and P5-C5 11 equivalents NHS addition portions
[0919] The reaction was carried out as per Example 64A noting that the MMAF-C5-NHS was added during the first addition and the subsequent two additions were carried out adding P5-C5-NHS. All other handling and purification processes were as Example 64A. [0920] Reaction 1—scFv-TCT1067: MMAF-C5-P5-C5; 11 equivalents MMAF-C5 NHS and 21 equivalents of P5-C5 NHS;
[0921] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size-exclusion chromatography. The scFv has a retention time of 18.1 min correlating to a MW of around 30 kDa. The conjugate eluted slightly earlier at 17.8 min indicating a larger molecular weight (due to varying drug loads), but as a single, sharp, monomeric peak, indicating no aggregation (
[0922] The DAR was also determined by Amino Acid Analysis (AAA) at Cambridge University's Protein and Nucleic Acid Chemistry Facility and the results are shown in Table 41.
TABLE-US-00050 TABLE 41 Summary of AAA results showing dual payload DARs Reaction: 1 scFv (TCT1067)-MMAF-C5/P5-C5 Integer fit of measured mole ratios to expected values Expected Observed Closeness of fit Amino acid value value to expected value Cys 6 not determined — Asp 14 14.80 within 5-10% Thr 17 17.10 better than 5% Ser 44 41.29 within 5-10% Glu 21 21.54 better than 5% Gly 45 47.85 within 5-10% Ala 16 16.20 better than 5% Val 0 excluded — Met 5 4.95 better than 5% Ile 8 7.66 better than 5% Leu 16 15.79 better than 5% Norleu std Tyr 14 13.82 better than 5% Phe 0 excluded — His 2 1.81 within 5-10% Lys 12 11.48 better than 5% Arg 6 5.70 within 5-10% Pro 0 excluded — Trp 9 (not determined) Total (used) 220 residues Average Total sample 0.223 nmoles 0.217 6.32 ug 6.15 Concentration 55.85 nmoles/ml 54.33 1580.85 ug/ml 1537.79 Total DAR MMAF (Phe) 0.137 0.61 0.7 P5C5 (Ava) 1.028 4.60 4.6
[0923] LC-MS analysis was performed as described in Example 64A.
[0924] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-MMAF-C5/P5-C5 are shown in
[0925] A major peak was observed in the TIC of the scFv (TCT1067)-MMAF-C5/P5-C5 sample eluting at 8.7-10.5 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 31332, 31964, 32597, 33230, 33963, 31135, 31769, 32499, 30307, 30940, 31673, 32306, 30113, 30746, 31478, 32109.
[0926] These were consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with several combinations of MMAF-C5 and P5-C5 as indicated in Table 42.
TABLE-US-00051 TABLE 42 Summary of LCMS of scFv (TCT1067)- MMAF-C5/P5-C5 dual payload (135) Esti- Observed mated Esti- Retention peak Added DAR mated time mass mass MMAF- DAR Sample (min) (m/z) (m/z) C5 P5-C5 scFv(TCT1067) 8.35 28215 0 0 0 TCT1067- 9.7 31332 3113 3 1 MMAF/C5-P5- 31964 3745 3 2 C5 ADC 32597 4378 3 3 33230 5011 3 4 9.4 31135 2916 2 2 31769 3550 2 3 32499 4280 2 4 33135 4916 2 5 8.9 30307 2088 1 2 30940 2721 1 3 31673 3454 2 3 32306 4087 1 5 8.7 30114 1895 0 3 30747 2528 0 4 31479 3260 0 5 32109 3890 0 6
[0927] Overall scFv (TCT1067)-MMAF-C5/P5-C5 Conclusions, Biophysical Data
[0928] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions with a high yield of high DAR conjugates. The resulting conjugates were concentrated to ˜1.5 g/ml and were stable in buffer for several weeks.
Example 70. Bioconjugation of a Maytansine-DM1 Derivative with a Linker onto a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[0929] Maytansine DM1-dPEG.sub.12-NHS (compound 90) was conjugated to scFv (TCT1067) to obtain conjugates (compound 124) with various DARs. The conditions identified and carried forward were:
TABLE-US-00052 TABLE 43 Reaction conditions for DM1-dPEG.sub.12-NHS - bioconjugations Type Condition Buffer PBS buffer at pH 8.0 and 0.1% Polysorbate-20 Co-solvent Anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation Thermomixer 1000 rpm Antibody 1 mg/ml concentration Maytansine 100 mM solution in 100% anhydrous filtered DMSO DM1- PEG(12)- NHS handling Maytansine Every 120 min DM1- PEG(12)- NHS addition rate Maytansine 8 equivalents DM1- PEG(12)- NHS addition portions
[0930] The reactions were set up as per Table 37 and Example 64A noting that, prior to adding the drug stock to the reaction, the total amount needed was diluted in 25% of the total volume of the DMSO required for the reaction. The drug addition was carried out by adding 16 equivalents of the NHS-drug DMSO form this new stock solution. Precipitation was visible at the completion of the reactions and that increased with increasing number of equivalents.
[0931] In this example, the set up was:
[0932] Reaction 1—scFv-TCT1067: DM1-dPEG.sub.12 NHS, 16 equivalents;
[0933] Reaction 2—scFv-TCT1067: DM1-dPEG.sub.12 NHS, 32 equivalents
[0934] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size-exclusion chromatography (
TABLE-US-00053 Reaction 1 1% aggregates Reaction 2 4% aggregates
[0935] LC-MS analysis was performed as described in Example 64A.
[0936] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data of scFv (TCT1067)-DM1-dPEG.sub.12 are shown in
[0937] In sample 1, a major peak was observed in the TIC of the scFv (TCT1067)-DM1-dPEG.sub.12 sample eluting at 11 min. The zero-charge deconvoluted mass spectrum for this peak produced a series of major peaks at m/z 37144, 38631 and 40079, which was consistent with the supplied theoretical mass of the scFv (TCT1067) molecule, together with 6, 7 and 8 additions of the Maytansine DM1 molecule. In sample 2, the conjugate eluted at 11.5 mins and the deconvoluted mass gave a peak at m/z 40123 which corresponds to the scFv (TCT1067) together with 8 additions of Maytansine DM1.
TABLE-US-00054 TABLE 44 Summary of the LC-MS of scFv (TCT1067)-DM1-dPEG.sub.12 (124) Observed Retention peak Added time mass mass Calculated Average Sample (min) (m/z) (m/z) DAR DAR scFv(TCT1067) 8.35 28215 0 0 TCT1067- 10.6 37144 9024 6 7 Maytansine- 10.9 38631 10528 7 PEG(12) DM1 11-11.5 40079 12032 8 ADC 1 TCT1067- 11.5-12.0 40123 12032 8 8 Maytansine- PEG(12) DM1 ADC 2
[0938] An SDS-PAGE was run (
[0939] Binding Activity of scFv (TCT1067)-DM1-dPEG.sub.12
[0940] ScFv (TCT1067)-DM1-dPEG.sub.12 (compound 124) was made and characterised as described above. Their binding affinities against immobilised HER2 target antigen was determined by Biacore SPR compared to the unmodified scFv as described in Example 64A.
[0941] The scFv (TCT1067)-DM1-dPEG.sub.12 DAR 8 had an association rate of 1.32×10.sup.4 M.sup.−1 s.sup.−1 and a dissociation rate of 3.28×10.sup.−5 s.sup.−1, giving an overall binding Kd of 2.48 nM. The scFv (TCT1067)-DM1-dPEG.sub.12 DAR 7 had an association rate of 1.95×10.sup.4 M.sup.−1 s.sup.−1 and a dissociation rate of 2.7×10.sup.−5 s.sup.−1, giving an overall binding Kd of 1.39 nM. Both conjugates had a moderately reduced association rate (due to reversible steric hindrance of the antibody binding site by the high number of attached payloads), but once bound, there was insignificant effect on the dissociation rate compared to the unmodified scFv (TCT1067) which had an association rate of 3.9×10.sup.6 M.sup.−1 s.sup.−1 and a dissociation rate of 3.7×10.sup.−5 s.sup.−1, with an overall binding Kd of 9.5 pM.
[0942] Overall scFv (TCT1067)-DM1-dPEG.sub.12 Conclusions, Biophysical Data
[0943] The conjugation conditions were optimised as detailed above. This optimisation allowed for controlled conjugation reactions to obtain conjugates with medium and high DAR. The purified conjugates were concentrated to ˜500 μg/ml in buffer.
[0944] The orthogonal techniques used for analysis are in agreement and support that an optimised scFv structure, exemplified by scFv (TCT1067), can be loaded with multiple drugs using lysine residues on the antibody and the conjugation can be controlled to obtain monomeric conjugates (as shown by SEC-HPLC) with the desired DAR whilst retaining binding affinity. On an SDS gel, the purified conjugates with a medium DAR (sample 1) run slightly higher and was more polydispersed, whereas for the high DAR (sample 2) there was a clear migration shift on the gel where the sample was clearly bigger in size than the control, unmodified scFv (TCT1067). These observations were further supported by the HPLC where the samples had progressively shorter retention times than TCT, eluting faster from the SEC column due to their increasing size.
Example 71. Bioconjugation of Auristatin F to an scFv which has a High Lysine Content which is not Optimally-Dispersed (‘Non-OptiLinked’) Leads to Aggregation and Lower than Desired Drug: Antibody Ratios
[0945] An scFv based on the panitumumab monoclonal antibody [SEQ ID 5] has a high affinity [U.S. Pat. No. 8,227,580B2] and possesses 8 Lysine residues that are predicted to be surface exposed, but are not in an optimal spatial configuration and are not in preferred locations compared to the example of scFv (TCT1067) with only 4 similar positions (see below). This panitumumab scFv (scFv (Pan)) was constructed, expressed and purified using established methodology [Bhatti M et al (2008) 122: 1155] and used in bioconjugation reactions under identical conditions to scFv (TCT1067) using the payload Auristatin F. The conditions used were:
TABLE-US-00055 TABLE 44A Reaction conditions for AF-C5-NHS bioconjugations Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 0.83 mg/ml concentration AF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling AF-C5-NHS every 120 min addition rate AF-C5-NHS 5 equivalents addition portions
TABLE-US-00056 Amino acid sequence of panitumumab single chain Fv [SEQ ID NO. 5] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRD RVTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVG DRTVITCQASQDISNYLNVVYQQKPGKAPKLLIYDASNLETGVPSRFSGS GSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIQNGSEQKLI SEEDLAAA
[0946] Amino acid sequence alignment of scFv (TCT1067), T against scFv (panitumumab), P. Lysine residues are in bold and commonly-positioned lysine residues are underlined. Panitumumab has 8 lysines that are in a significantly different configuration to the 12 that are present in the scFv (TCT1067).
TABLE-US-00057 T QVQLVQSGAEVKKPGESLKISCKGSGYS--FTSYWIAWVRQMPGKGLEYMGLIYPGDSDT P QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYS-GNT T KYSPSFQGQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCTDRTCAAWPEWLGV P NYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVT-----------GAFDI T WGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPP-SVSAAPGQKVTISCSGSSSNIGNNY P WGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRTVITCQASQ--DISNY T VSWYQQLPGTAPKLLIYGHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWD P LNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFD T YTLSGWVFGGGTKLTVL P HLP--LAFGGGTKVEIQ [T = SEQ ID NO. 6; P = SEQ ID NO. 7]
[0947] Low, medium and high DAR conjugation reaction conditions were set up as described in Example 67B and the conjugates analysed by SDS-PAGE (
TABLE-US-00058 TABLE 45 Comparison of a ‘non-OptiLinked’ scFv (panitumumab) vs an ‘OptiLinked scFv’ (TCT1067) Molar DAR equivalents distribution of Auristatin by LC-MS % aggregate F-C5 NHS and (mean by HPLC-SEC Reaction scFv used DAR) Yield (FIG. 71) 1 Panitumumab 5 0, 1, 2, 3, 4 (3) 44% 37% 2 Panitumumab 10 0, 1, 2, 3, 4, 5 26% 42% (3.4) 3 Panitumumab 25 None 0%* 95% 4 TCT1067 5 0, 1, 2, 3 (2) 65% 0% 5 TCT1067 10 2, 3, 4, 5, 6, 7 70% 0% (5) 6 TCT1067 25 7, 8, 9, 10, 11(9) 46% 0% *observed precipitation
Example 72. Cell Killing Potency and Specificity of a Medium and High Affinity scFv Conjugated to Medium and High DAR Payloads
Example 72A. scFv (TCT)-MMAF-C5, scFv (TCT1067)-MMAF-C5 and Trastuzumab-MMAF-C5 Conjugates (117), DAR 6.5
[0948] ScFv (TCT)-MMAF-C5, ScFv (TCT1067)-MMAF-C5 and Trastuzumab-MMAF-C5 conjugates (118) were made and characterised as described above (Examples 65 which had similar DARs as before. SKBr3, human breast cancer cell line, high HER2 expression levels, up to 1,000,000 receptors per cell [Lazar G A, et al Proc Natl Acad Sci USA. 2006, 103:4005-10] were grown in McCoy's 5A/10% FCS (complete media) at 37° C., 5% CO.sub.2 in a humidified atmosphere. When confluency was 70-80%, cells were washed with PBS (2×10 ml) and incubated with trypsin for 5-7 min. Complete McCoy's 5A was added and the cells were resuspended by pipetting. The cells were recovered by centrifugation (2 min, 2000 rpm), the supernatant was discarded, and the cells were resuspended in complete McCoy's 5A (5 ml). The cells were then counted using a haemocytometer and diluted accordingly. They were plated at 5000 cells/well (200 μl) using attachment factor and incubated overnight at 37° C., 5% CO.sub.2 in a humidified atmosphere. U87 is a non-HER2 expressing glioblastoma cell line [Zitron I M et al (2013) BMC Cancer 13:83] and was grown in a similar way, plated at 1000 cells/well (using DMEM media). BT474 is a HER2 expressing breast cancer cell line [Brockhoff G et al (2007) Cell Prolif 40:488-507] and was grown in a similar way, plated at 7500 cells/well (using RPMI media). NCI-N87 is a HER2 expressing gastric cancer cell line [Yamashita-Kashima Y et al (2013) Oncol. Rep 30:1087-93] and was grown in a similar way, plated at 7500 cells/well (using RPMI media).
[0949] The cells were exposed to the various ADCs diluted in complete media for 96 hours at 37° C., 5% CO.sub.2 in a humidified atmosphere. Cell viability was measured using the Promega Aqueous Celltitre96™ aqueous one solution cell proliferation kit (MTS reagent) according to manufacturer's instructions. Briefly, the media was removed and 100 μl of complete phenol red free media, pre-combined with MTS reagent, was added to the cells (20 μl of reagent per 100 μl of media). The plates were read on an ELISA plate reader at 490 nm after a 2 hr incubation in the dark (5% CO.sub.2, 37° C.).
[0950] The data (absorption units) were converted to % cell survival by using the untreated controls as the 100% cell survival and the Triton X-100 controls as the 100% cell death. The average absorption value for the latter was subtracted from all the rest of the data in order to get a suitable baseline. The averages were converted to survival and standard error values were obtained for each n value (as a % cell survival). The data were plotted and fitted to a dose-response sigmoidal logistic 3-parameter curve using the equation y=y.sub.0+a/(1+(x/x.sub.0)b) where, x.sub.0=IC50 and x.sub.0>0 and a=100 using GraphPad Prism. Experiments were repeated at least 3 times for each compound tested and a set or an average of the data was plotted and fitted to obtain a dose-response curve.
[0951] The data (
TABLE-US-00059 TABLE 46 Summary of Cell killing potency of scFv (TCT & TCT1067) & Trastuzumab MMAF-C5 conjugates (compounds 118) SKBr3 Cells BT474 Cells U87 Cells (HER2 +++) (HER2++) (HER2−) IC50 IC50 IC50 IC50 IC50 IC50 Sample μg/ml pM μg/ml pM μg/ml pM scFv (TCT)- 0.087 3.1 0.2 7.14 >5 NC MMAF-C5, DAR 6.6 scFv 0.007 250 0.008 286 >5 NC (TCT1067)- MMAF-C5, DAR 6.4 Trastuzumab- 0.004 26 0.01 67 >5 NC MMAF-C5, DAR 4 Free drug NC NC — >10,000 NC NC MMAF Unconjugated NC NC NC NC NC NC scFv Unconjugated 0.6 4000 NC NC NC NC trastuzumab NC = No cytotoxicity
Example 72B. scFv (TCT)-MMAF-C5, scFv (TCT1067)-MMAF-C5 and Trastuzumab-MMAF-C5 Conjugates (118), DAR 8
[0952] Cell killing assays were set up as described in Example 72A
[0953] The data (
TABLE-US-00060 TABLE 47 Summary of Cell killing potency of scFv (TCT & TCT1067) & Trastuzumab MMAF-C5 conjugates (compounds 118) SKBr3 Cells BT474 Cells U87 Cells (HER2 +++) (HER2++) (HER2−) IC50 IC50 IC50 IC50 IC50 IC50 Sample μg/ml pM μg/ml pM μg/ml pM scFv (TCT)- 0.0053 189 0.02 714 NC NC MMAF DAR 8 scFv 0.00091 32.5 0.001 35.7 NC NC (TCT1067)- MMAF DAR 8.7 Trastuzumab- 0.0051 34 0.05 336 NC NC MMAF, DAR 6 Free drug MMAF NC NC NC NC NC NC Unconjugated scFv NC NC NC NC NC NC Unconjugated 0.6 4000 NC NC NC NC trastuzumab NC = No cytotoxicity
Example 72C. scFv (TCT1067)-P5C5 and Trastuzumab-P5C5 Conjugates (71), DAR 10.6 and 12.5
[0954] Cell killing assays were set up as described in Example 72A.
[0955] The data (
TABLE-US-00061 TABLE 48 Summary of Cell killing potency of scFv (TCT1067) & Trastuzumab P5C5 ADCs (compounds 71) SKBr3 Cells BT474 Cells U87 Cells (HER2 +++) (HER2++) (HER2−) IC50 IC50 IC50 IC50 IC50 IC50 Sample μg/ml nM μg/ml nM μg/ml nM scFv (TCT1067)- 0.04 1.42 0.3 10.7 NC NC P5C5 DAR 12.5 scFv (TCT1067)- ND ND 0.5 17.85 NC NC P5C5 DAR 10.6 Trastuzumab- ND ND 0.2 1.33 NC NC P5C5, DAR 6 Free drug P5C5 — >33,000 — >3300 — 100,000 Unconjugated NC NC NC NC NC NC scFv Unconjugated 0.6 4 NC NC NC NC trastuzumab NC = No cytotoxicity, ND = Not determined
Example 72D. scFv (TCT1067)-Auristatin F-C5 at Low, Medium and High DARs and Trastuzumab-Auristatin F-C5 Conjugates (122)
[0956] Cell killing assays were set up as described in Example 72A
[0957] The data (
TABLE-US-00062 TABLE 49 Summary of Cell killing potency of scFv (TCT1067)-Auristatin F-C5 & Trastuzumab Auristatin F-C5 ADCs (compounds 122) SKBr3 Cells BT474 Cells NCI-N874 Cells U87 Cells (HER2 +++) (HER2++) (HER2++) (HER2−) IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 Sample μg/ml pM μg/ml pM μg/ml pM μg/ml pM scFv 0.00089 31.7 0.00115 39.2 0.00087 31.05 NC NC (TCT1067)- Auristatin F- C5, DAR 2.7 scFv 0.00101 36.1 0.00112 39.9 0.00036 12.85 NC NC (TCT1067)- Auristatin F- C5, DAR 6.2 scFv 0.00844 299 0.01082 386 0.0087 310 NC NC (TCT1067)- Auristatin F- C5, DAR 11.8 Trastuzumab- 0.003171 21 0.008193 54.6 0.0018 6.7 NC NC Auristatin F- C5, DAR 4.8 Free drug >5500 >2500 ND >12,500 Auristatin F Unconjugated NC NC NC NC NC NC NC NC scFv Unconjugated 0.6 4000 NC NC NC NC NC NC trastuzumab NC = No cytotoxicity, ND = Not determined
Example 72E. ScFv (TCT1067)-DM1-(dPEG.SUB.12.) and Trastuzumab-DM1-(dPEG.SUB.12.) Conjugates (124) Low, Medium and High DAR
[0958] Cell killing assays were set up as described in Example 72A
[0959] The data (
TABLE-US-00063 TABLE 50 Summary of Cell killing potency of scFv (TCT1067)-DM1- (dPEG.sub.12) & Trastuzumab-DM19(dPEG.sub.12) ADCs (compounds 124) SKBr3 Cells (HER2+++) U87 Cells (HER2−) Sample IC50 μg/ml IC50 nM IC50 μg/ml IC50 nM scFv (TCT1067)- 0.0417 1.49 0.641 22.9 DM1 DAR 3.5 scFv (TCT1067)- 0.0078 0.0278 0.419 14.94 DM1 DAR 5.5 scFv (TC11067)- 0.07606 2.7 0.355 12.66 DM1 DAR 8 Free drug — 47.63 — 50.7 DM1 Unconjugated scFv NC NC NC NC NC = No cytotoxicity
Example 72F. ScFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.SUB.9 .and Trastuzumab-MMAE-PAB-Cit-Val-dPEG.SUB.9 .Conjugates, DAR9 (121)
[0960] Cell killing assays were set up as described in Example 72A.
[0961] The data (
TABLE-US-00064 TABLE 51 Summary of Cell killing potency of scFv (TCT1067)- MMAE-PAB-Cit-Val-dPEG.sub.9 & Trastuzumab- MMAE-PAB-Cit-Val-dPEG.sub.9 ADCs (compounds 120) SKBr3 Cells U87 Cells NCI-N87 Cells (HER2 +++) (HER2−) (HER2++) IC50 IC50 IC50 IC50 IC50 IC50 Sample μg/ml nM μg/ml nM μg/ml nM scFv (TCT1067)- 0.04476 1.59 0.1 3.57 0.195 6.947 MMAE-PAB-Cit- Val-dPEG.sub.9, DAR 9 Trastuzumab- 0.0052 0.035 NC NC 0.067 0.45 MMAE-PAB-Cit- Val-dPEG.sub.9, DAR 4 Free drug MMAE NC NC NC NC ND ND Unconjugated scFv NC NC NC NC NC NC Unconjugated 0.6 4 NC NC NC NC trastuzumab NC = No cytotoxicity
Example 73. Demonstration that Antibody Fragment ADCs are Highly Potent at Lower Incubation Times
[0962] Cell killing assays were set up as described in Example 72A but the incubation time was shortened to 4 hours to mimic the reduced exposure time expected in vivo of antibody fragment-based ADCs. Two similar DAR (around 5) conjugates were compared (1) high affinity scFv (TCT1067)-AF-C5 conjugate, DAR 5.3 (2) Trastuzumab-AF-C5 conjugate, DAR 4.8. The results are shown in
TABLE-US-00065 TABLE 52 Cell killing potency of scFv (TCT1067)-AF-C5 conjugates, DAR 5.3 (compounds 122) for short and long incubations SKBr3 Cells (HER2+++) Sample IC50 μg/ml IC50 pM Free Auristatin 4 hours 0.00181 >100,000 Free Auristatin, 96 hours 0.00081 >10,000 ScFv (TCT1067)-AF-C5 DAR 5.3, 4 0.00181 64.4 hours ScFv (TCT1067)-AF-C5 DAR 5.3, 96 0.00081 28.8 hours Trastuzumab-AF-C5 DAR 4.8, 4 hours 0.0144 93.4 Trastuzumab-AF-C5 DAR 4.8, 96 hours 0.0029 19.3 NC = No cytotoxicity
Example 74. Demonstration that OptiLinked scFv-Drug Conjugates Penetrate into Human Tumour Xenografts More Rapidly than the Whole Immunoglobulin-Drug Conjugate with Equivalent Payloads
[0963] Mice. Female severe combined immunodeficient mice (Fox Chase SCID®, CB-17/Icr-Prkdcscid/IcrIcoCrl, Charles River Laboratories) were twelve weeks old with a body weight (BW) range of 15.3 to 18.4 grams on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services, who carried out this contracted R&D) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.
[0964] In Vivo Implantation and Tumor Growth. Xenografts were initiated with BT474 human breast carcinomas maintained at CR Discovery Services by serial subcutaneous transplantation in SCID mice. On the day of tumor implant, each test mouse received a 1 mm.sup.3 BT474 fragment implanted subcutaneously in the right flank, and tumor growth was monitored as the average size approached the target range of 400 to 600 mm.sup.3. Fifty days after tumor implantation, designated as Day 1 of the study, the animals were re-sorted into groups each consisting of two mice with individual tumor volumes of 405 to 600 mm.sup.3 and group mean tumor volumes of 466 to 503 mm.sup.3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumor Volume (mm.sup.3)=w.sup.2×l/2
where w=width and l=length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.
[0965] Therapeutic (test) Agents. All test agents were prepared as ready-to-dose dosing solutions at concentrations of 0.625 mg/mL. All dosing solutions were stored at 4° C. until dosed. All treatments were administered in a dosing volume of 8 mL/kg scaled to the body weights of the individual animals resulting in a dose of 5 mg/kg. Treatment. On Day 1 of the study, female SCID mice bearing established BT474 xenografts were dosed according to the treatment plan summarized in Table 53. All agents were administered intravenously (i.v.) via tail vein injection in a single dose on Day 1.
TABLE-US-00066 TABLE 53 Treatment plan for tumour uptake study using scFv-P5C5 conjugates (71) Dose Number of Tumours excised Group Agent (mg/kg) doses after (hours) 1 Trastuzumab-P5C5 5 1 2 2 scFv (TCT)-P5C5 5 1 2 3 scFv (TCT1067)- 5 1 2 P5C5
[0966] Endpoint. The study endpoint occurred on Day 1, two hours post dose.
[0967] Treatment-Related Side Effects. Test animals were weighed Day 1. Animals were observed frequently for overt signs of any adverse, treatment-related side effects. Individual body weight loss was monitored every other day and any animal whose weight exceeded the limits for acceptable body weight loss was euthanized. Group mean body weight loss also was monitored as per protocol. Acceptable toxicity for the maximum tolerated dose (MTD) was defined as a group mean body weight loss of less than 20% during the test.
[0968] Sampling. Samples were collected two hours post dose in the study for further analysis. Blood (full blood volume) was collected from all animals via terminal cardiac puncture under isoflurane anesthesia. Once collected, blood samples were processed for plasma using lithium heparin as the anticoagulant. Each plasma sample was then frozen and stored at −80° C. for analysis. Immediately following the collection of blood, tumors were collected. Tumors were place in formalin for approximately 24 hours at room temperature and then transferred to 70% ethanol. Tumors were then embedded in paraffin wax blocks and multiple slides of serial sections of each tumor were made.
[0969] Immunohistochemical analyses. Slides containing tumour sections were deparafinized by incubating in xylene for 2×5 minutes, rehydrated by incubating in 100% ethanol for 4×2 minutes and distilled water for 2×5 minutes. The slides were drained briefly by standing on absorbent tissue and hydrophobic pen (a ‘PAP’ pen) was used to draw a circle around each section, taking care not to touch the section. Each section was covered with 100-400 μl of blocking solution (1% BSA in TBS) and incubated for 1 hr in a humidified chamber. The blocking solution was flicked off and 100-400 μl of primary antibody (mouse anti-cemadotin monoclonal antibody, Example 33, 5 μg/ml) in the blocking buffer was applied and incubated overnight at 4° C. in a humidified chamber. Next, the antibody solution was removed and the sections were washed three times in TBS buffer for 5 mins each, then the secondary antibody (goat anti-mouse FITC conjugate, Thermo-Fisher 62-6511, 1:50 or anti-human FITC conjugate Thermo-Fisher 054211, 1:20) solution (in the blocking buffer) was added and incubated for 60 mins at room temperature in the dark. The antibody solution was removed and the sections washed three times in TBS buffer for 5 mins each. The sections were mounted with coverslips using a mounting medium. The mounting medium was allowed to set and the slides viewed under a fluorescent microscope, with digital images captured.
[0970]
Example 75A. Demonstration that OptiLinked scFv-Drug Conjugates have a Slower Plasma Pharmacokinetic Profile than that of the Unmodified scFv in Rodent Animal Models
[0971] Mice. Female BALB/c mice (BALB/cAnNCrl, Charles River) were eight weeks old with body weights ranging from 15.9 to 21.9 grams at the beginning of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and at 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services, who carried out this contracted R&D) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The Animal Care and Use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, which assures compliance with accepted standards for the care and use of laboratory animals.
[0972] Rats. Male Sprague-Dawley rats (sourced from Charles River, UK) were group housed in a temperature and light controlled facility on a 12 hour light/dark cycle with food and water available ad libitum. Rats selected for study inclusion were individually housed until completion of the study. All animals were subjected to health monitoring in accordance with the guidelines by the onsite home office registered veterinarian. All animal experimentation was covered under the UK Animals (Scientific Procedures) Act (1986) and EU directive 86/609/EEC. All such work was monitored by regular inspections of procedures and facilities by the onsite Veterinarian and UK Home Office inspectors. The study design required the surgical implantation of catheters into the jugular of male Sprague-Dawley rats. Rats were anaesthetised using the inhalation anaesthetic isoflurane and placed in dorsal recumbency. The right jugular vein was exposed and a loose ligature was placed caudally and the cranial end of vein was ligated. A small incision was made between the ligatures into which the catheter (polyethylene 50 tubing) was inserted. The catheter was secured in place by tying the loose ligature around the catheterised vessel. A small incision was made in the scapular region to serve as the exit site of the catheter. The catheter was subcutaneously tunnelled and exteriorised through the scapular incision. Patency of the catheter was tested, and the catheter was filled with a locking solution (heparinised saline) and sealed with a stainless steel pin. Post-operative monitoring of animals was performed according to Home Office good practice guidelines. Intravenous dosing was via the tail vein.
[0973] Therapeutic (test) Agents. All test agents were supplied as ready-to-dose dosing solutions. All dosing solutions were stored at 4° C. until dosed. All treatments were administered in a dosing volume scaled to the body weights of the individual animals to obtain the dosing concentration described in the treatment tables.
[0974] Treatment (mice). On Day 1 of the study, mice were divided into groups each consisting of eighteen mice (per test agent being evaluated), and dosing was initiated according to the treatment plan summarized in the treatment tables. All doses were administered intravenously (i.v.) by tail vein injection as described in the tables below.
[0975] Treatment (rats). On Day 1 of the study, rats were divided into groups of three animals (per test agent being evaluated), and dosing was initiated according to the treatment plan summarized in the tables below
[0976] Endpoint. The study endpoint occurred after the last sampling point, typically on Day 2 or 4, twenty-four or seventy-two hours post dose.
[0977] Treatment-Related Side Effects. Test animals were weighed twice on Day 1. Animals were observed frequently for overt signs of any adverse, treatment-related side effects. Individual body weight loss was monitored every other day and any animal whose weight exceeded the limits for acceptable body weight loss was euthanized. Group mean body weight loss also was monitored. Acceptable toxicity for the maximum tolerated dose (MTD) was defined as a group mean body weight loss of less than 20% during the test.
[0978] Sampling (mice). Blood (full blood volume) was collected from three animals per treatment group per timepoint. Samples were collected from all animals via terminal cardiac puncture under isoflurane anesthesia. Once collected, blood samples were collected into collection tubes containing lithium heparin or K2EDTA as anticoagulants and were processed for plasma at each timepoint. Each plasma sample was stored at −80° C. until used for analysis.
[0979] Sampling (rats). Serial venous blood samples (approx. 0.1-0.2 ml) were collected via the exteriorised jugular vein catheter at specified time points (0.5 to 72 hrs post dosing) and placed in heparinised containers. Prior to taking the blood samples the catheter was evacuated of heparinised saline to prevent dilution of the blood sample. Following each blood sample the volume of blood removed is replaced with an equal volume of heparinised saline via the catheter and sealed. Blood samples were centrifuged (5 minutes, 16,100 g, 4° C.) to separate the plasma. Plasma samples were transferred to fresh containers and promptly frozen and stored at −20° C. until used for analysis.
[0980] Quantification of test agents in plasma. An ELISA was carried out as described in Example 31 The detecting antibodies were (a) Anti-T7 tag to detect the scFv (total antibody), (b) Anti-Human Fab-specific to detect trastuzumab (total antibody), (c) Anti-MMAF (Concortis), Anti-MMAE (Concortis), anti-DM1 (Concortis) and anti-cemadotin (in-house mouse monoclonal antibody, Example 33 that also recognised P5C5 and AuristatinF), total ADC. The reference test agents were used to construct a calibration curve for which the plasma samples were compared to, in order to quantify the amount present. The concentration was plotted against time (average of three animals with standard error) and fitted to a 2-phase decay curve using GraphPad Prism to derive kinetic parameters.
Example 75B. Pharmacokinetic Analyses of ScFv (TCT)-MMAF-C5, Trastuzumab-MMAF-C5 Conjugates (118) and Unconjugated Antibodies in Mice
[0981] Mice were prepared, treated and plasma analysed as described in Example 75A. The dosing and sampling schedule is shown in Table 54.
TABLE-US-00067 TABLE 54 Pharmacokinetic dosing and analyses of scFv (TCT)- MMAF-C5, Trastuzumab-MMAF-C5 conjugates (compounds 118) and unconjugated antibodies in mice Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT)- 1 × 5 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- MMAF-C5 payload ELISA anti-T7 Tag Trastuzumab- 1 × 5 mg/kg 1, 2, 6, 24, 48, 72 ELISA anti- MMAF-C5 Human IgG ELISA anti- payload scFv (TCT) 1 × 5 mg/kg 2, 4, 6, 24 ELISA anti-T7 Tag
[0982] The pharmacokinetic plot is shown in
TABLE-US-00068 TABLE 55 Pharmacokinetic parameters of scFv (TCT)-MMAF- C5, Trastuzumab-MMAF-C5 conjugates (compounds 117) and unconjugated antibodies in mice Elimination half-life (hrs) Total Total Bioavailability (blood Test agent antibody ADC exposure, AUC, 24 hrs)* scFv (TCT)-MMAF- 2.84 2.78 40.88 C5 Trastuzumab- 20.11 24.93 1890 MMAF-C5 scFv (TCT) 2.78 — 2.9 *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 75C. Pharmacokinetic Analyses of scFv (TCT1067)-MMAF-C5, Trastuzumab-MMAF-C5 Conjugates (118) and Unconjugated Antibodies in Mice
[0983] Mice were prepared, treated and plasma analysed as described in Example 75A. The dosing and sampling schedule is shown in Table 56.
TABLE-US-00069 TABLE 56 Pharmacokinetic dosing and analyses of scFv (TCT)- MMAF-C5, Trastuzumab-MMAF-C5 conjugates (compounds 118) and unconjugated antibodies in mice Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT1067)- 1 × 5 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- MMAF-C5 payload ELISA anti-T7 Tag Trastuzumab- 1 × 5 mg/kg 1, 2, 6, 24, 48, 72 ELISA anti- MMAF-C5 Human IgG ELISA anti- payload scFv (TCT1067) 1 × 5 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti-T7 Tag
[0984] The pharmacokinetic plot is shown in
[0985] The slower clearance of the scFv (TCT1067)-MMAF-C5 conjugate leads to a significant blood exposure (as illustrated by the area under the clearance curve, (AUC)), showing a 15.5-fold increase in bioavailability, which in turn leads to a significant and effective tumour exposure. The MMAF-C5 conjugates also had a lower volume of distribution compared to the unmodified scFv which led to a higher bioavailability. The scFv (TCT1067)-MMAF-C5 conjugates were detected in the plasma via its T7 tag (detecting total antibody) and via the payload (detecting total ADC). The trastuzumab-MMAF-C5 conjugates were detected in the plasma using anti-human Fab (detecting total antibody) and via the payload (detecting total ADC). The clearance lines for the scFv (TCT1067)-MMAF-C5 conjugates were very similar suggesting that the scFv (TCT1067)-MMAF-C5 conjugates were stable in plasma and insignificant de-conjugation was occurring.
TABLE-US-00070 TABLE 57 Pharmacokinetic parameters of scFv (TCT)-MMAF- C5, Trastuzumab-MMAF-C5 conjugates (compounds 118) and unconjugated antibodies in mice Elimination half-life (hrs) Total Total Bioavailability (blood Test agent Antibody ADC exposure, AUC, 24 hrs)* scFv (TCT1067)- 3.34 2.62 126 MMAF-C5 Trastuzumab- 20.11 24.93 1890 MMAF-C5 scFv (TCT1067) 1.05 — 8.12 *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 75D. Pharmacokinetic Analyses of scFv (TCT)-P5C5, Trastuzumab-P5C5 Conjugates (71) and Unconjugated Antibodies in Mice
[0986] Mice were prepared, treated and plasma analysed as described in Example 75A The dosing and sampling schedule is shown in Table 58.
TABLE-US-00071 TABLE 58 Pharmacokinetic dosing and analyses of scFv (TCT)- P5C5, Trastuzumab-P5C5 conjugates (compounds 71) and unconjugated antibodies in mice Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT)-P5C5 1 × 5 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- payload ELISA anti-T7 Tag Trastuzumab- 1 × 5 mg/kg 1, 2, 6, 24, 48, 72 ELISA anti- P5C5 Human IgG ELISA anti- payload scFv (TCT) 1 × 5 mg/kg 2, 4, 6, 24 ELISA anti-T7 Tag
[0987] The pharmacokinetic plot is shown in
TABLE-US-00072 TABLE 59 Pharmacokinetic parameters of scFv (TCT1067)- P5C5, Trastuzumab-P5C5 conjugates (compounds 71) and unconjugated antibodies in mice Elimination half-life (hrs) Total Total Bioavailability (blood Test agent Antibody ADC exposure, AUC, 24 hrs)* scFv (TCT)-P5C5 4.69 4.99 215.35 Trastuzumab-P5C5 17.3 ND >150 scFv (TCT) 2.78 2.91 *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 75E. ScFv (TCT1067)-Auristatin F-C5, Trastuzumab-Auristatin F-C5 Conjugates (122) and Unconjugated Antibodies in Mice
[0988] Mice were prepared, treated and plasma analysed as described in Example 75A The dosing and sampling schedule is shown in Table 60.
TABLE-US-00073 TABLE 60 Pharmacokinetic dosing and analyses of scFv (TCT1067)- AF-C5, Trastuzumab-AF-C5 conjugates (compounds 122) and unconjugated antibodies in mice Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT1067)- 1 × 2 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- AF-C5 payload ELISA anti-T7 Tag scFv (TCT1067) 1 × 2 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti-T7 Tag
[0989] The pharmacokinetic plot is shown in
TABLE-US-00074 TABLE 61 Pharmacokinetic parameters of scFv (TCT1067)- AF-C5, Trastuzumab-AF-C5 conjugates (compounds 122) and unconjugated antibodies in mice Elimination half-life (hrs) Total Total Bioavailability (blood Test agent Antibody ADC exposure, AUC, 24 hrs)* scFv (TCT1067)-AF- 0.92 0.85 27.6 C5 scFv (TCT1067) 1.05 8.13 ND = not determined *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 75F. ScFv (TCT1067)-DM1(PEG12), Trastuzumab-DM1(PEG12) Conjugates (124) and Unconjugated Antibodies in Mice
[0990] Mice were prepared, treated and plasma analysed as described in Example 75A The dosing and sampling schedule is shown in Table 61.
TABLE-US-00075 TABLE 61 Pharmacokinetic and dosing analyses of scFv (TCT1067)- DM1(dPEG.sub.12) (compound 124) and unconjugated scFv in mice Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT1067)- 1 × 2 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- DM1(dPEG.sub.12) payload ELISA anti-T7 Tag scFv (TCT1067) 1 × 2 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti-T7 Tag
[0991] The pharmacokinetic plot is shown in
TABLE-US-00076 TABLE 62 Pharmacokinetic parameters of scFv (TCT1067)- DM1(dPEG.sub.12), Trastuzumab-DM1(dPEG.sub.12) (compounds 124) conjugates and unconjugated antibodies in mice Elimination half-life (hrs) Total Total Bioavailability (blood Test agent Antibody ADC exposure, AUC 24 hrs)* scFv (TCT1067)- 0.92 1.53 22.15 DM1(dPEG.sub.12) scFv (TCT1067) 1.05 — 8.13 *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 75G. ScFv (TCT1067)-P5C5 Conjugate (71) and Unconjugated Antibody in Rats
[0992] Rats were prepared, treated and plasma analysed as described in Example 75A The dosing and sampling schedule is shown in Table 63.
TABLE-US-00077 TABLE 63 Pharmacokinetic and dosing analyses of scFv (TCT1067)- P5C5 (compound 71) and unconjugated antibody in rats Administered Sampling times Test agent dose (hrs) Analysis scFv (TCT1067)- 1 × 4 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti- P5C5 payload ELISA anti-T7 Tag scFv (TCT1067) 1 × 4 mg/kg 0.5, 1, 2, 4, 8, 24 ELISA anti-T7 Tag
[0993] The pharmacokinetic plot is shown in
TABLE-US-00078 TABLE 64 Pharmacokinetic parameters of scFv (TCT1067)-P5C5 conjugate (compound 71) and unconjugated antibody in rats Elimination half-life (hrs) Total Total Bioavailability (blood Test agent Antibody ADC exposure, AUC 24 hrs)* scFv (TCT1067)- 0.512 2.2085 90.1 P5C5 scFv (TCT1067) 0.8 20.46 *Calculated from mean AUC in anti-payload and anti-protein detection (μghr/ml)
Example 76. OptiLinked scFv-Drug Conjugates Tumour Regression or Eradication Studies in Human Tumour Xenografts Models Compared to Equivalent Payload-Bearing Whole Immunoglobulin-Drug Conjugates and Controls
[0994] Mice. Female severe combined immunodeficient mice (Fox Chase SCID®, CB-17/Icr-Prkdcscid/IcrIcoCrl Charles River Laboratories) were Twelve weeks old with a body weight (BVV) range of 15.3 to 18.4 grams on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services, who carried out this contracted R&D) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The Animal Care and Use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.
[0995] In Vivo Implantation and Tumor Growth. Xenografts were initiated with (a) BT474 human breast carcinomas maintained at CR Discovery Services by serial subcutaneous transplantation in SCID mice. On the day of tumor implant, each test mouse received a 1 mm3 BT474 fragment implanted subcutaneously in the right flank, and tumor growth was monitored as the average size approached the target range of 110 to 144 mm.sup.3. Fifty days after tumor implantation, designated as Day 1 of the study, the animals were re-sorted into six groups each consisting of two mice with individual tumor volumes of 110 to 144 mm.sup.3 and group mean tumor volumes of 115 to 118 mm.sup.3. (b) Xenografts were initiated with a cell suspension of NCI-N87 tumour cells implanted subcutaneously in the right flank, and tumor growth was monitored as the average size approached the target range of 110 to 144 mm.sup.3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumor Volume (mm.sup.3)=width.sup.2×length/2
[0996] where width and length of the tumor was in mm. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.
[0997] Therapeutic Agents and treatment. All test agents were supplied as ready-to-dose dosing solutions and stored at 4° C. until used. All treatments were administered in a dosing volume scaled to the body weights of the individual animals to achieve the dosing concentration described in the respective treatment tables. All agents were administered intravenously (i.v.) via tail vein injection.
[0998] Endpoint. The study continued for up to 90 days or until the tumours reached a maximum size of 1000 mm.sup.3.
[0999] Treatment-Related Side Effects. Test animals were weighed Day 1. Animals were observed frequently for overt signs of any adverse, treatment-related side effects. Individual body weight loss was monitored every other day and any animal whose weight exceeded the limits for acceptable body weight loss was euthanized. Group mean body weight loss also was monitored as per protocol. Acceptable toxicity for the maximum tolerated dose (MTD) was defined as a group mean body weight loss of less than 20% during the test.
Example 76A. Tumour Growth Inhibition or Eradication in a BT474 Xenograft Model with scFv (TCT1067)-MMAF-C5, Trastuzumab-MMAF-C5 Conjugates (117) and Free MMAF Therapeutic Agents
[1000] BT474 tumours were set up as described in Example 76. The treatment plan for this experiment is described in Table 65
TABLE-US-00079 TABLE 65 Treatment plan for scFv (TCT1067)-MMAF-C5 (compound 118) compared to trastuzumab-MMAF-C5 benchmark Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml vol Every other 12 day 2 Free MMAF 1 Every other 12 day 3 scFv (TCT1067)- 0.05 Every other 12 MMAF-C5 day 4 scFv (TCT1067)- 0.5 Every other 12 MMAF-C5 day 5 scFv (TCT1067)- 2 Every other 12 MMAF-C5 day 6 Trastuzumab- 5 Once per 4 MMAF-C5 week 7 Trastuzumab- 1 Once per 4 MMAF-C5 week
[1001] Tumour volume (mm.sup.3) was plotted against time (
[1002] The results show that there is a clear dose-response relationship with all of the ADCs with the scFv (TCT1067)-MMAF-C5 2 mg/kg dosing regimen leading to complete (100%) cures (durable to almost 90 days) by day 30. The scFv (TCT1067)-MMAF-C5 0.5 mg/kg dosing regimen also led to complete (100%) cures (reached by 90 day) A similar response was seen, as expected with the Trastuzumab-MMAF-C5 5 mg/kg dosing regimen. However, the ability to give more payload with the scFv (TCT1067)-MMAF-C5 ADC, more frequently resulted in the tumours shrinking more rapidly, approximately twice as fast as the trastuzumab-MMAF-C5 ADC. The saline (vehicle) and free payload treated animal group tumours grew rapidly. The scFv (TCT1067)-MMAF-C5 treatment seemed to be better tolerated as seen from the increasing body weights with the antibody fragment ADC group of mice being up to 15% heavier than the IgG-MMAF-C5 ADC group of mice. An estimation of the therapeutic index for the scFv (TCT1067)-MMAF-C5 is at least 40 (at least 2 mg/kg, maximum tolerated dose/0.05 mg/kg minimum efficacious dose), compared to an approximate value of around 5 for the trastuzumab ADC.
Example 76B. Tumour Growth Inhibition or Eradication in a BT474 Xenograft Model with scFv (TCT)-MMAF-C5, Trastuzumab-MMAF-C5 Conjugates (Compounds 118) and Free MMAF Therapeutic Agents
[1003] BT474 tumours were set up as described in Example 76. The treatment plan for this experiment is described in Table 66.
TABLE-US-00080 TABLE 66 Treatment plan for scFv (TCT)-MMAF-C5 (118) compared to trastuzumab-MMAF-C5 benchmark Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml vol Every other 12 day 2 Free MMAF 1 Every other 12 day 3 scFv (TCT)- 0.5 Every other 12 MMAF-C5 day 4 scFv (TCT)- 2 Every other 12 MMAF-C5 day 5 Trastuzumab- 1 Once per 4 MMAF-C5 week 6 Trastuzumab- 5 Once per 4 MMAF-C5 week
[1004] Tumour volume (mm.sup.3) was plotted against time (
[1005] The medium affinity scFv (TCT)-MMAF-C5 conjugates were dosed more frequently to account for the more rapid pharmacokinetic clearance at three lower doses. The data from example 74A is included as a comparison.
[1006] The results show that there is a clear dose-response relationship with all of the ADCs with the scFv (TCT)-MMAF-C5 2 mg/kg dosing regimen leading to complete (100%) cures (durable to almost 90 days) by day 30. A similar response was seen, as expected with the Trastuzumab-MMAF-C5 5 mg/kg dosing regimen. However, the ability to give more payload with the scFv (TCT)-MMAF-C5 ADC, more frequently resulted in the tumours shrinking more rapidly. The saline (vehicle) and free payload treated animal group tumours grew rapidly. The scFv (TCT)-MMAF-C5 treatment seemed to be better tolerated as seen from the increasing body weights with the antibody fragment ADC group of mice being up to 20% heavier than the trastuzumab-MMAF-C5 ADC group of mice. Despite the 1000-fold difference in binding affinity (Example 63) between the two antibody fragment ADCs, the 2 mg/kg dosing regimens led to similar and rapid responses suggesting that binding affinity is not a critical factor (although there must be a minimum affinity) but the high payload loading and rapid penetration lead to high efficacy. At the lower dose of 0.5 mg/kg scFv (TCT)-MMAF-C5 the responses are inferior to the higher affinity scFv where the tumours start to regrow at day 40 with a cure rate of 50%
Example 76C. Tumour Growth Inhibition or Eradication in BT474 Xenograft Model with scFv (TCT1067)-P5C5 and Trastuzumab-P5C5 Conjugates (Compounds 71)
[1007] BT474 tumours were set up as described in Example 76. The treatment plan for this experiment is described in Table 67.
TABLE-US-00081 TABLE 67 Treatment plan for scFv (TCT1067)-P5C5 compared to trastuzumab-P5C5 benchmark (compounds 71) Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml vol Every other 12 day 2 scFv (TCT1067)-P5C5 5 Every other 12 day 3 Trastuzumab-P5C5 5 Once per 4 week
[1008] Tumour volume (mm.sup.3) was plotted against time (
[1009] The scFv (TCT1067)-MMAF 2 mg/kg dosing regimen which led to complete (100%) cures is also shown for comparison. The scFv (TCT1067)-P5C5 5 mg/kg dosing regimen led to an approximate 20-day tumour growth delay, however the Trastuzumab-P5C5 5 mg/kg dosing regimen led to a marginal, insignificant growth delay. Therefore the ability to give more payload with the scFv (TCT1067)-P5C5 ADC, more frequently resulted in the tumours shrinking more effectively than the trastuzumab-P5C5 ADC. The saline (vehicle) and free payload treated animal group tumours grew rapidly. The scFv (TCT1067)-P5C5 treatment seemed to be better tolerated as seen from the increasing body weights with the antibody fragment ADC group of mice being up to 20% heavier than the trastuzumab-P5-C5 ADC group of mice.
Example 76D. Tumour Growth Inhibition or Eradication in a BT474 Human Breast Cancer Xenograft Model with scFv (TCT1067) Conjugates at Two Different DARs
[1010] BT474 tumours were set up as described in Example 76. The treatment plan for this experiment is described in Table 68.
TABLE-US-00082 TABLE 68 Treatment plan for scFv (TCT1067)-Auristatin F-C5 conjugates (compounds 122) at two DARs Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml Every 4 days 4 2 scFv (TCT1067)-AF-C5 5 Every 4 days 4 (L) 3 scFv (TCT1067)-AF-C5 5 Every 4 days 4 (M)
[1011] Tumour volume (mm.sup.3) was plotted against time (
Example 76E. Tumour Growth Inhibition or Eradication in a BT474 Human Breast Cancer Xenograft Model with scFv (TCT1067)-AF-C5 Conjugates at Three Different DARs
[1012] BT474 tumours were set up as described in Example 76. The treatment plan for this experiment is described in Table 69.
TABLE-US-00083 TABLE 69 Treatment plan for scFv (TCT1067)-Auristatin F-C5 conjugates (compounds 122) at three DARs Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml Every 4 days 2 2 scFv (TCT1067)-AF-C5 5 Every 4 days 2 (L) 3 scFv (TCT1067)-AF-C5 5 Every 4 days 2 (M) 3 scFv (TCT1067)-AF-C5 5 Every 4 days 2 (H)
[1013] Tumour volume (mm.sup.3) was plotted against time (
Example 76F. ScFv (TCT1067)-MMAE-PAB-Cit-Val-dPEG.SUB.9., Trastuzumab-MMAE-PAB-Cit-Val-dPEG.SUB.9 .Conjugates (121) and Free MMAE in a NCI-N87 Human Gastric Cancer Xenograft Model
[1014] NCI-N87 tumours are set up as described in Example 76. The treatment plan for this experiment is described in Table 64.
TABLE-US-00084 TABLE 70 Treatment plan for scFv (TCT1067)-MMAE-PAB- Cit-Val-dPEG.sub.9 compared to trastuzumab-MMAE-PAB-Cit-Val- dPEG.sub.9 benchmark (compounds 121) Dose Therapeutic agent or administered Number No control (mg/kg) Schedule of doses 1 Vehicle (saline) 8 ml Every 4 days 8 2 scFv (TCT1067)- 5 Every 4 days 8 MMAE-PAB-Cit-Val- dPEG.sub.9 3 Trastuzumab-MMAE- 5 Once per 4 PAB-Cit-Val-dPEG.sub.9 week
Example 77 Bioconjugation of a TCO Derivative onto a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues for a 2-Step Tetrazine Click Reaction
[1015] TCO-PEG4-NHS (purchased from Jena Biosciences) was conjugated to scFv (TCT) to obtain 1 conjugate (compound 134) with medium DARs. The conditions identified and carried forward were:
TABLE-US-00085 Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 1 mg/ml concentration TCO-PEG4-NHS 100 mM solution in 100% anhydrous filtered DMSO handling TCO-PEG4-NHS every 120 min addition rate TCO-PEG4-NHS 8 equivalents addition portions
[1016] The reactions were carried out as per Example 64A.
[1017] In this example, the set up was:
[1018] Reaction 1—scFv (TCT): TCO-PEG4-NHS, 16 equivalents.
[1019] No visible precipitates were noticeable and the sample recovery was high. The sample was analysed by SDS-PAGE (
[1020] The LCMS, Total Ion Current (TIC) chromatograms and spectra and the deconvoluted data are shown in
[1021] A major peak was observed in the TIC of the scFv (TCT)-TCO-PEG4 sample eluting at 12.3 mins. The zero-charge deconvoluted mass spectrum for this peak produced peaks at m/z 30956 and 31372 corresponding to the theoretical mass of the scFv together with 7 and 8 additions of the TCO-PEG4 molecule. Therefore, the conjugate (compound 134) had an average DAR of 7.5.
[1022] A tetrazine-terminated linker-payload, such as the MMAF (117), can subsequently be conjugated in a second step to form an antibody drug conjugate (135).
Example 78—Preparation of SN38-(DNMEA)-PAB-Cit-Val-dPEG.SUB.5.-NHS Ester (140)
[1023] ##STR00074## ##STR00075##
[1024] To a stirred solution of DNMEA-SN38 98 (80 mg, 0.13 mmol) and Fmoc-Val-Cit-PAB-PNP 13 (0.14 g, 0.19 mmol) in DMF (2 ml), was added HOBt (34 mg, 0.25 mmol), pyridine (52 μl) and DIPEA (22 μl). The reaction mixture was stirred under N.sub.2 atmosphere at room temperature for 24 h. Solvents were evaporated in vacuo and the resulting residue was directly used for the next step. HRMS: ESI m/z Found 1135.0803 [M+H].sup.+ calculated 1135.2650 for C.sub.61H.sub.68N.sub.9O.sub.13.
[1025] A solution of Fmoc-Val-Cit-PAB-DNMEA-SN38 137 (90 mg, 0.08 mmol) in DMF (1.5 ml) and diethylamine (0.4 ml) was stirred for 3 hours at room temperature. The reaction mixture was then concentrated in vacuo and was directly used without further purification. HRMS: ESI m/z Found 913.0200 [M+H].sup.+ calculated 913.0220 for C.sub.46H.sub.58N.sub.9C.sub.11.
[1026] To a solution of H-Val-Cit-PAB-DNMEA-SN38 138 (70 mg, 0.08 mmol) in DMF (3 ml) was added DIPEA (40 μl) and Acid-dPEG.sub.5-NHS (40 mg, 0.09 mmol) at room temperature. The reaction mixture was stirred under N.sub.2 atmosphere for 16 h. The solvents were evaporated in vacuo, the obtained crude compound was used directly for the next step. HRMS: ESI m/z Found 1233.2537 [M+H].sup.+ calculated 1233.3600 for C.sub.60H.sub.82N.sub.9O.sub.19.
[1027] To a solution of Acid-dPEG.sub.5-Val-Cit-PAB-DNMEA-SN38 (90 mg, 0.07 mmol) in DMF (3 ml) was added DIPEA (63 μl) and TSTU (44 mg, 0.14 mmol) at room temperature and the reaction mixture was stirred under N.sub.2 atmosphere for 3 h. The solvents were evaporated in vacuo and the crude product was purified on Biotage flash purification system using C18 column to give the desired compound NHS-dPEG.sub.5-Val-Cit-PAB-DNMEA-SN38 140 HRMS: ESI m/z Found 1330.3479 [M+H]+ calculated 1330.4300 for C.sub.64H.sub.85N.sub.10O.sub.21
Example 79 Bioconjugation of an SN-38 Derivative with a Protease Cleavable Linker onto a High Affinity Single-Chain Fv Antibody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues
[1028] ##STR00076##
[1029] SN38-(DNMEA)-PAB-Cit-Val-dPEG.sub.5-NHS ester (140) was conjugated to scFv (TCT1067) to obtain conjugates (141) of various DARs.
[1030] The conditions used for the conjugations were:
TABLE-US-00086 Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 1 mg/ml concentration SN38-(DNMEA)- 50 mM solution in 100% anhydrous filtered DMSO PAB-Cit-Val- dPEG.sub.5-NHS handling SN38-(DNMEA)- every 90 min PAB-Cit-Val- dPEG.sub.5-NHS addition rate SN38-(DNMEA)- 5 equivalents for reaction 1; 10 equivalents for PAB-Cit-Val- reaction 2; 12.5 equivalents for reaction 3 and 4. dPEG.sub.5-NHS addition portions
[1031] The reactions were carried out as detailed in Example 64A.
[1032] In this example, the set up was:
[1033] Reaction 1—scFv (TCT1067): SN38-(DNMEA)-PAB-Cit-Val-dPEG.sub.5-NHS, 5 equivalents
[1034] Reaction 2—scFv (TCT1067): SN38-(DNMEA)-PAB-Cit-Val-dPEG.sub.5-NHS, 10 equivalents
[1035] Reaction 3—scFv (TCT1067): SN38-(DNMEA)-PAB-Cit-Val-dPEG.sub.5-NHS, 25 equivalents
[1036] Reaction 4—scFv (TCT1067): SN38-(DNMEA)-PAB-Cit-Val-dPEG.sub.5-NHS, 35 equivalents
[1037] The unconjugated and conjugated scFv (TCT1067) were analysed by HPLC size exclusion chromatography (
Example 80 Bioconjugation of Auristatin-C5-NHS onto a High Affinity Diabody Fragment Bearing Multiple, Well-Dispersed, Surface Lysine Residues AF-C5-NHS Ester (88) was Conjugated to Diabody (TCT) and as a Control to scFv (TCT) to Obtain Conjugates (Compound 122) of Various DARs
[1038] The reaction conditions used were as follows:
TABLE-US-00087 Type Condition Buffer Bicarbonate buffer with 150 mM NaCl at pH 8.8 Co-solvent anhydrous filtered DMSO at a final 20% (v/v) concentration Temperature 20° C. Agitation thermomixer 1000 rpm Antibody 0.71 mg/ml concentration AF-C5-NHS 50 mM solution in 100% anhydrous filtered DMSO handling AF-C5-NHS every 90 min addition rate AF-C5-NHS 7.5 equivalents addition portions
[1039] The reactions were carried out as detailed in Example 64A.
[1040] In this example, the set up was:
[1041] Reaction 1: diabody (TCT): AF-C5-NHS, 30 equivalents;
[1042] Reaction 2: scFv (TCT): AF-C5-NHS, 15 equivalents;
[1043] The SDS PAGE gel in