Antibody-SN-38 immunoconjugates with a CL2A linker
The present invention concerns improved methods and compositions for preparing SN-38 conjugates of proteins or peptides, preferably immunoconjugates of antibodies or antigen-binding antibody fragments. More preferably, the SN-38 is attached to the antibody or antibody fragment using a CL2A linker, with 1-12, more preferably 6 or less, most preferably 1-5 SN-38 moieties per antibody or antibody fragment. Most preferably, the immunoconjugate is prepared in large scale batches, with various modifications to the reaction scheme to optimize yield and recovery in large scale. Other embodiments concern optimized dosages and/or schedules of administration of immunoconjugate to maximize efficacy for disease treatment and minimize side effects of administration.
1. A method of delivering SN-38 to a target cell comprising:
a) producing a compound, CL2A-SN-38, of the structure,
by a reaction scheme as shown:
wherein the reaction scheme further comprises reacting SN-38 with tert-butyldimethylsilyl chloride (TBDMS-Cl) to produce 10-O-TBDMS-SN-38 (intermediate 4), wherein the reaction is carried out in dichloromethane solvent;
b) attaching an antibody or antigen-binding antibody fragment to the CL2A-SN-38 to produce a mAb-CL2A-SN-38; and
c) administering the mAb-CL2A-SN-38 to a subject.
2. The method of claim 1 , further comprising reacting 10-O-TBDMS-SN-38 (intermediate 4) with triphosgene and DMAP to make 10-O-TBDMS-SN-38-20-O-chloroformate (reactive intermediate 5), wherein the reaction is performed by adding triphosgene to a dichloromethane reaction mixture that contains intermediate 4, and the triphosgene is added in portions, to reduce exothermic reaction during large scale manufacture and maintain high reaction yield.
3. The method of claim 1 , further comprising precipitating Lys(MMT)-PABOH (intermediate 2) with heptane.
4. The method of claim 3 , further comprising assaying residual diethylamine in the precipitated intermediate 2 and purifying the precipitate by chromatography if the presence of diethylamine was detected.
5. The method of claim 1 , further comprising reacting azido-PEG-Lys(MMT)-PABO-CO-20-O—SN-38 (intermediate 7) with MCC-Yne (intermediate 8) by a copper catalyzed cycloaddition reaction to make MCC-PEG-Lys(MMT)-PABO-CO-20-O—SN-38 (intermediate 9), wherein the reaction is carried out for 14 h to improve product yield.
6. The method of claim 5 , wherein intermediate 9 is first purified by silica gel chromatography, followed by extraction with EDTA to remove copper.
7. The method of claim 1 , further comprising purifying the mAb-CL2A-SN-38 by tangential flow filtration (TFF).
8. The method of claim 7 , wherein the TFF is performed with a 50,000 dalton molecular weight cut-off membrane using 25- to 30-diafiltration volumes of buffer.
9. The method of claim 1 , further comprising formulating the mAb-CL2A-SN-38 in Good's biological buffer at a pH of 6.0 to 7.0, and lyophilizing the mAb-CL2A-SN-38 for storage.
10. The method of claim 9 , wherein the Good's biological buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (IVIES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and 1,4-piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7, preferably in the pH range of 6.5 to 7, and at a buffer concentration of 10-100 mM, preferably 25 mM.
11. The method of claim 10 , wherein the buffer is 25 mM MES buffer, pH 6.5.
12. The method of claim 1 , wherein the antibody is a bispecific antibody or a monoclonal antibody.
13. The method of claim 1 , wherein the antibody fragment is selected from the group consisting of F(ab′) 2 , F(ab) 2 , Fab′, Fab, Fv, and scFv.
14. The method of claim 1 , wherein the antibody or antibody fragment is attached to between 1 and 12 copies of CL2A-SN38.
15. The method of claim 1 , wherein the antibody or antibody fragment is attached to 6 to 8 copies of CL2A-SN38.
16. The method of claim 1 , wherein the antibody is an anti-cancer antibody, an anti-infectious disease antibody, or an anti-autoimmune disease antibody.
17. The method of claim 16 , wherein the antibody is selected from the group consisting of hLL1 (anti-CD74), hLL2 (anti-CD22), RFB4 (anti-CD22), hRS7 (anti-EGP-1), hPAM4 (anti-MUC5AC), KC4 (anti-mucin), hA19 (anti-CD19), hA20 (anti-CD20), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hMN-3 (anti-CEACAM6), hR1 (anti-IGF-1R), hMu-9 (anti-CSAp), Immu 31 (anti-AFP), CC49 (anti-TAG-72), J591 (anti-PSMA), HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), hL243 (anti-HLA-DR), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1), nivolumab (anti-PD-1), pidilizumab (anti-PD-1), MDX-1105 (anti-PD-L1), MEDI4736 (anti-PD-L1), BMS-936559 (anti-PD-L1), ipilimumab (anti-CTLA-4), tremelimumab (anti-CTLA-4), IMMU-H2B (anti-H2B), IMMU-H3 (anti-H3), IMMU-H4 (anti-H4), TAG-72, alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20), and trastuzumab (anti-ErbB2), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE).
18. The method of claim 16 , wherein the antibody is selected from the group consisting of P4/D10, Ab 75, Ab 76, Ab 77, CR6261, exbivirumab, felvizumab, foravirumab, motavizumab, palivizumab, panobacumab, rafivirumab, regavirumab, sevirumab, tivirumab, and urtoxazumab.
19. The method of claim 1 , wherein the antibody or antibody fragment binds to an antigen selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, ART-4, B7, Ba 733, BAGE, BrE3-antigen, BCL-1, BCL-2, BCL-6, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11A, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD34, CD37, CD38, CD40, CD40L, CD41a, CD43, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, H2B, H3, H4, HLA-DR, HM1.24, human chorionic gonadotropin (HCG), HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, bcl-2, bcl-6, Kras, gp45, and gp120.