COMBINED THERAPEUTIC USE OF ANTIBODIES AND IMMUNOGLOBULIN G-DEGRADING ENZYMES

Abstract

The invention relates to compositions comprising therapeutic antibodies, and uses and methods for increasing the potency of therapeutic antibodies. In particular, the invention provides a composition comprising (i) an agent which reduces Fc receptor binding of endogenous serum antibodies, and (ii) a therapeutic antibody, preferably a therapeutic antibody which is resistant to the agent. The therapeutic antibody may be administered to the subject after a set time interval, or the blood of the subject may be treated with the agent prior to administration of the therapeutic antibody.

Claims

1. A method of treating cancer in a subject, wherein the method comprises the steps: (i) administering to the subject an agent which reduces Fc receptor binding of endogenous serum antibodies, wherein said agent is EndoS, and (ii) after a set time interval, administering to the subject a therapeutic antibody, wherein said therapeutic antibody is trastuzumab, and wherein said therapeutic antibody comprises one or more glycoforms resistant to the activity of the agent in the Fc domain, said glycoforms comprising oligomannose-type glycans.

2. The method according to claim 1, wherein the cancer is selected from the group consisting of bladder cancer, lung cancer, breast cancer, melanoma, colon cancer, rectal cancer, non-Hodgkin's lymphoma, endometrial cancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer, leukemia, thyroid cancer and oesophageal cancer.

3. The method according to claim 1, wherein the set time interval is 1-2, 1-5, 1-10 or 1-20 days.

4. The method according to claim 1, wherein each of the glycans of the Fc domain contains at least 5 mannose residues.

5. The method according to claim 1, wherein: (a) each of the glycans of the Fc domain contains only two GlcNAc residues and three or more mannose residues; or (b) each of the glycans of the Fc domain contains Man.sub.5GlcNAc.sub.2,Man.sub.8GlcNAc.sub.2, or Man.sub.9GlcNAc.sub.2; or (c) the oligomannose-type glycoform is a mixture of oligomannose-type glycans; or (d) each of the glycans of the Fc domain contains between five and twenty mannose residues.

6. The method according to claim 1, wherein the cancer is breast cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0186] Additional preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following drawings and examples, wherein:

[0187] FIG. 1 shows binding of human IgG1 Fc to immobilized FcγRIIIa in the presence of PBS or increasing concentrations of human serum, treated or mock-treated with Endoglycosidase-S.

[0188] FIGS. 2A and 2B show mass spectrometric analysis of PNGase F release of N-linked glycans found on human serum IgG before EndoS digestion.

[0189] FIGS. 3A and 3B shows mass spectrometric analysis of PNGase F release of N-linked glycans found on human serum IgG after EndoS digestion. (Main ions are chloride adducts. Ions at m/z 1762 and 1924 are phosphate adducts; m/z 1519 is a fragment of m/z 1862.)

[0190] FIGS. 4A and 4B shows direct mass spectrometric analysis of glycans released by EndoS. (Ran as chloride adducts; m/z 1210, 1372 and 1534 are phosphate adducts; m/z 951, 1113 and 1275 are fragments.)

[0191] FIG. 5 shows resistance of oligomannose-type containing Fc glycoforms to EndoS mediated hydrolysis. Naturally glycosylated IgG1 Fc (a-c) and oligomannose-type (Man.sub.9GlcNAc.sub.2) Fc (d-f) were digested with either Endo-S(b, e) or Endo-H (c, f). Glycan structures shown are based on the most abundant isomeric species, consistent with known biosynthetic pathways. The cleavage of the core GlcNAcβ1.fwdarw.4GlcNAc bond results in the removal of a single GlcNAc and attached Fucose residue from complex glycans (predicted Δm/z=349.1), and a GlcNAc from oligomannose-type-type glycans (predicted Δm/z=203.1).

[0192] FIG. 6 shows the binding by ELISA of recombinant monoclonal IgG1 (mAb IgG CIIC1 which is a chimerical IgG with human Fc and mouse Fab) to immobilized FcγRIIIa is shown in the presence of buffer (PBS) or increasing concentrations of human serum. Binding was detected using a secondary antibody specific for the mouse Fab domain.

[0193] FIG. 7 shows EndoS mediated deactivation of serum leads to enhancement of a mAb IgG CIIC1 (a chimerical IgG with human Fc and mouse Fab) binding to FcγRIIIa. The binding of human IgG1 Fc containing oligomannose-type (Man.sub.5GlcNAc.sub.2) to FcγRIIIa is shown in the presence of PBS, serum, serum and Endo-S, serum and Endo-H, or serum and EndoS and Endo H. Data points represent the calculated mean of three independent measurements from a total of four experiments.

[0194] FIG. 8 shows a schematic representation of how EndoS abolishes affinity of bulk serum antibodies to FcγRs but not glycan engineered (oligomannose-type) antibodies.

BRIEF DESCRIPTION OF THE SEQUENCES

[0195] SEQ ID NO: 1 is an amino acid sequence of EndoS isolated from S. pyogenes AP1. SEQ ID NO: 2 is an amino acid sequence of EndoS isolated from S. pyogenes AP1, including a signal sequence.

EXAMPLES

[0196] The Principle of the Invention

[0197] FIG. 8 illustrates the principle of at least one aspect of the invention. In particular, FIG. 8 shows a schematic representation of how EndoS abolishes affinity of bulk serum antibodies to FcγRs by removing the “normal” Fc glycosylation. However, EndoS has no effect on glycan engineered (oligomannose-type) antibodies, and activation of FcRs is achieved. Under physiological conditions the majority of FcRs are bound by “irrelevant”, typically serum, Ig Fc. This places a hard limit on the effective concentration of available Fc receptors (FcγRs). In this embodiment Endo-S is used to significantly reduce the affinity of irrelevant serum antibodies to FcγRs but not of glycan engineered (oligomannose-type) monoclonal antibodies.

Example 1: Reduction of Serum IgG Binding to FcγRIIIa Increases the Binding of Resistant Therapeutic Antibody

[0198] In order to demonstrate that EndoS could be used to reduce serum IgG binding to FcγRIIIa the following experiment was performed. FcγRIIIa (158Val variant; R&D systems, Minneapolis, U.S.A.) at 2.5 μg/mL in PBS was coated on high-binding microtitre plates (3690, Corning, NY, U.S.A.) overnight at 4° C. Coated plates were washed with PBS containing 0.05% Tween 20 (Sigma-aldrich, U.S.A.) and blocked for 2 hours at room temperature with 3% BSA in PBS. Serial dilutions of human serum (H4522, Sigma-Aldrich, U.S.A.) or recombinant human IgG1 glycoforms bearing Man.sub.9GlcNAc.sub.2 or Man.sub.5GlcNAc.sub.2 (starting concentration of 0.1 mg/mL in PBS), was then added and allowed to bind for 2 hours at room temperature. Plates were washed five times with PBS containing 0.05% Tween and binding was detected using a HRP conjugated Fab fragment specific for murine IgG Fab (ab98659, Abcam, Cambridge, UK). TMB substrate (Thermo Scientific, Rockford, IL, U.S.A.) was used for colour development according to manufacturer's directions. Colour development was stopped by the addition of 2M H2504 and absorbance was measured at 450 nm on a Spectramax M5 (Molecular Devices, California, U.S.A.) multiwall plate reader. Serum was incubated overnight with 1 μg/mL of EndoS or PBS at 37° C. Control serum samples were mock treated with PBS and incubated overnight at 37° C. Data was processed and plotted using Prism (GraphPad software, California, U.S.A.). Apparent affinity was calculated as the concentration of oligomannose-type mAb corresponding to half-maximal binding on the ELISA binding curve. The binding of normal human sera was detected using labelled anti-human Fab. The results are shown in FIG. 1 and clearly demonstrate that EndoS treatment of human serum IgG results in a decrease in FcγRIIIa binding.

[0199] FIG. 6 demonstrates that reduced binding of a monoclonal antibody IgG CIIC1 (a chimerical IgG with human Fc, and mouse Fab which enables specific detection of the monoclonal against the larger pool of serum antibodies) to FcγRIIIa is observed with increasing concentrations of serum IgG. The binding was determined at increasing dilutions of sera (1:5, 1:10, 1:20 and 1:50) and in PBS for a range of monoclonal IgG concentrations (x-axis). Binding of the monoclonal antibody IgG CIIC1 was detected using HRP-conjugated anti-mouse (Fab)′2 antibody. FIG. 6 shows how serum IgG outcompetes the monoclonal antibody IgG CIIC1.

Serum IgG is Sensitive to EndoS Activity

[0200] In order to demonstrate that bisected glycans and sialylated structures were resistant to EndoS, compositional analysis of N-linked glycans found on human serum IgG was performed before (a) and after (b) EndoS digestion as described in the Materials and Methods section. The major, neutral bi-antennary structures found on native serum Ig (m/z at 1559, 1721, 1883) were entirely absent on IgG that has been exposed to EndoS. In contrast the corresponding bisected structures (m/z 1762, 1925, and 2087 in FIGS. 2A and 2B) represented a minor population prior to digestion with Endo-S but corresponded to the most abundant species on IgG following EndoS digestion.

[0201] Direct mass spectrometric analysis of the glycans released by EndoS was performed as described in the Materials and Methods Section. FIGS. 4A and 4B shows that neutral bi-antennary structures were hydrolysed (yielding products at m/z 1148, 1310, 1473) but no detectable bisecting structures were found in the released pool. Some mono-sialylated structures were found in the released pool (m/z 1566 and 1728) but were found as an enriched fraction on digested IgG (m/z 2078 and 2280), indicating these structures showed partial resistance to EndoS. Consistent with the resistance of neutral, bisected structures, the bisected, sialylated structure (m/z 2280) was significantly enriched as a fraction of residual glycans. Most of the sialylated glycans were released with EndoS.

[0202] Overall these data show bisected glycans are resistant to EndoS, sialylated structures are moderately resistant and the major population of bi-antennary glycans on IgG (lacking bisects and sialic acids) are entirely sensitive to EndoS.

Ability of Oligomannose-Type Antibodies to Resist Enzymatic Degradation by EndoS

[0203] To demonstrate that oligomannose-type antibodies could be generated that were resistant to EndoS the following experiments were performed.

[0204] FIG. 5 shows the resistance of oligomannose-type Fc glycoforms to EndoS mediated hydrolysis. Recombinant IgG-Fc domain was expressed in human embryonic kidney 293T cells either without FIG. 3 (a) or with (d) the inhibitor kifunensine as described in the Materials and Methods section. This produces a typical biantennary, complex-type glycan profile as shown in (a) similar to the profile seen in human sera as illustrated in FIGS. 2A and 2B or produces a purely oligomannose-type profile (d) if the inhibitor kifunensine is used. FIG. 5(b) shows how EndoS was able to completely cleave the complex-type glycans but had no effect on the oligomannose-type glycan (e). Digestion with EndoH demonstrated the reciprocal specificity of EndoH and how it is unable to cleave complex-type glycans as shown in FIG. 5(c) with complete hydrolysis seen of the oligomannose-type Fc glycoform as shown in FIG. 5(f).

[0205] In order to demonstrate that EndoS could be used to increase the apparent affinity of an oligomannose-type-mAb for FcγRIIIa the following experiment was performed. FcγRIIIa (158Val variant; R&D systems, Minneapolis, U.S.A.) at 2.5 μg/mL in PBS was coated on high-binding microtitre plates (3690, Corning, NY, U.S.A.) overnight at 4° C. Coated plates were washed with PBS containing 0.05% Tween 20 (Sigma-aldrich, U.S.A.) and blocked for 2 hours at room temperature with 3% BSA in PBS. Serial dilutions of human serum (H4522, Sigma-Aldrich, U.S.A.) or recombinant human IgG1 glycoforms bearing Man.sub.9GlcNAc.sub.2 or Man.sub.5GlcNAc.sub.2 (starting concentration of 0.1 mg/mL in PBS), was then added and allowed to bind for 2 hours at room temperature. Plates were washed five times with PBS containing 0.05% Tween and binding was detected using a HRP conjugated Fab fragment specific for murine IgG Fab (ab98659, Abcam, Cambridge, UK). TMB substrate (Thermo Scientific, Rockford, IL, U.S.A.) was used for colour development according to manufacturer's directions. Colour development was stopped by the addition of 2M H.sub.2SO.sub.4 and absorbance was measured at 450 nm on a Spectramax M5 (Molecular Devices, California, U.S.A.) multiwall plate reader. A 1:5 dilution of serum was incubated with 1:100 dilution of EndoS (1 mg/mL) or Endo H (500 U/μL) overnight at 37° C. Control serum samples were mock treated with PBS and incubated overnight at 37° C. Data was processed and plotted using Prism (GraphPad software, California, U.S.A.). Apparent affinity was calculated as the concentration of oligomannose-type mAb corresponding to half-maximal binding on the ELISA binding curve.

[0206] Consistent with the data from FIG. 1, FIG. 7 shows that the enzyme-free serum efficiently blocked the binding of the oligomannose-type mAb IgG CIIC1 to FcγRIIIa detected using HRP-conjugated anti-mouse Fab IgG. The binding was determined in the presence of only PBS, or in the presence of serum or serum plus combinations of EndoS and EndoH. In the absence of any endoglycosidase, the serum effectively outcompetes the oligomannose-type mAb IgG CIIC1. However, the addition of EndoS led to a dramatic increase in apparent affinity of oligomannose-type-mAb for FcγRIIIa with 50% receptor saturation achieved at approximately 0.05 μM of mAb, a level approaching that of mAb:FcR determined for IgG in the complete absence of serum and consistent with the reported value (0.08 μM) of this interaction (Kanda, Y., Yamada, T., Mori, K., Okazaki, A., Inoue, M., Kitajima-Miyama, K., Kuni-Kamochi, R., Nakano, R., Yano, K, Kakita, S., shitara, K. & Satoh, M. (2007). Glycobiology 17, 104-18).

[0207] This enhancement was a direct consequence of the differential glycosylation of the engineered monoclonal and natural serum Ab. This glycoform-dependence was confirmed by the addition of Endo H which led to a loss of detectable FcγRIIIa binding regardless of whether or not the competing sera had also been treated with EndoS. For example, the use of oligomannose-type antibody glycoforms enables the therapeutic antibody to be deactivated with Endo H as demonstrated in FIG. 7. EndoH could be utilised for the deactivation of the therapeutic antibody to control the time of activity in the body or to deactivate it in the event of an adverse patient reactions.

Materials and Methods

[0208] Protein Expression and Purification of IgG1 Oligomannose-Type Glycoforms Man.sub.9GlcNAc.sub.2 and Man.sub.5GlcNAc.sub.2.

[0209] Human IgG1 Fc (residues 240-440, following the numbering of Edelman et al.; GenBank accession no. J00228) was cloned into the pHLsec vector and transiently expressed in Human Embryonic Kidney cells as previously described (Aricescu et al. Acta Crystallogr D Biol Crystallogr. 2006 October; 62(Pt 10):1243-50. Epub 2006 Sep. 19) with DNA mixed with polyethyleneimine (PEI) in a mass ratio of 1:1.5, respectively. The Man.sub.9GlcNAc.sub.2 glycoform was obtained by transient expression in Human Embryonic Kidney 293T cells in the presence of 5 μM kifunensine, a class I α-mannosidase inhibitor (Chang V T, Crispin M, Aricescu A R, Harvey D J, Nettleship J E, Fennelly J A, Yu C, Boles K S, Evans E J, Stuart D I, Dwek R A, Jones E Y, Owens R J, Davis S J. Structure. 2007 March; 15(3):267-73), to generate IgG-Fc bearing the immature oligomannose-type N-linked glycan, Man.sub.9GlcNAc.sub.2. The Man.sub.5GlcNAc.sub.2 glycoform was obtained by transient expression in GlcNAc Transferase I-deficient Human Embryonic Kidney 293S cells Reeves, P. J., N. Callewaert, et al. (2002). Proc Natl Acad Sci USA 99 (21): 13419-13424. Cell supernatant was clarified five days following transfection and IgG-Fc was purified by immobilized metal affinity chromatography using Chelating Sepharose Fast Flow Ni.sup.2+-agarose beads (GE Healthcare, Buckinghamshire, UK). IgG-Fc was partially deglycosylated at 25° C. for 12 h using 75 μg mL.sup.−1 endoglycosidase H and then purified by size exclusion chromatography. Protein purity was assessed by SDS-PAGE analysis and typical yields of deglycosylated IgG were 20 mg L.sub.−1 cell culture.

[0210] Full length IgG1 antibodies bearing human IgG1 Fc domains was generated by cloning the Fab domains of the murine monoclonal antibody CIIC1 (Developmental Studies Hybridoma Bank, University of Iowa, Department of Biology, Iowa City, IA 52242) into pFUSE-CHIg-hG1 vector (Invivogen, San Diego, California, U.S.A.). Intact CIIC1 antibody with complex and oligomannose-type glycans was transiently expressed in Human Embryonic Kidney cells as described above. Successful expression of full length antibody was confirmed by SDS-PAGE analysis and also by ELISA assay for binding to mouse collagen Type II protein.

Enzymatic Release of N-Linked Glycans.

[0211] Oligosaccharides were released from bands containing approximately 10 μg of target glycoprotein that were excised from Coomassie blue-stained reducing SDS-PAGE gels, washed with alternating water and acetonitrile and dried in a vacuum centrifuge, followed by rehydration with 100 Units/ml of PNGase F (New England Biolabs, MA, U.S.A.) and incubation for 12 hours at 37° C. The enzymatically released N-linked glycans were eluted with water. Endoglycosidase digestion of glycans was performed by addition of 1 μg of recombinant EndoS (Purchased from Genovis A B, Lund, Sweden and also obtained from Professor Ben Davis, CRL, University of Oxford) or 1 μl of Endo H (500 U/μl, New England Biolabs, MA, U.S.A.) and incubation for 12 hours at 37° C.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Mass Spectrometry.

[0212] Aqueous solutions of the glycans generated by the aforementioned method were cleaned with a Nafion 117 membrane. Positive ion MALDI-TOF mass spectra were recorded with a Shimazu AXIMA TOF MALDI TOF/TOF fitted with delayed extraction and a nitrogen laser (337 nm). The acceleration voltage was 20 kV; the pulse voltage was 3200 V; and the delay for the delayed extraction ion source was 500 ns. Samples were prepared by adding 0.5 μL of an aqueous solution of the sample to the matrix solution (0.3 μL of a saturated solution of 2, 5-dihydroxybenzoic acid in acetonitrile) on the stainless steel target plate and allowing it to dry at room temperature. The sample/matrix mixture was then recrystallized from ethanol.

Example 2: Mouse Model

[0213] Cells from the cell line SKBR3 (a cell line with high expression of HER2) are introduced subcutaneously into mice. EndoS and a therapeutic antibody resistant to EndoS and directed towards HER2 (e.g. Herceptin) are introduced intravenously at a dose of week, for a period of 4 weeks. Control mice do not receive either (i) Endo S or (ii) the therapeutic antibody. The size of resultant tumours is measured by calipers.

Example 3: Delayed Administration of Therapeutic Antibody

[0214] A breast cancer subject is treated with 15 mg EndoS i.v. in a saline solution. Patient IgG glycosylation levels are monitored by purification of a sample of patient IgG and assessment of the IgG mass by SDS-PAGE. (Deglycosylation of IgG results in a reduction of mass of the IgG).

[0215] After a delay of 2 days, the subject is treated with 2 mg therapeutic antibody/kg body weight trastuzumab i.v.

Example 4: Extracorporeal Treatment of Blood

[0216] The blood of a breast cancer subject is passed through an Endo S column at a rate of 250 mL/hour for 4 hours and returned to the subject In this time, the glycosylated endogenous serum IgG levels drop to below 50% of the starting levels. The subject is subsequently treated with 2 mg/Kg trastuzumab i.v.