Multimeric Fc proteins
11352414 · 2022-06-07
Assignee
Inventors
- Farnaz Fallah-Arani (Slough, GB)
- Robert Anthony Griffin (Slough, GB)
- David Paul Humphreys (Slough, GB)
- Shirley Jane Peters (Slough, GB)
- Bryan John Smith (Slough, GB)
- Paul Edward Stephens (Slough, GB)
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P7/02
HUMAN NECESSITIES
C12P21/02
CHEMISTRY; METALLURGY
International classification
C07K1/00
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
Abstract
The invention relates to multimeric fusion proteins which bind to human Fc receptors. The invention also relates to therapeutic compositions comprising the proteins, and their use in the treatment of immune disorders.
Claims
1. A multimeric fusion protein comprising two or more polypeptide monomer units; wherein each polypeptide monomer unit comprises an antibody Fc-domain comprising two heavy chain Fc-regions; wherein each heavy chain Fc-region comprises any amino acid residue other than cysteine at position 309, and is fused at its C-terminal to a tailpiece of a naturally occurring IgM which causes the monomer units to assemble into a multimer; wherein each polypeptide monomer unit does not comprise an antibody variable region or an N-terminal fusion partner; and wherein each heavy chain Fc-region comprises either: IgG1 CH2 and CH3 domains comprising SEQ ID NO: 36, in which the leucine residue at position 234 has been substituted with a phenylalanine residue and the proline residue at position 331 has been substituted with a serine residue (L234F/P331S), IgG4 CH2 and CH3 domains comprising SEQ ID NO: 37, in which the phenylalanine residue at position 234 has been substituted with a leucine residue (F234L), IgG4 CH2 and CH3 domains comprising SEQ ID NO: 38, in which the phenylalanine residue at position 234 has been substituted with a leucine residue and the phenylalanine residue at position 296 has been substituted with a tyrosine residue (F234L/F296Y), IgG4 CH2 and CH3 domains comprising SEQ ID NO: 39, in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 330 has been substituted with an alanine residue (G327A/S330A), IgG4 CH2 and CH3 domains comprising SEQ ID NO: 40, in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (G327A/S331P), IgG4 CH2 and CH3 domains comprising SEQ ID NO: 41, in which the serine residue at position 330 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (S330A/S331P), or an IgG4 CH2 domain and an IgG1 CH3 domain comprising SEQ ID NO: 43, and wherein the EU numbering system is used for residue numbering, and further wherein the residue substitutions identified above are the only residue substitutions made.
2. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains from IgG1 in which the leucine residue at position 234 has been substituted with a phenylalanine residue and the proline residue at position 331 has been substituted with a serine residue (L234F/P331S).
3. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains comprising SEQ ID NO: 37, derived from IgG4 in which the phenylalanine residue at position 234 has been substituted with a leucine residue (F234L).
4. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains comprising SEQ ID NO: 38, derived from IgG4 in which the phenylalanine residue at position 234 has been substituted with a leucine residue and the phenylalanine residue at position 296 has been substituted with a tyrosine residue (F234L/F296Y).
5. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains comprising SEQ ID NO: 39, derived from IgG4 in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 330 has been substituted with an alanine residue (G327A/S330A).
6. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains comprising SEQ ID NO: 40, derived from IgG4 in which the glycine residue at position 327 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (G327A/S331P).
7. The multimeric fusion protein of claim 1, wherein each heavy chain Fc-region comprises CH2 and CH3 domains comprising SEQ ID NO: 41, derived from IgG4 in which the serine residue at position 330 has been substituted with an alanine residue and the serine residue at position 331 has been substituted with a proline residue (S330A/S331P).
8. A pharmaceutical composition comprising the multimeric fusion protein of claim 1, in combination with a pharmaceutically acceptable excipient, diluent or carrier.
9. The pharmaceutical composition according to claim 8 additionally comprising other active ingredients.
Description
FIGURE LEGENDS
(1)
(2)
(3) 2(a) Example amino acid sequences of a polypeptide chain of a polypeptide monomer unit. In each sequence, the tailpiece sequence is underlined, and any mutations are shown in bold and underlined. The hinge is in bold. In constructs comprising a CH4 domain from IgM, this region is shown in italics.
(4) 2(b) Example amino acid sequences for an Fc-multimer polypeptide chain comprising a CH2 and CH3 domain derived from IgG1 or a CH2 and CH3 domain derived from IgG4. In each sequence, the positions of difference between IgG1 and IgG4 are in bold and highlighted.
(5) 2(c) Example amino acid sequences for Fc-multimers designed to combine certain selected properties of IgG1 and certain selected properties of IgG4. The mutations are shown in bold and underlined.
(6) 2(d) Example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions engineered by domain exchange.
(7) 2(e) Example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions and additional mutations, that have been engineered to combine certain selected properties of IgG1 and certain selected properties of IgG4.
(8) 2(f) DNA and amino acid sequences of the B72.3 signal peptide. (i) DNA sequence, (ii) amino acid sequence.
(9) 2(g) Example DNA sequences for Fc-multimers.
(10)
(11) 3(a) Size exclusion chromatography traces showing the effect of IgG1/IgG4 CH3 domain exchange on hexamerisation of Fc-multimers.
(12) 3(b) Effect of point mutations in the CH3 domain on hexamerisation of IgG1 Fc IgM tp.
(13) 3(c) High levels of hexamerisation of IgG4 Fc IgM tp are achieved by point mutation Q355R.
(14) 3(d) Effect of mutagenesis of R355 to all other amino acids in IgG1 Fc IgM tp.
(15)
(16) 4(a) hIgG1 Fc-multimer IgM tp L309C binding to low density FcRn.
(17) 4(b) hIgG1 Fc-multimer IgM tp binding to low density FcRn.
(18) 4(c) hIgG1 Fc-multimer IgM tp L309C binding to high density FcRn.
(19) 4(d) hIgG1 Fc-multimer IgM tp binding to high density FcRn.
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29) (a) Single dose of 10 mg/kg Fc-multimer administered on day 3.
(30) (b) Four consecutive daily doses, 10 mg/kg per dose, administered on days 3, 4, 5, and 6.
(31) The group size for each point on the graph was n=6.
(32)
(33)
EXAMPLES
Example 1: Molecular Biology
(34) Fc-multimer DNA sequences were assembled using standard molecular biology methods, including PCR, restriction-ligation cloning, point mutagenesis (Quikchange) and Sanger sequencing. Expression constructs were cloned into expression plasmids (pNAFL, pNAFH) suitable for both transient and stable expression in CHO cells. Other examples of suitable expression vectors include pCDNA3 (Invitrogen).
(35) A diagram of an expression construct and multimeric fusion protein according to the invention is shown in
(36) Diagrams showing example amino acid sequences of a polypeptide chain of a polypeptide monomer unit are provided in
(37) IgG1/IgG4 Crossover Mutations
(38) Various Fc-multimer variants were constructed in which certain key amino acid residues in the Fc-domain were designed to match those found in IgG1, whilst other key amino acid residues were designed to match those found in IgG4. IgG1 and IgG4 differ from one another at seven positions in the CH2 domain and six positions in the CH3 domain as summarised in Table 4.
(39) TABLE-US-00004 TABLE 4 IgG1 IgG4 position amino acid amino acid mutation of mutation of number residue residue IgG1 to IgG4 IgG4 to IgG1 234 L F L234F F234L 268 H Q H268Q Q268H 274 K Q K274Q Q274K 296 Y F Y296F F296Y 327 A G A327G G327A 330 A S A330S S330A 331 P S P331S S331P 355 R Q R355Q Q355R 356 D E D356E E356D 358 L M L358M M358L 409 K R K409R R409K 419 Q E Q419E E419Q 445 P L P445L L445P
(40) Diagrams showing example amino acid sequences for an Fc-multimer polypeptide chain comprising a CH2 and CH3 domain derived from IgG1 or a CH2 and CH3 domain derived from IgG4 are provided in
(41) Diagrams showing example amino acid sequences for Fc-multimers designed to combine certain selected properties of IgG1 and certain selected properties of IgG4 are provided in
(42) It will be appreciated that a particular sequence of interest may be created using either the IgG1 or the IgG4 Fc-domain sequence as a starting point and making the relevant mutations. For example, an IgG4 CH2 domain with mutation F234L is the same as an IgG1 CH2 domain with mutations H268Q, K274Q, Y296F, A327G, A330S, and P331S.
(43) Fc-Region Domain Exchange
(44) Fc-multimer variants were also constructed comprising hybrid heavy chain Fc-regions in which the CH2 domain was derived from one particular IgG subclass and the CH3 domain was derived from a different IgG subclass. Diagrams showing example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions are provided in
(45) Diagrams showing example amino acid sequences for Fc-multimers with hybrid heavy chain Fc-regions and additional mutations, that have been designed to combine certain selected properties of IgG1 and certain selected properties of IgG4 are provided in
Example 2: Expression
(46) Small scale expression was performed using ‘transient’ expression of HEK293 or CHO cells transfected using lipofectamine or electroporation. Cultures were grown in shaking flasks or agitated bags in CD-CHO (Lonza) or ProCHO5 (Life Technologies) media at scales ranging from 50-2000 ml for 5-10 days. Cells were removed by centrifugation and culture supernatants were stored at 4° C. until purified. Preservatives were added to some cultures after removal of cells.
(47) The results demonstrated that the multimeric fusion proteins are expressed well.
(48) The signal peptide used to express the multimeric fusion proteins was found to have an impact on the level of expression achieved. A signal peptide from antibody B72.3 resulted in higher expression levels than an IL-2 signal peptide sequence described in the prior art.
(49) The DNA and amino acid sequences of the B72.3 signal peptide are shown in
Example 3: Purification and Analysis
(50) Fc-multimers were purified from culture supernatants after checking/adjusting pH to be ≥6.5, by protein A chromatography with step elution using a pH3.4 buffer. Eluate was immediately neutralised to ˜pH7.0 using 1M Tris pH8.5 before storage at 4° C. Analytical size exclusion chromatography was used to separate various multimeric forms of Fc-domains using S200 columns and fraction collection. Fractions were analysed and pooled after G3000 HPLC and reducing and non-reducing SDS-PAGE analysis. Endotoxin was tested using the limulus amoebocyte lysate (LAL) assay and samples used in assays were <1 EU/mg.
(51) The multimeric fusion proteins were found to be expressed and purified predominantly in hexameric form, with some protein in dodecameric and other forms. The results demonstrate that the proteins assemble effectively into multimers in the absence of cysteine at position 309.
(52) Purification of the multimeric fusion proteins in the presence of a preservative reduced the tendency to aggregate, producing improved preparations with more uniform structure. Examples of preservatives shown to be effective include thiol capping agents such as N-ethylmaleimide (NEM) and glutathione (GSH); and disulphide inhibiting agents such as ethylenediaminetetraacetic acid (EDTA).
Example 4: Role of the CH3 Domain in Fc-Multimer Assembly
(53) The extent of multimerisation was unexpectedly found to vary depending on the IgG subclass from which the Fc-region was derived. Fc-multimers comprising a CH2 domain and a CH3 domain derived from IgG1 assembled very efficiently into hexamers, with approximately 80% of the molecules being present in hexameric form. In contrast, Fc-multimers comprising a CH2 domain and a CH3 domain derived from IgG4 formed lower levels of hexamers. Investigation of Fc-multimers comprising hybrid Fc-regions in which the CH2 domain was derived from one particular IgG subclass and the CH3 domain was derived from a different IgG subclass revealed that the ability to form hexamers is encoded mainly by the CH3 domain. The presence of a CH3 domain derived from IgG1 significantly increases hexamerisation. Hybrid Fc-multimers in which the CH3 domain is derived from IgG1 and the CH2 domain is derived from IgG4 hexamerised just as efficiently as IgG1 wild-type, with approximately 80% of the molecules being found as hexamers. Thus, replacing the CH3 domain of IgG4 with that of IgG1 improves the levels of hexamerisation compared to wild type IgG4 Fc-multimers. The resulting hybrid has the advantage of high levels of hexamer formation whilst retaining many of the desirable properties of IgG4.
(54) The CH3 domains of IgG1 and IgG4 differ at six positions as described in Example 1. Starting with an Fc-multimer comprising a CH2 and CH3 domain derived from IgG1, each of these positions was mutated in turn, from the IgG1 residue to the IgG4 residue. The results demonstrated that the amino acid at position 355 is critical for hexamerisation. The amino acid found at position 355 in wild type IgG1, arginine, promotes efficient hexamerisation. That found in wild type IgG4, glutamine, results in lower hexamerisation. The other positions of difference between the IgG1 and IgG4 CH3 domains did not affect hexamerisation.
(55) In Fc-multimers comprising a CH3 domain derived from IgG4, substitution of the glutamine residue at position 355 with an arginine residue (Q355R) resulted in high levels of hexamerisation. Thus, the problem of lower hexamerisation of IgG4 Fc-multimers can be solved by a single amino acid substitution. This has the advantage that the resulting Fc-multimer assembles into hexamers with high efficiency whilst retaining the characteristic properties of IgG4.
(56) In IgG1 Fc multimers, substitution of arginine at position 355 with cysteine (R355C) increased hexamer formation beyond that of wild type IgG1. Although we do not wish to be bound by theory, this result suggests that a cysteine residue at position 355 may be capable of forming disulphide bonds with the cysteine in the tailpiece. Mutagenesis of R355 to all the other amino acids did not result in further enhancement of hexamer formation in IgG1 Fc-multimers.
Example 5: Affinity Measurements for Interactions of Fc-Multimers and FcR
(57) Affinity/avidity measurements for the interactions of multimeric fusion proteins and Fc receptors (FcR) including FcγR and FcRn can be performed using well known methods including surface plasmon resonance, competition ELISA and competition binding studies on FcR bearing cell lines. Soluble, extra-cellular domains (ECDs) of FcRs were used in surface plasmon resonance experiments by non-specific immobilisation or tag specific capture onto a BIAcore sensor chip on a Biacore T200. Human FcRn extracellular domain was provided as a non-covalent complex between the human FcRn alpha chain extracellular domain and β2 microglobulin. Multimeric fusion proteins were titrated over the receptors at a variety of concentrations and flow rates in order to best determine the strength of the interaction. Data was analysed using Biacore T200 Evaluation software.
(58)
(59) (a) human IgG1 Fc-multimer IgM tp L309C binding to low density FcRn.
(60) (b) human IgG1 Fc-multimer IgM tp binding to low density FcRn.
(61) (c) human IgG1 Fc-multimer IgM tp L309C binding to high density FcRn.
(62) (d) human IgG1 Fc-multimer IgM tp binding to high density FcRn.
(63) Constructs used in (a) and (c) contain a leucine to cysteine substitution at position 309.
(64) The results demonstrated that Fc-multimers comprising histidine at position 310 bind to human FcRn.
(65) A number of Fc-multimers were also generated which incorporated mutations thought to increase binding to human FcRn.
(66) Table 5 shows the dissociation constants for the binding of mutated monomeric human IgG1 Fc fragments to human FcRn at pH6.0. The mutations resulted in increased binding to human FcRn. However, the strength of the interaction of the monomeric fragments is still weak, with dissociation constants in the micromolar range. Multimerisation of the mutated Fc-domains, as described in the present invention, may confer an avidity benefit, so greatly improving the strength of the interaction.
(67) TABLE-US-00005 TABLE 5 Binding of mutated monomeric IgG1 Fc- fragments to human FcRn at pH 6.0 Sample mutation KD (M) KD (μM) IgG1 Fc, WT 9.78E−07 0.98 IgG1 Fc, L309S 1.25E−06 1.25 IgG1 Fc, Q311A 7.69E−07 0.77 IgG1 Fc, T307A 5.65E−07 0.57 IgG1 Fc, T307P 6.93E−07 0.69 IgG1 Fc, V308C 7.00E−07 0.70 IgG1 Fc, V308F 3.32E−07 0.33 IgG1 Fc, V308P 1.36E−07 0.14 IgG1 Fc, WT 1.07E−06 1.07
Example 6: Macrophage Phagocytosis of B Cell Targets
(68) An assay was designed to measure antibody-dependent phagocytosis of B cells by human macrophages. To prepare macrophages, human peripheral blood mononuclear cells (PBMC) were first isolated from fresh blood by density-gradient centrifugation. Monocytes were then selected by incubating the PBMCs for 1 hour at 37° C. in 6-well tissue culture coated plates, followed by removal of non-adherent cells. Adherent monocytes were differentiated into macrophages by 5 day culture in macrophage-colony stimulating factor (MCSF). Human B cells were then prepared from a separate (allogeneic) donor by isolation of PBMC followed by purification of B cells by negative selection using MACS (B cell isolation kit II, Miltenyi Biotech). In some assays, B cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes). Differentiated macrophages and B cells were co-cultured at a 1:5 ratio in the presence of anti-CD20 mAb (rituximab) to induce antibody-dependent phagocytosis of the B cells. Multimeric fusion proteins or controls were added at the indicated concentrations and the cells incubated at 37° C. 5% CO.sub.2 for 1-24 hrs. At the end of each time-point, cells were centrifuged and resuspended in FACS buffer at 4° C. to stop further phagocytosis and the B cells surface-stained with anti-CD19 allophycocyanin (APC) before analysis by flow cytometry. Macrophages were distinguished by their auto-fluorescence/side-scatter properties and B cells by their CFSE/CD19 labelling. CFSE-positive macrophages negative for CD19 labelling were assumed to contain engulfed B cells.
(69) The results demonstrated that the multimeric fusion proteins of the invention inhibit B cell depletion by human macrophages. (
(70) The demonstration that the dodecamer form and hexamer form are equally potent is of benefit for product manufacturing and safety, as there is no need for additional purification to remove trace amounts of dodecamer from hexamer.
(71) Flow cytometry analysis using CFSE stained B-cells confirmed that the mechanism of action is inhibition of macrophage phagocytosis, and not B-cell killing or apoptosis by other means.
(72) In order to assess the ability of any given Fc-multimer construct to inhibit macrophage phagocytosis, its activity was measured in the assay described herein above and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of each mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers.
(73) Results for Fc-multimers comprising single cross-over mutations at each of the eight positions of difference in the IgG1 and IgG4 Fc region are shown in
(74) Wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, potently inhibits macrophage phagocytosis of antibody-coated target cells. Two of the single mutations in IgG1 Fc multimers (A330S, K409R) have no effect on the potency of inhibition of phagocytosis. Six of the single mutations (L234F, H268Q, K274Q, Y296F, A327G and P331S) result in a modest reduction in the potency of inhibition of phagocytosis. The residues L234F and A327G are of particular interest as mutating these significantly reduces cytokine release (see Example 8), whilst maintaining relatively high potency in inhibition of phagocytosis. This combination of properties will be useful for the treatment of autoimmune disorders.
(75) Wild type IgG4 Fc-multimers inhibit macrophage phagocytosis of antibody-coated target cells, but less potently than IgG1 Fc-multimers. Two of the single mutations in IgG4 Fc-multimers (F234L and G327A) modestly increase the potency of inhibition of phagocytosis. Six of the mutations in IgG4 (Q268H, Q274K, F296Y, S330A, S331P, R409K) have no effect on inhibition of phagocytosis when mutated individually. The residues F234L and G327A are of particular interest as, whilst individually mutating either of these positions enhances potency of IgG4 Fc multimers in inhibition of phagocytosis, they have no effect on increasing cytokine production (see Example 8). This combination of properties will be useful for the treatment of autoimmune disorders.
(76) TABLE-US-00006 TABLE 6 potency of phagocytosis Fc-multimer inhibition IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG1 Fc IgM tp L309 L234F high IgG1 Fc IgM tp L309 H268Q high IgG1 Fc IgM tp L309 K274Q high IgG1 Fc IgM tp L309 Y296F high IgG1 Fc IgM tp L309 A327G high IgG1 Fc IgM tp L309 A330S IgG1-like IgG1 Fc IgM tp L309 P331S high IgG1 Fc IgM tp L309 K409R IgG1-like IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG4 Fc IgM tp L309 F234L medium IgG4 Fc IgM tp L309 Q268H IgG4-like IgG4 Fc IgM tp L309 Q274K IgG4-like IgG4 Fc IgM tp L309 F296Y IgG4-like IgG4 Fc IgM tp L309 G327A low IgG4 Fc IgM tp L309 S330A IgG4-like IgG4 Fc IgM tp L309 S331P IgG4-like IgG4 Fc IgM tp L309 R409K IgG4-like
(77) Results for further Fc-multimer constructs designed for use in the treatment of immune disorders are shown in
(78) Wild type IgG1 and IgG4 Fc-multimers inhibit macrophage phagocytosis of antibody-coated target cells more potently than IVIG.
(79) IgG1 Fc-multimers comprising L234F (which reduces cytokine production) or L234F and P331S (reduces cytokine production and C1Q binding, see Examples 8 and 15) have modestly reduced potency in inhibition of phagocytosis relative to wild type IgG1 Fc-multimers, but are still highly potent relative to wild type IgG4 Fc-multimers or IVIG.
(80) IgG4 Fc-multimers comprising the mutations F234L; F234L and F296Y; G327A and S331P; S330A and S331P; or G327A and S330A; have enhanced potency in inhibition of phagocytosis compared to wild type IgG4 Fc-multimers.
(81) TABLE-US-00007 TABLE 7 potency of phagocytosis Fc-multimer inhibition IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG1 Fc IgM tp L309 L234F high IgG1 Fc IgM tp L309 L234F P331S high Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp medium L309 IgG4 Fc IgM tp L309 F234L medium IgG4 Fc IgM tp L309 F234L F296Y medium IgG4 Fc IgM tp L309 G327A S330A medium IgG4 Fc IgM tp L309 G327A S331P medium IgG4 Fc IgM tp L309 S330A S331P medium
Example 7: THP1 Cell Phagocytosis of IgG FITC Beads
(82) THP1 cells were plated out at passage 7, counted and re-suspended at 5×10.sup.5 cells/ml. 200 μl of cells were added to each well of a 96-well flat bottom plate (1×10.sup.5 cells per well). Beads coated with rabbit IgG (Cambridge bioscience CAY500290-1 ea) were added directly to each well, mixed (1 in 10 dilution, 10 μl/well) and left for the time points: 1 h, 2 h, 4 h, 8 h. Zero time points were effected by adding beads to cells in ice cold buffer on ice. At the end of each time point, cells were centrifuged at 300 g for 3 mins. The cells were resuspended in FACS buffer containing a 1:20 dilution of trypan blue stock solution for 2 minutes. Cells were washed with 150 μl FACS buffer, centrifuged and resuspended in 200 μl FACS buffer and transferred to a round bottom plate ready for FACS. Cells were centrifuged once more and resuspended in 200 μl of FACS buffer before analysis by flow cytometry. THP1 cells were gated on forward and side-scatter and uptake of beads measured as FITC fluorescence.
Example 8: Human Whole Blood Cytokine Release Assay
(83) Fresh blood was collected from donors in lithium heparin vacutainers. The Fc-multimer constructs of interest or controls were serially diluted in sterile PBS to the indicated concentrations. 12.5 μl of Fc-multimer or control was added to the assay plates, followed by 237.5 μl of whole blood. The plate was incubated at 37° C. without CO.sub.2 supplementation for 24 hrs. Plates were centrifuged at 1800 rpm for 5 minutes and the serum removed for cytokine analysis. Cytokine analysis was performed by Meso Scale Discovery cytokine multiplex according to the manufacturer's protocol and read on a Sector Imager 6000.
(84) Results are shown in
(85) In order to assess the effect of any given Fc-multimer construct on cytokine release its activity was measured in the assay described herein above and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers.
(86) Results for Fc-multimers comprising single cross-over mutations at selected positions of difference in the IgG1 and IgG4 Fc region are shown in
(87) The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, stimulates the release of very significant levels of cytokines. The results shown are for IFNγ. Similar results were observed for TNFα.
(88) Two of the single mutations (L234F, A327G) significantly reduced cytokine release in IgG1 Fc-multimers. One of the single mutations (Y296F) produced a moderate reduction of cytokine release. One of the mutations (P331S) significantly increased cytokine release. Three of the mutations (H268Q, K274Q, A330S) had no effect on cytokine release.
(89) In marked contrast, wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, produced virtually no cytokine release. None of the single cross-over mutations had any effect on cytokine release by IgG4 Fc-multimers, not even those at positions shown to be important for cytokine release in the IgG1 Fc-multimers.
(90) TABLE-US-00008 TABLE 8 Fc-multimer stimulation of IFNγ release IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG1 Fc IgM tp L309 L234F low IgG1 Fc IgM tp L309 H268Q IgG1-like IgG1 Fc IgM tp L309 K274Q IgG1-like IgG1 Fc IgM tp L309 Y296F medium IgG1 Fc IgM tp L309 A327G low IgG1 Fc IgM tp L309 A330S IgG1-like IgG1 Fc IgM tp L309 P331S higher than IgG1 IgG1 Fc IgM tp L309 K409R higher than IgG1 IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG4 Fc IgM tp L309 F234L IgG4-like IgG4 Fc IgM tp L309 Q268H IgG4-like IgG4 Fc IgM tp L309 Q274K IgG4-like IgG4 Fc IgM tp L309 F296Y IgG4-like IgG4 Fc IgM tp L309 G327A IgG4-like IgG4 Fc IgM tp L309 S330A IgG4-like IgG4 Fc IgM tp L309 S331P IgG4-like IgG4 Fc IgM tp L309 R409K IgG4-like
(91) Results for further Fc-multimer constructs designed to modulate cytokine release are shown in
(92) The data shows that cytokine release by IgG1 Fc-multimers can be reduced to levels approximately equivalent to IVIG by inclusion of L234F alone or in combination with P331S. Such Fc-multimers may be useful for the treatment of immune disorders. All the IgG4 Fc-multimers containing mutations shown to have other useful properties, for example enhanced potency in phagocytosis (Example 6), retain virtually zero levels of cytokine release, and may thus be useful for the treatment of immune disorders.
(93) TABLE-US-00009 TABLE 9 Cytokine release by Fc-multimers Fc-multimer stimulation of IFNγ release IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG1 Fc IgM tp L309 L234F low IgG1 Fc IgM tp L309 L234F P331S medium Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp IgG4-like L309 IgG4 Fc IgM tp L309 F234L IgG4-like IgG4 Fc IgM tp L309 F234L F296Y IgG4-like IgG4 Fc IgM tp L309 G327A S330A IgG4-like IgG4 Fc IgM tp L309 G327A S331P IgG4-like IgG4 Fc IgM tp L309 S330A S331P IgG4-like
Example 9: Effect on IgG Recycling in Cells in Culture
(94) Cell-based assays were performed using Madin-Darby Canine Kidney (MDCK) II cells which had been stably transfected with a human FcRn and human 32M double gene vector with a Geneticin selection marker. A stable cell clone was selected that was able to recycle and transcytose human IgG and this was used for all subsequent studies. It will be referred to as MDCK II clone 15. Equivalent MDCK cell lines, transfected with either cynomolgus monkey (“clone 40”) or mouse FcRn have been generated in a similar way, for use in assays equivalent to the above.
(95) An in vitro assay was established to examine the ability of a multimeric fusion protein of the present invention to inhibit the IgG-recycling capabilities of FcRn. Briefly, MDCK II clone 15 cells were incubated with biotinylated human IgG (1 μg/ml) in the presence or absence of the multimeric fusion protein in an acidic buffer (pH 5.9) to allow binding to FcRn. After a pulse time of 60 mins, all excess protein was removed and the cells incubated in a neutral pH buffer (pH 7.2) which allowed release of surface-exposed, bound IgG into the supernatant. The inhibition of FcRn was followed using an MSD assay to detect the amount of IgG recycled and thus released into the supernatant.
(96) MDCK II clone 15 cells were plated at 15,000 cells per well in a 96 well plate and incubated overnight at 37° C., 5% CO.sub.2. The cells were incubated with 1 μg/ml of biotinylated human IgG (Jackson) in the presence and absence of the multimeric fusion protein in HBSS+(Ca/Mg) pH 5.9+1% BSA for 1 hour at 37° C., 5% CO.sub.2. The cells were washed with HBSS+pH 5.9 then incubated at 37° C., 5% CO2 for 2 hours in HBSS+pH 7.2. The lysates and supernatant were removed and analysed for total IgG using an MSD assay (using an anti-human IgG capture antibody (Jackson) and a streptavidin-sulpho tag reveal antibody (MSD)). The inhibition curve was analysed by non-linear regression (Graphpad Prism) to determine the EC.sub.50.
(97) The results demonstrated that the multimeric fusion proteins of the invention inhibit FcRn-mediated IgG recycling. (
(98) Table 10 shows the effects of mutations on the blocking activity of Fc-multimers. Three mutations that increase binding to FcRn, V308F, V308P and T307A, were tested in Fc-multimers comprising IgG1 Fc/IgM tailpiece or IgG4 Fc/IgM tailpiece. The results showed significant improvements in the ability of the Fc-multimers to block FcRn-mediated IgG intracellular uptake and IgG recycling. The data demonstrates the utility of these mutations for improving the potency of Fc-multimers comprising either IgG1 or IgG4 Fc-regions.
(99) Substitution of histidine at position 310 with leucine (H310L) was shown to destroy the ability of the Fc-multimers to block FcRn-mediated IgG intracellular uptake and IgG recycling. The results demonstrate that Fc-multimers which retain the histidine residue at position 310 have much better binding to FcRn, and are more potent blockers of IgG intracellular uptake and IgG recycling. The data confirms that the Fc-multimers of the invention may provide new improved therapeutic compositions with longer half-life and greater efficacy.
(100) TABLE-US-00010 TABLE 10 Effects of mutations on Fc-multimer blockade of IgG intracellular uptake and IgG recycling Blockade of IgG Blockade of IgG intracellular uptake recycling Fc-multimer mutants EC.sub.50 (μg/ml) EC.sub.50 (μg/ml) IgG1 IgM tail-piece, Wild-type 4.4 0.33 IgG1 IgM tail-piece, V308F 0.12 0.073 IgG1 IgM tail-piece, V308P 0.34 0.55 IgG1 IgM tail-piece, T307A 0.54 0.35 IgG1 IgM tail-piece, H310L No detectable Maximal 45% inhibition inhibition at 100 μg/ml IgG4 IgM tail-piece, Wild-type 5.9 1.0 IgG4 IgM tail-piece, V308F 0.42 0.56 IgG4 IgM tail-piece, V308P 0.51 0.42 IgG4 IgM tail-piece, T307A 0.50 Not tested
Example 10: Efficacy of Fc-Multimers in Acute ITP
(101) Efficacy of Fc-multimers was studied in a mouse model of ITP, in which platelet loss is induced by administration of anti-CD41. This antibody binds to glycoprotein IIb on the surface of the platelets, targeting them for destruction.
(102) ITP In Vivo Protocol
(103) A 5 μl blood sample was taken from the tails of the mice prior to dosing to obtain baseline platelet numbers.
(104) Mice were dosed i.v. with 1 mg/kg or 10 mg/kg Fc-multimers.
(105) An hour later 1 μg/mouse rat anti-mouse CD41 IgG1 antibody (MWReg30) was dosed i.p.
(106) Terminal cardiac puncture was performed 24 hours after anti-CD41 administration.
(107) FACs Staining Protocol
(108) 5 μl of blood was taken from the tail vein. For terminal samples blood was taken by cardiac puncture into a heparin tube and 5 μl was taken for staining. 100 μl of antibody cocktail was added to the 5 μl blood sample and was incubated at 4° C. in the dark for 20 minutes. 5 mls of FACs buffer was added.
(109) Each sample was diluted 1:4 to make a final volume of 200 μl in a ‘vee’ bottom plate and kept on ice until ready to acquire on the Becton Dickinson FACs Canto.
(110) TABLE-US-00011 TABLE 11 Antibody name/Clone Antibody Colour Dilution Supplier/Lot number CD45 (30-F11) PerCPCy5.5 1/400 Ebio E08336-1633 CD42d (1C2) PE 1/200 Ebio E14346-104 Fc block 1/200
FACS Acquisition
(111) A set volume of 150 μl of sample was collected at a flow rate of 1.5 μl/sec. The threshold was set at 200.
(112) Analysis was performed on FlowJo software. Platelet counts were derived from the CD45−/CD42d+ gated cells.
(113) Cell counts were corrected for sample dilution based on the fact that the initial 5 μl blood sample is diluted 1/4000 and 150 μl of this is run through the FACs machine which equates to 0.1875 μl of the original sample being analysed. 5/0.1875=multiplication factor of ×26.7 for platelet/μl.
(114) Reagents
(115) Rat anti mouse CD41 Functional grade purified (Ebiosciences, MWReg30, lot #E11914-1632)
(116) Endotoxin free PBS (Sigma, D8537)
(117) FACs buffer: 0.1% FCS, 2 mM EDTA
(118) Results
(119) Human multimeric fusion proteins (‘Fc-multimers’) at 1 mg/kg dose were well tolerated. However they were not efficacious at this dose in the model. Positive results were observed using 10 mg/kg human Fc-multimers.
(120) Fc-multimers with either an IgM or an IgA tailpiece significantly inhibited platelet decrease caused by the injection of 1 μg/mouse anti-CD41.
(121) The results demonstrated that Fc-multimers prevent platelet loss in an in vivo model of acute immune thrombocytopenia. Statistically significant reductions in platelet loss were achieved using human IgG1 Fc/IgM tailpiece multimers, both with and without L309C, and human IgG4 Fc/IgM tailpiece multimers with L309C, at a dose of 10 mg/kg. (
Example 11: Efficacy of Fc-Multimers in Chronic ITP
(122) Efficacy of Fc-multimers was studied in a mouse model of chronic ITP, in which platelet loss is induced by administration of anti-CD41 for a sustained period of time using minipumps.
(123) ITP In Vivo Protocol
(124) A 5 ul tail bleed was taken prior to dosing to obtain baseline platelet numbers. An alzet mini pump containing rat anti-mouse CD41 at a concentration of 82.5 ug/ml (57.75 ul rat anti-mouse CD41 Ab+642.25 ul PBS/BSA (1.5 mg/ml)) was implanted subcutaneously. The pumps have a flow rate of 0.5 ul/hour, dosing the equivalent of 0.99 ug of anti-CD41 per day.
(125) 5 ul tail bleeds to obtain platelet counts were done daily.
(126) At a time point when a steady state platelet count has been reached mice are dosed intravenously with 1 g/kg IVIg, or Fc-multimers at a range of doses.
(127) At 7 days a terminal cardiac puncture was performed.
(128) FACs Staining Protocol
(129) Take 5 ul of blood via the tail vein. For terminal samples take blood by cardiac puncture into a heparin tube and take 5 ul for staining.
(130) Add 100 ul of antibody cocktail and incubate at 4° C. in the dark for 20 minutes. Add 5 mls of FACs buffer
(131) Dilute each sample 1:4 to make a final volume of 200 ul in a v-bottom plate and keep on ice until ready to acquire on the BD FACs Canto.
(132) TABLE-US-00012 TABLE 12 Antibody name/Clone Antibody Colour Dilution Supplier/Lot number CD45 (30-F11) PerCPCy5.5 1/400 Ebio E08336-1633 CD42d (1C2) PE 1/200 Ebio E14346-104 Fc block 1/200
FACS Acquisition
(133) A set volume of 150 ul of sample will be collected at a flow rate of 1.5 ul/sec. The threshold will be set at 200.
(134) Analysis will be performed on FlowJo software. Platelet counts will be derived from the CD45−CD42d+ gated cells.
(135) Cell counts will be corrected for sample dilution based on the initial 5 ul blood sample is diluted 1/4000 and 150 ul of this is run through the FACs machine which equates to 0.1875 ul of the original sample being analysed. 5/0.1875=multiplication factor of ×26.7 for platelet/ul.
(136) Reagents
(137) Rat anti mouse CD41 Functional grade purified (Ebiosciences, MWReg30, lot #E11914-1632)
(138) Endotoxin free PBS (Sigma, D8537)
(139) FACs buffer: 0.1% FCS, 2 mM EDTA
(140) 10 mg/kg IgG1 IgM tp, (ID:PB0000238), EWBE-017553, 5.69 mg/mg,
(141) Endototoxin<0.35 EU/mg
(142) IgG1 Fc IgM tp L309C, (ID:PB0000198), EWBE-017400, 6.49 mg/ml, Endotoxin<0.46 EU/mg
(143) IVIg: Gammunex lot #26NK1 N1.
(144) The results demonstrated that the multimeric fusion proteins prevent platelet loss in an in vivo model of chronic thrombocytopenia.
Example 12: Disulphide Bond and Glycan Analysis of Hexameric Fc-Multimers by Mass Spectrometry
(145) Method
(146) Purified samples of hexameric Fc-multimers (100 ug) were denatured in the presence of 8M urea in 55 mM Tris-HCl pH8.0 and free thiols were capped by incubating with 22 mM iodoacetamide (IAM) for 60 minutes at 37° C. The urea concentration was reduced to 6M using ultrafiltration and protein was digested with a LysC/trypsin mix (Promega) for 3 hours at 37° C. The sample was further diluted with 5 volumes of buffer and the digestion continued overnight at 37° C. Peptides were collected, desalted with Waters Oasis HLB cartridges, dried using a centrifugal evaporator and reconstituted in water containing 0.2% formic acid (solvent A).
(147) Samples (7.5 uL, ˜7 ug) were loaded at 150 uL/min onto a 2.1×150 mm C18 column (Waters 1.7u PST 300A) equilibrated with solvent A and operated at 40° C. Peptides were eluted by a 60 min gradient to 50% solvent B (4:4:1 acetonitrile:1-propanol:water/0.2% formic acid) into a Waters Xevo mass spectrometer operated in MS.sup.E+ve-ion mode. MS.sup.E data, which consists of alternating scans of low and high collision energy, was collected over the range 100-1900 m/z. during elution. After running, the digests were reduced by adding 10 mM Tris(hydroxypropyl)phosphine (THP) solution directly to the autosampler vial and incubating for >1 hr at room temperature. Reduced samples were then analysed a second time.
(148) MS.sup.E data was searched against the relevant Fc-multimer sequence using Waters BiopharmaLynx™ (BPL). The proportion of free disulphide thiols was calculated from the ratio of IAM-labelled to free peptide in the THP reduced digest. Glycan profiles were determined from the various glycopeptide isoforms detected in the digests.
(149) Results
(150) The results for glycan analysis of Fc-multimers (IgG1 Fc/IgM tailpiece), with and without L309C, are shown in Table 13. Glycan structures are shown in Table 8.
(151) The data demonstrates high occupancy of N297, the glycosylation site in the IgG1 Fc-region, with less than 10% free asparagine residues being found at this position. Glycosylation at N297 was mainly fucosylated biantennary complex, primarily G0F. Occupancy of the IgM tailpiece site, N563, was about 50%, higher than the level of about 20% found in native IgM. Glycosylation at N563 was mainly high mannose.
(152) TABLE-US-00013 TABLE 13 Glycan analysis Occu- pancy G0F-N G2-F G1-F G0-F M6 M5 IgG1 Fc CH2 93% 14% 5% 20% 38% 1% 15% IgM tp N297 L309C Tail 59% <1% 7% 5% 2% 24% 20% piece N563 IgG1 Fc CH2 91% 14% <1% 26% 34% <1% 15% IgM tp N297 Tail 45% 1% 4% 3% <1% 14% 22% piece N563
(153) Results for analysis of interchain disulphide bonds are shown in Table 14. Similar results were also obtained for intrachain disulphide bonds. The data demonstrated that a high proportion of the cysteine residues in the Fc-multimers are disulphide bonded. There was no evidence for significant amounts of scrambled disulphide bonding, and all expected dipeptides were found at high levels before reduction.
(154) TABLE-US-00014 TABLE 14 Interchain Disulphide bonds DSB (%) ‘Free’ thiol (%) IgG1 Fc Hinge 97 3 IgM tp CH2 89 10 L309C L309C-L309C Tail piece 84 14 C575-C575 IgG1 Hinge 96 4 IgM tp Tail piece 80 19 C575-C575
Example 13: Binding of Fc-Multimers to C1q
(155) Binding of Fc-multimers to C1q was measured by enzyme-linked immunosorbent assay (ELISA), using a C1q ELISA kit from Abnova Corporation, catalogue number: KA1274, lot number: V14-111723527. The Fc-multimer constructs were titrated in five-fold dilutions from 500 μg/ml through to 4 μg/ml. 100 μl of each Fc-multimer construct was added to the appropriate well, and agitated for one hour to enable binding. The assay was then carried out according to the manufacturer's protocol and analysed on a plate reader at an absorbance of 450 nm.
(156) In order to assess the binding of any given Fc-multimer construct to C1q, its activity was measured and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers.
(157) The results are shown in
(158) The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, binds strongly to C1 q. In contrast, wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, binds very poorly to C1 q. The dominant residue defining C1q binding in the Fc-multimers was found to be proline at position 331 (P331). Substitution of this proline residue with serine (P331S) effectively reduced C1q binding in Fc-multimers with a CH2 domain derived from IgG1. The converse mutant, S331P, increased C1q binding in Fc-multimers with a CH2 domain derived from IgG4.
(159) TABLE-US-00015 TABLE 15 Binding of Fc-multimers to C1q Fc-multimer C1q binding IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG1 Fc IgM tp L309 L234F IgG1-like IgG1 Fc IgM tp L309 P331S low IgG1 Fc IgM tp L309 L234F P331S IgG4-like Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp IgG4-like L309 IgG4 Fc IgM tp L309 F234L IgG4-like IgG4 Fc IgM tp L309 F234L F296Y IgG4-like IgG4 Fc IgM tp L309 G327A S330A IgG4-like IgG4 Fc IgM tp L309 G327A S331P medium IgG4 Fc IgM tp L309 S330A S331P medium
Example 14: Platelet Activation
(160) Platelet activation by Fc-multimers was analysed by flow cytometry. Two-fold dilutions of Fc-multimer were prepared in RMPI medium and transferred to FACS tubes. The final concentration was from 100 μg/ml down to 3.12 μg/ml. 5 μl fresh whole blood (from a minimum of 2 human donors) was added per tube. Platelets were gated using anti-CD42b labelled Mab and activation followed with Mabs against markers: CD62p, CD63 and PAC-1. (Becton Dickinson, BD CD42b APC Cat:551061, BD CD62p PE Cat:550561, BD CD63 PE-Cy-7 Cat:561982, BD PAC-1 FITC Cat:340507). Cells were fixed by addition of 500 μl paraformaldehyde 1% before analysis by flow-cytometry.
(161) CD62p was found to be the most sensitive of the three markers tested.
(162) In order to assess the platelet activation by any given Fc-multimer construct, its activity was measured and compared with the activities of IgG1 and IgG4 wild type Fc-multimers. The activity of the mutant was then summarised as “IgG1-like”, “high”, “medium”, “low”, or “IgG4-like”, based on a visual comparison of its concentration vs. effect curve with those obtained for IgG1 and IgG4 wild type Fc-multimers.
(163) Results for induction of CD62p expression are shown in
(164) The results demonstrated that wild type IgG1 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG1, results in significant levels of platelet activation.
(165) We have shown in Example 8 that two mutations are useful for reducing cytokine release from IgG1 Fc-multimer, L234F and A327G. Of these two mutations, L234F has much reduced levels of platelet activation compared to IgG1 wild-type Fc-domain, whereas an Fc-multimer with the A327G mutation retains significant levels of platelet activation. Thus L234F is a very useful mutation—it reduces cytokine release and platelet activation with only minor loss of potency in the inhibition of macrophage phagocytosis.
(166) Addition of P331S (shown in Example 13 to be a useful C1q reducing mutation) to L234F (L234F P331 S) results in low levels of platelet activation. This double mutant is a means of achieving low cytokine, low platelet activation and zero C1q binding. Thus this combination of mutations is particularly useful and is expected to provide new therapies for the treatment of immune disorders.
(167) L234F may be dominant over A327G since the triple L234F A327G P331S mutant has low platelet activation.
(168) Wild type IgG4 Fc-multimer, comprising a CH2 and a CH3 domain derived from IgG4, results in virtually no platelet activation.
(169) Switching the CH3 domain to that of IgG1 retains these reduced levels of platelet activation.
(170) The F234L mutation has low levels of platelet activation (and enhanced potency)—but the platelet activation is increased in comparison with wild type IgG4 Fc-multimer.
(171) F296Y can be combined with F234L without additionally increasing platelet activation. This observation is important since F234L F296Y has increased potency compared to IgG4 wild type Fc-multimers. Thus this combination of mutations is particularly useful and is expected to provide new therapies for the treatment of immune disorders.
(172) G327A mutation does not increase platelet activation. This observation is surprising in view of the results for the reverse mutation A327G in IgG1 Fc-multimers, which retained high levels of platelet activation.
(173) Certain double mutants (G327A S330A, G327A S331P and S330A S331P) which also have enhanced potency over IgG4 WT have very low levels of platelet activation (IgG4 like) and are expected to provide new therapies for the treatment of immune disorders.
(174) TABLE-US-00016 TABLE 16 Platelet activation by Fc-multimers Fc-multimer CD62p IgG1 Fc IgM tp L309 the standard for “IgG1-like” IgG4 Fc IgM tp L309 the standard for “IgG4-like” IgG1 Fc IgM tp L309 L234F low IgG1 Fc IgM tp L309 L234F P331S IgG4-like IgG1 Fc IgM tp L309 A327G IgG1-like IgG1 Fc IgM tp L309 A327G A330S P331S IgG1-like IgG1 Fc IgM tp L309 A327G P331S IgG1-like IgG1 Fc IgM tp L309 L234F A327G P331S medium Hybrid Fc IgG4-CH2 IgG1-CH3 IgM tp low L309 IgG4 Fc IgM tp L309 F234L medium IgG4 Fc IgM tp L309 F234L F296Y medium IgG4 Fc IgM tp L309 G327A S330A IgG4-like IgG4 Fc IgM tp L309 G327A S331P IgG4-like IgG4 Fc IgM tp L309 S330A S331P low
Example 15: Engineering of Fc-Multimer Variants
(175) The previous examples illustrate that Fc-multimers have been created that are particularly suitable for use in the treatment of immune disorders. The Fc-multimers were engineered with the aim of generating Fc multimers which possess the following properties:
(176) Inhibition of Macrophage Phagocytosis of Antibody Coated Target Cells.
(177) The potency of the Fc-multimer should be as high as possible.
(178) Cytokine Release.
(179) Stimulation of cytokine release by the Fc-multimer should be as low as possible.
(180) C1q Binding.
(181) Binding of the Fc-multimer to C1q should be as low as possible.
(182) Platelet Activation.
(183) Platelet activation by the Fc-multimer should be as low as possible.
(184) However, the work in the previous examples has illustrated that it may be necessary to compromise between maximum potency and slightly lower potency in order to achieve reduced side effects.
(185) Wild type IgG1 Fc-multimer comprising a CH2 and CH3 domain derived from IgG1 without any additional mutations may be less suitable for use in the treatment of immune disorders because, although it displays high potency of phagocytosis inhibition, it also shows high levels of unwanted side effects, measured by cytokine release, C1q binding and platelet activation.
(186) Wild type IgG4 Fc-multimer comprising a CH2 and CH3 domain derived from IgG4, produces very low levels of unwanted side effects although its potency is low relative to that of IgG1. Notwithstanding, the potency of wild type IgG4 Fc-multimer is still significantly higher than that of IVIG, as shown in
(187) Fc-multimers have been designed which combine the desirable properties of both IgG1 and IgG4 wild type Fc-multimers, without the undesirable properties. These Fc-multimers display effective levels of potency, whilst reducing unwanted side effects to a tolerable level as shown below in Table 17. These Fc-multimers are expected to be particularly useful for use in the treatment of immune disorders.
(188) TABLE-US-00017 TABLE 17 phagocytosis IFNγ C1q platelet Fc-multimer inhibition release binding activation wild type IgG1 the the the the Fc-multimer standard standard standard standard for “IgG1- for “IgG1- for “IgG1- for “IgG1- like” like” like” like” wild type IgG4 the the the the Fc-multimer standard standard standard standard for “IgG4- for “IgG4- for “IgG4- for “IgG4- like” like” like” like” IgG1 Fc IgM tp high medium IgG4-like IgG4-like L309 L234F P331S Hybrid Fc IgG4-CH2 medium IgG4-like IgG4-like low IgG1-CH3 IgM tp L309 IgG4 Fc IgM tp medium IgG4-like IgG4-like medium L309 F234L IgG4 Fc IgM tp medium IgG4-like IgG4-like medium L309 F234L F296Y IgG4 Fc IgM tp medium IgG4-like IgG4-like IgG4-like L309 G327A S330A IgG4 Fc IgM tp medium IgG4-like medium IgG4-like L309 G327A S331P IgG4 Fc IgM tp medium IgG4-like medium IgG4-like L309 S330A S331P