WO2012133782A1 - 抗原結合分子の血漿中滞留性と免疫原性を改変する方法 - Google Patents
抗原結合分子の血漿中滞留性と免疫原性を改変する方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/524—CH2 domain
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/71—Decreased effector function due to an Fc-modification
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- C07K2317/00—Immunoglobulins specific features
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- C07K2317/72—Increased effector function due to an Fc-modification
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to an Fc region of an antigen-binding molecule comprising an antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen varies depending on the ion concentration condition, and an Fc region having an FcRn-binding activity under neutral pH conditions.
- the present invention relates to a method for improving the pharmacokinetics of a living body to which an antigen-binding molecule has been administered by modifying, or a method for reducing the immune response of an antigen-binding molecule.
- the present invention also relates to an antigen-binding molecule that has improved pharmacokinetics when administered to a living body or reduced immune response by the living body.
- the present invention relates to a method for producing the antigen-binding molecule and a pharmaceutical composition comprising the antigen-binding molecule as an active ingredient.
- Non-patent Documents 1 and 2 various technologies have been developed as technologies applicable to second-generation antibody drugs, such as technologies that improve effector function, antigen binding ability, pharmacokinetics, stability, or reduce immunogenicity risk, etc. has been reported (Non-Patent Document 3). Since antibody drugs generally have a very high dose, it is considered that it is difficult to produce a subcutaneously administered preparation and that the production cost is high. As a method for reducing the dose of the antibody drug, a method for improving the pharmacokinetics of the antibody and a method for improving the affinity between the antibody and the antigen can be considered.
- Non-patent Documents 4 and 5 As a method for improving the pharmacokinetics of an antibody, artificial amino acid substitution in the constant region has been reported (Non-patent Documents 4 and 5). Affinity maturation technique (Non-patent Document 6) has been reported as a technique for enhancing antigen-binding ability and antigen-neutralizing ability. By introducing mutations into amino acids such as CDR regions of variable regions, binding activity to antigens is reported. Can be enhanced. In vitro biological activity can be improved or the dose can be reduced by enhancing the antigen binding ability, and further, the drug efficacy in vivo can be improved (Non-patent Document 7). .
- the amount of antigen that can be neutralized per antibody molecule depends on the affinity, and it is possible to neutralize the antigen with a small amount of antibody by increasing the affinity, and increase the affinity of the antibody by various methods. Is possible (Non-Patent Document 6). Furthermore, if it is possible to covalently bind to an antigen and make the affinity infinite, it is possible to neutralize one molecule of antigen (two antigens in the case of bivalence) with one molecule of antibody. However, the conventional methods limit the stoichiometric neutralization reaction of one molecule of antigen (two antigens in the case of bivalent) with one molecule of antibody. It was impossible to neutralize.
- Non-patent Document 9 there is a limit to the effect of increasing affinity.
- a neutralizing antibody in order to maintain the neutralizing effect for a certain period of time, it is necessary to administer an amount of antibody equal to or greater than the amount of antigen produced in vivo during that period.
- the affinity maturation technique alone has a limit in reducing the required antibody dose. Therefore, in order to maintain the antigen neutralizing effect for a target period with an amount of antibody equal to or less than the amount of antigen, it is necessary to neutralize a plurality of antigens with one antibody.
- Patent Document 1 an antibody that binds to an antigen in a pH-dependent manner has been recently reported.
- a pH-dependent antigen-binding antibody that binds strongly to antigen under neutral conditions in plasma and dissociates from antigen under acidic conditions within endosomes can dissociate from antigen within endosomes.
- a pH-dependent antigen-binding antibody can bind to an antigen again when the antibody is recycled into the plasma by FcRn after dissociating the antigen, so multiple antigens can be repeated with one pH-dependent antigen-binding antibody. It becomes possible to combine.
- the retention of the antigen in plasma is very short compared to the antibody that is recycled by binding to FcRn.
- the plasma retention of the antibody-antigen complex becomes long as with the antibody. Therefore, when the antigen binds to the antibody, the plasma retention is rather prolonged, and the plasma antigen concentration increases.
- IgG antibody has a long plasma retention by binding to FcRn.
- the binding between IgG and FcRn is observed only under acidic conditions (pH 6.0), and the binding is hardly observed under neutral conditions (pH 7.4).
- IgG antibodies are nonspecifically taken up by cells, but return to the cell surface by binding to FcRn in endosomes under acidic conditions in endosomes and dissociate from FcRn in neutral conditions in plasma. When a mutation is introduced into the Fc region of IgG and the binding to FcRn under acidic conditions is lost, the antibody is not recycled from the endosome into the plasma, so that the antibody retention in the plasma is significantly impaired.
- IgG antibodies are not recycled into plasma, plasma retention is conversely impaired.
- an antibody in which binding to mouse FcRn is observed under neutral conditions (pH 7.4) by introducing an amino acid substitution into IgG1 the retention of the antibody in plasma deteriorates.
- introduction of amino acid substitutions into IgG1 improves human FcRn binding under acidic conditions (pH 6.0), but at the same time, binding to human FcRn under neutral conditions (pH 7.4) appears to be observed. It has been reported that the antibody retention in the antibody did not improve and the change in the plasma retention was not observed when the antibody was administered to cynomolgus monkeys (Non-patent Documents 10, 11 and 12).
- the antibody plasma is increased by increasing the binding to human FcRn under acidic conditions without increasing the binding to human FcRn under neutral conditions (pH 7.4).
- the focus is only on improving the retention in the medium, so far the benefits of introducing amino acid substitutions into the Fc region of IgG antibodies and increasing binding to human FcRn under neutral conditions (pH 7.4) Not reported.
- the affinity of the antibody for the antigen is improved, the disappearance of the antigen from the plasma cannot be promoted.
- the above-mentioned pH-dependent antigen-binding antibody is also effective as a method for promoting the disappearance of an antigen from plasma as compared with a normal antibody (Patent Document 1).
- a pH-dependent antigen-binding antibody can bind to multiple antigens with a single antibody and promote the disappearance of the antigen from the plasma compared to normal antibodies.
- no antibody engineering technique has been reported so far that further improves the effect of repeatedly binding to the antigen of this pH-dependent antigen-binding antibody and the effect of promoting the disappearance of the antigen from plasma.
- the immunogenicity of an antibody drug is very important in terms of plasma retention, effectiveness and safety when the antibody drug is administered to humans.
- Undesirable events such as the production of antibodies against the administered antibody drug in the human body, the disappearance of the antibody drug in the plasma is accelerated, the effectiveness is reduced, and a hypersensitivity reaction is caused and the safety is affected. (Non-patent Document 13).
- Fc ⁇ receptor an Fc ⁇ receptor that acts by binding to the Fc region of an IgG antibody.
- Fc ⁇ R is expressed on the cell membrane of dendritic cells, NK cells, macrophages, neutrophils, adipocytes, etc., and by binding the Fc region of IgG, it gives an active or inhibitory intracellular signal to immune cells. It is known to communicate.
- Non-patent Document 14 As the protein family of human Fc ⁇ R, isoforms of Fc ⁇ RIa, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, and Fc ⁇ RIIIb have been reported, and allotypes of each have been reported (Non-patent Document 14). As the allotype of human Fc ⁇ RIIa, two types, 131st Arg (hFc ⁇ RIIa (R)) and His (hFc ⁇ RIIa (H)), have been reported. Two types of human Fc ⁇ RIIIa allotypes, 158th, Val (hFc ⁇ RIIIa (V)) and Phe (hFc ⁇ RIIIa (F)) have been reported. Moreover, as a protein family of mouse Fc ⁇ R, Fc ⁇ RI, Fc ⁇ RIIb, Fc ⁇ RIII, and Fc ⁇ RIV have been reported (Non-patent Document 15).
- Human Fc ⁇ R is classified into Fc ⁇ RIa, Fc ⁇ RIIa, Fc ⁇ RIIIa and Fc ⁇ RIIIb which are active receptors and Fc ⁇ RIIb which is an inhibitory receptor.
- mouse Fc ⁇ R is classified into Fc ⁇ RI, Fc ⁇ RIII, and Fc ⁇ RIV that are active receptors, and Fc ⁇ RIIb that is an inhibitory receptor.
- Fc ⁇ R When activated Fc ⁇ R is cross-linked with immune complex, it induces phosphorylation of intracellular receptor or immunoreceptor tyrosine-based activating motifs (ITAMs) contained in FcR common ⁇ -chain, which is an interaction partner, and is a signal transmitter Activating SYK and initiating an activation signal cascade causes an inflammatory immune response (Non-patent Document 15).
- ITAMs immunoreceptor tyrosine-based activating motifs
- Non-Patent Document 15 Non-Patent Document 16
- Centering on antibodies introduced with mutations at these sites various mutants having binding properties to Fc ⁇ R have been studied so far, and Fc region mutants having higher affinity for active Fc ⁇ R have been obtained ( Patent Document 2, Patent Document 3, Patent Document 4, and Patent Document 5).
- Fc ⁇ RIIb which is an inhibitory Fc ⁇ R
- Fc ⁇ RIIb is the only Fc ⁇ R expressed in B cells (Non-patent Document 18). It has been reported that the initial immunity of B cells is suppressed by the interaction of the Fc region of the antibody with Fc ⁇ RIIb (Non-patent Document 19). In addition, when Fc ⁇ RIIb and B cell receptor (BCR) on B cells are cross-linked via immune complexes in blood, B cell activation is suppressed and B cell antibody production is suppressed. It is reported that it is suppressed (Non-patent Document 20).
- ITIM immunoreceptor tyrosine-based inhibitory motif
- Fc ⁇ RIIb is expected as a technique for directly reducing immunogenicity against antibody drugs.
- a molecule (Ex4 / Fc) in which mouse IgG1 is fused to Exendin-4 (Ex4), a heterologous protein for mice, does not produce antibodies when administered to mice, but by modifying Ex4 / Fc
- An antibody against Ex4 is produced by administration of a molecule (Ex4 / Fc mut) that does not bind to Fc ⁇ RIIb on B cells (Non-patent Document 24). This result suggests that Ex4 / Fc binds to Fc ⁇ RIIb on B cells, thereby suppressing the production of mouse antibodies against Ex4 by B cells.
- Fc ⁇ RIIb is also expressed on dendritic cells, macrophages, activated neutrophils, mast cells, and basophils. Also in these cells, Fc ⁇ RIIb inhibits the function of active Fc ⁇ R such as phagocytosis and release of inflammatory cytokines and suppresses inflammatory immune responses (Non-patent Document 25).
- Fc ⁇ RIIb knockout mice humoral immunity is not properly controlled (Non-patent Document 26), sensitivity to collagen-induced arthritis (CIA) is increased (Non-patent Document 27), or lupus-like symptoms are exhibited. It has been reported that the symptoms of the syndrome such as Goodpasture (Goodpasture) are present (Non-patent Document 28).
- Fc ⁇ RIIb has been reported to be associated with human autoimmune diseases.
- SLE systemic lupus erythematosus
- Non-patent Document 34 Non-patent Document 35
- Fc ⁇ RIIb is thought to play a role in controlling autoimmune diseases and inflammatory diseases, mainly involving B cells, and controls autoimmune diseases and inflammatory diseases. Is a promising target molecule.
- Non-patent Document 36 It is known that IgG1, which is mainly used as a commercially available antibody drug, strongly binds not only to Fc ⁇ RIIb but also to active Fc ⁇ R.
- Development of antibody drugs with immunosuppressive properties compared to IgG1 by using the Fc region with enhanced binding to Fc ⁇ RIIb or improved Fc ⁇ RIIb binding selectivity compared to active Fc ⁇ R There is a possibility.
- the activation of B cells may be inhibited by using an antibody having a variable region that binds to BCR and Fc that has enhanced binding to Fc ⁇ RIIb (Non-patent Document 37).
- Fc ⁇ RIIb is known to have a structure that is 93% identical to that of Fc ⁇ RIIa, which is one of the active Fc ⁇ Rs, and has a very similar structure. Furthermore, Fc ⁇ RIIa has a genetic polymorphism, that is, an H type in which the 131st amino acid of the second Ig domain is His and an R type that is Arg, and the interaction with the antibody is different (non-patent document 38). Therefore, in order to create an Fc region that specifically binds to Fc ⁇ RIIb, the binding activity to Fc ⁇ RIIb is not increased or decreased while increasing the binding activity to Fc ⁇ RIIb. It is considered that the most difficult task is to impart the property of selectively improving the activity to the Fc region of an antibody.
- Non-patent Document 39 An example in which the specificity of binding to Fc ⁇ RIIb has been increased by introducing an amino acid modification in the Fc region has been reported so far (Non-patent Document 39).
- mutants were produced in which binding to Fc ⁇ RIIb was maintained rather than Fc ⁇ RIIa of both gene polymorphisms.
- binding to Fc ⁇ RIIb was reduced as compared to native IgG1. For this reason, it is considered difficult for these mutants to actually cause an immunosuppressive reaction via Fc ⁇ RIIb beyond IgG1.
- Non-patent Document 37 the binding to Fc ⁇ RIIb was enhanced by adding modifications such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody.
- S267E / L328F modifications such as S267E / L328F, G236D / S267E, and S239D / S267E to the Fc region of the antibody.
- the antibody introduced with the mutation of S267E / L328F bound most strongly to Fc ⁇ RIIb, but this mutant maintained the binding of Fc ⁇ RIa and Fc ⁇ RIIa to the H type at the same level as that of natural IgG1.
- Non-patent Document 25 Even if the binding to Fc ⁇ RIIb is enhanced as compared to IgG1, cells such as platelets that do not express Fc ⁇ RIIb and express Fc ⁇ RIIa (Non-patent Document 25) are not enhanced binding to Fc ⁇ RIIb, but Fc ⁇ RIIa. Only the effect of enhanced binding to is considered to be affected. For example, in systemic lupus erythematosus, there is a report that platelets are activated by an Fc ⁇ RIIa-dependent mechanism, and platelet activation correlates with severity (Non-patent Document 40). Furthermore, according to another report, this modification increased the binding of Fc ⁇ RIIa to the R-type by several hundred times as much as the binding to Fc ⁇ RIIb.
- Non-Patent Document 41 The H type and R type of Fc ⁇ RIIa are observed at almost the same frequency in Caucasian and African-American (Non-Patent Document 41, Non-Patent Document 42). From these facts, it is considered that there are certain limitations in using antibodies with enhanced binding to Fc ⁇ RIIa R for the treatment of autoimmune diseases. Even if the binding to Fc ⁇ RIIb is enhanced compared to that of active Fc ⁇ R, the fact that the binding to any of the polymorphisms of Fc ⁇ RIIa is enhanced from the viewpoint of use as a therapeutic agent for autoimmune diseases. Cannot be overlooked.
- Fc-mediated binding increased for any gene polymorphism of Fc ⁇ RIIa compared to natural IgG? It is important that the binding to Fc ⁇ RIIb is preferably reduced and enhanced. However, there have been no reports of mutants having such properties so far, and their development has been sought.
- Antigen presentation is an immune mechanism in which antigen-presenting cells such as macrophages and dendritic cells take in foreign and endogenous antigens such as bacteria and degrade them, and then present a part of them to the cell surface. is there.
- the presented antigen is recognized by T cells and activates cellular immunity and humoral immunity.
- Non-patent Document 43 As antigen presentation in dendritic cells, antigens taken into cells as immune complexes (complexes formed from multivalent antibodies and antigens) are degraded by lysosomes, and antigen-derived peptides are presented to MHC class II There is a route. FcRn plays an important role in this pathway. When using FcRn-deficient dendritic cells or immune complexes that do not bind to FcRn, antigen presentation and the resulting T cell activity It has been reported that no conversion occurs (Non-patent Document 43).
- a foreign animal antigen protein When a foreign animal antigen protein is administered to a normal animal, antibodies against the administered antigen protein are frequently produced. For example, when a soluble human IL-6 receptor that is a heterologous protein is administered to a mouse, a mouse antibody against the soluble human IL-6 receptor is produced. However, even when a human IgG1 antibody, which is a heterologous protein, is administered to mice, mouse antibodies against human IgG1 antibodies are hardly produced. This difference is considered to be due to the disappearance rate of the administered heterologous protein in plasma.
- human IgG1 antibody since human IgG1 antibody has the ability to bind to mouse FcRn under acidic conditions, human IgG1 antibody incorporated into endosomes is mediated by mouse FcRn as well as mouse antibody. Receive recycling. Therefore, when human IgG1 antibody is administered to normal mice, its disappearance from plasma is very slow. On the other hand, soluble human IL-6 receptor disappears rapidly after administration because it does not undergo recycling through mouse FcRn. On the other hand, as shown in Reference Example 4, production of a mouse antibody against soluble human IL-6R antibody was observed in normal mice administered with soluble human IL-6 receptor, whereas human IgG1 antibody No normal production of mouse antibody against human IgG1 antibody is observed in normal mice administered with. That is, in mice, the soluble human IL-6 receptor, which disappears faster, is more immunogenic than the human IgG1 antibody, which disappears slowly.
- heterologous proteins soluble human IL-6 receptor or human IgG1 antibody
- the heterologous protein taken up by the antigen-presenting cell is processed in the cell, then associates with the MHC class II molecule, and is transported onto the cell membrane.
- antigen presentation to antigen-specific T cells for example, T cells that specifically respond to soluble human IL-6 receptor or human IgG1 antibody
- antigen-specific T cells are activated.
- antigen presentation to antigen-specific T cells for example, T cells that specifically respond to soluble human IL-6 receptor or human IgG1 antibody
- Non-Patent Documents 10, 11 and 12 If the retention in plasma deteriorates due to enhanced binding to FcRn under neutral conditions (pH 7.4) of the antigen-binding molecule, it is possible that the antigen-binding molecule disappears faster and the immunogenicity may increase. It is done.
- FcRn is expressed in antigen-presenting cells and has been reported to be involved in antigen presentation.
- MBP myelin basic protein
- MBP-Fc an antigen-binding molecule
- an antigen-binding molecule that contains an antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen changes depending on the ion concentration condition, and an Fc region that has binding activity to FcRn under pH neutral conditions, Although it has been found that elimination can be promoted from plasma, the retention of antigen-binding molecules in plasma and immunogenicity by enhancing the binding activity to FcRn under neutral pH conditions of the Fc region The effects of have not been fully studied so far. As the present inventors proceeded with research, by increasing the binding activity to FcRn under the pH neutral range of the Fc region, the retention of the antigen-binding molecule in plasma was reduced (pharmacokinetics deteriorated). It has been found that there is a problem that the immunogenicity of the antigen-binding molecule is increased (the immune response to the antigen-binding molecule is exacerbated).
- the present invention has been made in view of such a situation, and the object thereof is an antigen-binding domain in which the binding activity of an antigen-binding molecule to an antigen varies depending on ion concentration conditions, and a pH neutral condition.
- Another object of the present invention is to provide an antigen-binding molecule comprising an antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen varies depending on the condition of the ion concentration, and an Fc region having a binding activity to FcRn under pH neutral conditions. It is intended to provide a method for reducing the immune response of an antigen-binding molecule by modifying the Fc region. Another object of the present invention is to provide an antigen-binding molecule that has improved pharmacokinetics when administered to a living body or reduced immune response by the living body. Furthermore, the present invention aims to provide a method for producing the antigen-binding molecule, and also to provide a pharmaceutical composition containing the antigen-binding molecule as an active ingredient.
- the inventors of the present invention have made extensive studies to achieve the above-described object.
- the antigen-binding domain in which the binding activity of the antigen-binding molecule to the antigen varies depending on the ion concentration condition, and the pH neutral condition. It was found that an antigen-binding molecule containing an Fc region having binding activity to FcRn forms a heterocomplex (FIG. 48) that includes the antigen-binding molecule / bimolecular FcRn / active Fc ⁇ receptor. It has been found that body formation adversely affects pharmacokinetics and immune responses.
- the present inventors have found an antigen-binding molecule having the above-mentioned properties and a method for producing the same, and a medicament containing the antigen-binding molecule or the antigen-binding molecule produced by the production method according to the present invention as an active ingredient. Completion of the present invention by discovering superior properties compared to conventional antigen-binding molecules, such as improved pharmacokinetics and reduced immune response by the administered organism when the composition is administered did.
- the present invention provides the following.
- the Fc region of an antigen-binding molecule comprising an antigen-binding domain whose binding activity to an antigen varies depending on the condition of the ion concentration and an Fc region having binding activity to FcRn under the neutral pH condition, Any of the following methods comprising: changing to an Fc region that does not form a heterocomplex comprising two molecules of FcRn and one molecule of active Fc ⁇ receptor under the conditions of (a) a method for improving the pharmacokinetics of an antigen binding molecule, or (b) a method for reducing the immunogenicity of an antigen-binding molecule, [2] Modification to the Fc region that does not form the heterocomplex results in that the Fc region has a lower binding activity to the active Fc ⁇ receptor than the binding activity of the Fc region of natural human IgG to the active Fc ⁇ receptor.
- An amino acid represented by EU numbering of the Fc region; 234 of amino acid Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr or Trp, 235 of the amino acid Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, one of Val or Arg, 236 of the amino acid Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr, 237 of the amino acid Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg, 238 of the amino acid is Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg, 239th amino
- the amino acid at position 248 250 of the amino acid is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, 252 of the amino acid is Phe, Trp, or Tyr, Thr, the amino acid at position 254 Glu for the 255th amino acid
- the 256th amino acid is Asp, Asn, Glu, or Gln
- 257 of the amino acid is one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val
- the amino acid at position 258 is His, Ala for the amino acid at position 265 286 of the amino acid is either Ala or Glu
- the antigen binding domain is an antigen binding domain whose binding activity changes such that the binding activity to the antigen under the condition of low calcium ion concentration is lower than the binding activity to the antigen under the condition of high calcium ion concentration.
- the method according to [14], [16] The method according to any one of [1] to [13], wherein the antigen-binding domain is an antigen-binding domain whose binding activity to an antigen varies depending on pH conditions.
- the antigen-binding domain, wherein the antigen-binding domain has a binding activity that changes such that the antigen-binding activity in the acidic pH range is lower than the antigen-binding activity in the neutral pH range.
- the method according to [23], [25] The method according to any one of [20] to [22], wherein the antigen-binding domain is an antigen-binding domain whose binding activity to an antigen varies depending on pH conditions; [26] The antigen-binding domain wherein the binding activity changes such that the binding activity to the antigen in the acidic pH range is lower than the binding activity to the antigen in the neutral pH range.
- the method of, [27] The method according to any one of [20] to [26], wherein the antigen-binding domain is a variable region of an antibody, [28]
- the amino acid at position 248 250 of the amino acid is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, 252 of the amino acid is Phe, Trp, or Tyr, Thr, the amino acid at position 254 Glu for the 255th amino acid 256 of the amino acid is Asp, Asn, Glu, or Gln, 257 of the amino acid is Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val, The amino acid at position 258 is His, Ala for the amino acid at position 265 286 of the amino acid is Ala or Glu, The amino acid at position 289 is His, 297 of the amino acid is Ala, Ala at position 303 Ala for the amino acid at position 305 307 of the amino acid is Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr, 308 of
- Antigen-binding molecules of [38] The antigen-binding molecule according to any one of [29] to [37], wherein the antigen-binding domain is a variable region of an antibody, [39]
- the present invention also relates to a kit for use in the method of the present invention comprising the antigen binding molecule of the present invention or the antigen binding molecule produced by the production method of the present invention.
- the present invention also relates to an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule, which contains as an active ingredient the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention.
- the present invention also relates to a method for treating an immunoinflammatory disease, comprising a step of administering to the subject an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention.
- the present invention also relates to the use of the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention in the production of a pharmacokinetics improving agent for an antigen-binding molecule or an immunogenicity reducing agent for an antigen-binding molecule. .
- the present invention also relates to an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention for use in the method of the present invention.
- the present invention provides a method for improving the pharmacokinetics of an antigen-binding molecule or a method for reducing the immunogenicity of an antigen-binding molecule. According to the present invention, it is possible to perform treatment with an antibody without causing an undesirable event in a living body as compared with a normal antibody.
- FIG. 6 is a graph showing changes in plasma concentration when Fv4-IgG1 or Fv4-IgG1-F1 is administered intravenously or subcutaneously to normal mice. It is the figure which showed that Fv4-IgG1-F157 in the state couple
- FIG. 4 shows that Fv4-IgG1-F157 in a state bound to human FcRn binds to human Fc ⁇ RIIa (R).
- FIG. 4 is a view showing that Fv4-IgG1-F157 in a state bound to human FcRn binds to human Fc ⁇ RIIa (H).
- FIG. 4 is a view showing that Fv4-IgG1-F157 bound to human FcRn binds to human Fc ⁇ RIIb.
- FIG. 4 is a view showing that Fv4-IgG1-F157 in a state bound to human FcRn binds to human Fc ⁇ RIIIa (F). It is the figure which showed that Fv4-IgG1-F157 in the state couple
- Fv4-IgG1-F157 in a state bound to human FcRn binds to mouse Fc ⁇ RIIb. It is the figure which showed that Fv4-IgG1-F157 in the state couple
- FIG. 4 shows changes in plasma concentrations of Fv4-IgG1-F21, Fv4-IgG1-F140, Fv4-IgG1-F157, and Fv4-IgG1-F424 in human FcRn transgenic mice. It is the figure which showed the plasma level transition of Fv4-IgG1 and Fv4-IgG1-F760 in a human FcRn transgenic mouse.
- FIG. 3 is a graph showing changes in plasma concentrations of mPM1-mIgG1-mF14, mPM1-mIgG1-mF38, mPM1-mIgG1-mF39, and mPM1-mIgG1-mF40 in normal mice.
- B is an enlarged view of A. It is a figure which shows the antibody titer of the mouse antibody produced with respect to Fv4-IgG1-F939 after 3 days, 7 days, 14 days, 21 days, and 28 days after being administered to a human FcRn transgenic mouse. It is a figure which shows the antibody titer of the mouse
- FIG. 2 is a graph showing plasma kinetics of soluble human IL-6 receptor in normal mice and antibody titers of mouse antibodies against soluble human IL-6 receptor in mouse plasma.
- FIG. 2 is a graph showing plasma kinetics of soluble human IL-6 receptor in normal mice administered with anti-mouse CD4 antibody and antibody titer of mouse antibody against soluble human IL-6 receptor in mouse plasma. It is a figure which shows the plasma dynamics of the anti- IL-6 receptor antibody in a normal mouse.
- FIG. 3 is a view showing the structure of heavy chain CDR3 of the Fab fragment of 6RL # 9 antibody determined by X-ray crystal structure analysis. It is a figure which shows transition of the antibody concentration in the plasma of H54 / L28-IgG1, 6RL # 9-IgG1, and FH4-IgG1 in normal mice.
- the solid line is a chromatogram of an antibody containing the human Vk5-2 sequence (heavy chain: CIM_H, SEQ ID NO: 108 and light chain: hVk5-2, SEQ ID NO: 4), and the broken line is an antibody having the hVk5-2_L65 sequence (heavy chain: The chromatogram of CIM_H (sequence number: 108), light chain: hVk5-2_L65 (sequence number: 107)) is represented.
- FIG. 1 It is a figure which shows alignment and EU numbering of the constant region arrangement
- FIG. 4 is a schematic diagram showing the action of an Fc region having a binding activity to FcRn under a pH neutral range condition and a selective binding activity to inhibitory Fc ⁇ R, two molecules of FcRn, and one molecule of Fc ⁇ R. Only one of the two polypeptides constituting the FcRn-binding domain has an FcRn-binding activity under pH neutral conditions, and the other has no FcRn-binding activity under pH neutral conditions. It is a schematic diagram showing the action of the region, bimolecular FcRn, and single molecule Fc ⁇ R.
- the horizontal axis represents the amino acid site represented by Kabat numbering.
- the vertical axis represents the ratio of amino acid distribution.
- the horizontal axis represents the amino acid site represented by Kabat numbering.
- the vertical axis represents the ratio of amino acid distribution. It is a graph showing the time course of Fv4-IgG1-F947 and Fv4-IgG1-F1326 concentration in mouse plasma when Fv4-IgG1-F947 and Fv4-IgG1-F1326 are administered to human FcRn transgenic mice.
- the horizontal axis represents the relative binding activity value of each PD ⁇ variant to Fc ⁇ RIIb
- the vertical axis represents the relative binding activity value of each PD variant to Fc ⁇ RIIa R type.
- Binding to each Fc ⁇ R of IL6R-F652 (modified Fc in which Pro at position 238 represented by EU numbering was replaced by Asp), which is an antibody before the introduction of the alteration, using the amount of binding of each PD variant to each Fc ⁇ R as a control
- the value obtained by dividing by the amount value and further multiplying by 100 was taken as the value of the relative binding activity of each PD variant to each Fc ⁇ R.
- the plot F652 in the figure shows the value of IL6R-F652.
- the vertical axis represents the relative binding activity value of Fc ⁇ RIIb to each modified product introduced into GpH7-B3 without P238D modification
- the horizontal axis represents each modified product introduced into IL6R-F652 with P238D modification.
- Relative binding activity values for Fc ⁇ RIIb are shown.
- the value of the binding amount of each variant to Fc ⁇ RIIb was divided by the value of the binding amount of the antibody to Fc ⁇ RIIb before the introduction of the modification, and the value obtained by multiplying by 100 was used as the relative binding activity value.
- the crystal structure of the Fc (P238D) / Fc ⁇ RIIb extracellular region complex and the model structure of the Fc (WT) / Fc ⁇ RIIb extracellular region complex are based on the distance between C ⁇ atoms to the Fc ⁇ RIIb extracellular region and FcFCH2 domain A.
- FIG. 5 is a diagram showing Glu at position 233 represented by EU numbering of Fc Chain A in the crystal structure of the Fc (P238D) / Fc ⁇ RIIb extracellular region complex and its peripheral residues in the Fc ⁇ RIIb extracellular region.
- FIG. 3 is a diagram showing Ala at position 330 represented by EU numbering of Fc Chain A in the crystal structure of the Fc (P238D) / Fc ⁇ RIIb extracellular region complex and its peripheral residues in the extracellular region of Fc ⁇ RIIb.
- the crystal structures of Fc (P238D) / Fc ⁇ RIIb extracellular region complex and Fc (WT) / Fc ⁇ RIIIa extracellular region complex are superimposed on Fc Chain B by the least square method based on the distance between C ⁇ atoms, It is the figure which showed the structure of Pro of the 271st position represented by EU numbering of Fc * Chain * B.
- the “antigen” is not limited to a specific structure as long as it includes an epitope to which an antigen-binding domain binds.
- the antigen can be inorganic or organic.
- Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9 , ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1
- an epitope that means an antigenic determinant present in an antigen means a site on the antigen to which an antigen-binding domain in the antigen-binding molecule disclosed in the present specification binds.
- an epitope can be defined by its structure.
- the epitope can also be defined by the binding activity to the antigen in the antigen-binding molecule that recognizes the epitope.
- the antigen is a peptide or polypeptide
- the epitope can be specified by the amino acid residues constituting the epitope.
- the epitope is a sugar chain
- the epitope can be specified by a specific sugar chain structure.
- a linear epitope is an epitope including an epitope whose primary amino acid sequence is recognized.
- Linear epitopes typically include at least 3, and most commonly at least 5, such as about 8 to about 10, 6 to 20 amino acids in a unique sequence.
- a conformational epitope is, in contrast to a linear epitope, an epitope in which the primary sequence of the amino acid containing the epitope is not a single defining component of the recognized epitope (eg, the primary sequence of amino acids does not necessarily define the epitope).
- a conformational epitope may include an increased number of amino acids relative to a linear epitope.
- the antibody recognizes the three-dimensional structure of the peptide or protein. For example, when a protein molecule is folded to form a three-dimensional structure, certain amino acid and / or polypeptide backbones that form a conformational epitope are juxtaposed to allow the antibody to recognize the epitope.
- Methods for determining the conformation of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope® Mapping® Protocols in Methods Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).
- a test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule.
- a linear peptide comprising an amino acid sequence constituting the extracellular domain of IL-6R is synthesized.
- the peptide can be chemically synthesized.
- it can be obtained by genetic engineering techniques using a region encoding an amino acid sequence corresponding to the extracellular domain in IL-6R cDNA.
- the binding activity between a linear peptide consisting of an amino acid sequence constituting the extracellular domain and a test antigen-binding molecule containing an antigen-binding domain for IL-6R is evaluated.
- the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen.
- the binding activity to the linear peptide can be revealed based on the level of inhibition by the linear peptide in the binding of the antigen-binding molecule to IL-6R-expressing cells. These tests can reveal the binding activity of the antigen-binding molecule to the linear peptide.
- test antigen-binding molecule containing an antigen-binding domain for IL-6R recognizes a three-dimensional epitope.
- cells expressing IL-6R are prepared.
- a test antigen-binding molecule containing an antigen-binding domain for IL-6R comes into contact with an IL-6R-expressing cell, it binds strongly to the cell, while the IL-6R extracellular domain to which the antigen-binding molecule is immobilized
- substantially not binding means 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.
- a test antigen-binding molecule containing an antigen-binding domain to IL-6R to IL-6R-expressing cells for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory ( 1988) 359-420). That is, it can be evaluated according to the principle of ELISA or FACS (fluorescence-activated cell sorting) using IL-6R-expressing cells as antigens.
- the binding activity of a test antigen-binding molecule containing an antigen-binding domain to IL-6R to IL-6R-expressing cells is quantitatively evaluated by comparing the signal level generated by the enzyme reaction. That is, a test polypeptide aggregate is added to an ELISA plate on which IL-6R-expressing cells are immobilized, and a test antigen-binding molecule bound to the cell is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule.
- test antigen-binding molecule binding activity against IL-6R-expressing cells can be compared by preparing a dilution series of test antigen-binding molecules and determining the antibody binding titer (titer) against IL-6R-expressing cells. Can be done.
- the binding of the test antigen-binding molecule to the antigen expressed on the cell surface suspended in a buffer or the like can be detected by a flow cytometer.
- a flow cytometer For example, the following devices are known as flow cytometers.
- EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)
- the following method may be mentioned as an example of a suitable method for measuring the binding activity of an antigen-binding molecule containing an antigen-binding domain to IL-6R to an antigen.
- staining is performed with a FITC-labeled secondary antibody that recognizes a test antigen-binding molecule reacted with cells expressing IL-6R.
- the antigen-binding molecule is adjusted to a desired concentration and used. For example, it can be used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
- fluorescence intensity and cell number are measured by FACSCalibur (BD).
- the amount of antibody bound to the cells is reflected in the fluorescence intensity obtained by analysis using CELL QUEST Software (BD), that is, the value of Geometric Mean. That is, by obtaining the value of Geometric Mean, the binding activity of the test antigen binding molecule represented by the binding amount of the test antigen binding molecule can be measured.
- BD CELL QUEST Software
- test antigen-binding molecule containing an antigen-binding domain for IL-6R shares an epitope with a certain antigen-binding molecule can be confirmed by competition for the same epitope.
- Competition between antigen-binding molecules is detected by a cross-blocking assay or the like.
- a competitive ELISA assay is a preferred cross-blocking assay.
- the IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competitive antigen binding molecule and then the test antigen. A binding molecule is added.
- the amount of test antigen binding molecule bound to the IL-6R protein in the well is indirectly correlated with the binding ability of candidate competitive antigen binding molecules that compete for binding to the same epitope. That is, the higher the affinity of the competitive antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the well coated with IL-6R protein.
- the amount of the test antigen binding molecule bound to the well via the IL-6R protein can be easily measured by labeling the antigen binding molecule in advance.
- biotin labeled antigen binding molecules are measured by using an avidin peroxidase conjugate and an appropriate substrate.
- a cross-blocking assay using an enzyme label such as peroxidase is particularly referred to as a competitive ELISA assay.
- Antigen-binding molecules can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.
- the competitive antigen binding molecule binds the test antigen binding molecule comprising the antigen binding domain to IL-6R.
- the test antigen binding molecule binds to substantially the same epitope as the competitive antigen binding molecule or binds to the same epitope if it can block at least 20%, preferably at least 20-50%, more preferably at least 50% Antigen-binding molecule that competes for.
- binding activity for example, it can be measured by comparing the binding activity of a test antigen-binding molecule and a control antigen-binding molecule against a linear peptide into which a mutation has been introduced in the above-mentioned ELISA format.
- the binding activity to the mutant peptide bound to the column is quantified, and the antigen-binding molecule eluted in the eluate after the test antigen-binding molecule and the control antigen-binding molecule are allowed to flow through the column.
- a method for adsorbing a mutant peptide on a column as a fusion peptide with GST, for example, is known.
- the identified epitope is a steric epitope
- cells that express IL-6R and cells that express IL-6R in which a mutation has been introduced into the epitope are prepared.
- a test antigen-binding molecule and a control antigen-binding molecule are added to a cell suspension in which these cells are suspended in an appropriate buffer such as PBS.
- an FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added to the cell suspension washed with an appropriate buffer.
- the fluorescence intensity and the number of cells stained with the labeled antibody are measured by FACSCalibur (BD).
- the concentration of the test antigen-binding molecule and the control antigen-binding molecule is adjusted to a desired concentration by appropriately diluting with a suitable buffer and used. For example, it is used at any concentration between 10 ⁇ g / ml and 10 ng / ml.
- the amount of the labeled antibody bound to the cells is reflected in the fluorescence intensity obtained by analysis using CELL
- “substantially does not bind to mutant IL-6R-expressing cells” can be determined by the following method. First, a test antigen-binding molecule and a control antigen-binding molecule bound to a cell expressing mutant IL-6R are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. By calculating this comparison value ( ⁇ Geo-Mean) based on the following formula from the value of Geometric Mean in the presence and absence of polypeptide aggregates, the rate of increase in fluorescence intensity due to binding of antigen-binding molecules Can be requested.
- ⁇ Geo-Mean Geo-Mean (in the presence of polypeptide aggregate) / Geo-Mean (in the absence of polypeptide aggregate)
- Geometric Mean comparison value (mutated IL-6R molecule ⁇ Geo-Mean value) that reflects the amount of binding of the test antigen binding molecule to the mutant IL-6R expressing cell obtained by the analysis to the IL-6R expressing cell of the test antigen binding molecule Compare with the ⁇ Geo-Mean comparison value that reflects the amount of binding.
- concentration of the test antigen-binding molecule used in determining the ⁇ Geo-Mean comparison value for the mutant IL-6R-expressing cell and the IL-6R-expressing cell may be prepared at the same or substantially the same concentration. Particularly preferred.
- An antigen-binding molecule that has been confirmed in advance to recognize an epitope in IL-6R is used as a control antigen-binding molecule.
- the ⁇ Geo-Mean comparison value of the test antigen-binding molecule for the mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30% of the ⁇ Geo-Mean comparison value of the test antigen-binding molecule for the IL-6R-expressing cells. %, Particularly preferably less than 15%, “substantially does not bind to mutant IL-6R-expressing cells”.
- the calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in CELL QUEST Software User's Guide (BD biosciences).
- the epitope of the test antigen-binding molecule and the control antigen-binding molecule can be evaluated to be the same if it can be substantially equated by comparing the comparison values.
- Antigen-binding domain in the present specification, a domain having any structure can be used as long as it binds to an antigen of interest.
- examples of such domains include, for example, variable regions of antibody heavy and light chains, modules called A domains of about 35 amino acids contained in Avimer, a cell membrane protein present in vivo (WO2004 / 044011, WO2005 / 040229), Adnectin (WO2002 / 032925) containing 10Fn3 domain, a domain that binds to the protein in fibronectin, a glycoprotein expressed on the cell membrane, IgG that constitutes a three-helix bundle consisting of 58 amino acids of ProteinA Affibody with binding domain as scaffold (WO1995 / 001937), on the molecular surface of ankyrin repeat (AR), which has a structure in which a turn containing 33 amino acid residues and two antiparallel helix and loop subunits are stacked repeatedly DARPins (Designed Ankyrin Repeat proteins) (WO2002
- WO2003 / 029462 which are four loop regions that support one side of a barrel structure in which eight antiparallel strands highly conserved in lipocalin molecules such as hil gelatinase-associated lipocalin (NGAL) A leucine-rich-repeat (LRR) rich in leucine residues of the variable lymphocyte receptor (VLR), which has no immunoglobulin structure, as an acquired immune system for jawless eels such as lampreys and eels ))
- LRR leucine-rich-repeat
- VLR variable lymphocyte receptor
- the recessed area (WO2008 / 016854) of the parallel sheet structure inside the horseshoe-shaped structure in which the modules are repeatedly stacked.
- antigen-binding domain of the present invention include antigen-binding domains comprising antibody heavy and light chain variable regions.
- antigen binding domains include “scFv (single chain Fv)”, “single chain antibody”, “Fv”, “scFv2 (single chain Fv 2)”, “Fab” or “F ( ab ′) 2 ”and the like are preferable.
- the antigen-binding domain in the antigen-binding molecule of the present invention can bind to the same epitope.
- the same epitope can be present in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example. Further, it can be present in a protein consisting of amino acids 20 to 365 of the amino acid sequence shown in SEQ ID NO: 1.
- the antigen binding domains in the antigen binding molecules of the present invention can bind to different epitopes.
- different epitopes can exist in a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example. Further, it can be present in a protein consisting of amino acids 20 to 365 of the amino acid sequence shown in SEQ ID NO: 1.
- the specific specific, specifically one molecule of molecules that bind refers to a state that does not show any significant binding to molecules other than molecules of the other party to one or more binding thereof. Moreover, it is used also when an antigen binding domain is specific with respect to a specific epitope among several epitopes contained in a certain antigen. When an epitope to which an antigen binding domain binds is contained in a plurality of different antigens, an antigen binding molecule having the antigen binding domain can bind to various antigens containing the epitope.
- an antibody refers to an immunoglobulin that is naturally occurring or produced by partial or complete synthesis.
- the antibody can be isolated from natural resources such as plasma and serum in which it naturally exists, or from the culture supernatant of hybridoma cells producing the antibody, or partially or completely by using techniques such as genetic recombination Can be synthesized.
- Preferred examples of antibodies include immunoglobulin isotypes and subclasses of those isotypes.
- human immunoglobulins nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known. Of these isotypes, the antibody of the present invention may include IgG1, IgG2, IgG3, and IgG4.
- a method for producing an antibody having a desired binding activity is known to those skilled in the art.
- a method for producing an antibody that binds to IL-6R (anti-IL-6R antibody) is exemplified.
- Antibodies that bind to antigens other than IL-6R can be appropriately prepared according to the following examples.
- the anti-IL-6R antibody can be obtained as a polyclonal or monoclonal antibody using known means.
- a monoclonal antibody derived from a mammal can be suitably prepared.
- Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques.
- the monoclonal antibodies of the present invention include “humanized antibodies” and “chimeric antibodies”.
- Monoclonal antibody-producing hybridomas can be prepared, for example, as follows by using known techniques. That is, a mammal is immunized according to a normal immunization method using IL-6R protein as a sensitizing antigen. The resulting immune cells are fused with known parental cells by conventional cell fusion methods. Next, hybridomas that produce anti-IL-6R antibodies can be selected by screening monoclonal antibody-producing cells by conventional screening methods.
- the production of a monoclonal antibody is performed as follows, for example.
- the IL-6R gene whose nucleotide sequence is disclosed in SEQ ID NO: 2
- the IL-6R protein represented by SEQ ID NO: 1 used as a sensitizing antigen for antibody acquisition can be obtained.
- an appropriate host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector.
- the desired human IL-6R protein is purified from the host cell or culture supernatant by a known method.
- a soluble form of IL-6R from the culture supernatant for example, a soluble form as described by Mullberg et al. (J. Immunol.
- IL-6R polypeptide sequence represented by SEQ ID NO: 1 which is IL-6R
- a protein consisting of amino acids 1 to 357 is expressed instead of the IL-6R protein represented by SEQ ID NO: 1. Is done. Purified natural IL-6R protein can also be used as a sensitizing antigen as well.
- the purified IL-6R protein can be used as a sensitizing antigen used for immunization against mammals.
- a partial peptide of IL-6R can also be used as a sensitizing antigen.
- the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector for expression.
- the region and size of IL-6R peptide used as a partial peptide are not particularly limited to a specific embodiment.
- any sequence can be selected from amino acid sequences corresponding to amino acids 20 to 357 in the amino acid sequence of SEQ ID NO: 1.
- the number of amino acids constituting the peptide to be sensitized antigen is preferably at least 5 or more, for example 6 or more, or 7 or more. More specifically, a peptide having 8 to 50, preferably 10 to 30 residues can be used as a sensitizing antigen.
- a fusion protein obtained by fusing a desired partial polypeptide or peptide of IL-6R protein with a different polypeptide can be used as a sensitizing antigen.
- an antibody Fc fragment or a peptide tag can be suitably used.
- a vector that expresses a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into the expression vector as described above. The method for producing the fusion protein is described in Molecular® Cloning® 2nd® ed.
- the mammal immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.
- rodent animals such as mice, rats, hamsters, rabbits, monkeys and the like are preferably used.
- the above animals are immunized with a sensitizing antigen.
- immunization is performed by administering a sensitizing antigen intraperitoneally or subcutaneously to a mammal.
- a sensitized antigen diluted with PBS (Phosphate-Buffered Saline) or physiological saline at an appropriate dilution ratio is mixed with a normal adjuvant, for example, Freund's complete adjuvant, and emulsified, if desired.
- the sensitizing antigen is administered to the mammal several times every 4 to 21 days.
- an appropriate carrier can be used during immunization with the sensitizing antigen.
- a partial peptide having a low molecular weight when used as a sensitizing antigen, it may be desirable to immunize the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.
- a carrier protein such as albumin or keyhole limpet hemocyanin.
- a hybridoma that produces a desired antibody can be prepared as follows using DNA immunization.
- DNA immunization a sensitized antigen is expressed in vivo in the immunized animal to which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal.
- This is an immunization method in which immune stimulation is given.
- the following advantages are expected in DNA immunization. -Maintains the structure of membrane proteins such as IL-6R and can be given immune stimulation-No need to purify immune antigens
- DNA expressing IL-6R protein is administered to an immunized animal.
- DNA encoding IL-6R can be synthesized by a known method such as PCR.
- the obtained DNA is inserted into an appropriate expression vector and administered to an immunized animal.
- the expression vector for example, a commercially available expression vector such as pcDNA3.1 can be suitably used.
- a method for administering a vector to a living body a generally used method can be used.
- DNA immunization is performed by introducing gold particles adsorbed with an expression vector into cells of an immunized animal individual using a gene gun.
- an antibody recognizing IL-6R can also be produced using the method described in International Publication WO2003 / 104453.
- immune cells are collected from the mammal and subjected to cell fusion. Spleen cells can be used as preferred immune cells.
- Mammalian myeloma cells are used as the cells fused with the immune cells.
- the myeloma cell is preferably provided with an appropriate selection marker for screening.
- a selectable marker refers to a trait that can (or cannot) survive under certain culture conditions.
- Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
- HGPRT deficiency hypoxanthine-guanine-phosphoribosyltransferase deficiency
- TK deficiency thymidine kinase deficiency
- Cells having HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity).
- HGPRT-deficient or TK-deficient cells can be selected in a medium containing 6 thioguanine, 8 azaguanine (hereinafter abbreviated as 8AG), or 5 'bromodeoxyuridine, respectively.
- 8AG 8 azaguanine
- 5 'bromodeoxyuridine normal cells that incorporate these pyrimidine analogs into DNA die.
- cells deficient in these enzymes that cannot take up these pyrimidine analogs can survive in selective media.
- G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by a neomycin resistance gene.
- gentamicin analogs gentamicin analogs
- myeloma cells suitable for cell fusion are known.
- Examples of such myeloma cells include P3 (P3x63Ag8.653) (J.JImmunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1- 7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 ( Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133) and the like can be suitably used.
- P3x63Ag8.653 J.JImmunol. (1979) 123 (4)
- cell fusion between the immune cells and myeloma cells is performed according to a known method such as the method of Köhler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, for example, the cell fusion can be performed in a normal nutrient culture medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary such as dimethyl sulfoxide is optionally added to increase the fusion efficiency.
- the usage ratio of immune cells and myeloma cells can be set arbitrarily.
- the number of immune cells is preferably 1 to 10 times that of myeloma cells.
- the culture medium used for the cell fusion for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture are used. Serum replacement fluid such as fetal serum (FCS) can be suitably added.
- FCS fetal serum
- a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60%. It is added at a concentration of (w / v).
- a desired fused cell is formed by gently mixing the mixture.
- cell fusion agents and the like that are undesirable for the growth of hybridomas can be removed by repeating the operation of adding the appropriate culture solution listed above and removing the supernatant by centrifugation.
- the hybridoma thus obtained can be selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine).
- a HAT culture solution a culture solution containing hypoxanthine, aminopterin and thymidine.
- the culture using the HAT culture solution can be continued for a time sufficient for cells other than the desired hybridoma (non-fused cells) to die (usually, sufficient time is several days to several weeks).
- screening and single cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.
- the hybridoma thus obtained can be selected by using a selective culture solution corresponding to the selection marker possessed by the myeloma used for cell fusion.
- a selective culture solution corresponding to the selection marker possessed by the myeloma used for cell fusion.
- cells having HGPRT or TK deficiency can be selected by culturing in a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can selectively proliferate in the HAT medium.
- the culture using the HAT culture solution is continued for a time sufficient for cells other than the desired hybridoma (non-fusion cells) to die.
- a desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas producing the desired antibody can be performed by conventional limiting
- Desired antibody screening and single cloning can be suitably performed by a screening method based on a known antigen-antibody reaction.
- a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface.
- Such monoclonal antibodies can be screened, for example, by FACS (fluorescence-activated cell sorting).
- FACS fluorescence-activated cell sorting
- cells expressing IL-6R are prepared.
- Preferred cells for screening are mammalian cells in which IL-6R is forcibly expressed.
- a non-transformed mammalian cell used as a host cell as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, a hybridoma that produces an IL-6R monoclonal antibody can be obtained by selecting a hybridoma that produces an antibody that does not bind to host cells but binds to IL-6R forced expression cells.
- the binding activity of the antibody to the immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA.
- IL-6R-expressing cells are immobilized in the well of an ELISA plate.
- the culture supernatant of the hybridoma is brought into contact with the immobilized cells in the well, and an antibody that binds to the immobilized cells is detected.
- the monoclonal antibody is derived from a mouse
- the antibody bound to the cell can be detected by an anti-mouse immunoglobulin antibody.
- a hybridoma that produces a desired antibody having an ability to bind to an antigen selected by these screenings can be cloned by a limiting dilution method or the like.
- the hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution.
- the hybridoma can also be stored for a long time in liquid nitrogen.
- the hybridoma is cultured according to a usual method, and a desired monoclonal antibody can be obtained from the culture supernatant.
- a hybridoma can be administered to a mammal compatible therewith and allowed to proliferate, and a monoclonal antibody can be obtained from the ascites.
- the former method is suitable for obtaining a highly pure antibody.
- An antibody encoded by an antibody gene cloned from antibody-producing cells such as the hybridoma can also be suitably used.
- An antibody encoded by the gene is expressed by incorporating the cloned antibody gene into a suitable vector and introducing it into a host. Methods for isolation of antibody genes, introduction into vectors, and transformation of host cells are already established by, for example, Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767- 775). As described below, methods for producing recombinant antibodies are also known.
- cDNA encoding a variable region (V region) of an anti-IL-6R antibody is obtained from a hybridoma cell that produces the anti-IL-6R antibody.
- V region variable region
- RNA is extracted from the hybridoma.
- the following method can be used. -Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) -AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)
- Extracted mRNA can be purified using mRNA “Purification” Kit (manufactured by GE Healthcare Bioscience) or the like.
- kits for extracting total mRNA directly from cells such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience) are also commercially available.
- mRNA can be obtained from the hybridoma.
- CDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase.
- cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation).
- the desired cDNA fragment is purified from the obtained PCR product and then ligated with vector DNA.
- a desired recombinant vector can be prepared from Escherichia coli that has formed the colony. Then, whether or not the recombinant vector has the target cDNA base sequence is confirmed by a known method such as the dideoxynucleotide chain termination method.
- cDNA is synthesized using RNA extracted from a hybridoma cell as a template to obtain a 5′-RACE cDNA library.
- a commercially available kit such as SMART® RACE® cDNA® amplification kit is appropriately used.
- the antibody gene is amplified by PCR using the obtained 5′-RACE® cDNA library as a template.
- Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as IsoIStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics).
- primers capable of amplifying genes encoding ⁇ 1, ⁇ 2a, ⁇ 2b, ⁇ 3 as heavy chains and ⁇ chain and ⁇ chain as light chains are provided. Can be used.
- a primer that anneals to a portion corresponding to a constant region close to the variable region is generally used as the 3 ′ primer.
- the primer attached to the 5 ′ RACE cDNA library preparation kit is used as the 5 ′ primer.
- an immunoglobulin comprising a combination of a heavy chain and a light chain
- Desired antibodies can be screened using the reconstituted immunoglobulin binding activity to IL-6R as an index.
- the binding of the antibody to IL-6R is more preferably specific.
- Antibodies that bind to IL-6R can be screened, for example, as follows; (1) contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) a step of detecting binding between an IL-6R-expressing cell and an antibody, and (3) a step of selecting an antibody that binds to the IL-6R-expressing cell.
- a method for detecting the binding between an antibody and IL-6R-expressing cells is known. Specifically, the binding between the antibody and the IL-6R-expressing cell can be detected by a technique such as FACS described above. In order to evaluate the binding activity of the antibody, a fixed specimen of IL-6R-expressing cells can be appropriately used.
- a panning method using a phage vector is also preferably used as an antibody screening method using binding activity as an index.
- an antibody gene is obtained from a polyclonal antibody-expressing cell group as a heavy chain and light chain subclass library
- a screening method using a phage vector is advantageous.
- Genes encoding the variable regions of the heavy chain and the light chain can form a single chain Fv (scFv) by ligating with an appropriate linker sequence.
- scFv single chain Fv
- the phage encoding the antigen can be recovered to recover the DNA encoding scFv having the desired binding activity. By repeating this operation as necessary, scFv having a desired binding activity can be concentrated.
- the cDNA is digested with a restriction enzyme that recognizes restriction enzyme sites inserted at both ends of the cDNA.
- a preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene.
- a restriction enzyme that gives a sticky end.
- An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector.
- a chimeric antibody is obtained.
- the chimeric antibody means that the origin of the constant region and the variable region are different.
- a heterologous chimeric antibody such as mouse-human
- a human-human homologous chimeric antibody is also included in the chimeric antibody of the present invention.
- a chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance.
- a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately arranged on the 5 ′ side of an expression vector holding a DNA encoding a desired antibody constant region (C region).
- a chimeric antibody expression vector is constructed by fusing both digested with the same combination of restriction enzymes in-frame.
- the antibody gene is incorporated into an expression vector so that it is expressed under the control of the expression control region.
- An expression control region for expressing an antibody includes, for example, an enhancer and a promoter.
- An appropriate signal sequence can also be added to the amino terminus so that the expressed antibody is secreted extracellularly.
- a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences are added.
- the expressed polypeptide can be cleaved at the carboxyl terminal portion of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide.
- an appropriate host cell is transformed with this expression vector, whereby a recombinant cell expressing a DNA encoding an anti-IL-6R antibody can be obtained.
- DNAs encoding antibody heavy chains (H chains) and light chains (L chains) are each incorporated into separate expression vectors.
- An antibody molecule having an H chain and an L chain can be expressed by co-transfecting the same host cell with a vector in which an H chain and an L chain are incorporated.
- host cells can be transformed by incorporating DNAs encoding H and L chains into a single expression vector (see International Publication WO 1994/011523).
- host cells and expression vectors for producing antibodies by introducing an isolated antibody gene into a suitable host are known. Any of these expression systems can be applied to isolate the antigen binding domain of the present invention.
- animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, the following cells can be exemplified as animal cells.
- Mammalian cells CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, HEK (human embryonic kidney) 293, etc.
- Amphibian cells Xenopus oocytes, etc.
- Insect cells sf9, sf21, Tn5, etc.
- Nicotiana such as Nicotiana tabacum
- Callus cultured cells can be used as appropriate for transformation of plant cells.
- -Yeast Saccharomyces genus such as Saccharomyces serevisiae, Pichia genus such as methanol-utilizing yeast (Pichia pastoris)-Filamentous fungi: Aspergillus genus such as Aspergillus niger
- antibody gene expression systems using prokaryotic cells are also known.
- bacterial cells such as E. coli (E.coli) and Bacillus subtilis can be used as appropriate.
- An expression vector containing the target antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, a desired antibody can be obtained from the culture of the transformed cells.
- transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding a desired antibody has been introduced.
- an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein that is uniquely produced in milk.
- a protein secreted in milk for example, goat ⁇ casein can be used.
- the DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat.
- the desired antibody can be obtained as a fusion protein with milk protein from milk produced by a transgenic goat (or its progeny) born from a goat that has received the embryo.
- hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced from the transgenic goat (Bio / Technology (1994), 12 (7), 699-702). .
- polypeptide assembly described in the present specification When the polypeptide assembly described in the present specification is administered to humans, it is a genetically modified form that has been artificially modified for the purpose of reducing the heterologous antigenicity to humans as an antigen-binding domain in the assembly.
- An antigen-binding domain derived from an antibody can be appropriately employed.
- the recombinant antibody includes, for example, a humanized antibody. These modified antibodies are appropriately produced using known methods.
- variable region of an antibody used to generate an antigen binding domain in a polypeptide assembly described herein typically comprises three complementarity determining regions (four) sandwiched between four framework regions (FR). complementarity-determining (region); (CDR).
- CDRs are regions that substantially determine the binding specificity of an antibody.
- the amino acid sequence of CDR is rich in diversity.
- the amino acid sequences constituting FR often show high identity even among antibodies having different binding specificities. Therefore, it is generally said that the binding specificity of a certain antibody can be transplanted to another antibody by CDR grafting.
- Humanized antibodies are also referred to as reshaped human antibodies.
- non-human animals for example, humanized antibodies obtained by grafting mouse antibody CDRs to human antibodies are known.
- General genetic recombination techniques for obtaining humanized antibodies are also known.
- Overlap-Extension-PCR is known as a method for transplanting mouse antibody CDRs into human FRs.
- PCR extension the base sequence which codes CDR of the mouse antibody which should be transplanted is added to the primer for synthesize
- a human FR comprising an amino acid sequence having high identity with the FR amino acid sequence adjacent to the mouse CDR to be transplanted.
- the base sequences to be linked are designed to be connected to each other in frame.
- Human FRs are synthesized individually by each primer.
- a product in which DNA encoding mouse CDR is added to each FR is obtained.
- the base sequences encoding mouse CDRs of each product are designed to overlap each other.
- the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed with each other to perform a complementary chain synthesis reaction. By this reaction, human FRs are linked via the mouse CDR sequence.
- a human-type antibody expression vector can be prepared by inserting the DNA obtained as described above and a DNA encoding the human antibody C region into an expression vector so as to be fused in frame. After the recombinant vector is established by introducing the integration vector into the host, the recombinant cell is cultured, and the humanized antibody is expressed by expressing the DNA encoding the humanized antibody. (See European Patent Publication EP239400, International Publication WO1996 / 002576).
- the CDR forms a favorable antigen-binding site when linked via CDR.
- a human antibody FR can be suitably selected.
- FR amino acid residues can be substituted so that the CDR of the reshaped human antibody forms an appropriate antigen-binding site.
- amino acid sequence mutations can be introduced into FRs by applying the PCR method used for transplantation of mouse CDRs into human FRs.
- partial nucleotide sequence mutations can be introduced into primers that anneal to the FR.
- a nucleotide sequence mutation is introduced into the FR synthesized by such a primer.
- a mutant FR sequence having a desired property can be selected by measuring and evaluating the antigen-binding activity of a mutant antibody substituted with an amino acid by the above method (Cancer Res., (1993) 53, 851-856). .
- transgenic animals having all repertoires of human antibody genes are used as immunized animals, and DNA immunization is performed. Desired human antibodies can be obtained.
- the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method.
- Phages expressing scFv that bind to the antigen can be selected.
- the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined.
- the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector, whereby an expression vector can be prepared.
- the human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody.
- These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).
- the amino acid positions assigned to CDRs and FRs of antibodies are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. , 1987 and 1991.
- Kabat Sequences of Proteins of Immunological Interest
- the amino acids in the variable region follow the Kabat numbering
- the amino acids in the constant region are EU numbering according to the amino acid position of Kabat. It is expressed according to
- the ion concentration refers to the metal ion concentration.
- Metal ion means group I such as alkali metal and copper group excluding hydrogen, group II such as alkaline earth metal and zinc group, group III excluding boron, group IV excluding carbon and silicon, It refers to ions of elements belonging to Group A, Group VIII, Group V, Group VI and Group VII, such as Iron and Platinum, and metal elements such as antimony, bismuth and polonium. Metal atoms have the property of releasing valence electrons and becoming cations, which is called ionization tendency. A metal having a large ionization tendency is considered to be chemically active.
- An example of a metal ion suitable for the present invention is calcium ion.
- Calcium ions are involved in the regulation of many life phenomena, such as contraction of muscles such as skeletal muscle, smooth muscle and myocardium, activation of leukocyte movement and phagocytosis, activation of platelet deformation and secretion, lymphocytes, etc. , Activation of mast cells such as histamine secretion, cellular response via catecholamine alpha receptor and acetylcholine receptor, exocytosis, release of transmitter from neuronal terminals, neuronal axon flow, etc. Ions are involved.
- intracellular calcium ion receptors are troponin C, calmodulin, parvalbumin, myosin light chain, etc., which have multiple calcium ion binding sites and are thought to be derived from a common origin in molecular evolution. Many of its binding motifs are also known. For example, cadherin domain, EF hand contained in calmodulin, C2 domain contained in Protein kinase C, Gla domain contained in blood clotting protein FactorIX, C-type lectin contained in asialoglycoprotein receptor and mannose binding receptor, LDL receptor The included A domain, annexin, thrombospondin type 3 domain and EGF-like domain are well known.
- the calcium ion concentration condition when the metal ion is calcium ion, the calcium ion concentration condition includes a low calcium ion concentration condition and a high calcium ion concentration condition.
- the binding activity changes depending on the calcium ion concentration condition means that the binding activity of the antigen-binding molecule to the antigen changes depending on the difference between the low calcium ion concentration and the high calcium ion concentration.
- the binding activity of an antigen-binding molecule to an antigen under a high calcium ion concentration condition is higher than the binding activity of the antigen-binding molecule to an antigen under low calcium ion concentration conditions.
- the high calcium ion concentration is not particularly limited to a unique numerical value, but may preferably be a concentration selected from 100 ⁇ M to 10 ⁇ m. In another embodiment, the concentration may be selected from 200 ⁇ M to 5 ⁇ M. In another embodiment, the concentration may be selected between 400 ⁇ M and 3 ⁇ mM, and in another embodiment, the concentration may be selected between 200 ⁇ M and 2 ⁇ mM. Furthermore, it may be a concentration selected from between 400 ⁇ M and 1 ⁇ mM. Particularly preferred is a concentration selected from 500 ⁇ M to 2.5 ⁇ mM, which is close to the calcium ion concentration in plasma (blood) in vivo.
- the low calcium ion concentration is not particularly limited to a unique numerical value, but may preferably be a concentration selected from 0.1 ⁇ M to 30 ⁇ M. In another embodiment, the concentration may be selected from between 0.2 ⁇ M and 20 ⁇ M. In another embodiment, the concentration may be selected between 0.5 ⁇ M and 10 ⁇ M, and in another embodiment, the concentration may be selected between 1 ⁇ M and 5 ⁇ M. It can also be a concentration selected between 2 ⁇ M and 4 ⁇ M. Particularly preferred is a concentration selected from 1 ⁇ M to 5 ⁇ M close to the ionized calcium concentration in the early endosome in vivo.
- the binding activity to the antigen under the condition of low calcium ion concentration is lower than the binding activity to the antigen under the condition of high calcium ion concentration is a calcium ion concentration selected from 0.1 ⁇ M to 30 ⁇ M of the antigen binding molecule.
- the binding activity to the antigen is weaker than the binding activity to the antigen at a calcium ion concentration selected between 100 ⁇ M and 10 ⁇ mM.
- the antigen-binding molecule has a weaker binding activity against an antigen at a calcium ion concentration selected between 0.5 ⁇ M and 10 ⁇ M than the binding activity against an antigen at a calcium ion concentration selected between 200 ⁇ M and 5 ⁇ mM.
- the antigen binding activity at the calcium ion concentration in the early endosome in the living body is weaker than the antigen binding activity at the calcium ion concentration in the plasma in the living body, specifically, the antigen It means that the binding activity of the binding molecule to the antigen at a calcium ion concentration selected from between 1 ⁇ M to 5 ⁇ M is weaker than the binding activity to the antigen at a calcium ion concentration selected from between 500 ⁇ M to 2.5 ⁇ M.
- Whether or not the binding activity of the antigen-binding molecule to the antigen is changed depending on the condition of the metal ion concentration can be determined, for example, by using a known measurement method as described in the above-mentioned binding activity section. For example, in order to confirm that the binding activity of an antigen-binding molecule to an antigen under a high calcium ion concentration condition changes higher than the binding activity of the antigen-binding molecule to an antigen under a low calcium ion concentration condition, The binding activity of the antigen binding molecule to the antigen under conditions of low and high calcium ion concentrations is compared.
- the expression “the binding activity to the antigen under the condition of low calcium ion concentration is lower than the binding activity to the antigen under the condition of high calcium ion concentration” means that the antigen binding molecule binds to the antigen under the condition of high calcium ion concentration. It can also be expressed that the activity is higher than the binding activity to the antigen under low calcium ion concentration conditions.
- “the binding activity to the antigen under the condition of low calcium ion concentration is lower than the binding activity to the antigen under the condition of high calcium ion concentration” is referred to as “the antigen binding ability under the condition of low calcium ion concentration is high calcium ion concentration.
- the antigen-binding ability under the calcium ion concentration condition may be weaker than the antigen-binding ability under the high calcium ion concentration condition.
- Conditions other than the calcium ion concentration when measuring the binding activity to the antigen can be appropriately selected by those skilled in the art and are not particularly limited.
- measurement can be performed under the conditions of HEPES buffer and 37 ° C.
- it can be measured using Biacore (GE Healthcare).
- the antigen is a soluble antigen
- the binding activity to the soluble antigen can be measured by flowing the antigen as an analyte to the chip on which the antigen-binding molecule is immobilized.
- the antigen is a membrane-type antigen
- it is possible to evaluate the binding activity to the membrane-type antigen by flowing the antigen-binding molecule as an analyte to the chip on which the antigen is immobilized. It is.
- the binding activity to the antigen under the condition of low calcium ion concentration is weaker than the binding activity to the antigen under the condition of high calcium ion concentration
- the binding activity to the antigen under the low calcium ion concentration condition and high calcium is not particularly limited, but preferably the ratio of KD under the condition of low calcium ion concentration to the antigen and KD under the condition of high calcium ion concentration.
- the value of (Ca 3 ⁇ M) / KD (Ca 2 mM) is 2 or more, more preferably the value of KD (Ca 3 ⁇ M) / KD (Ca 2 mM) is 10 or more, more preferably KD (Ca 3 ⁇ M)
- the value of / KD (Ca 2 mM) is 40 or more.
- the upper limit of the value of KD (Ca 3 ⁇ M) / KD (Ca 2 mM) is not particularly limited, and may be any value such as 400, 1000, 10000, etc., as long as it can be produced by a person skilled in the art.
- KD dissociation constant
- apparent KD ApparentKDdissociation constant
- KD dissociation constant
- apparent KD apparent dissociation constant
- dissociation rate constant kd dissociation rate constant
- kd dissociation rate constant
- KD dissociation constant
- the ratio of kd (low calcium concentration condition) / kd (high calcium concentration condition), which is the ratio of rate constants, is preferably 2 or more, more preferably 5 or more, and even more preferably 10 or more. More preferably, it is 30 or more.
- the upper limit of the value of Kd (conditions for low calcium concentration) / kd (conditions for high calcium concentration) is not particularly limited, and may be any value such as 50, 100, 200, etc. as long as it can be produced by the common general knowledge of those skilled in the art.
- kd dissociation rate constant
- apparent kd Apparent dissociation rate constant
- kd (dissociation rate constant) and apparent kd can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), a flow cytometer or the like can be used. Is possible.
- Biacore GE healthcare
- a flow cytometer or the like can be used. Is possible.
- the conditions other than the calcium concentration are preferably the same.
- an antigen-binding domain or antibody whose binding activity to an antigen under conditions of low calcium ion concentration is lower than the binding activity to antigen under conditions of high calcium ion concentration is the following step (a )
- To (c) can be obtained by screening an antigen-binding domain or antibody.
- an antigen-binding domain or antibody that has an activity of binding to an antigen under conditions of low calcium ion concentration which is one aspect provided by the present invention, lower than that of antigen under conditions of high calcium ion concentration is the following step (a )
- To (c) can be obtained by screening antigen binding domains or antibodies or libraries thereof.
- (a) contacting an antigen-binding domain or antibody or a library thereof in a high calcium concentration condition with an antigen;
- an antigen-binding domain or antibody that has an activity of binding to an antigen under conditions of low calcium ion concentration which is one aspect provided by the present invention, lower than that of antigen under conditions of high calcium ion concentration is the following step (a )
- To (d) can be obtained by screening antigen binding domains or antibodies or libraries thereof.
- step (a) contacting the antigen-binding domain or library of antibodies with the antigen under low calcium concentration conditions; (b) selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a), (c) binding the antigen-binding domain or antibody selected in step (b) to an antigen under high calcium concentration conditions; (d) A step of isolating the antigen binding domain or antibody bound to the antigen in the step (c).
- an antigen-binding domain or antibody that has an activity of binding to an antigen under conditions of low calcium ion concentration which is one aspect provided by the present invention, lower than that of antigen under conditions of high calcium ion concentration is the following step (a ) To (c). (a) contacting an antigen-binding domain or antibody library with a column immobilized with an antigen under high calcium concentration conditions; (b) a step of eluting the antigen-binding domain or antibody bound to the column in the step (a) from the column under a low calcium concentration condition, (c) A step of isolating the antigen-binding domain or antibody eluted in the step (b).
- an antigen-binding domain or antibody that has an activity of binding to an antigen under conditions of low calcium ion concentration which is one aspect provided by the present invention, lower than that of antigen under conditions of high calcium ion concentration is the following step (a ) To (d). (a) passing an antigen-binding domain or antibody library through a column immobilized with an antigen under a low calcium concentration condition; (b) recovering the antigen-binding domain or antibody eluted without binding to the column in the step (a), (c) a step of binding the antigen-binding domain or antibody recovered in the step (b) to an antigen under high calcium concentration conditions, (d) A step of isolating the antigen binding domain or antibody bound to the antigen in the step (c).
- an antigen-binding domain or antibody that has an activity of binding to an antigen under conditions of low calcium ion concentration which is one aspect provided by the present invention, lower than that of antigen under conditions of high calcium ion concentration is the following step (a ) To (d).
- step (a) contacting an antigen-binding domain or library of antibodies with an antigen under high calcium concentration conditions; (b) obtaining an antigen-binding domain or antibody bound to the antigen in the step (a), (c) placing the antigen-binding domain or antibody obtained in step (b) under low calcium concentration conditions, (d) A step of isolating an antigen-binding domain or antibody whose antigen-binding activity is weaker than the criterion selected in the step (b) in the step (c).
- the above process may be repeated twice or more. Therefore, according to the present invention, in the above-described screening method, the condition of low calcium ion concentration obtained by the screening method further comprising the step of repeating the steps (a) to (c) or (a) to (d) twice or more.
- An antigen-binding domain or antibody is provided which has a lower binding activity to the antigen in the antigen than binding activity to the antigen under conditions of high calcium ion concentration.
- the number of times the steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is usually within 10 times.
- the antigen-binding activity of the antigen-binding domain or antibody under low calcium concentration conditions is not particularly limited as long as the ion-binding calcium concentration is between 0.1 ⁇ M and 30 ⁇ M.
- an antigen-binding activity between 0.5 ⁇ M and 10 ⁇ M can be mentioned.
- a more preferable ionized calcium concentration includes an ionized calcium concentration in an early endosome in a living body, and specifically includes an antigen binding activity at 1 ⁇ M to 5 ⁇ M.
- the antigen-binding activity of the antigen-binding domain or antibody under high calcium concentration conditions is not particularly limited as long as the ion-binding calcium concentration is between 100 ⁇ M and 10 ⁇ m.
- the preferable ionized calcium concentration is 200 ⁇ M to 5 ⁇ m.
- Antigen-binding activity between More preferable ionized calcium concentration includes ionized calcium concentration in plasma in a living body, specifically, antigen-binding activity at 0.5 to 2.5 mM.
- the antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art.
- the antigen-binding activity of an antigen-binding domain or antibody is KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate: dissociation rate constant), or apparent Kd (Apparent dissociation: apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plot, FACS and the like can be used.
- the step of selecting an antigen-binding domain or antibody whose antigen-binding activity under a high calcium concentration condition is higher than that under a low calcium concentration condition is that the antigen-binding activity under a low calcium concentration condition is high This is the same meaning as the step of selecting an antigen-binding domain or antibody lower than the antigen-binding activity below.
- the difference between the antigen binding activity under the high calcium concentration condition and the antigen binding activity under the low calcium concentration condition is not particularly limited,
- the antigen binding activity under a high calcium concentration condition is 2 times or more of the antigen binding activity under a low calcium concentration condition, more preferably 10 times or more, and more preferably 40 times or more.
- the antigen-binding domain or antibody of the present invention to be screened by the above screening method may be any antigen-binding domain or antibody.
- the above-described antigen-binding domain or antibody can be screened.
- an antigen binding domain or antibody having a natural sequence may be screened, or an antigen binding domain or antibody having an amino acid sequence substituted may be screened.
- the antigen-binding domain or antibody of the present invention comprises an antigen-binding domain containing at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen depending on ion concentration conditions. It can be obtained from a library mainly composed of a plurality of antigen-binding molecules having different sequences.
- the ion concentration include a metal ion concentration and a hydrogen ion concentration.
- library refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules, or nucleic acids and polynucleotides encoding these sequences.
- sequences of a plurality of antigen-binding molecules or a plurality of fusion polypeptides comprising antigen-binding molecules contained in the library are not single sequences but are fusion polypeptides comprising antigen-binding molecules or antigen-binding molecules having different sequences from each other.
- the term “differing in sequence from each other” in the description of a plurality of antigen-binding molecules having different sequences means that the sequences of individual antigen-binding molecules in the library are different from each other. That is, the number of different sequences in the library reflects the number of independent clones having different sequences in the library, and is sometimes referred to as “library size”. In a normal phage display library, the number is 10 6 to 10 12 , and the library size can be increased to 10 14 by applying a known technique such as a ribosome display method. However, the actual number of phage particles used during phage library panning selection is typically 10 to 10,000 times larger than the library size.
- the term “different from each other” in the present invention means that the sequences of individual antigen-binding molecules in the library from which the number of library equivalents is excluded are different from each other, more specifically, antigen-binding molecules having different sequences from each other.
- 10 6 to 10 14 molecules, preferably 10 7 to 10 12 molecules, more preferably 10 8 to 10 11 , particularly preferably 10 8 to 10 10 are present.
- the term “plurality” in the description of a library mainly composed of a plurality of antigen-binding molecules of the present invention is, for example, an antigen-binding molecule, a fusion polypeptide, a polynucleotide molecule, a vector or a virus of the present invention.
- antigen-binding molecules of the invention there are two or more antigen-binding molecules of the invention that are substantially the same, preferably the same sequence, except for flexible residues or specific variant amino acids at very diverse amino acid positions exposed on the surface
- the sequences are substantially the same, preferably the same except for the base encoding the flexible residue, or the base encoding a particular variant amino acid at a very diverse amino acid position exposed on the surface. If there are two or more polynucleotide molecules of the present invention, there are a plurality of polynucleotide molecules of the present invention.
- the term “consisting mainly of” in the description of the library mainly composed of a plurality of antigen-binding molecules of the present invention means that the antigen binding to the antigen depends on the condition of ion concentration among the number of independent clones having different sequences in the library. It reflects the number of antigen-binding molecules with different molecular binding activities. Specifically, it is preferable that at least 10 4 antigen-binding molecules exhibiting such binding activity exist in the library. More preferably, the antigen-binding domain of the present invention can be obtained from a library having at least 10 5 antigen-binding molecules exhibiting such binding activity.
- the antigen-binding domain of the present invention can be obtained from a library in which at least 10 6 antigen-binding molecules exhibiting such binding activity are present.
- the antigen-binding domain of the present invention can be obtained from a library in which at least 10 7 antigen-binding molecules exhibiting such binding activity are present.
- the antigen-binding domain of the present invention can be obtained from a library in which at least 10 8 antigen-binding molecules exhibiting such binding activity are present.
- it can be suitably expressed as a ratio of antigen-binding molecules having different binding activities of antigen-binding molecules to the antigen depending on ion concentration conditions.
- the antigen-binding domain of the present invention comprises 0.1% to 80%, preferably 0.5% to 60%, more preferably 0.5% to 60% of the number of independent clones having different binding sequences in the library. Preferably from 1% to 40%, more preferably from 2% to 20%, particularly preferably from 4% to 10%.
- a fusion polypeptide, a polynucleotide molecule or a vector it can be expressed by the number of molecules or a ratio in the whole molecule as described above.
- a virus as described above, it can be expressed by the number of virus individuals and the ratio in the whole individual.
- the amino acid that changes the binding activity of the antigen-binding domain to the antigen depending on the condition of the calcium ion concentration may be prepared in any way, for example, when the metal ion is calcium In the case of ion concentration, pre-existing antibodies, pre-existing libraries (such as phage libraries), hybridomas obtained from immunization to animals, or antibodies or libraries prepared from B cells from immunized animals
- pre-existing antibodies, pre-existing libraries such as phage libraries
- hybridomas obtained from immunization to animals or antibodies or libraries prepared from B cells from immunized animals
- These antibodies and libraries are amino acids that can chelate calcium (for example, aspartic acid and glutamic acid) and antibodies or libraries in which non-natural amino acid mutations have been introduced (amino acids that can chelate calcium (for example, aspartic acid and glutamic acid) or non-natural amino acids. It is possible to use a library in which the content of natural amino acids is increased, an amino acid capable of chelating calcium
- an amino acid that changes the binding activity of an antigen-binding molecule to an antigen depending on the condition of the ion concentration as described above for example, when the metal ion is a calcium ion, if it is an amino acid that forms a calcium-binding motif, Any type.
- Calcium binding motifs are well known to those skilled in the art and have been described in detail (eg, Springer et al. (Cell (2000) 102, 275-277), Kawasaki and Kretsinger (Protein f Prof. (1995) 2, 305-490) Moncrief et al. (J. Mol. Evol. (1990) 30, 522-562), Chauvaux et al. (Biochem. J.
- any known calcium-binding motif such as C-type lectin such as ASGPR, CD23, MBR, DC-SIGN and the like can be included in the antigen-binding molecule of the present invention.
- a calcium-binding motif contained in the antigen-binding domain described in SEQ ID NO: 4 can also be mentioned.
- an amino acid having a metal chelate action can be suitably used as an example of an amino acid that changes the binding activity of the antigen-binding molecule to the antigen depending on the condition of the calcium ion concentration.
- amino acids having metal chelating action include, for example, serine (Ser (S)), threonine (Thr (T)), asparagine (Asn (N)), glutamine (Gln (Q)), aspartic acid (Asp (D) ) And glutamic acid (Glu (E)) and the like.
- the position of the antigen-binding domain containing the amino acid is not limited to a specific position. As long as the binding activity of the antigen-binding molecule to the antigen is changed depending on the condition of calcium ion concentration, the heavy chain variable region that forms the antigen-binding domain or It can be at any position in the light chain variable region. That is, the antigen-binding domain of the present invention is mainly composed of antigen-binding molecules having different sequences from each other, wherein the amino acid that changes the binding activity of the antigen-binding molecule to the antigen depending on the calcium ion concentration condition is contained in the antigen-binding domain of the heavy chain. Can be obtained from a library.
- the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, wherein the amino acid is contained in CDR3 of the heavy chain.
- the antigen-binding domain of the present invention comprises antigens having different sequences from each other, wherein the amino acids are contained at positions 95, 96, 100a and / or 101 represented by the Kabat numbering of heavy chain CDR3. It can be obtained from a library consisting primarily of binding molecules.
- the antigen-binding domain of the present invention has an amino acid sequence that changes the binding activity of an antigen-binding molecule to an antigen depending on the condition of calcium ion concentration. It can be obtained from a library consisting primarily of different antigen binding molecules. In another embodiment, the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, wherein the amino acid is contained in CDR1 of the light chain. In another embodiment, the antigen-binding domain of the present invention is an antigen-binding molecule having a different sequence from each other, which is contained in positions 30, 31, and / or 32 represented by Kabat numbering of light chain CDR1. It can be obtained from the main library.
- the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different amino acid sequences, the amino acid residues of which are contained in the light chain CDR2.
- the antigen-binding domain of the present invention can be obtained from a library mainly composed of antigen-binding molecules having different sequences from each other, the amino acid residues of which are contained in the light chain CDR3.
- the antigen-binding domain of the present invention is obtained from a library mainly composed of antigen-binding molecules having different sequences, wherein the amino acid residue is contained in position 92 represented by Kabat numbering of light chain CDR3. obtain.
- the antigen-binding domain of the present invention is an antigen-binding domain in which the amino acid residues are contained in two or three CDRs selected from CDR1, CDR2 and CDR3 of the light chain described above. It can be obtained as a different embodiment of the present invention from a library consisting mainly of molecules. Furthermore, the antigen-binding domain of the present invention includes the amino acid residue at any one or more of positions 30, 31, 32, 50, and / or 92 represented by Kabat numbering of the light chain. It can be obtained from a library mainly composed of antigen-binding molecules having different sequences.
- the framework sequence of the light chain and / or heavy chain variable region of the antigen-binding molecule comprises a human germline framework sequence.
- the antigen binding molecule of the invention will have little or no immunogenic response when administered to a human (eg, treatment of disease). It is thought not to cause.
- the phrase “an moth containing a germline sequence” of the present invention means that a part of the framework sequence of the present invention is identical to a part of any human germline framework sequence. Means.
- the heavy chain FR2 sequence of the antigen-binding molecule of the present invention is a sequence in which the heavy chain FR2 sequences of a plurality of different human germline framework sequences are combined
- the "germline sequence of the present invention” Is an antigen binding molecule.
- V-Base http://vbase.mrc-cpe.cam.ac.uk/
- a preferred arrangement is the work area.
- These framework region sequences can be appropriately used as germline sequences contained in the antigen-binding molecule of the present invention. Germline sequences can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798) Williams and Winter (Eur. J. Immunol. (1993) 23, 1456 -1461) and Cox et al. (Nat. Genetics (1994) 7, 162-168)).
- a suitable germline sequence can be appropriately selected from V ⁇ classified into 7 subgroups, V ⁇ classified into 10 subgroups, and VH classified into 7 subgroups.
- VH1 subgroups eg, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45).
- VH1-46, VH1-58, VH1-69) VH2 subgroups (eg VH2-5, VH2-26, VH2-70)
- VH3 subgroups VH3-7, VH3-9, VH3-11, VH3 -13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49 , VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74), VH4 subgroup (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39,
- the fully human Vk sequence is not limited to the following, for example, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, which are classified into the Vk1 subgroup, L1, L19, L22, L23, L24, O2, O4, O8, O12, O14, O18, Vk2 subgroup A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, O11 A11, A27, L2, L6, L10, L16, L20, L25, B3 classified into Vk4 subgroup, B2 classified into Vk5 subgroup (in this specification, Vk5-2 A10, A14, A26 etc. (Kawasaki et al. (Eur. J.
- the fully human VL sequence is not limited to the following, but for example, V1-2, V1-3, V1-4, V1-5, V1-7, V1- 9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-20, V1-22, VL1 subgroup V2-1, V2-6, V2 -7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V3-2, V3-3, V3-4, classified into VL3 subgroup V4-1, V4-2, V4-3, V4-4, V4-6 classified into VL4 subgroup, V5-1, V5-2, V5-4, V5-6 etc. classified into VL5 subgroup (Kawasaki et al. (Genome Res. (1997) 7, 250-261)) is preferable.
- framework sequences differ from each other by the difference in one or more amino acid residues.
- These framework sequences can be used together with the “at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen depending on ion concentration conditions” of the present invention.
- Examples of fully human frameworks used in conjunction with the “at least one amino acid residue that alters the binding activity of an antigen binding molecule to an antigen under ion concentration conditions” of the present invention are not limited to this.
- Others include KOL, NEWM, REI, EU, TUR, TEI, LAY, POM and the like (for example, Kabat et al. (1991) and Wu et al. (J. Exp. Med. (1970) 132, 211-250)).
- germline sequences are expected to eliminate adverse immune responses in most individuals. It has been. Affinity maturation steps that occur during normal immune responses frequently result in somatic mutations in the variable regions of immunoglobulins. These mutations occur mainly around CDRs whose sequences are hypervariable, but also affect residues in framework regions. These framework mutations are not present in germline genes and are unlikely to be immunogenic in patients. On the other hand, the normal human population is exposed to the majority of framework sequences expressed by germline genes, and as a result of immune tolerance, these germline frameworks are less immunogenic in patients Alternatively, it is expected to be non-immunogenic. In order to maximize the potential for immune tolerance, the gene encoding the variable region can be selected from a set of functional germline genes that normally exist.
- an antigen-binding molecule in which the amino acid that changes the binding activity of an antigen-binding molecule to an antigen according to the condition of the calcium ion concentration of the present invention is contained in the aforementioned framework sequence, site-directed mutagenesis (Kunkel et al. (Proc (Natl. Acad. Sci. USA (1985) (82), (488-492)) and Overlap extension (PCR) can be used as appropriate.
- a light chain variable region selected as a framework sequence that includes at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen depending on calcium ion concentration conditions and a randomized variable region sequence library
- a library containing a plurality of antigen-binding molecules having different sequences from each other according to the present invention can be prepared by combining the heavy chain variable regions prepared as above.
- the ion concentration is a calcium ion concentration
- a light chain variable region sequence and a randomized variable region sequence library described in SEQ ID NO: 4 (Vk5-2) A library combining the prepared heavy chain variable region is preferable.
- the amino acid sequence is added to the light chain variable region sequence selected as a framework sequence that contains at least one amino acid residue that changes the binding activity of the antigen-binding molecule to the antigen depending on the calcium ion concentration conditions. It is also possible to design such that various amino acids are included as residues other than the residues. In the present invention, such a residue is referred to as a flexible residue. As long as the binding activity of the antigen-binding molecule of the present invention to the antigen varies depending on the condition of ion concentration, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR and / or FR sequences of the heavy and / or light chain.
- the ion concentration is a calcium ion concentration
- the ion concentration is a calcium ion concentration
- Table 1 or Examples include the amino acid residues listed in Table 2.
- flexible residues are light and heavy chains having several different amino acids that are presented at that position when comparing amino acid sequences of known and / or natural antibodies or antigen binding domains.
- Kabat Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) provides in determining the highly diverse positions of known and / or natural antibodies. The data to be used is valid.
- the amino acid is preferably from about 2 to about 20, preferably from about 3 to about 19, preferably from about 4 to about 18, preferably from 5 to 17, preferably from 6 to 16, preferably from 7 to 7 at certain positions. If there is a diversity of 15, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 possible different amino acid residues, the position is very diverse.
- an amino acid position is preferably at least about 2, preferably at least about 4, preferably at least about 6, preferably at least about 8, preferably about 10, and preferably about 12 possible different amino acids. Can have residue diversity.
- the ion concentration is the calcium ion concentration
- SEQ ID NO: 5 SEQ ID NO: 1
- SEQ ID NO: 6 SEQ ID NO: 2
- SEQ ID NO: 7 Vk3
- a library combining a heavy chain variable region prepared as a randomized variable region sequence library is preferable.
- Non-limiting examples of the amino acid residues include amino acid residues contained in the light chain CDR1.
- non-limiting examples of the amino acid residues include amino acid residues contained in the light chain CDR2.
- an amino acid residue contained in CDR3 of the light chain is also exemplified.
- amino acid residues in which the amino acid residue is included in CDR1 of the light chain positions 30, 31 and / or represented by EU numbering in the CDR1 of the light chain variable region
- An amino acid residue at position 32 is mentioned.
- a non-limiting example of the amino acid residue that is included in the light chain CDR2 is the amino acid residue at position 50 represented by Kabat numbering in the light chain variable region CDR2.
- the amino acid residue is contained in the light chain CDR3, and a non-limiting example of the amino acid residue is the amino acid residue at position 92 represented by Kabat numbering in the light chain variable region CDR3.
- these amino acid residues may be included alone as long as these amino acid residues form a calcium-binding motif and / or the binding activity of the antigen-binding molecule to the antigen varies depending on the calcium ion concentration conditions.
- two or more of these amino acids may be included in combination.
- troponin C, calmodulin, parvalbumin, myosin light chain, etc. which have multiple calcium ion binding sites and are thought to be derived from a common origin in molecular evolution, are known to contain the binding motif. It is also possible to design light chain CDR1, CDR2 and / or CDR3.
- cadherin domain for the above purpose, EF hand contained in calmodulin, C2 domain contained in Protein kinase C, Gla domain contained in blood coagulation protein FactorIX, C-type lectin contained in asialoglycoprotein receptor and mannose binding receptor, The A domain, annexin, thrombospondin type 3 domain and EGF-like domain contained in the LDL receptor can be appropriately used.
- a light chain variable region into which at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen is changed according to the above ion concentration conditions is combined with a heavy chain variable region prepared as a randomized variable region sequence library Even in the case, as described above, it is possible to design such that the flexible residue is included in the sequence of the light chain variable region.
- the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR and / or FR sequences of the heavy and / or light chain.
- the ion concentration is a calcium ion concentration
- non-limiting examples of flexible residues introduced into the light chain variable region sequence include amino acid residues described in Table 1 or Table 2.
- a randomized variable region library is preferably mentioned.
- Known methods are appropriately combined with the method for producing the randomized variable region library.
- an antibody gene derived from an animal immunized with a specific antigen, an infectious disease patient, a human who has been vaccinated, has increased blood antibody titer, a cancer patient, or an autoimmune disease lymphocyte.
- the originally constructed immune library can be suitably used as a randomized variable region library.
- the V gene in genomic DNA ⁇ and the CDR sequence of the reconstructed functional V gene are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of an appropriate length.
- the synthesized library can also be suitably used as a randomized variable region library. In this case, since the diversity of the heavy chain CDR3 gene sequence is observed, it is also possible to replace only the CDR3 sequence.
- the criterion for creating amino acid diversity in the variable region of an antigen binding molecule is to provide diversity at the amino acid residues at positions exposed on the surface of the antigen binding molecule.
- a surface exposed position is a position that can be exposed and / or contacted with an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule. Generally speaking, it is the CDR.
- the position exposed on the surface is determined using coordinates from a three-dimensional model of the antigen binding molecule using a computer program such as the Insight II program (Accelrys).
- the position exposed on the surface can be determined using algorithms known in the art (eg, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400), Connolly (J. Appl. Cryst. (1983) 16, 1983). 548-558)).
- the determination of the position exposed on the surface can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies.
- software that can be used for such a purpose, SYBYL biopolymer module software (Tripos Associates) is preferably mentioned.
- the “size” of the probe used in the calculation is set to a radius of about 1.4 angstroms or less.
- Pacios Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1 , 46-53).
- a naive library consisting of a naive sequence that is an antibody sequence constructed from an antibody gene derived from a lymphocyte of a healthy person and having no bias in its repertoire is also a randomized variable region library.
- a naive sequence that is an antibody sequence constructed from an antibody gene derived from a lymphocyte of a healthy person and having no bias in its repertoire is also a randomized variable region library.
- the antigen-binding domain of the present invention can be obtained from a library containing a plurality of antigen-binding molecules of different sequences of the present invention by combining with a light chain variable region prepared as a randomized variable region sequence library.
- the ion concentration is a calcium ion concentration
- SEQ ID NO: 9 (6RL # 9-IgG1) or SEQ ID NO: 10 (6KC4-1 # 85-IgG1)
- the light chain variable region prepared as a randomized variable region sequence library it can be prepared by appropriately selecting from light chain variable regions having germline sequences.
- a heavy chain variable region sequence described in SEQ ID NO: 9 (6RL # 9-IgG1) or SEQ ID NO: 10 (6KC4-1 # 85-IgG1) and a light chain variable region having a germline sequence A combined library is preferred.
- sequence of the heavy chain variable region selected as a framework sequence containing in advance “at least one amino acid residue that changes the binding activity of the antigen-binding molecule to the antigen depending on the ion concentration conditions” is flexible. It can also be designed to include residues. As long as the binding activity of the antigen-binding molecule of the present invention to the antigen varies depending on the condition of ion concentration, the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR and / or FR sequences of the heavy and / or light chain.
- a non-limiting example of a flexible residue introduced into the heavy chain variable region sequence described in SEQ ID NO: 9 is examples include all amino acid residues of the chains CDR1 and CDR2, as well as amino acid residues of CDR3 other than positions 95, 96 and / or 100a of the heavy chain CDR3.
- flexible residues introduced into the heavy chain variable region sequence described in SEQ ID NO: 10 (6KC4-1 # 85-IgG1), all amino acid residues of heavy chain CDR1 and CDR2
- Other amino acid residues of CDR3 other than positions 95 and / or 101 of heavy chain CDR3 are also mentioned.
- a heavy chain variable region introduced with the above-described “at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen depending on ion concentration conditions” and a light chain variable region prepared as a randomized variable region sequence library
- a library containing a plurality of antigen binding molecules with different sequences can also be generated.
- the ion concentration is the calcium ion concentration
- a specific residue of the heavy chain variable region exhibits the binding activity of the antigen-binding molecule to the antigen depending on the condition of the calcium ion concentration.
- a library combining a heavy chain variable region sequence substituted with at least one amino acid residue to be changed and a light chain variable region prepared as a randomized variable region sequence library or a light chain variable region having a germline sequence Preferably mentioned.
- the amino acid residues include amino acid residues contained in heavy chain CDR1.
- non-limiting examples of the amino acid residues include amino acid residues contained in heavy chain CDR2.
- an amino acid residue contained in heavy chain CDR3 is also exemplified.
- amino acid residue is included in CDR3 of the heavy chain, and non-limiting examples of amino acid residues include those at positions 95, 96, 100a and / or 101 represented by Kabat numbering in CDR3 of the heavy chain variable region.
- amino acids include amino acids.
- these amino acid residues may be included alone as long as these amino acid residues form a calcium-binding motif and / or the binding activity of the antigen-binding molecule to the antigen varies depending on the calcium ion concentration conditions.
- two or more of these amino acids may be included in combination.
- Light chain variable region or reproductive region prepared as a heavy chain variable region and randomized variable region sequence library introduced with at least one amino acid residue that changes the binding activity of the antigen-binding molecule to the antigen depending on the ion concentration conditions.
- a flexible residue is included in the sequence of the heavy chain variable region as described above.
- the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues may be included in the heavy chain CDR and / or FR sequences.
- a randomized variable region library can also be suitably used as the CDR1, CDR2 and / or CDR3 amino acid sequence of the heavy chain variable region other than the amino acid residue that changes the binding activity of the antigen-binding molecule to the antigen depending on the ion concentration conditions.
- a germline sequence is used as the light chain variable region, for example, SEQ ID NO: 5 (Vk1), SEQ ID NO: 6 (Vk2), SEQ ID NO: 7 (Vk3), SEQ ID NO: 8 (Vk4) Germline sequences such as can be mentioned as non-limiting examples.
- any amino acid can be preferably used as long as it forms a calcium-binding motif.
- An amino acid having an electron donating property may be mentioned.
- Preferred examples of such an electron-donating amino acid include serine, threonine, asparagine, glutamine, aspartic acid and glutamic acid.
- the ion concentration conditions refer to hydrogen ion concentration conditions or pH conditions.
- the condition of the concentration of protons that is, the nuclei of hydrogen atoms
- the pH is defined as -log10aH +. If the ionic strength in the aqueous solution is low (eg, less than 10 ⁇ 3 ), aH + is approximately equal to the hydrogen ion strength. For example, since the ion product of water at 25 ° C.
- an aqueous solution having a pH lower than 7 is acidic
- an aqueous solution having a pH higher than 7 is alkaline.
- the pH condition is a high hydrogen ion concentration or low pH, that is, a pH acidic range condition, and a low hydrogen ion concentration or high pH, that is, a neutral pH range.
- the binding activity changes depending on the pH condition.
- the binding activity of the antigen-binding molecule to the antigen depends on the difference in conditions between high hydrogen ion concentration or low pH (pH acidic range) and low hydrogen ion concentration or high pH (pH neutral range). That changes.
- the binding activity of the antigen-binding molecule to the antigen in the neutral pH condition is higher than the binding activity of the antigen-binding molecule to the antigen in the acidic pH condition.
- the binding activity of the antigen-binding molecule to the antigen in the acidic pH range is higher than the binding activity of the antigen-binding molecule to the antigen in the neutral pH range.
- the neutral pH range is not particularly limited to a unique value, but can be preferably selected from pH 6.7 to pH 10.0.
- the pH can be selected between pH 6.7 and pH 9.5.
- the pH can be selected from between pH 7.0 and pH 9.0, and in other embodiments, the pH can be selected from between pH 7.0 and pH 8.0.
- Particularly preferred is pH 7.4, which is close to the pH in plasma (blood) in vivo.
- the acidic pH range is not particularly limited to an unambiguous numerical value, but may preferably be selected from pH 4.0 to pH 6.5.
- the pH can be selected between pH 4.5 and pH 6.5.
- the pH can be selected from between pH 5.0 and pH 6.5, and in another embodiment, the pH can be selected from between pH 5.5 and pH 6.5.
- Particularly preferred is pH 5.8 that is close to the ionized calcium concentration in the early endosome in vivo.
- the antigen-binding activity of an antigen-binding molecule at a high hydrogen ion concentration or low pH is lower than that at a low hydrogen ion concentration or high pH (pH neutral range).
- the antigen-binding activity of an antigen-binding molecule at a pH selected between pH 4.0 and pH 6.5 is greater than the antigen-binding activity at a pH selected between pH 6.7 and pH 10.0. It means weak.
- the antigen-binding molecule has an antigen-binding activity at a pH selected between pH 4.5 and pH 6.5, compared to an antigen-binding activity at a pH selected between pH 6.7 and pH 9.5.
- the binding activity of the antigen-binding molecule to the antigen at a pH selected from between pH 5.0 and pH 6.5 is selected from between pH 7.0 and pH 9.0. It means that it is weaker than the binding activity to the antigen.
- the antigen-binding molecule has an antigen-binding activity at a pH selected between pH 5.5 and pH 6.5, and has an antigen-binding activity at a pH selected between pH 7.0 and pH 8.0. Means weaker. Particularly preferably, it means that the antigen-binding activity at the pH in the early endosome in vivo is weaker than the antigen-binding activity at the pH in plasma in vivo, specifically, the antigen-binding molecule at pH 5.8. It means that the binding activity to the antigen is weaker than the binding activity to the antigen at pH 7.4.
- Whether or not the binding activity of the antigen-binding molecule to the antigen is changed depending on the pH condition can be determined by using, for example, a known measurement method as described in the above-mentioned section of binding activity. That is, the binding activity is measured under different pH conditions in the measurement method. For example, in order to confirm that the binding activity of an antigen-binding molecule to an antigen under neutral pH conditions changes higher than the binding activity of the antigen-binding molecule to an antigen under acidic pH conditions, The binding activity of the antigen binding molecule to the antigen under acidic and pH neutral conditions is compared.
- the expression “the binding activity to an antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range is lower than the binding activity to antigen under conditions of low hydrogen ion concentration or high pH ie pH neutral” is Expressing that the binding activity of an antigen-binding molecule to an antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range, is higher than the binding activity to antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range You can also.
- the binding activity to an antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range is lower than the binding activity to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral”.
- the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range is weaker than the ability to bind to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral pH.
- High hydrogen ion concentration or low pH or pH acidic binding activity to antigen is lower than low hydrogen ion concentration or high pH or pH neutral antigen binding activity
- binding activity to antigens under low pH, ie acidic pH range Sometimes referred to as weakly than the binding affinity to the antigen under the condition of frequency.
- the conditions other than the hydrogen ion concentration or pH when measuring the binding activity to the antigen can be appropriately selected by those skilled in the art and are not particularly limited.
- measurement can be performed under the conditions of HEPES buffer and 37 ° C.
- it can be measured using Biacore (GE Healthcare).
- the antigen is a soluble antigen
- the binding activity to the soluble antigen can be measured by flowing the antigen as an analyte to the chip on which the antigen-binding molecule is immobilized.
- the antigen is a membrane-type antigen
- it is possible to evaluate the binding activity to the membrane-type antigen by flowing the antigen-binding molecule as an analyte to the chip on which the antigen is immobilized. It is.
- the antigen-binding molecule of the present invention as long as the binding activity to the antigen in the condition of high hydrogen ion concentration or low pH, ie pH acidic range is weaker than the binding activity to the antigen in the condition of low hydrogen ion concentration or high pH, pH neutral range
- the ratio of the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH or pH acidic range and the binding activity to the antigen under conditions of low hydrogen ion concentration or high pH or pH neutral range is not particularly limited, but preferably Is the ratio of KD (dissociation constant) in the condition of high hydrogen ion concentration or low pH, ie pH acidic range, to KD (pH 5.
- the value of / KD (pH 7.4) is 2 or more, more preferably the value of KD (pH 5.8) / KD (pH 7.4) is 10 or more, more preferably KD (pH 5.8) / KD
- the value of (pH 7.4) is 40 or more.
- the upper limit of the value of KD (pH5.8) / KD (pH7.4) is not particularly limited, and may be any value such as 400, 1000, 10000, etc., as long as it can be produced by a person skilled in the art.
- KD dissociation constant
- apparent KD ApparentKDdissociation constant
- KD dissociation constant
- apparent KD apparent dissociation constant
- the ratio of the binding activity of the antigen-binding molecule of the present invention to the antigen in the condition of high hydrogen ion concentration or low pH, that is, pH acidic range, and the binding activity to the antigen in the condition of low hydrogen ion concentration or high pH, pH neutral range As another index to be shown, for example, kd (Dissociation rate constant) which is a dissociation rate constant can also be suitably used.
- kd dissociation rate constant
- KD dissociation constant
- the value of hydrogen ion concentration or the ratio of kd (dissociation rate constant) at high pH, i.e. neutral pH condition is preferably the value of kd (in acidic pH condition) / kd (in neutral pH condition) It is 2 or more, more preferably 5 or more, further preferably 10 or more, more preferably 30 or more.
- Kd (in the acidic pH range) / kd (in the neutral pH range) value is not particularly limited, and any value such as 50, 100, 200, etc., as long as it can be prepared by the common general knowledge of those skilled in the art But you can.
- kd dissociation rate constant
- apparent kd Apparent dissociation rate constant
- kd (dissociation rate constant) and apparent kd can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), a flow cytometer or the like can be used. Is possible.
- Biacore GE healthcare
- a flow cytometer or the like can be used. Is possible.
- the conditions other than the hydrogen ion concentration, that is, pH are preferably the same.
- the binding activity to an antigen under conditions of high hydrogen ion concentration or low pH, that is, pH acidic range is binding to antigen under conditions of low hydrogen ion concentration or high pH, that is, pH neutral range.
- An antigen-binding domain or antibody having a lower activity can be obtained by screening an antigen-binding domain or antibody comprising the following steps (a) to (c).
- the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range which is one aspect provided by the present invention, is binding to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range.
- Antigen-binding domains or antibodies with lower activity can be obtained by screening antigen-binding domains or antibodies or libraries thereof comprising the following steps (a) to (c).
- step (a) contacting an antigen-binding domain or antibody or a library thereof in a neutral pH range with an antigen; (b) placing the antigen-binding domain or antibody bound to the antigen in the step (a) under conditions of acidic pH range; (c) A step of isolating the antigen-binding domain or antibody dissociated in the step (b).
- binding activity to an antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range which is one aspect provided by the present invention, is binding to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range.
- Antigen-binding domains or antibodies with lower activity can be obtained by screening antigen-binding domains or antibodies or libraries thereof comprising the following steps (a) to (d).
- step (a) contacting the antigen-binding domain or antibody library with the antigen under acidic pH conditions; (b) selecting an antigen-binding domain or antibody that does not bind to the antigen in step (a), (c) a step of binding the antigen-binding domain or antibody selected in the step (b) to an antigen under pH neutral conditions; (d) A step of isolating the antigen binding domain or antibody bound to the antigen in the step (c).
- the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range which is one aspect provided by the present invention, is binding to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range.
- An antigen-binding domain or antibody having a lower activity can be obtained by a screening method including the following steps (a) to (c).
- step (a) contacting an antigen-binding domain or an antibody library with a column immobilized with an antigen under neutral pH conditions; (b) a step of eluting the antigen-binding domain or antibody bound to the column in the step (a) from the column under pH acidic conditions; (c) A step of isolating the antigen-binding domain or antibody eluted in the step (b).
- the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range which is one aspect provided by the present invention, is binding to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range.
- An antigen-binding domain or antibody having a lower activity can be obtained by a screening method including the following steps (a) to (d).
- step (a) passing an antigen-binding domain or an antibody library through an antigen-immobilized column under acidic pH conditions; (b) recovering the antigen-binding domain or antibody eluted without binding to the column in the step (a), (c) a step of binding the antigen-binding domain or antibody recovered in the step (b) to an antigen under pH neutral conditions; (d) A step of isolating the antigen binding domain or antibody bound to the antigen in the step (c).
- the binding activity to the antigen under conditions of high hydrogen ion concentration or low pH, ie pH acidic range which is one aspect provided by the present invention, is binding to antigen under conditions of low hydrogen ion concentration or high pH, ie pH neutral range.
- An antigen-binding domain or antibody having a lower activity can be obtained by a screening method including the following steps (a) to (d).
- step (a) contacting the antigen-binding domain or antibody library with the antigen under neutral pH conditions; (b) obtaining an antigen-binding domain or antibody bound to the antigen in the step (a), (c) a step of placing the antigen-binding domain or antibody obtained in the step (b) under conditions of an acidic pH range; (d) A step of isolating an antigen-binding domain or antibody whose antigen-binding activity is weaker than the criterion selected in the step (b) in the step (c).
- the above process may be repeated twice or more. Therefore, according to the present invention, in the above-described screening method, the conditions (a) to (c) or (a) to (d) may be repeated in the acidic pH range obtained by the screening method further comprising the step of repeating the step twice or more.
- An antigen-binding domain or antibody is provided that has a lower binding activity for the antigen than the binding activity for the antigen in the neutral pH range.
- the number of times the steps (a) to (c) or (a) to (d) are repeated is not particularly limited, but is usually within 10 times.
- the antigen-binding domain or the antigen-binding activity of the antibody in a high hydrogen ion concentration condition or low pH is not particularly limited as long as the antigen-binding activity is between pH 4.0 and 6.5.
- Preferable pH includes antigen binding activity between pH 4.5 and 6.6.
- an antigen binding activity between pH 5.0 and 6.5, and further an antigen binding activity between pH 5.5 and 6.5 can be mentioned.
- More preferable pH includes the pH in the early endosome in the living body, specifically, the antigen binding activity at pH 5.8.
- the antigen-binding activity of the antigen-binding domain or antibody at low hydrogen ion concentration conditions or high pH is not particularly limited as long as the antigen-binding activity is between 6.7 and 10, but the preferred pH is pH May have an antigen binding activity between 6.7 and 9.5.
- an antigen binding activity between pH 7.0 and 9.5, and further an antigen binding activity between pH 7.0 and 8.0 can be mentioned.
- a more preferable pH is a pH in plasma in a living body, specifically, an antigen binding activity at a pH of 7.4.
- the antigen-binding activity of an antigen-binding domain or antibody can be measured by methods known to those skilled in the art, and conditions other than the ionized calcium concentration can be appropriately determined by those skilled in the art.
- the antigen-binding activity of an antigen-binding domain or antibody is KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate: dissociation rate constant), or apparent Kd (Apparent dissociation: apparent dissociation rate constant), etc. These can be measured by methods known to those skilled in the art, and for example, Biacore® (GE® healthcare), Scatchard plot, FACS and the like can be used.
- an antigen-binding domain or antibody having an antigen-binding activity at a low hydrogen ion concentration or at a high pH, ie, pH neutral range is higher than an antigen-binding activity at a high hydrogen ion concentration, or at a low pH, pH acidic condition.
- the step of selecting an antigen-binding domain or an antibody whose antigen-binding activity under conditions of high hydrogen ion concentration or low pH, ie pH acidic range, is lower than antigen binding activity under conditions of low hydrogen ion concentration or high pH, ie pH neutral range Means the same.
- the antigen binding activity at low hydrogen ion concentration or high pH or pH neutral conditions is higher than the antigen binding activity at high hydrogen ion concentration or low pH or pH acidic conditions, it is at low hydrogen ion concentration or high pH or pH
- the antigen binding activity in is at least 2 times the antigen binding activity at high hydrogen ion concentration or low pH, ie, acidic pH range, more preferably 10 times or more, and more preferably 40 times or more.
- the antigen-binding domain or antibody of the present invention to be screened by the above screening method may be any antigen-binding domain or antibody.
- the above-described antigen-binding domain or antibody can be screened.
- an antigen binding domain or antibody having a natural sequence may be screened, or an antigen binding domain or antibody having an amino acid sequence substituted may be screened.
- the antigen-binding domain or antibody of the present invention to be screened by the above screening method may be prepared in any way, for example, a pre-existing antibody, a pre-existing library (such as a phage library), an animal Antibodies or libraries prepared from hybridomas obtained from immunization or B cells from immunized animals, amino acids with side chain pKa of 4.0-8.0 (such as histidine and glutamic acid) and unnatural amino acid mutations in these antibodies and libraries Introduced antibodies or libraries (amino acids with side chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or unnatural amino acid content increased or amino acids with side chain pKa of 4.0-8.0 at specific locations ( (For example, histidine and glutamic acid) or unnatural amino acid mutation library Etc.) can be used.
- a pre-existing antibody such as a phage library
- a method for obtaining an antigen-binding domain or antibody having an antigen-binding activity higher than the antigen-binding activity in a low pH or acidic pH range for example, at least one of the amino acid in the antigen-binding domain or antibody as described in WO2009 / 125825, Amino acids with side chain pKa of 4.0-8.0 (eg histidine or glutamic acid) or unnatural amino acid mutations or in antigen binding domains or antibodies, amino acids with side chain pKa of 4.0-8.0 (eg histidine And an antigen-binding molecule or antibody in which an unnatural amino acid is inserted.
- a person skilled in the art can appropriately determine the number of amino acids to be substituted with amino acids (for example, histidine and glutamic acid) whose side chain has a pKa of 4.0-8.0, or unnatural amino acids, or the number of amino acids to be inserted.
- amino acids for example, histidine and glutamic acid
- side chain has a pKa of 4.0-8.0, or unnatural amino acids, or the number of amino acids to be inserted.
- amino acid eg histidine or glutamic acid
- amino acid eg histidine or glutamic acid
- one amino acid eg histidine or glutamic acid
- one amino acid eg histidine or glutamic acid
- two or more amino acids with side chain pKa of 4.0-8.0 (eg histidine or glutamic acid) or unnatural amino acids
- side chain pKa of 4.0-8.0 The above amino acids (for example, histidine and glutamic acid) and unnatural amino acids can be inserted.
- substitution of amino acids with side chain pKa of 4.0-8.0 for example, histidine or glutamic acid
- non-natural amino acids for example, amino acids with side chain pKa of 4.0-8.0
- insertion of amino acids with side chain pKa of 4.0-8.0 for example, histidine or glutamic acid
- unnatural amino acids for example, amino acids with side chain pKa of 4.0-8.0
- other amino acid deletions, additions, insertions and / or substitutions can be performed simultaneously.
- Substitution with amino acids with side chain pKa of 4.0-8.0 or non-natural amino acids or amino acids with side chain pKa of 4.0-8.0 (eg histidine or glutamic acid) or insertion of unnatural amino acids
- An amino acid for example, histidine or glutamic acid
- whose side chain pKa is 4.0-8.0 or an unnatural amino acid which can be randomly performed by a method such as histidine or the like in which alanine in alanine scanning is replaced by histidine or the like KD (pH acidic range) / KD (pH neutral range) or kd (pH acidic range) / kd from antigen-binding domains or antibodies in which substitution or insertion mutations are randomly introduced compared to before the mutation
- An antigen-binding molecule having an increased (pH neutral range) value can be selected.
- an amino acid having a side chain pKa of 4.0-8.0 for example, histidine or glutamic acid
- a non-natural amino acid is mutated, and the antigen binding activity in the acidic pH range is an antigen in the neutral pH range.
- antigen-binding molecules with lower than binding activity include, for example, antigen binding in the neutral pH range after mutation to amino acids (eg, histidine or glutamic acid) whose side chain has a pKa of 4.0-8.0 or unnatural amino acids
- antigen-binding molecules whose activity is equivalent to that of an amino acid whose side chain has a pKa of 4.0-8.0 for example, histidine or glutamic acid
- an antigen-binding activity in the neutral pH range before mutation to an unnatural amino acid It is done.
- an amino acid having a side chain pKa of 4.0-8.0 (for example, histidine or glutamic acid) or an antigen-binding molecule after mutation of an unnatural amino acid is an amino acid having a side chain pKa of 4.0-8.0 (for example, histidine Or glutamic acid) or an antigen-binding molecule equivalent to that of the unnatural amino acid before the mutation, an amino acid whose side chain has a pKa of 4.0-8.0 (for example, histidine or glutamic acid) or an unnatural amino acid before the mutation
- the antigen-binding activity of an antigen-binding molecule is 100%
- the antigen-binding activity of the antigen-binding molecule after mutation of an amino acid (for example, histidine or glutamic acid) whose side chain is pKa 4.0-8.0 or an unnatural amino acid is It means at least 10% or more, preferably 50% or more, more preferably 80% or more, more preferably 90% or more.
- an antigen-binding molecule When the antigen-binding activity of an antigen-binding molecule is reduced by substitution or insertion into an amino acid (for example, histidine or glutamic acid) whose side chain has a pKa of 4.0-8.0 or an unnatural amino acid, Antigen binding activity due to substitution, deletion, addition and / or insertion of multiple amino acids, etc., before substitution or insertion of amino acids (eg histidine or glutamic acid) whose side chain has a pKa of 4.0-8.0 or unnatural amino acids It can be made equivalent to antigen binding activity.
- amino acids for example, histidine or glutamic acid
- substitution, deletion, addition and / or insertion of one or more amino acids after substitution or insertion of an amino acid for example, histidine or glutamic acid
- an amino acid for example, histidine or glutamic acid
- antigen-binding molecules whose binding activity is equivalent by performing.
- the antigen-binding molecule is a substance containing an antibody constant region
- another preferred embodiment of the antigen-binding molecule whose antigen-binding activity in the acidic pH range is lower than the antigen-binding activity in the neutral pH range is the antigen-binding molecule.
- a method in which the antibody constant region contained in is modified.
- the constant region described in SEQ ID NO: 11, 12, 13, or 14 is preferably exemplified.
- the antigen-binding domain or antibody of the present invention to be screened by the above screening method may be prepared in any way, for example, the condition of ion concentration Is a hydrogen ion concentration condition or pH condition, a pre-existing antibody, a preexisting library (such as a phage library), a hybridoma obtained from immunization of an animal, or B from an immunized animal
- Antibodies or libraries produced from cells antibodies or libraries in which amino acid (for example, histidine or glutamic acid) whose side chain pKa is 4.0-8.0 or unnatural amino acid mutations are introduced into these antibodies or libraries (side chain pKa is Amino acids that are 4.0-8.0 (eg histidine and glutamic acid) and non-natural Library pKa of the side chain to increase library or specific part of the content has mutated amino acids (e.g., histidine
- a light chain variable region and a randomized variable region sequence library into which “at least one amino acid residue that changes the binding activity of an antigen-binding molecule to an antigen depending on hydrogen ion concentration conditions” is introduced
- a library containing a plurality of antigen-binding molecules having different sequences from each other according to the present invention can also be prepared by combining with the prepared heavy chain variable region.
- amino acid residue is an amino acid residue contained in the light chain CDR1.
- amino acid residues include amino acid residues contained in the light chain CDR2.
- amino acid residues contained in CDR3 of the light chain is also exemplified.
- amino acid residues in which the amino acid residue is contained in CDR1 of the light chain examples include amino acid residues at positions 31, 32 and / or 34.
- amino acid residues that is included in the light chain CDR2 of the amino acid residue examples include amino acid residues at positions 54, 55 and / or 56.
- amino acid residue is contained in the light chain CDR3, as non-limiting examples of amino acid residues, the 89 position, 90 position, 91 position, 92 position represented by Kabat numbering in the CDR3 of the light chain variable region, Examples include amino acid residues at positions 93, 94 and / or 95A.
- these amino acid residues can be included alone, or two or more of these amino acid residues can be combined as long as the binding activity of the antigen-binding molecule to the antigen changes depending on the hydrogen ion concentration conditions. Can be included.
- a flexible residue is included in the sequence of the light chain variable region.
- the number and position of the flexible residues are not limited to a specific embodiment. That is, one or more flexible residues can be included in the CDR and / or FR sequences of the heavy and / or light chain.
- non-limiting examples of flexible residues introduced into the light chain variable region sequence include the amino acid residues listed in Table 3 or Table 4.
- the amino acid sequence of the light chain variable region other than the amino acid residue or flexible residue that changes the binding activity of the antigen-binding molecule to the antigen depending on the condition of hydrogen ion concentration is Vk1 (SEQ ID NO: 5) as a non-limiting example.
- Germline sequences such as Vk2 (SEQ ID NO: 6), Vk3 (SEQ ID NO: 7), Vk4 (SEQ ID NO: 8) can be preferably used.
- any amino acid residue can be suitably used.
- Specific examples of such amino acid residues include And amino acids having a pKa of the side chain of 4.0-8.0.
- amino acids having such electron donating properties in addition to natural amino acids such as histidine or glutamic acid, histidine analogs (US2009 / 0035836) or m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21) Or a non-natural amino acid such as 3,5-I2-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17), 3761-2768) is preferably exemplified.
- Particularly preferred examples include amino acids having a side chain pKa of 6.0 to 7.0, and histidine is a preferred example of such an amino acid having an electron donating property.
- a cell-free translation system (Clover Direct (Protein Express) in which a tRNA with a non-natural amino acid linked to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also suitable. Used.
- a randomized variable region library is preferably mentioned.
- Known methods are appropriately combined with the method for producing the randomized variable region library.
- an antibody gene derived from an animal immunized with a specific antigen, an infectious disease patient, a human who has been vaccinated, has increased blood antibody titer, a cancer patient, or an autoimmune disease lymphocyte.
- the originally constructed immune library can be suitably used as a randomized variable region library.
- the V gene in genomic DNA or the reconstructed functional V gene CDR sequence includes a sequence encoding a codon set of an appropriate length.
- Synthetic libraries substituted with oligonucleotide sets can also be suitably used as randomized variable region libraries.
- the criterion for creating amino acid diversity in the variable region of an antigen binding molecule is to provide diversity at the amino acid residues at positions exposed on the surface of the antigen binding molecule.
- a surface exposed position is a position that can be exposed to the surface and / or contacted with an antigen based on the structure, structural ensemble, and / or modeled structure of the antigen-binding molecule. Generally speaking, it is its CDR.
- the position exposed on the surface is determined using coordinates from a three-dimensional model of the antigen binding molecule using a computer program such as the Insight II program (Accelrys).
- the position exposed on the surface can be determined using algorithms known in the art (eg, Lee and Richards (J. Mol. Biol. (1971) 55, 379-400), Connolly (J. Appl. Cryst. (1983) 16, 548-558)).
- the determination of the position exposed on the surface can be performed using software suitable for protein modeling and three-dimensional structural information obtained from antibodies.
- software that can be used for such a purpose, SYBYL biopolymer module software (Tripos Associates) is preferably mentioned.
- the “size” of the probe used in the calculation is set to a radius of about 1.4 angstroms or less.
- Pacios Comput. Chem. (1994) 18 (4), 377-386 and J. Mol. Model. (1995) 1 , 46-53).
- a naive library consisting of a naive sequence that is an antibody sequence constructed from an antibody gene derived from a lymphocyte of a healthy person and having no bias in its repertoire is also a randomized variable region library.
- FcRn Unlike the Fc ⁇ receptor, which belongs to the immunoglobulin superfamily, human FcRn is structurally similar to a major tissue incompatibility complex (MHC) class I polypeptide, and has a class I MHC molecule and 22-29% sequence. Have identity (Ghetie et al., Immunol. Today (1997) 18 (12), 592-598). FcRn is expressed as a heterodimer consisting of a transmembrane ⁇ or heavy chain complexed with a soluble ⁇ or light chain ( ⁇ 2 microglobulin).
- MHC tissue incompatibility complex
- the ⁇ chain of FcRn consists of three extracellular domains ( ⁇ 1, ⁇ 2, ⁇ 3), and the short cytoplasmic domain tethers the protein to the cell surface.
- the ⁇ 1 and ⁇ 2 domains interact with the FcRn binding domain in the Fc region of antibodies (Raghavan et al. (Immunity (1994) 1, 303-315).
- FcRn is expressed in the maternal placenta or yolk sac of mammals, which is involved in the transfer of IgG from the mother to the fetus.
- the small intestine of rodent neonates that express FcRn is involved in the movement of maternal IgG across the brush border epithelium from colostrum or milk ingested FcRn.
- FcRn is expressed in many other tissues as well as various endothelial cell lines across many species. It is also expressed on human adult vascular endothelium, muscle vasculature, and liver sinusoidal capillaries.
- FcRn is thought to play a role in maintaining the plasma concentration of IgG by binding to IgG and recycling it to serum.
- the binding of FcRn to IgG molecules is usually strictly pH dependent, with optimal binding observed in the acidic pH range below 7.0.
- Human FcRn having a polypeptide containing the signal sequence represented by SEQ ID NO: 15 as a precursor is human ⁇ 2-microglobulin in vivo (SEQ ID NO: 16 describes the polypeptide containing the signal sequence). And form a complex.
- soluble human FcRn forming a complex with ⁇ 2-microglobulin is produced by using a usual recombinant expression technique. The binding activity of the Fc region of the present invention to soluble human FcRn forming a complex with such ⁇ 2-microglobulin can be evaluated.
- human FcRn refers to a form that can bind to the Fc region of the present invention, and examples include a complex of human FcRn and human ⁇ 2-microglobulin.
- Fc region Fc region comprises an amino acid sequence derived from the constant region of the antibody heavy chain.
- the Fc region is a part of the heavy chain constant region of the antibody including the hinge, CH2 and CH3 domains from the N-terminus of the hinge region of the papain cleavage site at approximately 216 amino acids represented by EU numbering.
- the binding activity of the Fc region provided by the present invention to FcRn, particularly human FcRn can be measured by methods known to those skilled in the art, as described in the above-mentioned binding activity section. Conditions can be appropriately determined by those skilled in the art.
- Antigen binding activity and human FcRn binding activity of an antigen binding molecule are KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate kd (Dissociation rate). Alternatively, it can be evaluated as apparent kd (Apparent dissociation).
- KD Dissociation constant
- apparent KD Apparent dissociation constant
- dissociation rate kd dissociation rate
- it can be evaluated as apparent kd (Apparent dissociation).
- Biacore GE healthcare
- Scatchard plot flow cytometer, etc.
- the conditions other than pH for measuring the binding activity of the Fc region of the present invention to human FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009125825, it can be measured under conditions of MES buffer and 37 ° C.
- the binding activity of the Fc region of the present invention to human FcRn can be measured by methods known to those skilled in the art, and can be measured using, for example, Biacore (GE Healthcare).
- the measurement of the binding activity between the Fc region of the present invention and human FcRn was carried out by using the Fc region or Fc region containing the human FcRn, Fc region or Fc region, respectively. It can be evaluated by flowing the antigen-binding molecule of the invention as an analyte.
- the pH neutral range as a condition having the binding activity between the Fc region and FcRn contained in the antigen-binding molecule of the present invention usually means pH 6.7 to pH 10.0.
- the pH neutral range is preferably a range indicated by any pH value from pH 7.0 to pH 8.0, preferably pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, It is selected from 7.9 and 8.0, and particularly preferably has a pH of 7.4 which is close to the pH in plasma (blood) in vivo.
- pH 7.0 can be used instead of pH 7.4.
- the acidic pH range as a condition having binding activity between the Fc region and FcRn contained in the antigen-binding molecule of the present invention usually means pH 4.0 to pH 6.5. Preferably, it means pH 5.5 to pH 6.5, and particularly preferably means pH 5.8 to pH 6.0 which is close to the pH in the early endosome in vivo.
- the temperature used for the measurement conditions the binding affinity between the human FcRn-binding domain and human FcRn may be evaluated at any temperature from 10 ° C to 50 ° C. Preferably, a temperature between 15 ° C. and 40 ° C. is used to determine the binding affinity between the human FcRn binding domain and human FcRn. More preferably, from 20 ° C.
- 35 ° C. such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 ° C. are also used to determine the binding affinity between the human FcRn binding domain and human FcRn.
- a temperature of 25 ° C. is a non-limiting example of an embodiment of the present invention.
- the human FcRn binding activity of natural human IgG1 is KD 1.7 ⁇ M in the acidic pH range (pH 6.0), but almost no activity in the neutral pH range It cannot be detected. Accordingly, in a preferred embodiment, the human FcRn binding activity in the acidic pH range is KDM20 ⁇ M or higher, and the human FcRn binding activity in the neutral pH range includes an antigen-binding molecule equivalent to or higher than that of natural human IgG.
- the antigen-binding molecule of the present invention having human FcRn binding activity in the acidic range and neutral pH range can be screened.
- the antigen-binding molecule of the present invention comprises an antigen-binding molecule having a human FcRn binding activity in the acidic pH range of KD 2.0 ⁇ M or higher and a human FcRn binding activity in the neutral pH range of KD 40 ⁇ M or higher.
- the antigen binding of the present invention comprising an antigen-binding molecule having a human FcRn binding activity in the acidic pH range of KD 0.5 ⁇ M or higher and a human FcRn binding activity in the neutral pH range of KD 15 ⁇ M or higher. Molecules can be screened.
- the KD value is determined by the method described in The Journal of Immunology (2009) 182: 7663-7671 (an antigen-binding molecule is immobilized on a chip and human FcRn is flowed as an analyte).
- an Fc region having human FcRn binding activity in the acidic pH range and neutral pH range is preferred.
- the domain can be used as it is if it is an Fc region having human FcRn binding activity in the acidic pH range and neutral pH range in advance. If the domain has no or weak human FcRn binding activity in the acidic pH range and / or neutral pH range, an Fc region with binding activity to the desired human FcRn is obtained by modifying the amino acid in the antigen-binding molecule. However, by modifying an amino acid in the human Fc region, an Fc region having binding activity for a desired human FcRn in the acidic pH range and / or neutral pH range can also be suitably obtained.
- an Fc region having a binding activity to a desired human FcRn can also be obtained by modifying an amino acid in the Fc region having a human FcRn binding activity in the acidic pH range and / or neutral pH range in advance. Amino acid modification of the human Fc region that results in such desired binding activity can be found by comparing human FcRn binding activity in the acidic pH range and / or neutral pH range before and after the amino acid modification. Those skilled in the art can appropriately modify amino acids using known techniques.
- “modification of amino acid” or “amino acid modification” of the Fc region includes modification to an amino acid sequence different from the amino acid sequence of the starting Fc region.
- the modified variant of the starting Fc region can bind to human FcRn in the acidic pH range (thus, the starting Fc region does not necessarily require binding activity to human FcRn under pH neutral conditions)
- Any Fc region can be used as the starting domain.
- an Fc region of an IgG antibody that is, a natural type Fc region is preferably exemplified.
- a modified Fc region further modified by using an Fc region that has already been modified as a starting Fc region can also be suitably used as the modified Fc region of the present invention.
- the starting Fc region can mean the polypeptide itself, a composition comprising the starting Fc region, or the amino acid sequence encoding the starting Fc region.
- the starting Fc region can include the Fc regions of known IgG antibodies produced recombinantly as outlined in the antibody section.
- the origin of the starting Fc region can be obtained from any organism or person, including but not limited to a non-human animal.
- any organism suitably includes an organism selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cows, horses, camels, and non-human primates.
- the starting Fc region can also be obtained from a cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human.
- the starting Fc region can be obtained from human IgG1, but is not limited to a particular subclass of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be appropriately used as the starting Fc region.
- the Fc region of any class or subclass of IgG from any of the aforementioned organisms can preferably be used as the starting Fc region.
- Examples of naturally occurring IgG variants or engineered forms are described in the known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4 ), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009 / 086320, WO2008 / 092117, WO2007 / 041635, and WO2006 / 105338) Not.
- the modification include one or more mutations, for example, a mutation substituted with an amino acid residue different from the amino acid of the starting Fc region, or insertion of one or more amino acid residues with respect to the amino acid of the starting Fc region or the starting Fc region. Deletion of one or more amino acids from these amino acids.
- the amino acid sequence of the modified Fc region includes an amino acid sequence including at least a part of the Fc region that does not occur in nature.
- Such variants necessarily have less than 100% sequence identity or similarity with the starting Fc region.
- the variant has about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting Fc region, more preferably about 80% to less than 100%, more preferably about 85% to 100%.
- amino acid difference between the starting Fc region and the modified Fc region of the invention there is at least one amino acid difference between the starting Fc region and the modified Fc region of the invention.
- the amino acid difference between the starting Fc region and the modified Fc region can also be suitably specified, particularly by the amino acid difference in which the position of the amino acid residue represented by the aforementioned EU numbering is specified.
- a cell-free translation system (Clover Direct (Protein Express) in which a tRNA with a non-natural amino acid linked to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also suitable. Used.
- the Fc region having binding activity to human FcRn in the neutral pH range contained in the antigen-binding molecule of the present invention can be obtained by any method. Specifically, the human IgG-type immunoglobulin used as the starting Fc region can be obtained. By modifying the amino acid, an Fc region having binding activity to human FcRn in a neutral pH range can be obtained.
- Preferred Fc regions of IgG type immunoglobulin for modification include, for example, Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and variants thereof).
- the amino acid at any position can be modified. If the antigen-binding molecule contains the Fc region of human IgG1 as the human Fc region, modifications that have the effect of enhancing the binding to human FcRn in the neutral pH range over the binding activity of the starting Fc region of human IgG1 are included. It is preferable.
- amino acids that can be modified include EU numbering 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343, 345 , 360, 362, 370, 375-378, 380, 382, 385-387, 389, 396, 414, 416, 423, 424, 426, 438 And amino acids at positions 440 and 442. More specifically, for example, amino acid modifications as shown in Table 5 can be mentioned. These amino acid modifications enhance binding to human FcRn in the pH neutral range of the Fc region of IgG immunoglobulin.
- a modification that enhances binding to human FcRn even in the neutral pH range is appropriately selected.
- Fc region variant amino acids for example, represented by EU numbering 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 360, 376 , 380, 382, 384, 385, 386, 386, 387, 389, 424, 428, 433, 434 and 436.
- EU numbering of the Fc region Met is the amino acid at position 237.
- the amino acid at position 248 250 of the amino acid is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, 252 of the amino acid is Phe, Trp, or Tyr, Thr, the amino acid at position 254 Glu for the 255th amino acid
- the 256th amino acid is Asp, Asn, Glu, or Gln
- 257 of the amino acid is one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val
- the amino acid at position 258 is His, Ala for the amino acid at position 265 286 of the amino acid is either Ala or Glu
- an antigen-binding molecule is used as the broadest meaning of a molecule containing an antigen-binding domain and an Fc region. Specifically, as long as they exhibit binding activity against an antigen, various molecules can be used. The type is included.
- an antibody is an example of a molecule in which an antigen-binding domain is bound to an Fc region.
- Antibodies can include single monoclonal antibodies (including agonist and antagonist antibodies), human antibodies, humanized antibodies, chimeric antibodies, and the like. Further, when used as an antibody fragment, an antigen-binding domain and an antigen-binding fragment (for example, Fab, F (ab ′) 2, scFv and Fv) can be preferably mentioned.
- a scaffold molecule in which a three-dimensional structure such as an existing stable ⁇ / ⁇ barrel protein structure is used as a scaffold, and only a part of the structure is made into a library for the construction of an antigen-binding domain is also an antigen of the present invention. It can be included in the binding molecule.
- the antigen-binding molecule of the present invention can contain at least part of the Fc region that mediates binding to FcRn and binding to Fc ⁇ receptor.
- the antigen binding molecule can be an antibody or an Fc fusion protein.
- a fusion protein refers to a chimeric polypeptide comprising a polypeptide comprising a first amino acid sequence linked to a polypeptide having a second amino acid sequence that is not naturally linked in nature.
- the fusion protein may comprise an amino acid sequence encoding at least a portion of an Fc region (eg, a portion of an Fc region that confers binding to FcRn or a portion of an Fc region that confers binding to an Fc ⁇ receptor), and, for example, ligand binding of a receptor
- a non-immunoglobulin polypeptide comprising an amino acid sequence encoding a receptor binding domain of the domain or ligand can be included.
- the amino acid sequences can be in separate proteins that are brought together into the fusion protein, or they can usually be in the same protein, but are put into a new rearrangement in the fusion polypeptide. Fusion proteins can be made, for example, by chemical synthesis or by recombinant techniques that create and express a polynucleotide in which the peptide region is encoded in the desired relationship.
- Each domain of the present invention can be directly linked by a polypeptide bond or linked via a linker.
- the linker any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (for example, the protein disclosed in Protein Engineering (1996) 9 (3), 299-305) can be used. In this case, a peptide linker is preferable.
- the length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably Is 20 amino acids or less), particularly preferably 15 amino acids.
- Synthetic chemical linkers are commonly used for cross-linking peptides such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) Suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene Glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy
- linker When a plurality of linkers linking each domain are used, the same type of linker can be used, or different types of linkers can also be used.
- a linker having a peptide tag such as a His tag, an HA tag, a myc tag, or a FLAG tag
- bonds together by a hydrogen bond, a disulfide bond, a covalent bond, an ionic interaction, or the combination of these bonds can also be utilized suitably.
- the affinity between CH1 and CL of an antibody is used, or the Fc region originating from the above-mentioned bispecific antibody is used for the association of hetero Fc regions.
- a disulfide bond formed between domains can also be suitably used.
- the polynucleotides encoding the domains are linked in-frame.
- Methods for ligating polynucleotides in-frame include known methods such as restriction fragment ligation, fusion PCR, and overlap PCR, and these methods can be used alone or in combination as appropriate for the production of the antigen-binding molecule of the present invention. Can be used.
- the terms “linked”, “fused”, “linked” or “fused” are used interchangeably. These terms refer to linking elements or components such as two or more polypeptides so as to form one structure by all means including the above-described chemical bonding means or recombinant techniques.
- Fusion in frame means that when two or more elements or components are polypeptides, two or more open reading frames to form a continuous longer reading frame so as to maintain the correct reading frame of the polypeptide.
- a bimolecular Fab is used as an antigen-binding domain
- an antibody that is an antigen-binding molecule of the present invention in which the antigen-binding domain and the Fc region are linked in-frame by a peptide bond without using a linker is suitable for the present application.
- Fc ⁇ receptor Fc ⁇ receptor refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and includes virtually any member of the protein family encoded by the Fc ⁇ receptor gene. means.
- this family includes Fc ⁇ RI (CD64) including isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; isoforms Fc ⁇ RIIa (including allotypes H131 and R131), Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2) and Fc ⁇ RIIc Fc ⁇ RII (CD32); and isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2), and any undiscovered human Fc ⁇ Rs or Fc ⁇ R isoforms Although allotype is also included, it is not limited to these.
- Fc ⁇ R may be derived from any organism, including but not limited to humans, mice, rats, rabbits and monkeys.
- Mouse Fc ⁇ Rs include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16) and Fc ⁇ RIII-2 (Fc ⁇ RIV, CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes However, it is not limited to these.
- Suitable examples of such Fc ⁇ receptors include human Fc ⁇ RI (CD64), Fc ⁇ RIIa (CD32), Fc ⁇ RIIb (CD32), Fc ⁇ RIIIa (CD16) and / or Fc ⁇ RIIIb (CD16).
- the polynucleotide sequence and amino acid sequence of human Fc ⁇ RI are SEQ ID NO: 25 (NM_000566.3) and 26 (NP_000557.1), respectively.
- the polynucleotide sequence and amino acid sequence of human Fc ⁇ RIIa are respectively SEQ ID NO: 27 (BC020823). .1) and 28 (AAH20823.1) (allotype R131 is a sequence in which the 166th amino acid of SEQ ID NO: 28 is substituted with Arg), the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIb are SEQ ID NO: 29, respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIIa are SEQ ID NO: 31 (BC033678.1) and 32 (AAH33678.1), respectively, and the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIIb.
- the sequences are described in SEQ ID NOs: 33 (BC128562.1) and 34 (AAI28563.1), respectively (the RefSeq registration numbers are shown in parentheses).
- allotype V158 is used in Reference Example 27 and the like, allotype F158 is used unless otherwise specified, as indicated by Fc ⁇ RIIIaV. It is not construed as limiting.
- Fc ⁇ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies, in addition to the FACS and ELISA formats described above, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and surface plasmon It can be confirmed by the BIACORE method using the resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
- SPR resonance
- Fc ligand or “effector ligand” means a molecule, preferably a polypeptide, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of the Fc ligand to Fc preferably induces one or more effector functions.
- Fc ligands include Fc receptor, Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein G and viral Fc ⁇ R. However, it is not limited to these.
- Fc ligands also include Fc receptor homologues (FcRH), a family of Fc receptors homologous to Fc ⁇ R (Davisaviet al., (2002) Immunological Reviews 190, 123-136). Fc ligands can also include undiscovered molecules that bind to Fc.
- FcRH Fc receptor homologues
- Fc ⁇ R Fc receptor homologues
- Fc ligands can also include undiscovered molecules that bind to Fc.
- Fc ⁇ RIa including Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc (CD64) and isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2) and Fc ⁇ RIII (CD16) of IgG
- the binding ⁇ chain associates with a common ⁇ chain having ITAM that transmits an activation signal into the cell.
- ITAM is contained in the own cytoplasmic domain of isoforms Fc ⁇ RIIa (including allotypes H131 and R131) and Fc ⁇ RII (CD32) containing Fc ⁇ RIIc.
- Fc ⁇ receptor having the ability to transmit an activation signal as described above is also referred to as an active Fc ⁇ receptor in the present invention.
- the cytoplasmic domain of Fc ⁇ RIIb contains ITIM that transmits inhibitory signals.
- the activation signal from BCR is suppressed by cross-linking of Fc ⁇ RIIb and B cell receptor (BCR), resulting in suppression of BCR antibody production.
- BCR B cell receptor
- macrophages phagocytic ability and ability to produce inflammatory cytokines are suppressed by cross-linking of Fc ⁇ RIII and Fc ⁇ RIIb.
- the Fc ⁇ receptor having the ability to transmit an inhibitory signal as described above is also called an inhibitory Fc ⁇ receptor in the present invention.
- ALPHA screen is implemented based on the following principle by ALPHA technology using two beads of donor and acceptor.
- a molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity.
- a photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted.
- the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
- an antigen-binding molecule containing a biotin-labeled Fc region is bound to a donor bead, and an Fc ⁇ receptor tagged with glutathione S-transferase (GST) is bound to an acceptor bead.
- GST glutathione S-transferase
- polypeptide aggregates with wild-type Fc region interact with Fc ⁇ receptors to produce a signal of 520-620 nm.
- Antigen binding molecules comprising untagged Fc region variants compete with the interaction between an antigen binding molecule having a native Fc region and the Fc ⁇ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from competition.
- biotinylate antigen-binding molecules such as antibodies using Sulfo-NHS-biotin or the like.
- a method of tagging Fc ⁇ receptor with GST it is expressed in a cell or the like held in a vector in which a fusion gene in which a polynucleotide encoding Fc ⁇ receptor and a polynucleotide encoding GST are fused in frame is operatively linked
- a method of purification using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
- the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
- Kinetics association rate constant (ka) and dissociation rate constant (kd) are obtained from the sensorgram curve, and affinity (KD) is obtained from the ratio of the constants.
- an inhibition measurement method is also preferably used. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
- antigen binding can be achieved by the formation of a heterocomplex comprising the Fc region contained in the antigen-binding molecule, four molecules of FcRn and one molecule of active Fc ⁇ receptor.
- pharmacokinetics retention in plasma
- immune response immune response against the administered antigen-binding molecule as follows:
- FcRn is expressed on immune cells in addition to various active Fc ⁇ receptors, and the formation of such a quaternary complex on an immune cell indicates that affinity for immune cells is increased.
- antigen-presenting cells by improving and further associating the intracellular domain, the internalization signal is enhanced and the uptake into immune cells is promoted.
- antigen-presenting cells it is suggested that the formation of a quaternary complex on the cell membrane of antigen-presenting cells may facilitate the incorporation of antigen-binding molecules into antigen-presenting cells.
- antigen-binding molecules taken up by antigen-presenting cells are degraded in lysosomes within the antigen-presenting cells and presented to T cells.
- formation of the above four-component complex on the cell membrane of the antigen-presenting cell may promote the uptake of the antigen-binding molecule into the antigen-presenting cell, thereby deteriorating the retention of the antigen-binding molecule in plasma.
- an immune response can be induced (exacerbated).
- an antigen-binding molecule with reduced ability to form such a four-component complex is administered to a living body, the retention of the antigen-binding molecule in plasma is improved, and induction of an immune response by the living body is suppressed. Can be considered.
- antigen-binding molecules that inhibit the formation of the complex on immune cells including such antigen-presenting cells the following three types may be mentioned.
- the antigen-binding molecule of aspect 1 forms a ternary complex by binding to two FcRn molecules, but does not form a complex including active Fc ⁇ R (FIG. 49).
- the binding activity to active Fc ⁇ R is natural.
- An Fc region lower than the binding activity of the Fc region to the active Fc ⁇ R can be produced by modifying the amino acid of the natural Fc region as described above. Whether or not the binding activity of the modified Fc region to the active Fc ⁇ R is lower than the binding activity of the natural Fc region to the active Fc ⁇ R can be appropriately performed using the method described in the above-mentioned section of binding activity.
- Active Fc ⁇ receptors include Fc ⁇ RIa (including Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc (CD64), Fc ⁇ RIIa (including allotypes R131 and H131), and isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (allotypes Fc ⁇ RIIIb-III and Rc)
- Fc ⁇ RIII CD16
- NA2 containing NA2.
- the pH conditions for measuring the binding activity between the Fc region and the Fc ⁇ receptor contained in the antigen-binding molecule of the present invention can be appropriately selected from pH acidic range or pH neutral range.
- the pH neutral range as a condition for measuring the binding activity between the Fc region and Fc ⁇ receptor contained in the antigen-binding molecule of the present invention usually means pH 6.7 to pH 10.0.
- Preferably in the range indicated by any pH value between pH 7.0 and pH 8.0 preferably selected from pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0.
- Particularly preferred is pH 7.4, which is close to the pH in plasma (blood) in vivo.
- the acidic pH range as a condition having the binding activity between the Fc region and Fc ⁇ receptor contained in the antigen-binding molecule of the present invention usually means pH 4.0 to pH 6.5. Preferably, it means pH 5.5 to pH 6.5, and particularly preferably means pH 5.8 to pH 6.0 which is close to the pH in the early endosome in vivo.
- the temperature used for the measurement conditions the binding affinity between the Fc region and the human Fc ⁇ receptor can be evaluated at any temperature between 10 ° C. and 50 ° C. Preferably, a temperature of 15 ° C. to 40 ° C. is used to determine the binding affinity between the human Fc region and the Fc ⁇ receptor. More preferably from 20 ° C.
- 35 ° C. such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 ° C. Is also used to determine the binding affinity between the Fc region and the Fc ⁇ receptor.
- a temperature of 25 ° C. is a non-limiting example of an embodiment of the present invention.
- the binding activity of the Fc region variant to the active Fc ⁇ receptor is lower than the binding activity of the natural Fc region to the active Fc ⁇ receptor.
- the binding activity to any human Fc ⁇ receptor is lower than the binding activity of the natural Fc region to these human Fc ⁇ receptors.
- the binding activity of the antigen-binding molecule containing the Fc region variant is 95% or less, preferably 90%, compared to the binding activity of the antigen-binding molecule containing the natural Fc region as a control.
- % Or less 85% or less, 80% or less, 75% or less, particularly preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2%
- the binding activity is 1% or less.
- the native Fc region the starting Fc region can be used, and the Fc regions of different isotypes of the wild-type antibody can also be used.
- the binding activity to the natural active Fc ⁇ R is preferably the binding activity of human IgG1 to the Fc ⁇ receptor.
- the binding activity to the Fc ⁇ receptor can be reduced by expressing an antigen-binding molecule containing an Fc region having binding activity to the Fc ⁇ receptor in a host to which no sugar chain is added, such as Escherichia coli. Can do.
- an antigen-binding molecule having an Fc region of an IgG monoclonal antibody can be used as appropriate.
- the structure of the Fc region is SEQ ID NO: 1 (A is added to the N terminus of RefSeq registration number AAC82527.1), 2 (A is added to the N terminus of RefSeq registration number AAB59393.1), 3 (RefSeq registration number CAA27268.1) ), 4 (RefSeq registration number AAB59394.1, N appended to the end of A).
- an antigen-binding molecule containing the Fc region of an antibody of a specific isotype as a test substance, by using the antigen-binding molecule having the Fc region of the IgG monoclonal antibody of the specific isotype as a control, The effect of the binding activity on the Fc ⁇ receptor by the antigen-binding molecule containing the Fc region is verified. As described above, an antigen-binding molecule containing an Fc region verified to have a high binding activity to the Fc ⁇ receptor is appropriately selected.
- the binding activity to active Fc ⁇ R is lower than the binding activity of the natural Fc region to active Fc ⁇ R.
- amino acids in the Fc region any one or more of the amino acids 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, 328, and 329 represented by EU numbering are the natural Fc region. Suitable examples include Fc regions that have been modified to different amino acids, but the modification of the Fc region is not limited to the above-described modifications.
- the Fc region described in Current Opinion in Biotechnology (2009) 20 (6), 685-691 Glycan (N297A, N297Q), IgG1-L234A / L235A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-L234F / L235E / P331S, IgG1- Modifications such as S267E / L328F, IgG2-V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, IgG4-L236E, and G236R / L328R described in WO 2008/092117
- the amino acid represented by EU numbering of the Fc region; 234 of amino acid Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr or Trp, 235 of the amino acid Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, one of Val or Arg, 236 of the amino acid Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr, 237 of the amino acid Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg, 238 of the amino acid is Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg, 239th amino acid Gln, His
- Mode 2 An antigen-binding molecule that has an FcRn-binding activity under neutral pH conditions, and has an Fc region that has a higher binding activity to the inhibitory Fc ⁇ R than the binding activity to the active Fc ⁇ receptor.
- the antigen-binding molecule of embodiment 2 can form a complex containing these four components by binding to two molecules of FcRn and one molecule of inhibitory Fc ⁇ R.
- one molecule of antigen-binding molecule can bind only to one molecule of Fc ⁇ R, one molecule of antigen-binding molecule cannot bind to other active Fc ⁇ R in a state of binding to inhibitory Fc ⁇ R (FIG. 50).
- antigen-binding molecules taken into cells while bound to inhibitory Fc ⁇ R are recycled onto the cell membrane to avoid degradation in the cells (Immunity (2005) 23, 503-514). That is, it is considered that an antigen-binding molecule having a selective binding activity to the inhibitory Fc ⁇ R cannot form a hetero complex including the active Fc ⁇ R causing the immune response and two molecules of FcRn.
- Active Fc ⁇ receptors include Fc ⁇ RIa (including Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc (CD64), Fc ⁇ RIIa (including allotypes R131 and H131), and isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (allotypes Fc ⁇ RIIIb-III and Rc) Preferred examples include Fc ⁇ RIII (CD16) containing NA2.
- Fc ⁇ RIIb (including Fc ⁇ RIIb-1 and Fc ⁇ RIIb-2) is a preferred example of an inhibitory Fc ⁇ receptor.
- the binding activity to the inhibitory Fc ⁇ R is higher than the binding activity to the active Fc ⁇ receptor means that the binding activity of the Fc region variant to Fc ⁇ RIIb is any one of Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa and / or Fc ⁇ RIIIb. It means higher than the binding activity to Fc ⁇ receptor.
- the binding activity of the antigen-binding molecule containing the Fc region variant to Fc ⁇ RIIb is 105% or more of the binding activity of any one of Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa and / or Fc ⁇ RIIIb to the human Fc ⁇ receptor.
- the binding activity to Fc ⁇ RIIb is all higher than Fc ⁇ RIa, Fc ⁇ RIIa (including allotypes R131 and H131) and Fc ⁇ RIIIa (including allotypes V158 and F158). Since Fc ⁇ RIa has an extremely high affinity for native IgG1, it is considered that the binding is saturated by a large amount of endogenous IgG1 in vivo. However, it is considered possible to inhibit the formation of the complex.
- an antigen-binding molecule having an Fc region of an IgG monoclonal antibody can be used as appropriate.
- the structure of the Fc region is as follows: SEQ ID NO: 11 (A added to the N terminus of RefSeq registration number AAC82527.1), 12 (A added to the N terminus of RefSeq registration number AAB59393.1), 13 (RefSeq registration number CAA27268.1) ), 14 (RefSeq registration number AAB59394.1, N appended to the end of A).
- an antigen-binding molecule containing the Fc region of an antibody of a specific isotype as a test substance, by using the antigen-binding molecule having the Fc region of the IgG monoclonal antibody of the specific isotype as a control, The effect of the binding activity on the Fc ⁇ receptor by the antigen-binding molecule containing the Fc region is verified. As described above, an antigen-binding molecule containing an Fc region verified to have a high binding activity to the Fc ⁇ receptor is appropriately selected.
- an Fc region having selective binding activity to inhibitory Fc ⁇ R 238 or 328 amino acids represented by EU numbering among the amino acids of the Fc region are natural Fc regions.
- Preferred examples include Fc regions that have been modified to different amino acids.
- the Fc region having selective binding activity to the inhibitory Fc ⁇ receptor the Fc region or modification described in US 2009/0136485 can be appropriately selected.
- 238 amino acids represented by EU numbering in the Fc region are modified as any one or more of Asp and 328 amino acids represented by EU numbering.
- Preferred examples include Fc regions.
- substitution of Pro at position 238 represented by EU numbering with Asp, and amino acid at position 237 represented by EU numbering are at position 237 represented by Trp and EU numbering.
- Amino acid is Phe
- amino acid at position 267 represented by EU numbering is Val
- amino acid at position 267 represented by EU numbering is Gln
- amino acid at position 268 represented by EU numbering is Asn
- 271 is represented by EU numbering
- the amino acid at the position is Gly
- the amino acid at position 326 represented by EU numbering is Leu
- the amino acid at position 326 represented by EU numbering is Gln
- the amino acid at position 326 represented by EU numbering is represented by Glu
- EU numbering is represented by EU numbering.
- the amino acid at position 237 represented by EU numbering is Leu, the amino acid at position 237 represented by EU numbering is Met, the amino acid at position 237 represented by EU numbering is Tyr, and the amino acid at position 330 represented by EU numbering is Lys, the amino acid at position 330 represented by EU numbering is Arg, the amino acid at position 233 represented by EU numbering is Asp, the amino acid at position 268 represented by EU numbering is Asp, and the amino acid at position 268 represented by EU numbering
- the antigen-binding molecule of aspect 3 can form a ternary complex by binding to one molecule of FcRn and one molecule of Fc ⁇ R, but a heterocomplex containing two molecules of FcRn and one molecule of Fc ⁇ R is Not formed (FIG. 51).
- One of the two polypeptides constituting the Fc region contained in the antigen-binding molecule of this embodiment 3 has binding activity to FcRn under pH neutral conditions, and the other polypeptide has pH neutral pH range.
- An Fc region originating from a bispecific antibody (bispecific antibody) can be used as appropriate as an Fc region that does not have an activity of binding to FcRn under conditions.
- Bispecific antibodies are two types of antibodies that have specificity for different antigens.
- An IgG-type bispecific antibody can be secreted by hybrid hybridoma (quadroma) produced by fusing two hybridomas producing IgG antibody (Milstein et al. (Nature (1983) 305, 537-540)) .
- the antigen-binding molecule of the above-described embodiment 3 is produced using a recombinant technique as described in the above-mentioned antibody section, a gene encoding a polypeptide constituting the two types of target Fc regions is introduced into the cell. A method of co-expressing them can be employed.
- the produced Fc region has one of the two polypeptides constituting the Fc region having binding activity to FcRn under pH neutral conditions, and the other polypeptide under pH neutral condition conditions.
- the Fc region that does not have FcRn binding activity and the two polypeptides that constitute the Fc region constitute an Fc region that has FcRn binding activity under pH neutral conditions.
- An Fc region in which both of the two polypeptides have no binding activity to FcRn under pH neutral conditions is a mixture having a ratio of the number of molecules of 2: 1: 1. It is difficult to purify an antigen-binding molecule containing the desired combination of Fc regions from three types of IgG.
- an antigen-binding molecule containing a heterogeneous Fc region is preferentially added by modifying the CH3 domain constituting the Fc region with an appropriate amino acid substitution.
- the amino acid side chain present in the CH3 domain of one heavy chain is replaced with a larger side chain (knob), and the amino acid side present in the CH3 domain of the other heavy chain
- WO 1996027011, Ridgway et al. Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat. Biotech. (1998) 16, 77 677-681).
- bispecific antibodies by using a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region.
- a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region are known. That is, by altering the amino acid residues that form the interface in the two polypeptides constituting the Fc region, the association of the polypeptides constituting the Fc region having the same sequence is inhibited, and two Fc regions having different sequences
- a method of controlling the formation of a polypeptide aggregate that constitutes can be employed for the production of bispecific antibodies (WO2006 / 106905). Such a method can also be employed in producing the antigen-binding molecule of aspect 3 of the present invention.
- two polypeptides constituting the Fc region originating from the above bispecific antibody can be used as appropriate. More specifically, the two polypeptides constituting the Fc region, wherein 349 amino acids represented by EU numbering in the amino acid sequence of one of the polypeptides are Cys, 366 amino acids are Trp, and the other Two polypeptides characterized in that 356 amino acids represented by EU numbering are Cys, 366 amino acids are Ser, 368 amino acids are Ala, and 407 amino acids are Val. Is preferably used.
- the Fc region includes two polypeptides constituting the Fc region, and 409 amino acids represented by EU numbering in the amino acid sequence of one of the polypeptides are Asp.
- Two polypeptides characterized in that 399 amino acids represented by EU numbering in the amino acid sequence of the other polypeptide are Lys are preferably used.
- 409 amino acids may be Glu instead of Asp
- 399 amino acids may be Arg instead of Lys.
- Asp may be suitably added as 360 amino acids or Asp as 392 amino acids.
- the Fc region includes two polypeptides constituting the Fc region, and 370 amino acids represented by EU numbering in the amino acid sequence of one of the polypeptides are Glu And two polypeptides characterized in that the 357 amino acids represented by EU numbering in the amino acid sequence of the other polypeptide are Lys.
- the Fc region includes two polypeptides constituting the Fc region, and 439 amino acids represented by EU numbering in the amino acid sequence of one of the polypeptides.
- Two polypeptides that are Glu and have 356 amino acids represented by EU numbering in the amino acid sequence of the other polypeptide are preferably used.
- the Fc region is any of the following embodiments in which these are combined: Two polypeptides constituting the Fc region, wherein 409 amino acids represented by EU numbering in the amino acid sequence of one polypeptide are Asp, 370 amino acids are Glu, and the other polypeptide amino acid sequence Among the two polypeptides characterized in that 399 amino acids represented by EU numbering are Lys and 357 amino acids are Lys (in this embodiment, Asp instead of Glu of 370 amino acids represented by EU numbering) It may be 392 amino acids Asp instead of 370 amino acids Glu represented by EU numbering), Two polypeptides constituting the Fc region, wherein 409 amino acids represented by EU numbering of the amino acid sequence of one polypeptide are Asp, 439 amino acids are Glu, and the other polypeptide amino acid sequence Among them, two polypeptides characterized in that 399 amino acids represented by EU numbering are Lys and 356 amino acids are Lys (in this embodiment, 360 instead of
- two polypeptides constituting the Fc region wherein 356 amino acids represented by EU numbering in the amino acid sequence of one of the polypeptides are Lys. Also, two polypeptides characterized in that 435 amino acids represented by EU numbering in the amino acid sequence of the other polypeptide are Arg and 439 amino acids are Glu are also preferably used.
- two polypeptides constituting the Fc region wherein 356 amino acids represented by EU numbering among the amino acid sequences of one of the polypeptides are Lys, 357 Two polypeptides, wherein the amino acid is Lys, 370 amino acids represented by EU numbering in the amino acid sequence of the other polypeptide are Glu, 435 amino acids are Arg, and 439 amino acids are Glu are also preferably used.
- antigen-binding molecules of modes 1 to 3 can all reduce immunogenicity and improve plasma retention compared to antigen-binding molecules that can form a four-component complex. Be expected.
- Reduced immune response Whether or not the immune response to the antigen-binding molecule of the present invention has been modified can be evaluated by measuring the response of a living body administered with a pharmaceutical composition containing the antigen-binding molecule as an active ingredient.
- the biological response includes mainly cellular immunity (induction of cytotoxic T cells that recognize peptide fragments of antigen-binding molecules bound to MHC class I) and humoral immunity (induction of production of antibodies that bind to antigen-binding molecules).
- cellular immunity induction of cytotoxic T cells that recognize peptide fragments of antigen-binding molecules bound to MHC class I
- humoral immunity induction of production of antibodies that bind to antigen-binding molecules.
- immunogenicity antibody production against an administered antigen-binding molecule is called immunogenicity.
- In vivo immune response can be evaluated by measuring the antibody titer when an antigen-binding molecule is administered to a living body. For example, when the antibody titer when the antigen binding molecule of A and B is administered to a mouse is measured and the antibody titer of the antigen binding molecule of A is higher than that of B, or when administered to multiple mice, When the incidence of individuals with increased antibody titers is higher when the antigen-binding molecule of A is administered, it is judged that A is more immunogenic than B.
- the antibody titer can be measured by using a method for measuring a molecule that specifically binds to an administered molecule using ELISA, ECL, or SPR known in the art (J. Pharm. Biomed. Anal. (2011) 55 (5), 878-888).
- human peripheral blood mononuclear cells isolated from a donor and antigen-binding molecules can be evaluated in vitro.
- helper T cells that react and react or proliferate or the amount of cytokines produced (Clin. Immunol. (2010) 137 (1), 5-14, Drugs R D. (2008) 9 (6), 385-396).
- the antigen-binding molecules of A and B were evaluated by such an in vitro immunogenicity test, the antigen-binding molecule of A was more reactive than B, or it was evaluated with multiple donors.
- the antigen-binding molecule of A has a higher positive rate of reaction, it is judged that A is more immunogenic than B.
- antigen binding molecules having FcRn binding activity in the neutral pH range are four molecules of two molecules of FcRn and one molecule of Fc ⁇ R on the cell membrane of antigen-presenting cells. It is considered that an immune response is easily induced because the uptake into antigen-presenting cells is promoted because it is possible to form a hetero-complex containing.
- Phosphorylation sites exist in the intracellular domains of Fc ⁇ R and FcRn. In general, phosphorylation of the intracellular domain of a receptor expressed on the cell surface occurs when the receptors associate with each other, and internalization of the receptor occurs due to the phosphorylation.
- Heterocomplex formation involving the Fc ⁇ R / bimolecular FcRn / IgG quadratic is thought to occur on antigen-presenting cells that express both Fc ⁇ R and FcRn, thereby increasing the amount of antibody molecules taken up by the antigen-presenting cells. There is a possibility that the immunogenicity will deteriorate as a result.
- the uptake into the antigen-presenting cell is reduced, and as a result There is a possibility of improving the originality.
- the concentration of ions is reduced so that the binding activity to the antigen in the acidic pH range is lower than the binding activity to the antigen in the neutral pH range.
- an antigen-binding molecule containing an antigen-binding domain whose binding activity to an antigen changes depending on conditions and an Fc region having binding activity to human FcRn under neutral pH conditions it is applied to cells in the body.
- the reasons why the increased uptake increases the number of antigens that can be bound by a single antigen-binding molecule and the promotion of the disappearance of plasma antigen concentration can be explained, for example, as follows: It is.
- an antibody in which an antigen-binding molecule binds to a membrane antigen when administered into a living body, the antibody binds to the antigen, and then is taken into an endosome in the cell by internalization while being bound to the antigen. Thereafter, the antibody that has transferred to the lysosome while bound to the antigen is degraded by the lysosome together with the antigen. Disappearance in plasma through internalization is called antigen-dependent disappearance and has been reported for many antibody molecules (Drug Discov Today (2006) 11 (1-2), 81-88).
- IgG molecules taken into endosomes by pinocytosis bind to human FcRn expressed in endosomes under acidic conditions in endosomes.
- IgG molecules that fail to bind to human FcRn are degraded in the subsequently transferred lysosomes.
- IgG molecules bound to human FcRn migrate to the cell surface. Since IgG molecules dissociate from human FcRn under neutral conditions in plasma, the IgG molecules are recycled again into plasma.
- an antibody in which an antigen-binding molecule binds to a soluble antigen the antibody administered in vivo binds to the antigen, and then the antibody is taken into the cell while bound to the antigen. Many of the antibodies taken up into cells move to the cell surface after binding to FcRn in the endosome. Under neutral conditions in plasma, the antibody dissociates from human FcRn and is therefore released extracellularly.
- an antibody containing a normal antigen-binding domain whose binding activity to the antigen does not change depending on the condition of ion concentration such as pH is released outside the cell while bound to the antigen, and cannot bind to the antigen again. Therefore, like an antibody that binds to a membrane antigen, a normal single-molecule IgG antibody that does not change its binding activity to an antigen depending on ion concentration conditions such as pH cannot bind to three or more antigens.
- An antibody that binds to an antigen in a pH-dependent manner that binds strongly to the antigen under neutral pH conditions in plasma, and dissociates from the antigen under acidic pH conditions in the endosome Antibodies that bind under neutral pH conditions and dissociate under acidic pH conditions) and antigens bind to antigens under high calcium ion concentrations in plasma, and low calcium ion concentrations in endosomes
- An antibody that binds to an antigen in a calcium ion concentration-dependent manner an antibody that binds to an antigen under a high calcium ion concentration condition and dissociates under a low calcium ion concentration condition It is possible to dissociate from the antigen within the endosome.
- An antibody that binds to an antigen in a pH-dependent manner or an antigen that binds to an antigen in a calcium ion concentration-dependent manner can be bound to the antigen again after being dissociated and then recycled into plasma by FcRn. It is. Therefore, one molecule of antibody can repeatedly bind to a plurality of antigen molecules.
- the antigen bound to the antigen-binding molecule is dissociated from the antibody in the endosome and is not recycled into the plasma but is degraded in the lysosome.
- An antibody that binds to an antigen in a pH-dependent manner that binds strongly to the antigen under neutral pH conditions in plasma, and dissociates from the antigen under acidic pH conditions in the endosome Antibodies that bind under neutral pH conditions and dissociate under acidic pH conditions) and antigens bind to antigens under high calcium ion concentrations in plasma, and low calcium ion concentrations in endosomes
- An antibody that binds to an antigen in a calcium ion concentration-dependent manner an antibody that binds to an antigen under a high calcium ion concentration condition and dissociates under a low calcium ion concentration condition).
- the antigen binds to the antigen in a pH-dependent manner (binding under neutral pH conditions and dissociates under acidic pH conditions), or it binds to the antigen in a calcium ion concentration-dependent manner.
- an antibody that binds binds under conditions of high calcium ion concentration and dissociates under conditions of low calcium ion concentration
- the rate of disappearance is thought to be equal to the rate of uptake of the antibody and its antibody-antigen complex into cells by nonspecific endocytosis.
- IgG type immunoglobulin which is an embodiment of an antigen-binding molecule has almost no binding activity to FcRn in the neutral pH range.
- the present inventors can bind an IgG type immunoglobulin having binding activity to FcRn in a neutral pH range to FcRn present on the cell surface, and bind to FcRn present on the cell surface. It was considered that IgG type immunoglobulin was taken up into cells depending on FcRn. The rate of uptake into cells via FcRn is faster than the rate of uptake into cells by nonspecific endocytosis.
- the rate of disappearance of the antigen by the antigen-binding molecule can be further increased by imparting the binding ability to FcRn in the neutral pH range. That is, an antigen-binding molecule capable of binding to FcRn in the neutral pH range sends antigens into cells more rapidly than natural IgG immunoglobulins, dissociates antigens in endosomes, and again enters the cell surface or plasma. It is recycled, where it binds to the antigen again and is taken up into the cell via FcRn.
- By increasing the binding ability to FcRn in the neutral pH range it is possible to increase the rotational speed of this cycle, so that the rate of elimination of the antigen from plasma is increased.
- the binding activity of the antigen-binding molecule to the antigen in the acidic pH range than the binding activity to the antigen in the neutral pH range, it is possible to further increase the rate at which the antigen disappears from the plasma.
- the increase in the number of cycles resulting from increasing the rotational speed of this cycle is thought to increase the number of antigen molecules that can be bound by a single antigen-binding molecule.
- the antigen-binding molecule of the present invention consists of an antigen-binding domain and an FcRn-binding domain, and the FcRn-binding domain does not affect the binding to the antigen, and also depends on the type of antigen even when considered from the above mechanism Without binding the antigen binding activity (binding ability) to the antigen in the acidic pH range or low calcium ion concentration conditions of the antigen-binding molecule in the neutral pH or high calcium ion concentration conditions.
- uptake of an antigen into a cell” by an antigen-binding molecule means that the antigen is taken up into the cell by endocytosis.
- “promoting uptake into cells” means that the rate at which an antigen-binding molecule bound to an antigen in the plasma is taken into the cell is promoted and / or the taken-up antigen is in the plasma. This means that the amount recycled is reduced.
- an antigen-binding molecule having a binding activity to human FcRn in the pH neutral range, or a binding activity to the human FcRn and having a binding activity to the antigen in the acidic pH range is lower than the binding activity to the antigen in the pH neutral range.
- Antigen-binding molecules that do not have human FcRn-binding activity in the neutral pH range, or antigen-binding molecules that have a lower binding activity to the antigen in the acidic pH range than the antigen-binding molecule in the neutral pH range It is only necessary that the rate of uptake into cells is promoted.
- the antigen-binding molecule of the present invention preferably has a higher rate of uptake into cells than natural human IgG, and more preferably than natural human IgG. preferable. Therefore, in the present invention, whether or not the antigen-binding molecule has promoted uptake of the antigen into the cell can be determined by whether or not the uptake rate of the antigen into the cell has increased.
- the rate of antigen uptake into cells can be measured, for example, by adding an antigen-binding molecule and an antigen to a culture medium containing human FcRn-expressing cells and measuring the decrease in the concentration of the antigen in the culture medium over time. It can be calculated by measuring the amount of antigen taken up into cells expressing FcRn over time.
- whether the antigen-binding molecule has promoted the uptake of the antigen into the cell can be determined, for example, by whether the rate of disappearance of the antigen present in the plasma is accelerated, or by the administration of the antigen-binding molecule. It can also be confirmed by measuring whether the antigen concentration is reduced.
- natural human IgG means unmodified human IgG, and is not limited to a specific subclass of IgG. This means that human IgG1, IgG2, IgG3, or IgG4 can be used as “natural human IgG” as long as it can bind to human FcRn in the acidic pH range.
- the “native human IgG” may be human IgG1.
- antigen elimination ability in plasma refers to an antigen present in plasma when the antigen-binding molecule is administered into the living body or when the antigen-binding molecule is secreted into the living body. The ability to disappear from inside. Therefore, in the present invention, “the antigen-dissolving ability of the antigen-binding molecule increases in plasma” means that when an antigen-binding molecule is administered, human FcRn binding activity in the neutral pH range of the antigen-binding molecule is increased, or In addition to the increase in the binding activity to human FcRn, the rate at which the antigen disappears from plasma compared to before the binding activity to the antigen in the acidic pH range is lower than the binding activity to the antigen in the neutral pH range.
- Whether or not the antigen-dissolving ability of the antigen-binding molecule in the plasma has increased is determined, for example, by administering the soluble antigen and the antigen-binding molecule in vivo and measuring the plasma concentration of the soluble antigen after administration. It is possible to judge by this. Increase the binding activity of the antigen-binding molecule to human FcRn in the neutral pH range, or increase the binding activity to the human FcRn in addition to the binding activity to the antigen in the acidic pH range than the binding activity to the antigen in the neutral pH range.
- the soluble antigen may be an antigen-binding molecule-bound antigen or an antigen-binding molecule-unbound antigen, and the concentrations thereof are “plasma antigen-binding molecule-bound antigen concentration” and “plasma antigen-binding molecule”, respectively. It can be determined as “unbound antigen concentration” (the latter is synonymous with “plasma free antigen concentration”).
- “Plasma total antigen concentration” means the total concentration of antigen-binding molecule-bound antigen and antigen-bound molecule-unbound antigen, or “plasma free antigen concentration” which is the concentration of antigen-bound molecule-unbound antigen.
- the soluble antigen concentration can be determined as “the total antigen concentration in plasma”.
- Various methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art, as described herein below.
- “improved pharmacokinetics”, “improved pharmacokinetics”, and “excellent pharmacokinetics” are “improved plasma (blood) retention”, “plasma (blood) retention”. It can be rephrased as “improvement”, “excellent plasma (blood) retention”, “longer plasma (blood) retention”, and these terms are used interchangeably.
- the pharmacokinetics is improved means that an antigen-binding molecule is administered to humans or non-human animals such as mice, rats, monkeys, rabbits, dogs, etc., and disappears from plasma (for example, cells Until the antigen-binding molecule is unable to return to the plasma due to degradation in the body), and the time until the antigen-binding molecule is degraded and disappears. It also includes a period of time during which plasma stays in plasma in a state where it can bind to an antigen (for example, a state where an antigen-binding molecule is not bound to an antigen). Human IgG having a natural Fc region can bind to FcRn derived from a non-human animal.
- administration can be preferably performed using a mouse.
- a mouse Methodhods Mol. Biol. (2010) 602, 93-104 in which the original FcRn gene is disrupted and expressed with a transgene for the human FcRn gene is also described below.
- it can be used for administration.
- “improving pharmacokinetics” also includes increasing the time until an antigen-binding molecule that is not bound to an antigen (antigen-unbound antigen-binding molecule) is degraded and disappears. Even if an antigen-binding molecule is present in plasma, if the antigen is already bound to the antigen-binding molecule, the antigen-binding molecule cannot bind to a new antigen. Therefore, if the time during which the antigen-binding molecule is not bound to the antigen becomes longer, the time for binding to the new antigen becomes longer (the opportunity for binding to the new antigen increases), and the antigen binds to the antigen-binding molecule in vivo.
- the time during which the antigen is not bound can be reduced, and the time during which the antigen is bound to the antigen-binding molecule can be lengthened. If the disappearance of the antigen from the plasma can be accelerated by administration of the antigen-binding molecule, the plasma concentration of the non-antigen-binding antigen-binding molecule will increase, and the time that the antigen will bind to the antigen-binding molecule will be longer. Become.
- “improvement of pharmacokinetics of antigen-binding molecule” means improvement of pharmacokinetic parameters of any non-antigen-binding antigen-binding molecule (increase in plasma half-life, increase in mean plasma residence time, Either a decrease in plasma clearance), or an increase in the time during which the antigen is bound to the antigen-binding molecule after administration of the antigen-binding molecule, or accelerated disappearance of the antigen from the plasma by the antigen-binding molecule, including.
- Judgment by measuring any of the parameters of the antigen-binding molecule or non-antigen-binding antigen-binding molecule such as plasma half-life, mean plasma residence time, and plasma clearance (pharmacokinetics, understanding through exercises (Nanzan Hall)) Is possible.
- plasma half-life a plasma half-life
- mean plasma residence time a plasma residence time
- plasma clearance pharmacokinetics, understanding through exercises (Nanzan Hall)
- the parameters are appropriately evaluated by noncompartmental analysis according to the attached procedure manual. be able to.
- Measurement of the plasma concentration of an antigen-binding molecule that is not bound to an antigen can be performed by a method known to those skilled in the art, for example, as measured in Clin. Pharmacol. (2008) 48 (4), 406-417. Can be used.
- the pharmacokinetics is improved includes that the time during which the antigen is bound to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule. Whether or not the time during which the antigen is bound to the antigen-binding molecule is prolonged after administration of the antigen-binding molecule is determined by measuring the plasma concentration of the free antigen, or the free antigen concentration relative to the total antigen concentration. It is possible to judge based on the time until the ratio increases.
- the plasma concentration of free antigen not bound to the antigen-binding molecule or the ratio of the free antigen concentration to the total antigen concentration can be determined by methods known to those skilled in the art. For example, it can be determined using the method used in Pharm. Res. (2006) 23 (1), 95-103.
- an antigen exhibits some function in vivo
- whether the antigen is bound to an antigen-binding molecule (antagonist molecule) that neutralizes the function of the antigen is evaluated based on whether the function of the antigen is neutralized. It is also possible to do. Whether the antigen function is neutralized can be assessed by measuring any in vivo marker that reflects the antigen function.
- Whether an antigen is bound to an antigen-binding molecule (agonist molecule) that activates the function of the antigen can be evaluated by measuring some in vivo marker that reflects the function of the antigen.
- Measurement of free antigen in plasma, measurement of ratio of plasma free antigen to total plasma antigen, measurement of in vivo marker, etc. are not particularly limited, but fixed after administration of antigen-binding molecule It is preferably performed after a lapse of time.
- the time after a fixed time has elapsed since the administration of the antigen-binding molecule is not particularly limited, and can be determined by a person skilled in the art according to the nature of the administered antigen-binding molecule. 1 day after administration of the molecule, 3 days after administration of the antigen binding molecule, 7 days after administration of the antigen binding molecule, 14 days after administration of the antigen binding molecule, For example, 28 days after administration.
- plasma antigen concentration means “total plasma antigen concentration”, which is the sum of antigen-binding molecule-bound antigen and antigen-binding molecule-unbound antigen, or antigen-binding molecule-unbound antigen concentration. It is a concept that includes any of the “plasma free antigen concentrations”.
- the plasma total antigen concentration is compared to the case where human IgG containing a natural Fc region as a human FcRn binding domain is administered as a reference antigen binding molecule, or compared to the case where the antigen binding molecule of the present invention is not administered.
- administration of the antigen-binding molecule of the present invention it can be reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or more.
- the antigen elimination efficiency per antigen-binding molecule is higher, and when the C value is larger, the antigen elimination efficiency per antigen-binding molecule is lower.
- the antigen / antigen binding molecule molar ratio can be calculated as described above.
- the antigen / antigen binding molecule molar ratio is 2 times, 5 times by administration of the antigen binding molecule of the present invention, compared to the case of administering a reference antigen binding molecule containing a natural human IgG ⁇ ⁇ Fc region as a human FcRn binding domain, It can be reduced by 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1,000 times or more.
- natural human IgG1, IgG2, IgG3, or IgG4 is preferably a natural human IgG for reference natural human IgG applications compared to an antigen binding molecule for human FcRn binding activity or in vivo binding activity.
- a reference antigen-binding molecule containing the same antigen-binding domain as the target antigen-binding molecule and a natural human IgG-Fc region as a human FcRn-binding domain can be used as appropriate.
- natural human IgG1 is used for reference natural human IgG applications that are compared to antigen-binding molecules for human FcRn binding activity or in vivo binding activity.
- the decrease in plasma total antigen concentration or antigen / antibody molar ratio can be evaluated as described in Examples 4, 5 and 12. More specifically, if the antigen-binding molecule does not cross-react with mouse counterpart antigen, human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories, Methods Methods Mol. Biol. (2010) 602, 93-104) And can be evaluated by either antigen-antibody co-injection model or steady-state antigen injection model. If an antigen-binding molecule cross-reacts with a mouse counterpart, it can be assessed by simply injecting the antigen-binding molecule into human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories).
- mice are administered a mixture of antigen-binding molecules and antigens.
- mice are implanted with an infusion pump filled with an antigen solution to achieve a constant plasma antigen concentration, and then antigen binding molecules are injected into the mouse.
- Test antigen binding molecules are administered at the same dose. Plasma total antigen concentration, plasma free antigen concentration, and plasma antigen-binding molecule concentration are measured at appropriate time points using methods known to those skilled in the art.
- the long-term plasma antigen concentration is determined to be 2 days, 4 days, 7 days, 14 days, 28 days, 56 days after administration of the antigen binding molecule, or It is determined after 84 days by measuring the total or free antigen concentration and the antigen / antigen binding molecule molar ratio in plasma. Whether a reduction in plasma antigen concentration or antigen / antigen binding molecule molar ratio is achieved by an antigen binding molecule according to the present invention is assessed at any one or more of the time points described above. Can be determined.
- the short-term effect of the present invention can be evaluated.
- the short-term plasma antigen concentration is 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours after administration of the antigen-binding molecule. , 12 hours, or 24 hours later, by measuring the total or free antigen concentration and the antigen / antigen binding molecule molar ratio in plasma.
- the administration route of the antigen-binding molecule of the present invention can be selected from intradermal injection, intravenous injection, intravitreal injection, subcutaneous injection, intraperitoneal injection, parenteral injection, and intramuscular injection.
- mice eg., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.
- monkeys eg, cynomolgus monkeys
- “improvement of pharmacokinetics of antigen-binding molecule, improvement of plasma retention” means that any pharmacokinetic parameter when an antigen-binding molecule is administered to a living body is improved (the half-life in plasma is reduced). Any increase (increase in mean plasma residence time, decrease in plasma clearance, bioavailability)) means that the plasma concentration of the antigen-binding molecule at an appropriate time after administration is improved. It can be judged by measuring any of the parameters such as plasma half-life, mean plasma residence time, plasma clearance, bioavailability, etc. of antigen-binding molecules (under understanding by pharmacokinetic exercises (Nanzan Hall)). is there.
- mice normal mice and human FcRn transgenic mice
- rats, monkeys, rabbits, dogs, humans, etc. the plasma concentration of antigen-binding molecules is measured, and each parameter is calculated, It can be said that the pharmacokinetics of the antigen-binding molecule is improved when the plasma half-life is increased or when the average plasma residence time is increased.
- These parameters can be measured by methods known to those skilled in the art. For example, using the pharmacokinetic analysis software WinNonlin (Pharsight), the parameters are appropriately evaluated by noncompartmental analysis according to the attached procedure manual. be able to.
- antigen binding molecules having FcRn binding activity in the neutral pH range are four molecules of two molecules of FcRn and one molecule of Fc ⁇ R on the cell membrane of antigen-presenting cells. Since it is possible to form a complex containing, the uptake into antigen-presenting cells is promoted, and thus it is considered that the retention in plasma is reduced and the pharmacokinetics are deteriorated.
- Phosphorylation sites exist in the intracellular domains of Fc ⁇ R and FcRn. In general, phosphorylation of the intracellular domain of a receptor expressed on the cell surface occurs when the receptors associate with each other, and internalization of the receptor occurs due to the phosphorylation.
- Heterocomplex formation involving the Fc ⁇ R / bimolecular FcRn / IgG quadratic is thought to occur on antigen-presenting cells that express both Fc ⁇ R and FcRn, thereby increasing the amount of antibody molecules taken up by the antigen-presenting cells. It is possible that the pharmacokinetics may increase as a result.
- the uptake into the antigen-presenting cell is reduced, and as a result, the drug There is a possibility that the dynamics will improve.
- a polynucleotide encoding an antigen-binding domain whose binding activity varies depending on the conditions selected as described above is a single polynucleotide. After being released, the polynucleotide is inserted into an appropriate expression vector.
- the antigen-binding domain is an antibody variable region
- the cDNA is encoded by a restriction enzyme that recognizes restriction enzyme sites inserted at both ends of the cDNA after cDNA encoding the variable region is obtained. Digested.
- a preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antigen-binding molecule gene. Furthermore, in order to insert one copy of the digested fragment into the vector in the correct direction, it is preferable to insert a restriction enzyme that gives a sticky end.
- a polynucleotide encoding the antigen-binding molecule is incorporated into an expression vector in such a manner that it is operably linked to a control sequence.
- the control sequence includes, for example, an enhancer and a promoter.
- An appropriate signal sequence can also be linked to the amino terminus so that the expressed antigen binding molecule is secreted extracellularly.
- a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as the signal sequence, but other suitable signal sequences can be linked.
- the expressed polypeptide can be cleaved at the carboxyl terminal portion of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, an appropriate host cell is transformed with this expression vector, whereby a recombinant cell expressing a polynucleotide encoding a desired antigen-binding molecule can be obtained.
- the method for producing the antigen-binding molecule of the present invention from the recombinant cell can be produced according to the method described in the above-mentioned antibody section.
- the variant of the polynucleotide is appropriately expressed. Inserted into the vector.
- a polynucleotide sequence encoding the antigen-binding molecule of the present invention screened by using a synthetic library or an immune library produced from a non-human animal as a randomized variable region library Preferred is a variant in which is humanized.
- a method for producing a humanized antigen-binding molecule variant a method similar to the method for producing a humanized antibody described above may be employed.
- the binding affinity of the antigen-binding molecule of the present invention screened by using a synthetic library or a naive library as a randomized variable region library is increased (affinity maturation).
- Preferred examples include those in which such modifications are made to the isolated polynucleotide sequence.
- Such variants include CDR mutagenesis (Yang et al. (J. Mol. Biol. (1995) 254, 392-403)), chain shuffling (Marks et al. (Bio / Technology (1992) 10, 779-783)).
- Use of E. coli mutagenized strains Loow et al. (J. Mol. Biol.
- examples of the antigen-binding molecule produced by the production method of the present invention include antigen-binding molecules containing an Fc region, but various variants can be used as the Fc region.
- a polynucleotide encoding such a variant of the Fc region and a polynucleotide encoding an antigen-binding molecule whose binding activity varies depending on the conditions selected as described above have a heavy chain linked in-frame.
- a polynucleotide encoding an antigen-binding molecule is also preferably exemplified as one embodiment of the variant of the present invention.
- the Fc region for example, IgG1 represented by SEQ ID NO: 11 (AAC82527.1 with Ala added to the N terminus), IgG2 represented by SEQ ID NO: 12 (N AAB59393.1 with Ala added to the end, IgG3 represented by SEQ ID NO: 13 (CAA27268.1), IgG4 represented by SEQ ID NO: 14 (AAB59394.1 with Ala added to N-terminus), etc.
- the Fc constant region of the antibody is preferably mentioned. The reason why the retention of IgG molecules in plasma is relatively long (disappearance from plasma is slow) is due to the function of FcRn, particularly human FcRn, known as the salvage receptor for IgG molecules.
- IgG molecules taken into endosomes by pinocytosis bind to FcRn expressed in endosomes, particularly human FcRn, under acidic conditions in endosomes.
- IgG molecules that could not bind to FcRn, particularly human FcRn proceed to lysosomes where they are degraded, but IgG molecules that bind to FcRn, particularly human FcRn, migrate to the cell surface and under neutral conditions in plasma FcRn, especially human FcRn Dissociates from the blood and returns to the plasma again.
- FcRn normal antibodies and antibody-antigen complexes can be isolated by nonspecific endocytosis. It is taken up by cells and transported to the cell surface by binding to FcRn, particularly human FcRn, under conditions of acidic pH in endosomes.
- FcRn especially human FcRn, transports antibodies from the endosome to the cell surface, so FcRn, particularly part of human FcRn, is thought to be also present on the cell surface. Dissociates from FcRn, particularly human FcRn, so that the antibody is recycled into the plasma.
- the Fc region having binding activity to human FcRn in the neutral pH range contained in the antigen-binding molecule of the present invention can be obtained by any method. Specifically, the human IgG-type immunoglobulin used as the starting Fc region can be obtained. By modifying the amino acid, an Fc region having binding activity to human FcRn in a neutral pH range can be obtained.
- Preferred Fc regions of IgG type immunoglobulin for modification include, for example, Fc regions of human IgG (IgG1, IgG2, IgG3, or IgG4, and variants thereof).
- the amino acid at any position can be modified. If the antigen-binding molecule contains the Fc region of human IgG1 as the human Fc region, modifications that have the effect of enhancing the binding to human FcRn in the neutral pH range over the binding activity of the starting Fc region of human IgG1 are included. It is preferable.
- amino acids that can be modified include EU numbering 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325, 327-339, 341, 343, 345 , 360, 362, 370, 375-378, 380, 382, 385-387, 389, 396, 414, 416, 423, 424, 426, 438 And amino acids at positions 440 and 442. More specifically, for example, amino acid modifications as shown in Table 5 can be mentioned. These amino acid modifications enhance binding to human FcRn in the pH neutral range of the Fc region of IgG immunoglobulin.
- a modification that enhances binding to human FcRn even in the neutral pH range is appropriately selected.
- Fc region variant amino acids for example, represented by EU numbering 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 360, 376 , 380, 382, 384, 385, 386, 386, 387, 389, 424, 428, 433, 434 and 436.
- EU numbering of the Fc region Met is the amino acid at position 237.
- the amino acid at position 248 250 of the amino acid is Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, 252 of the amino acid is Phe, Trp, or Tyr, Thr, the amino acid at position 254 Glu for the 255th amino acid
- the 256th amino acid is Asp, Asn, Glu, or Gln
- 257 of the amino acid is one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val
- the amino acid at position 258 is His, Ala for the amino acid at position 265 286 of the amino acid is either Ala or Glu
- the present invention is not limited to a specific principle, but in addition to the above modifications, a heterocomplex comprising an Fc region contained in an antigen-binding molecule, four molecules of FcRn, and an active Fc ⁇ receptor.
- a method for producing an antigen-binding molecule comprising modification of the Fc region so that is not formed is provided.
- the following three types can be mentioned.
- (Mode 1) An antigen-binding molecule having an FcRn-binding activity under neutral pH conditions, and comprising an Fc region that has a lower binding activity to the active Fc ⁇ R than to the active Fc ⁇ R of the natural Fc region. These antigen binding molecules form a ternary complex by binding to two molecules of FcRn, but do not form a complex including active Fc ⁇ R (FIG. 49).
- the Fc region whose binding activity to the active Fc ⁇ R is lower than the binding activity to the active Fc ⁇ R of the natural Fc region can be produced by modifying the amino acid of the natural Fc region as described above. Whether or not the binding activity of the modified Fc region to the active Fc ⁇ R is lower than the binding activity of the natural Fc region to the active Fc ⁇ R can be appropriately performed using the method described in the above-mentioned section of binding activity.
- the binding activity of the Fc region variant to the active Fc ⁇ receptor is lower than the binding activity of the natural Fc region to the active Fc ⁇ receptor.
- the binding activity to any of the human Fc ⁇ receptors is lower than the binding activity of the natural Fc region to these human Fc ⁇ receptors.
- the binding activity of the antigen-binding molecule containing the Fc region variant is 95% or less, preferably 90%, compared to the binding activity of the antigen-binding molecule containing the natural Fc region as a control.
- % Or less 85% or less, 80% or less, 75% or less, particularly preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2%
- the binding activity is 1% or less.
- the native Fc region the starting Fc region can be used, and the Fc regions of different isotypes of the wild-type antibody can also be used.
- an antigen-binding molecule having an Fc region of an IgG monoclonal antibody can be used as appropriate.
- the structure of the Fc region is as follows: SEQ ID NO: 11 (A added to the N terminus of RefSeq registration number AAC82527.1), 12 (A added to the N terminus of RefSeq registration number AAB59393.1), 13 (RefSeq registration number CAA27268.1) ), 14 (RefSeq registration number AAB59394.1, N appended to the end of A).
- an antigen-binding molecule containing the Fc region of an antibody of a specific isotype as a test substance, by using the antigen-binding molecule having the Fc region of the IgG monoclonal antibody of the specific isotype as a control, The effect of the binding activity on the Fc ⁇ receptor by the antigen-binding molecule containing the Fc region is verified. As described above, an antigen-binding molecule containing an Fc region verified to have a high binding activity to the Fc ⁇ receptor is appropriately selected.
- an Fc region whose binding activity to active Fc ⁇ R is lower than the binding activity to the active Fc ⁇ R of the natural Fc region 234 represented by EU numbering among the amino acids of the Fc region. , 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329, one or more of the amino acids has been altered to a different amino acid from the natural Fc region.
- the Fc region is preferably exemplified, but the modification of the Fc region is not limited to the above modification.
- the deglycosyl chain (N297A, N297Q described in Current Opinion in Biotechnology (2009) 20 (6), 685-691) ), IgG1-L234A / L235A, IgG1-A325A / A330S / P331S, IgG1-C226S / C229S, IgG1-C226S / C229S / E233P / L234V / L235A, IgG1-L234F / L235E / P331S, IgG1-S267E / L328F, IgG2- Modification of V234A / G237A, IgG2-H268Q / V309L / A330S / A331S, IgG4-L235A / G237A / E318A, IgG4-L236E, etc., and G236R / L328R, L23 described in
- the amino acid represented by EU numbering of the Fc region; 234 of amino acid Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr or Trp, 235 of the amino acid Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, one of Val or Arg, 236 of the amino acid Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr, 237 of the amino acid Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg, 238 of the amino acid is Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg, 239th amino acid Gln, His
- Mode 2 Antigen-binding molecule that has FcRn-binding activity under neutral pH conditions, and has an Fc region that has a higher binding activity to inhibitory Fc ⁇ R than binding activity to active Fc ⁇ receptor. Molecules can form complexes containing these four by binding to two molecules of FcRn and one molecule of inhibitory Fc ⁇ R. However, since one molecule of antigen-binding molecule can bind only to one molecule of Fc ⁇ R, one molecule of antigen-binding molecule cannot bind to other active Fc ⁇ R in a state of binding to inhibitory Fc ⁇ R (FIG. 50).
- the binding activity to the inhibitory Fc ⁇ R is higher than the binding activity to the active Fc ⁇ receptor means that the binding activity of the Fc region variant to Fc ⁇ RIIb is any one of Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa and / or Fc ⁇ RIIIb. It means higher than the binding activity to Fc ⁇ receptor.
- the binding activity of the antigen-binding molecule containing the Fc region variant to Fc ⁇ RIIb is 105% or more of the binding activity of any one of Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIIa and / or Fc ⁇ RIIIb to the human Fc ⁇ receptor.
- an antigen-binding molecule having an Fc region of an IgG monoclonal antibody can be used as appropriate.
- the structure of the Fc region is as follows: SEQ ID NO: 11 (A added to the N terminus of RefSeq registration number AAC82527.1), 12 (A added to the N terminus of RefSeq registration number AAB59393.1), 13 (RefSeq registration number CAA27268.1) ), 14 (RefSeq registration number AAB59394.1, N appended to the end of A).
- an antigen-binding molecule containing the Fc region of an antibody of a specific isotype as a test substance, by using the antigen-binding molecule having the Fc region of the IgG monoclonal antibody of the specific isotype as a control, The effect of the binding activity on the Fc ⁇ receptor by the antigen-binding molecule containing the Fc region is verified. As described above, an antigen-binding molecule containing an Fc region verified to have a high binding activity to the Fc ⁇ receptor is appropriately selected.
- an Fc region having selective binding activity to inhibitory Fc ⁇ R 238 or 328 amino acids represented by EU numbering among the amino acids of the Fc region are natural Fc regions.
- Preferred examples include Fc regions that have been modified to different amino acids.
- the Fc region having selective binding activity to the inhibitory Fc ⁇ receptor the Fc region or modification described in US 2009/0136485 can be appropriately selected.
- 238 amino acids represented by EU numbering in the Fc region are modified as any one or more of Asp and 328 amino acids represented by EU numbering.
- Preferred examples include Fc regions.
- substitution of Pro at position 238 represented by EU numbering with Asp, and amino acid at position 237 represented by EU numbering are at position 237 represented by Trp and EU numbering.
- Amino acid is Phe
- amino acid at position 267 represented by EU numbering is Val
- amino acid at position 267 represented by EU numbering is Gln
- amino acid at position 268 represented by EU numbering is Asn
- 271 is represented by EU numbering
- the amino acid at the position is Gly
- the amino acid at position 326 represented by EU numbering is Leu
- the amino acid at position 326 represented by EU numbering is Gln
- the amino acid at position 326 represented by EU numbering is represented by Glu
- EU numbering is represented by EU numbering.
- the amino acid at position 237 represented by EU numbering is Leu, the amino acid at position 237 represented by EU numbering is Met, the amino acid at position 237 represented by EU numbering is Tyr, and the amino acid at position 330 represented by EU numbering is Lys, the amino acid at position 330 represented by EU numbering is Arg, the amino acid at position 233 represented by EU numbering is Asp, the amino acid at position 268 represented by EU numbering is Asp, and the amino acid at position 268 represented by EU numbering
- One of the two polypeptides constituting the Fc region has binding activity to FcRn under neutral pH conditions, and the other has binding ability activity to FcRn under neutral pH conditions.
- Antigen-binding molecule containing Fc region not possessed The antigen-binding molecule of Mode 3 can form a ternary complex by binding to one molecule of FcRn and one molecule of Fc ⁇ R, but two molecules of FcRn and one molecule of Fc ⁇ R. Heterocomplexes containing these four members do not form (FIG. 51).
- One of the two polypeptides constituting the Fc region contained in the antigen-binding molecule of this embodiment 3 has binding activity to FcRn under pH neutral conditions, and the other polypeptide has pH neutral pH range.
- An Fc region originating from a bispecific antibody can be used as appropriate as an Fc region that does not have an activity of binding to FcRn under conditions.
- Bispecific antibodies are two types of antibodies that have specificity for different antigens.
- An IgG-type bispecific antibody can be secreted by hybrid hybridoma (quadroma) produced by fusing two hybridomas producing IgG antibody (Milstein et al. (Nature (1983) 305, 537-540)). .
- the antigen-binding molecule of the above-described embodiment 3 is produced using a recombinant technique as described in the above-mentioned antibody section, a gene encoding a polypeptide constituting the two types of target Fc regions is introduced into the cell. A method of co-expressing them can be employed.
- the produced Fc region has one of the two polypeptides constituting the Fc region having binding activity to FcRn under pH neutral conditions, and the other polypeptide under pH neutral condition conditions.
- the Fc region that does not have FcRn binding activity and the two polypeptides that constitute the Fc region constitute an Fc region that has FcRn binding activity under pH neutral conditions.
- An Fc region in which both of the two polypeptides have no binding activity to FcRn under pH neutral conditions is a mixture having a ratio of the number of molecules of 2: 1: 1. It is difficult to purify an antigen-binding molecule containing the desired combination of Fc regions from three types of IgG.
- an antigen-binding molecule containing a heterogeneous Fc region is preferentially added by modifying the CH3 domain constituting the Fc region with an appropriate amino acid substitution.
- the amino acid side chain present in the CH3 domain of one heavy chain is replaced with a larger side chain (knob), and the amino acid side present in the CH3 domain of the other heavy chain
- WO 1996027011, Ridgway et al. Protein Engineering (1996) 9, 617-621), Merchant et al. (Nat. Biotech. (1998) 16, 77 677-681).
- bispecific antibodies by using a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region.
- a method for controlling the association of polypeptides or heterologous multimers composed of polypeptides for the association of two polypeptides constituting the Fc region are known. That is, by altering the amino acid residues that form the interface in the two polypeptides constituting the Fc region, the association of the polypeptides constituting the Fc region having the same sequence is inhibited, and two Fc regions having different sequences
- a method of controlling the formation of a polypeptide aggregate that constitutes can be employed for the production of bispecific antibodies (WO2006 / 106905). Such a method can also be employed in producing the antigen-binding molecule of aspect 3 of the present invention.
- two polypeptides constituting the Fc region originating from the above bispecific antibody can be used as appropriate. More specifically, the two polypeptides constituting the Fc region, the amino acid sequence of one of the polypeptides represented by EU numbering at position 349 is Cys and the position at position 366 is Trp. In the amino acid sequence of the other polypeptide, the amino acid at position 356 represented by EU numbering is Cys, the amino acid at position 366 is Ser, the amino acid at position 368 is Ala, and the amino acid at position 407 is Val. Two polypeptides are preferably used.
- the Fc region includes two polypeptides constituting the Fc region, and an amino acid at position 409 represented by EU numbering in the amino acid sequence of one of the polypeptides.
- Two polypeptides that are Asp and are characterized in that the amino acid at position 399 represented by EU numbering in the amino acid sequence of the other polypeptide is Lys.
- the amino acid at position 409 may be Glu instead of Asp
- the amino acid at position 399 may be Arg instead of Lys.
- Asp may be suitably added as the 360th amino acid or Asp as the 392rd amino acid.
- the Fc region includes two polypeptides constituting the Fc region, and the amino acid at position 370 represented by EU numbering in the amino acid sequence of one of the polypeptides is Two polypeptides characterized in that the amino acid at position 357 represented by EU numbering in the amino acid sequence of the other polypeptide is Lys are preferably used.
- the Fc region includes two polypeptides constituting the Fc region, and the amino acid at position 439 represented by EU numbering in the amino acid sequence of one of the polypeptides.
- Is preferably Glu
- the Fc region is any of the following embodiments in which these are combined:
- amino acid sequence two polypeptides characterized in that the amino acid at position 399 represented by EU numbering is Lys and the amino acid at position 357 is Lys (in this embodiment, the amino acid at position 370 represented by EU numbering) Asp instead of Glu, or Asp of the amino acid at position 392 instead of Glu at amino acid position 370 represented by EU numbering),
- the two polypeptides constituting the Fc region wherein the amino acid sequence of one of the polypeptides is Asp, the amino acid at position 409 represented by EU numbering is Gsp, the amino acid at position 439 is Glu, and the other polypeptide
- two polypeptides characterized in that the amino acid at position 399 represented by EU numbering is Lys and the amino acid at position 356 is Lys (in this embodiment, the amino acid at position 439 represented by EU numbering).
- Glu In place of Glu, it may be Asp of amino acid at position 360, Asp of amino acid at position 392 represented by EU numbering or Asp of amino acid at position 439),
- Two polypeptides characterized in that the amino acid at position 357 represented by EU numbering in the amino acid sequence is Lys, and the amino acid at position 356 is Lys, or
- polypeptides there are two polypeptides (this is characterized in that, in the amino acid sequence of the other polypeptide, the amino acid at position 399 represented by EU numbering is Lys, the amino acid at position 357 is Lys, and the amino acid at position 356 is Lys.
- the amino acid at position 370 represented by EU numbering may not be substituted with Glu, and further, the amino acid at position 370 is not substituted with Glu, and the Asp or 439 position is substituted for Glu at the amino acid position 439.
- Asp of the amino acid at position 392 instead of Glu of the amino acid of Are preferably used.
- two polypeptides constituting the Fc region wherein the amino acid at position 356 represented by EU numbering in the amino acid sequence of one of the polypeptides is Lys.
- two polypeptides characterized in that, in the amino acid sequence of the other polypeptide, the amino acid at position 435 represented by EU numbering is Arg and the amino acid at position 439 is Glu are also preferably used.
- antigen-binding molecules of modes 1 to 3 can all reduce immunogenicity and improve plasma retention compared to antigen-binding molecules that can form a four-component complex. Be expected.
- a cell-free translation system (Clover Direct (Protein Express) in which a tRNA with a non-natural amino acid linked to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also suitable. Used.
- a polynucleotide encoding a variant of the Fc region to which an amino acid mutation as described above is added and a polynucleotide encoding an antigen-binding molecule whose binding activity varies depending on the conditions selected as described above are in frame.
- a polynucleotide encoding an antigen-binding molecule having a linked heavy chain is also produced as one embodiment of the variant of the present invention.
- a cell into which a vector operably linked to a polynucleotide encoding an antigen-binding domain whose binding activity varies depending on the ion concentration conditions linked in-frame with the polynucleotide encoding the Fc region is introduced.
- a method for producing an antigen-binding molecule comprising recovering an antigen-binding molecule from a culture solution.
- a vector in which a polynucleotide encoding an Fc region operably linked in advance in a vector and a polynucleotide encoding an antigen-binding domain whose binding activity changes depending on ion concentration conditions is introduced.
- a method of producing an antigen binding molecule comprising recovering the antigen binding molecule from a culture of cultured cells.
- an existing neutralizing antibody against a pharmaceutical composition -soluble antigen When an existing neutralizing antibody against a pharmaceutical composition -soluble antigen is administered, it is expected that the persistence in plasma is increased by binding the antigen to the antibody.
- Antibodies generally have a long half-life (1-3 weeks), whereas antigens generally have a short half-life (1 day or less). Therefore, an antigen bound to an antibody in plasma has a significantly longer half-life than when the antigen is present alone.
- administration of an existing neutralizing antibody results in an increase in plasma antigen concentration.
- Such cases have been reported for neutralizing antibodies targeting various soluble antigens. For example, IL-6 (J. Immunotoxicol.
- the administration of the existing neutralizing antibody has been reported to increase the total plasma antigen concentration by about 10 to 1000 times from the baseline (the degree of increase varies depending on the antigen).
- the total antigen concentration in plasma means the concentration as the total amount of antigens present in plasma, that is, expressed as the sum of the antibody-bound and antibody-unbound antigen concentrations.
- the plasma total antigen concentration increase. This is because in order to neutralize the soluble antigen, a plasma antibody concentration that exceeds at least the plasma total antigen concentration is required. In other words, when the plasma total antigen concentration increases by 10 to 1000 times, the plasma total antigen concentration does not increase even when the plasma antibody concentration (ie, antibody dose) is neutralized. This means that 10 to 1000 times are required. On the other hand, if the plasma total antigen concentration can be reduced by 10 to 1000 times compared to the existing neutralizing antibody, the dose of the antibody can be reduced by the same amount. Thus, an antibody that can eliminate soluble antigen from plasma and reduce the total antigen concentration in plasma is significantly more useful than existing neutralizing antibodies.
- the binding activity to an antigen in the acidic pH range is lower than the binding activity to the antigen in the pH neutral range condition.
- an antigen-binding molecule containing an FcRn-binding domain such as an antibody-binding domain with an antigen-binding domain whose binding activity changes and an antibody constant region that additionally has binding activity to human FcRn under conditions of neutral pH, is administered in vivo
- the reason why the number of antigens that can be bound by a single antigen-binding molecule and the disappearance of plasma antigen concentration are promoted by promoting the uptake of cells into cells is explained as follows: Is possible.
- an antibody that binds to a membrane antigen when administered into a living body, the antibody binds to the antigen, and is then taken into the endosome in the cell by internalization together with the antigen while bound to the antigen. Thereafter, the antibody that has transferred to the lysosome while bound to the antigen is degraded by the lysosome together with the antigen. Disappearance in plasma through internalization is called antigen-dependent disappearance and has been reported for many antibody molecules (Drug Discov Today (2006) 11 (1-2), 81-88).
- one molecule of an IgG antibody When one molecule of an IgG antibody binds to an antigen bivalently, it is internalized with one molecule of antibody bound to two molecules of antigen, and is degraded as it is by lysosomes. Therefore, in the case of a normal antibody, one molecule of IgG antibody cannot bind to three or more molecules of antigen. For example, in the case of a single molecule IgG antibody having neutralizing activity, it is not possible to neutralize 3 or more antigens.
- IgG molecules taken into endosomes by pinocytosis bind to human FcRn expressed in endosomes under acidic conditions in endosomes.
- IgG molecules that fail to bind to human FcRn are degraded in the subsequently transferred lysosomes.
- IgG molecules bound to human FcRn migrate to the cell surface. Since IgG molecules dissociate from human FcRn under neutral conditions in plasma, the IgG molecules are recycled again into plasma.
- an antibody in which an antigen-binding molecule binds to a soluble antigen the antibody administered in vivo binds to the antigen, and then the antibody is taken into the cell while bound to the antigen. Many of the antibodies taken up into cells move to the cell surface after binding to FcRn in the endosome. Under neutral conditions in plasma, the antibody dissociates from human FcRn and is therefore released extracellularly.
- an antibody containing a normal antigen-binding domain whose binding activity to the antigen does not change depending on the condition of ion concentration such as pH is released outside the cell while bound to the antigen, and cannot bind to the antigen again. Therefore, like an antibody that binds to a membrane antigen, an ordinary single molecule IgG antibody whose binding activity to the antigen does not change depending on ion concentration conditions such as pH cannot bind to three or more antigens.
- An antibody that binds to an antigen in a pH-dependent manner that binds strongly to the antigen under neutral pH conditions in plasma, and dissociates from the antigen under acidic pH conditions in the endosome Antibodies that bind under neutral pH conditions and dissociate under acidic pH conditions) and antigens bind to antigens under high calcium ion concentrations in plasma, and low calcium ion concentrations in endosomes
- An antibody that binds to an antigen in a calcium ion concentration-dependent manner an antibody that binds to an antigen under a high calcium ion concentration condition and dissociates under a low calcium ion concentration condition It is possible to dissociate from the antigen within the endosome.
- An antibody that binds to an antigen in a pH-dependent manner or an antigen that binds to an antigen in a calcium ion concentration-dependent manner can be bound to the antigen again after being dissociated and then recycled into plasma by FcRn. It is. Therefore, one molecule of antibody can repeatedly bind to a plurality of antigen molecules.
- the antigen bound to the antigen-binding molecule is dissociated from the antibody in the endosome and is not recycled into the plasma but is degraded in the lysosome.
- An antibody that binds to an antigen in a pH-dependent manner that binds strongly to the antigen under neutral pH conditions in plasma, and dissociates from the antigen under acidic pH conditions in the endosome Antibodies that bind under neutral pH conditions and dissociate under acidic pH conditions) and antigens bind to antigens under high calcium ion concentrations in plasma, and low calcium ion concentrations in endosomes
- An antibody that binds to an antigen in a calcium ion concentration-dependent manner an antibody that binds to an antigen under a high calcium ion concentration condition and dissociates under a low calcium ion concentration condition).
- the antigen binds to the antigen in a pH-dependent manner (binding under neutral pH conditions and dissociates under acidic pH conditions), or it binds to the antigen in a calcium ion concentration-dependent manner.
- an antibody that binds binds under conditions of high calcium ion concentration and dissociates under conditions of low calcium ion concentration
- the rate of disappearance is thought to be equal to the rate of uptake of the antibody and its antibody-antigen complex into cells by nonspecific endocytosis.
- the antigen that does not dissociate from the antibody in the endosome is also recycled into the plasma together with the antibody.
- the rate of antigen disappearance is limited by the rate of uptake into cells by nonspecific endocytosis.
- FcRn transports antibodies from the endosome to the cell surface, it is considered that a part of FcRn is also present on the cell surface.
- IgG type immunoglobulin which is an embodiment of an antigen-binding molecule has almost no binding activity to FcRn in the neutral pH range.
- the present inventors can bind an IgG type immunoglobulin having binding activity to FcRn in a neutral pH range to FcRn present on the cell surface, and bind to FcRn present on the cell surface. It was considered that IgG type immunoglobulin was taken up into cells depending on FcRn. The rate of uptake into cells via FcRn is faster than the rate of uptake into cells by nonspecific endocytosis.
- the rate of disappearance of the antigen by the antigen-binding molecule can be further increased by imparting the binding ability to FcRn in the neutral pH range. That is, an antigen-binding molecule capable of binding to FcRn in the neutral pH range sends antigens into cells more rapidly than natural IgG immunoglobulins, dissociates antigens in endosomes, and again enters the cell surface or plasma. It is recycled, where it binds to the antigen again and is taken up into the cell via FcRn. By increasing the binding ability to FcRn in the neutral pH range, it is possible to increase the rotational speed of this cycle, so that the rate of elimination of the antigen from plasma is increased.
- the increase in the number of cycles resulting from increasing the rotational speed of this cycle may increase the number of antigen molecules that can be bound by one molecule of an antigen-binding molecule.
- the antigen-binding molecule of the present invention consists of an antigen-binding domain and an FcRn-binding domain, and the FcRn-binding domain does not affect the binding to the antigen, and also depends on the type of antigen even when considered from the above mechanism Without binding the antigen binding activity (binding ability) to the antigen in the acidic pH range or low calcium ion concentration conditions of the antigen-binding molecule in the neutral pH or high calcium ion concentration conditions.
- the antigen-binding molecule of the present invention is considered to exhibit an effect superior to conventional therapeutic antibodies in terms of reduction of side effects caused by the antigen, increase in the dose of the antibody, and improvement of the in vivo kinetics of the antibody. .
- FIG. 1 shows the mechanism by which soluble antigen disappears from plasma by administration of a pH-dependent antigen-binding antibody that has enhanced binding to FcRn at neutral pH compared to existing neutralizing antibodies.
- Existing neutralizing antibodies that do not have a pH-dependent antigen-binding ability are slowly taken up by nonspecific interactions with cells after binding to soluble antigens in plasma. The complex between the neutralizing antibody and the soluble antigen taken into the cell moves to acidic endosomes and is recycled into the plasma by FcRn.
- a pH-dependent antigen-binding antibody that has enhanced binding to FcRn under neutral conditions binds to a soluble antigen in plasma, and then rapidly enters cells that express FcRn on the cell membrane. It is captured.
- the soluble antigen bound to the pH-dependent antigen-binding antibody is dissociated from the antibody due to the pH-dependent binding ability in the acidic endosome.
- the soluble antigen dissociated from the antibody then moves to lysosomes and undergoes degradation due to proteolytic activity.
- the antibody dissociated from the soluble antigen is recycled onto the cell membrane by FcRn and released again into plasma. The antibody thus recycled to be free can bind again to another soluble antigen.
- Antigen-binding antibodies can transfer a large amount of soluble antigen to lysosomes to reduce plasma total antigen concentration.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising the antigen-binding molecule of the present invention, the antigen-binding molecule produced by the modification method of the present invention, or the antigen-binding molecule produced by the production method of the present invention.
- the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention has a higher effect of lowering the antigen concentration in plasma than that of a normal antigen-binding molecule by administration of the antigen-binding molecule. It is useful as a pharmaceutical composition because the immune response due to the above and the pharmacokinetics in the living body are modified.
- the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
- the pharmaceutical composition usually refers to a drug for treatment or prevention of a disease, or examination / diagnosis.
- the pharmaceutical composition of the present invention can be formulated using methods known to those skilled in the art. For example, it can be used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or in the form of suspension injections.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative Or in combination with binders and the like as appropriate, and can be formulated by mixing in unit dosage forms generally required for accepted pharmaceutical practice.
- the amount of the active ingredient in these preparations is set so as to obtain an appropriate volume within the indicated range.
- a sterile composition for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
- a vehicle such as distilled water for injection.
- the aqueous solution for injection include isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride).
- Appropriate solubilizers such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) can be used in combination.
- oily liquid examples include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can be used in combination as a solubilizing agent. It can also be formulated with buffers (eg, phosphate buffer and sodium acetate buffer), soothing agents (eg, procaine hydrochloride), stabilizers (eg, benzyl alcohol and phenol), and antioxidants.
- buffers eg, phosphate buffer and sodium acetate buffer
- soothing agents eg, procaine hydrochloride
- stabilizers eg, benzyl alcohol and phenol
- antioxidants antioxidants.
- the prepared injection solution is usually filled into an appropriate ampoule.
- the pharmaceutical composition of the present invention is preferably administered by parenteral administration.
- parenteral administration for example, an injection, nasal administration, pulmonary administration, or transdermal administration composition is administered.
- it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose of the pharmaceutical composition containing the antigen-binding molecule can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per time. Alternatively, for example, a dose of 0.001 to 100000 mg per patient can be set, but the present invention is not necessarily limited to these values.
- the dose and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.
- the present invention also provides a kit for use in the method of the present invention comprising at least the antigen-binding molecule of the present invention.
- the kit may be packaged with a pharmaceutically acceptable carrier, a medium, instructions describing the method of use, and the like.
- the present invention also relates to an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule, which contains as an active ingredient the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention.
- an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule which contains as an active ingredient the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention.
- an agent for improving the pharmacokinetics of an antigen-binding molecule or an agent for reducing the immunogenicity of an antigen-binding molecule which contains as an active ingredient the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention.
- the present invention also relates to a method for treating an immunoinflammatory disease, comprising a step of administering an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention to a subject (subject).
- the present invention also relates to the use of the antigen-binding molecule of the present invention or the antigen-binding molecule produced by the production method of the present invention in the production of a pharmacokinetics improving agent for an antigen-binding molecule or an immunogenicity reducing agent for an antigen-binding molecule. .
- the present invention also relates to an antigen-binding molecule of the present invention or an antigen-binding molecule produced by the production method of the present invention for use in the method of the present invention.
- amino acids contained in the amino acid sequences described in the present invention are modified after translation (for example, modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art). In some cases, even if the amino acid is post-translationally modified as such, it is naturally included in the amino acid sequence described in the present invention.
- Example 1 Effect of pH-dependent human IL-6 receptor-binding human antibody on plasma retention and immunogenicity by enhancing binding to human FcRn under neutral conditions Soluble form from plasma
- the FcRn-binding domain such as the Fc region (Nat. Rev. Immunol. (2007) 7 (9), 715-25) of the antigen-binding molecule such as an antibody that interacts with FcRn is neutral in pH. It is important to have binding activity for FcRn in the region.
- FcRn binding domain mutants having a binding activity to FcRn in the neutral pH range of the FcRn binding domain have been studied.
- the FcRn binding activity of F1 to F600 created as an Fc variant is evaluated in the neutral pH range, and the disappearance of antigens from plasma is accelerated by enhancing the binding activity to FcRn in the neutral pH range.
- the binding molecule has excellent plasma retention in vivo and low immunogenicity.
- Non-Patent Documents 10, 11 and 12 when cynomolgus monkey is administered with an antibody that has been found to bind to human FcRn under neutral conditions (pH 7.4), the plasma retention of the antibody does not improve and the plasma retention It has been reported that no change was observed in sex (Non-Patent Documents 10, 11 and 12).
- FcRn is expressed in antigen-presenting cells and has been reported to be involved in antigen presentation.
- MBP myelin basic protein
- MBP-Fc an antigen-binding molecule
- the effect of antigen-binding molecules on plasma retention and immunogenicity by enhancing FcRn binding under neutral conditions has not been fully investigated.
- the antigen-binding molecule When developing an antigen-binding molecule as a pharmaceutical product, it is preferable that the antigen-binding molecule has a long plasma retention and a low immunogenicity.
- Fv4-IgG1, VH3-IgG1-F1 (SEQ ID NO: 37) consisting of -CK (SEQ ID NO: 36) and Fv4-IgG1-F1, VH3-IgG1-F157 (SEQ ID NO: 38) consisting of VL3-CK and VL3 Fv4-IgG1-F157, VH3-IgG1-F20 (SEQ ID NO: 39) consisting of -CK and Fv4-IgG1-F20, VH3-IgG1-F21 (SEQ ID NO: 40) consisting of VL3-CK and VL3-CK Fv4-IgG1-F21 was produced by the method shown in Reference Example 1 and Reference Example 2.
- FcRn diluted solution and running buffer (as a reference solution) were injected, and mouse FcRn was allowed to interact with the antibody captured on the sensor chip.
- running buffer 50 mmol / L sodium phosphate, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, pH 7.4 was used, and each buffer was also used for dilution of FcRn. 10 mmol / L glycine-HCl, pH1.5 was used for regeneration of the sensor chip. All measurements were performed at 25 ° C.
- the KD (M for each antibody mouse FcRn) was calculated. Biacore T100 Evaluation Software (GE Healthcare) was used for calculation of each parameter. As a result, the KD (M) of IgG1 was not detected, whereas the KD (M) of the prepared IgG1-F1 was 1.06E-06 (M). The produced IgG1-F1 was shown to have enhanced binding activity to mouse FcRn under pH neutral conditions (pH 7.4).
- a calibration curve sample containing anti-human IL-6 receptor antibody with a plasma concentration of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 ⁇ g / mL and a mouse plasma measurement sample diluted 100 times or more were prepared.
- reaction of reaction with Biotinylated Anti-human IL-6 R Antibody (R & D) for 1 hour at room temperature and then Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 1 hour at room temperature is the TMB One Component HRP Microwell Substrate (BioFX Laboratories) was used as a substrate.
- the absorbance at 450 nm of the reaction solution in each well whose reaction was stopped by adding 1N-Sulfuric acid (Showa Chemical) was measured with a microplate reader.
- the antibody concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).
- FIG. 2 shows the pH-dependent human IL-6 receptor-binding antibody concentration in plasma after intravenously or subcutaneously administering pH-dependent human IL-6 receptor-binding human antibody to normal mice. From the results of FIG. 2, plasma was obtained when Fv4-IgG1-F1 with enhanced binding to mouse FcRn was administered intravenously under neutral conditions compared to intravenously administered Fv4-IgG1. It was shown that the retention was worse. On the other hand, Fv4-IgG1 administered subcutaneously showed the same retention in plasma as when administered intravenously.
- Example 2 Production of a human IL-6 receptor-binding mouse antibody having binding activity to mouse FcRn under pH neutral range conditions It has binding activity to mouse FcRn under pH neutral range conditions by the following method. A mouse antibody was produced.
- natural mouse IgG1 (SEQ ID NO: 44, hereinafter referred to as mIgG1) is used as the heavy chain constant region
- natural mouse kappa (SEQ ID NO: 45, hereinafter referred to as mk1) is used as the light chain constant region.
- mIgG1 an expression vector having the base sequences of heavy chain mPM1H-mIgG1 (SEQ ID NO: 46) and light chain mPM1L-mk1 (SEQ ID NO: 47) was prepared.
- mPM1-mIgG1 which is a human IL-6R-binding mouse antibody, consisting of mPM1H-mIgG1 and mPM1L-mk1 was prepared.
- mPM1 antibody capable of binding to mouse FcRn under pH neutral range conditions The prepared mPM1-mIgG1 is a mouse antibody containing a natural mouse Fc region, and has a pH neutral range. Has no binding activity to mouse FcRn under conditions. Thus, amino acid modification was introduced into the heavy chain constant region of mPM1-mIgG1 in order to confer binding activity to mouse FcRn under neutral pH conditions.
- amino acid substitution in which Thr at position 252 represented by EU numbering of mPM1H-mIgG1 was replaced with Tyr, amino acid substitution in which Thr at position 256 represented by EU numbering was replaced by Glu, and EU numbering MPM1H-mIgG1-mF38 (SEQ ID NO: 50) to which an amino acid substitution in which Asn at position 434 was substituted with Trp was added was prepared.
- mPM1-mIgG1-mF3 consisting of mPM1H-mIgG1-mF3 and mPM1L-mk1 was prepared as a mouse IgG1 antibody having binding to mouse FcRn under neutral pH conditions. .
- Example 3 Binding experiment to FcRn and Fc ⁇ R of antigen-binding molecule having Fc region
- Example 1 by increasing the binding to FcRn under neutral conditions of antigen-binding molecule, plasma retention and immunogen It was confirmed that the sex deteriorated. Since native IgG1 has no binding activity to human FcRn in the neutral region, it was thought that the retention in plasma and immunogenicity deteriorated by conferring binding to FcRn under neutral conditions .
- FcRn binding domain and Fc ⁇ R binding domain The Fc region of an antibody has a binding domain for FcRn and a binding domain for Fc ⁇ R. It has already been reported that two FcRn binding domains exist at two positions in the Fc region, and two FcRn molecules can simultaneously bind to the Fc region of one antibody molecule (Nature (1994) 372 (6504), 379-383). ). On the other hand, binding domains for Fc ⁇ R also exist in two places in the Fc region, but it is thought that two molecules of Fc ⁇ R cannot bind simultaneously.
- active Fc ⁇ R is expressed on the cell membranes of many immune cells such as tree cells, NK cells, macrophages, neutrophils and adipocytes. Furthermore, FcRn has been reported to be expressed in immune cells such as antigen-presenting cells such as dendritic cells, macrophages, and monocytes in humans (J. Immunol. (2001) 166 (5), 3266-3276). Since normal natural IgG1 cannot bind to FcRn but only to Fc ⁇ R in the neutral pH range, natural IgG1 binds to antigen-presenting cells by forming a Fc ⁇ R / IgG1 bipartite complex.
- Phosphorylation sites exist in the intracellular domains of Fc ⁇ R and FcRn.
- phosphorylation of the intracellular domain of a receptor expressed on the cell surface occurs when the receptors associate with each other, and internalization of the receptor occurs due to the phosphorylation.
- IgG1 forms a Fc ⁇ R / IgG1 bipartite complex on antigen-presenting cells, it does not associate with the intracellular domain of Fc ⁇ R, but it does not bind to FcRn under neutral pH conditions.
- the IgG molecule has a complex containing the four members of Fc ⁇ R / bimolecular FcRn / IgG, the association of the three intracellular domains of Fc ⁇ R and FcRn occurs, thereby causing Fc ⁇ R / bimolecular FcRn
- Fc ⁇ R / bimolecular FcRn There was a possibility that internalization of heterocomplexes including four of / IgG was induced.
- Heterocomplex formation involving Fc ⁇ R / bimolecular FcRn / IgG is considered to occur on antigen-presenting cells that express both Fc ⁇ R and FcRn, thereby causing the antibody molecule to be taken up by antigen-presenting cells. It was considered that the retention was deteriorated and the immunogenicity was further deteriorated.
- antigen-binding molecules containing FcRn-binding domains such as Fc regions that have FcRn-binding activity under neutral pH conditions have so far been found in immune cells such as antigen-presenting cells that express both Fc ⁇ R and FcRn.
- immune cells such as antigen-presenting cells that express both Fc ⁇ R and FcRn.
- the ability to form a Fc ⁇ R / bimolecular FcRn / IgG quaternary complex depends on whether the antigen-binding molecule containing Fc region has FcRn binding activity under neutral pH conditions at the same time against Fc ⁇ R and FcRn. Judgment can be made based on whether or not they can be combined. Therefore, according to the following method, a simultaneous binding experiment to FcRn and Fc ⁇ R in the Fc region contained in the antigen-binding molecule was performed.
- Fv4-IgG1-F157 was shown to be capable of binding to mouse Fc ⁇ RI, Fc ⁇ RIIb, Fc ⁇ RIII, and Fc ⁇ RIV simultaneously with binding to human FcRn.
- human antibodies having binding activity to human FcRn under neutral pH conditions bind to human FcRn, and at the same time, human Fc ⁇ RIa, Fc ⁇ RIIa (R), Fc ⁇ RIIa (H), Fc ⁇ RIIb, Fc ⁇ RIIIa ( It was shown that it can also bind to various human Fc ⁇ Rs such as F), mouse Fc ⁇ RI, Fc ⁇ RIIb, Fc ⁇ RIII, Fc ⁇ RIV and various mouse Fc ⁇ Rs.
- human antibodies and mouse antibodies having binding activity to mouse FcRn under neutral pH conditions can bind to mouse FcRn and simultaneously to various mouse Fc ⁇ Rs.
- the Fc region of human and mouse IgG has a binding region to FcRn and a binding region to Fc ⁇ R, but they do not interfere with each other, and one molecule of Fc and two molecules of FcRn, one molecule. It has been shown that it is possible to form heterocomplexes containing the four members of the molecule Fc ⁇ R.
- antigen-binding molecules having binding activity to FcRn in the neutral pH range can be incorporated into antigen-presenting cells by forming a hetero complex that includes one active Fc ⁇ R and two FcRn molecules. It was thought that plasma retention was worsened, and immunogenicity was further deteriorated.
- a mutation is introduced into an antigen-binding molecule having binding activity to FcRn in the neutral pH range, and an antigen-binding molecule having a reduced ability to form such a four-component complex is prepared.
- the retention of the antigen-binding molecule in plasma can be improved, and induction of an immune response by the living body can be suppressed (ie, immunogenicity can be reduced).
- the following three types may be mentioned as preferred modes of antigen-binding molecules that are taken into cells without forming such a complex.
- Antigen-binding molecule that has binding activity to FcRn under neutral pH conditions and has selective binding activity to inhibitory Fc ⁇ R
- the antigen-binding molecule in aspect 2 is composed of two molecules of FcRn and one molecule. By binding to the inhibitory Fc ⁇ R, a complex containing these four components can be formed. However, since the antigen-binding molecules of one molecule can not only bind Fc ⁇ R one molecule can not bind to other active Fc ⁇ R while antigen-binding molecules of one molecule bound to inhibitory Fc gamma R.
- an antigen-binding molecule taken into a cell while bound to an inhibitory Fc ⁇ R is recycled onto the cell membrane to avoid degradation in the cell (Immunity (2005) 23, 503-514). That is, it is considered that an antigen-binding molecule having a selective binding activity to inhibitory Fc ⁇ R cannot form a complex including an active Fc ⁇ R that causes an immune response.
- Antigen-binding molecules that do not have an antigen
- the antigen-binding molecule of aspect 3 can form a ternary complex by binding to one molecule of FcRn and one molecule of Fc ⁇ R, but two molecules of FcRn and one molecule of Fc ⁇ R. Does not form a heterocomplex containing.
- the antigen-binding molecules of these embodiments 1 to 3 all have improved plasma retention and an immunogen compared to an antigen-binding molecule that can form a complex comprising four molecules of two molecules of FcRn and one molecule of Fc ⁇ R. It is expected to be possible to reduce the sex.
- Example 4 Evaluation of plasma retention of human antibodies having binding activity to human FcRn in the neutral pH range and lower binding activity to human and mouse Fc ⁇ R than that of natural Fc ⁇ R binding domain (4-1) Production of antibody having binding activity to human Fc ⁇ R lower than that of natural Fc ⁇ R binding domain and binding to human IL-6 receptor in a pH-dependent manner Among the three modes shown in Example 3,
- the antigen-binding molecule of aspect 1, that is, an antigen-binding molecule having binding activity to FcRn under neutral pH conditions and having a binding activity to active Fc ⁇ R lower than that of the natural Fc ⁇ R binding domain is as follows: Was made as follows.
- Fv4-IgG1-F21 and Fv4-IgG1-F157 produced in Example 1 have binding activity to human FcRn under pH neutral conditions, and bind to human IL-6 receptor in a pH-dependent manner. It is an antibody.
- These amino acid sequences were modified by reducing the binding to mouse Fc ⁇ R by amino acid substitution in which Ser at position 239 represented by EU numbering was substituted with Lys.
- VH3-IgG1-F140 SEQ ID NO: 51 in which Ser at position 239 represented by EU numbering of the amino acid sequence of VH3-IgG1-F21 was substituted with Lys was produced.
- VH3-IgG1-F424 (SEQ ID NO: 52) in which Ser at position 239 represented by EU numbering of the amino acid sequence of VH3-IgG1-F157 was substituted with Lys was produced.
- Fv4-IgG1-F140 and Fv4-IgG1-F424 containing these heavy chains and VL3-CK light chain were produced.
- mouse Fc ⁇ Rs (R & D sytems, Sino Biological) (hereinafter referred to as mouse Fc ⁇ Rs) and antibody
- the binding activity of was evaluated.
- the antibody to be tested was captured on protein L (ACTIGEN) immobilized on a sensor chip CM4 (GE Healthcare) by an amine coupling method.
- a diluted solution of mouse Fc ⁇ Rs and a running buffer used as a blank were injected to interact with the antibody captured on the sensor chip.
- the binding activity to mouse Fc ⁇ Rs can be expressed by the relative binding activity to mouse Fc ⁇ Rs.
- the amount of change in the sensorgram before and after the antibody was captured by Protein L was defined as X1.
- mouse Fc ⁇ Rs interacted with the antibody, and the value obtained by dividing the binding activity of mouse Fc ⁇ Rs expressed as the amount of change ( ⁇ A1) in the sensorgram before and after that divided by the capture amount (X) of each antibody was 1500 times
- the value obtained by subtracting the binding activity of mouse Fc ⁇ Rs, expressed as the amount of change in sensorgram ( ⁇ A2) before and after interaction of the running buffer with the antibody captured by Protein L, was captured for each antibody.
- the value obtained by multiplying the value divided by (X) by 1500 (Y) was defined as the binding activity of mouse Fc ⁇ Rs (Formula 1).
- a calibration curve sample containing anti-human IL-6 receptor antibody with a plasma concentration of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 ⁇ g / mL and a mouse plasma measurement sample diluted 100 times or more were prepared.
- reaction of reaction with Biotinylated Anti-human IL-6 R Antibody (R & D) for 1 hour at room temperature and then Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 1 hour at room temperature is the TMB One Component HRP Microwell Substrate (BioFX Laboratories) was used as a substrate.
- the absorbance at 450 nm of the reaction solution in each well whose reaction was stopped by adding 1N-Sulfuric acid (Showa Chemical) was measured with a microplate reader.
- the antibody concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).
- FIG. 14 shows the pH-dependent human IL-6 receptor binding antibody concentration in plasma after intravenous administration of the pH-dependent human IL-6 receptor binding antibody to human FcRn transgenic mice. From the results shown in FIG. 14, it was confirmed that Fv4-IgG1-F140, which has lower binding to mouse Fc ⁇ R than Fv4-IgG1-F21, has improved plasma retention compared to Fv4-IgG1-F21. Similarly, Fv4-IgG1-F424, which has lower binding to mouse Fc ⁇ R compared to Fv4-IgG1-F157, was found to have an increased plasma retention compared to Fv4-IgG1-F157.
- an antibody having a binding to human FcRn under neutral pH conditions and having a binding to Fc ⁇ R lower than that of a normal Fc ⁇ R binding domain is higher than that of an antibody having a normal Fc ⁇ R binding domain. It was also shown that the plasma retention was high.
- the antigen-binding molecule is human under pH neutral conditions. It is also considered that the formation of the quaternary complex described in Example 3 was inhibited because it has an FcRn-binding domain that has FcRn-binding activity and lower Fc ⁇ R-binding activity than the natural Fc ⁇ R-binding domain. That is, it is considered that Fv4-IgG1-F21 and Fv4-IgG1-F157 that form a four-component complex on the cell membrane of antigen-presenting cells are likely to be taken into antigen-presenting cells.
- Example 5 Evaluation of plasma retention of human antibody having binding to human FcRn in neutral pH range and not binding to mouse Fc ⁇ R (5-1) Preparation of a human antibody that binds to human IL-6 receptor in a pH-dependent manner without binding activity to human and mouse Fc ⁇ Rs A pH-dependent human IL-6 receptor that does not have a binding activity to human and mouse Fc ⁇ Rs In order to produce a human antibody that binds to the antibody, antibody production was performed as follows.
- VH3-IgG1-F821 (SEQ ID NO: 57) having no binding activity to human and mouse Fc ⁇ R by amino acid substitution in which Leu at position 235 was substituted with Arg and amino acid substitution at which Ser at position 239 was substituted with Lys, VH3-IgG1-F939 (SEQ ID NO: 58) and VH3-IgG1-F1009 (SEQ ID NO: 59) were generated.
- Fv4-IgG1, Fv4-IgG1-F11, Fv4-IgG1-F890, Fv4-IgG1-F947, Fv4-IgG1-F760 containing these heavy chain and VL3-CK light chain Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 were produced.
- the binding activity of the antibody containing -IgG1-F1009 as the heavy chain and L (WT) -CK as the light chain at pH 7.4 to mouse Fc ⁇ R was measured. The measured results are shown in Table 9 below.
- Fv4-IgG1-F760, Fv4-IgG1-F821, Fv4-IgG1-F939 and Fv4-IgG1-F1009 are Fv4-IgG1, Fv4-IgG1-F11, Fv4-IgG1-F890 and Compared with Fv4-IgG1-F947, it was shown that the binding to mouse Fc ⁇ R was decreased without affecting the binding activity to human FcRn.
- Plasma was obtained by immediately centrifuging the collected blood at 4 ° C. and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer set to ⁇ 20 ° C. or lower until measurement was performed.
- the anti-human IL-6 receptor antibody concentration in mouse plasma was measured by ELISA as in the method of Example 4. The results are shown in FIG. Fv4-IgG1-F760, which reduced the binding activity of Fv4-IgG1 to mouse Fc ⁇ R, showed almost the same plasma retention as Fv4-IgG1-F11, and decreased plasma retention by reducing the binding activity to Fc ⁇ R The improvement effect was not seen.
- the antibody was administered once at 1 mg / kg. Blood was collected at 15 minutes, 7 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 21 days and 28 days after administration of anti-human IL-6 receptor antibody. Plasma was obtained by immediately centrifuging the collected blood at 4 ° C. and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer set to ⁇ 20 ° C. or lower until measurement was performed.
- the anti-human IL-6 receptor antibody concentration in mouse plasma was measured by ELISA as in the method of Example 4.
- the results are shown in FIG. Fv4-IgG1-F821, which reduced the binding activity of Fv4-IgG1-F11 to mouse Fc ⁇ R, showed almost the same plasma retention as Fv4-IgG1-F11.
- Fv4-IgG1-F939 which reduced the binding activity of Fv4-IgG1-F890 to mouse Fc ⁇ R, was found to have improved plasma retention compared to Fv4-IgG1-F890.
- Fv4-IgG1-F1009 in which the binding activity of Fv4-IgG1-F947 to mouse Fc ⁇ R was reduced, was found to have improved plasma retention compared to Fv4-IgG1-F947.
- Example 6 Evaluation of plasma retention of a mouse antibody having binding to mouse FcRn in the neutral pH range but not binding to mouse Fc ⁇ R (6-1) Preparation of mouse antibody that binds to human IL-6 receptor without binding activity to mouse Fc ⁇ R In Examples 4 and 5, the mouse has binding activity to human FcRn under neutral pH conditions. It was shown that an antigen-binding molecule containing an Fc ⁇ R binding domain whose binding activity to Fc ⁇ R is lower than that of a natural Fc ⁇ R binding domain has improved plasma retention in human FcRn transgenic mice.
- mPM1H-mIgG1-mF38 produced in Example 2, the amino acid substitution in which Pro at position 235 represented by EU numbering was replaced with Lys and the amino acid substitution in which Ser at position 239 was replaced with Lys, mPM1H-mIgG1-mF40 (SEQ ID NO: 60) is the amino acid sequence of mPM1H-mIgG1-mF14.
- mPM1H-mIgG1-mF39 SEQ ID NO: 61
- a soluble human IL-6 receptor solid-phase plate in which 100 ⁇ L of these calibration curve samples and plasma measurement samples were dispensed into each well was allowed to stand at room temperature for 2 hours.
- the color reaction of the reaction solution obtained by reacting with Anti-Mouse IgG-Peroxidase antibody (SIGMA) for 1 hour at room temperature and then reacting with Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) for 1 hour at room temperature is TMB One Component HRP Microwell Substrate ( BioFX Laboratories) was used as a substrate.
- FIG. 17 shows changes in plasma antibody concentration in normal mice after intravenous administration measured by this method.
- mPM1-mIgG1-mF40 having no binding to mouse Fc ⁇ R was found to have improved plasma retention compared to mPM1-mIgG1-mF38.
- mPM1-mIgG1-mF39 which has no binding to mouse Fc ⁇ R, was found to have improved plasma retention compared to mPM1-mIgG1-mF14.
- antibodies having Fc ⁇ R binding domains that have binding to mouse FcRn under neutral pH conditions and do not have binding activity to mouse Fc ⁇ R are more normal in normal mice than antibodies having normal Fc ⁇ R binding domains. It was shown that the plasma retention was high.
- the antigen-binding molecule having the property of the mode 1 with respect to the antigen-binding molecule has high plasma retention.
- the present invention is not bound by any particular theory, it is believed that the increase in plasma retention observed here was due to selective inhibition of uptake into immune cells such as antigen-presenting cells, As a result, it is expected that the induction of immune response can be inhibited.
- a humanized antibody (anti-human IL-6 receptor) that has a binding to human FcRn in a neutral pH range and has a binding activity to human Fc ⁇ R that is lower than that of a natural Fc ⁇ R binding domain
- Antigen binding molecule of mode 1 ie, binding activity to FcRn under neutral pH condition, binding activity to active Fc ⁇ R is binding of natural Fc ⁇ R binding domain
- in vitro T cell responses to the antigen-binding molecule were evaluated by the following method.
- the antibody to be tested was captured on protein L (ACTIGEN) immobilized on an appropriate amount by the amine coupling method on Sensor chip CM4 (GE Healthcare).
- a diluted solution of human Fc ⁇ Rs and a running buffer used as a blank were injected and allowed to interact with the antibody captured on the sensor chip.
- As a running buffer 20 mmol / L ACES, 150 mmol / L NaCl, 0.05% (w / v) Tween 20, pH 7.4 was used, and this buffer was also used for dilution of human Fc ⁇ Rs.
- 10 mmol / L Glycine-HCl, pH 1.5 was used for regeneration of the sensor chip. All measurements were performed at 25 ° C.
- the binding activity to human Fc ⁇ Rs can be expressed by the relative binding activity to human Fc ⁇ Rs.
- the amount of change in the sensorgram before and after the antibody was captured by Protein L was defined as X1.
- human Fc ⁇ Rs was allowed to interact with the antibody, and the value obtained by dividing the human Fc ⁇ Rs binding activity expressed as the amount of change ( ⁇ A1) in the sensorgram before and after that by the capture amount (X) of each antibody was 1500 times.
- the value obtained by multiplying the value divided by (X) by 1500 (Y) was defined as the binding activity of human Fc ⁇ Rs (Formula 2).
- CD8 + and CD25 hi T cells were removed and each donor's PBMC resuspended in AIMV medium (Invitrogen) containing 3% inactivated human serum to 2 ⁇ 10 6 / mL was allowed to 2 ⁇ 10 6 cells were added per well to the well plate. After culturing at 37 ° C. and 5% CO 2 for 2 hours, cells to which each test substance was added at final concentrations of 10, 30, 100, and 300 ⁇ g / mL were cultured for 8 days. At 6, 7, and 8 days of culture, BrdU (Bromodeoxyuridine) was added to 150 ⁇ L of the cell suspension in culture transferred to a round bottom 96-well plate, and the cells were further cultured for 24 hours.
- AIMV medium Invitrogen
- BrdU incorporated into the nucleus of cells cultured with BrdU is stained with anti-CD3, CD4 and CD19 antibodies (BD bioscience) at the same time as staining with the BrdU Flow Kit (BD bioscience) according to the attached standard protocol Antigens (CD3, CD4 and CD19) were stained. Subsequently, the ratio of BrdU positive CD4 + T cells was detected by BD FACS Calibur or BD FACS CantII (BD). On the 6th, 7th, and 8th days of culture, the ratio of BrdU positive CD4 + T cells at each final concentration of 10, 30, 100, and 300 ⁇ g / mL of the test substance was calculated, and the average value thereof was calculated.
- FIG. 18 shows the proliferative response of CD4T + cells to Fv4-IgG1-F21 and Fv4-IgG1-F140 in PBMCs of 5 human donors from which CD8 + and CD25 hi T cells have been removed.
- Fv4-IgG1-F140 has the property of Embodiment 1 with a lower binding activity to human Fc ⁇ R than Fv4-IgG1-F21. Based on the above results, immunogenicity to antigen-binding molecules that have binding to FcRn under neutral pH conditions and have a lower binding activity to human Fc ⁇ R than that of the native Fc ⁇ R binding domain. It was suggested that can be suppressed.
- Example 8 A humanized antibody (anti-human) comprising an antigen-binding domain having a binding activity to human FcRn under neutral pH conditions and a binding activity to human Fc ⁇ R lower than that of a natural Fc ⁇ R-binding domain.
- A33 antibody 8-1) Production of hA33-IgG1 As shown in Example 7, since the immunoreactivity of human PBMC to Fv4-IgG1-F21 is originally low, the binding activity to Fc ⁇ R is binding to the natural Fc ⁇ R binding domain. It was suggested that it is not suitable for evaluating suppression of immune responses against Fv4-IgG1-F140 containing an antigen-binding domain that is less active.
- hA33-IgG1 humanized A33 antibody
- hA33-IgG1 has been confirmed to produce anti-antibodies in 33-73% subjects in clinical trials (Hwang et al. (Methods (2005) 36, 3-10) and Walle et al. (Expert Opin. Bio. Ther. ( 2007) 7 (3), 405-418)).
- hA33-IgG1 decreases the binding activity to Fc ⁇ R against the molecule that has enhanced the binding activity to FcRn in the neutral pH range of hA33-IgG1 It was thought that the immunogenicity reduction effect by inhibiting the formation of the four-component complex was easy to detect.
- hA33H (SEQ ID NO: 62) as the heavy chain variable region and hA33L (SEQ ID NO: 63) as the light chain variable region of the humanized A33 antibody is known information (British Journal of Cancer (1995) 72, 1364-1372 )
- natural human IgG1 (SEQ ID NO: 11, hereinafter referred to as IgG1) as the heavy chain constant region
- natural human kappa (SEQ ID NO: 64, hereinafter referred to as k0) as the light chain constant region.
- k0 natural human kappa
- HA33H-IgG1-F21 (SEQ ID NO: 65) was produced by substituting the amino acid at position 434 represented by EU numbering from Asn to Tyr. Using the method of Reference Example 2, hA33H-IgG1-F21 as a heavy chain and hA33L-k0 as a light chain as an A33-binding antibody having binding activity to human FcRn under neutral pH conditions -IgG1-F21 was produced.
- Example 7 (8-4) Evaluation of immunogenicity of various A33-binding antibodies by in vitro T-cell assay Immunogenicity against hA33-IgG1-F21 and hA33-IgG1-F140 prepared using the same method as in Example 7. Evaluation was made.
- the healthy volunteer who is a donor is not the same individual as the healthy volunteer from which PBMC used in Example 7 was isolated. That is, donor A in Example 7 and donor A in this test are healthy volunteers of different individuals. The test results are shown in FIG. In FIG.
- hA33-IgG1-F21 having binding to human FcRn in the neutral pH range, and hA33-IgG1-F140 containing an Fc ⁇ R binding domain that has lower binding activity to human Fc ⁇ R than the binding activity of the native Fc ⁇ R binding domain.
- Donor C, D, and F are immune responses against hA33-IgG1-F21, as no response to hA33-IgG1-F21 from PBMC isolated from donors C, D, and F has been observed compared to negative controls It is thought that it is a donor who does not cause a rash.
- Example 9 In vitro immunogenicity evaluation of a humanized antibody (anti-human A33 antibody) having a binding activity to human FcRn and having no binding activity to human Fc ⁇ R under neutral pH conditions (9-1) Preparation of an A33-binding antibody having strong binding activity to human FcRn under pH neutral conditions
- the amino acid at position 252 represented by EU numbering for hA33H-IgG1 was substituted from Met to Tyr.
- the amino acid at position 286 represented by EU numbering is substituted from Asn to Glu
- the amino acid at position 307 represented by EU numbering is replaced from Thr to Gln
- the amino acid at position 311 represented by EU numbering is replaced from Gln.
- HA33H-IgG1-F698 (SEQ ID NO: 67) was produced by the method of Reference Example 1 by substituting the amino acid at position 434 represented by EU numbering from Asn to Tyr.
- hA33-IgG1-F698 containing hA33H-IgG1-F698 as a heavy chain and hA33L-k0 as a light chain was produced as a human A33-binding antibody with strong binding activity to human FcRn under pH neutral conditions. .
- Example 4 the binding activity of VH3 / L (WT) -IgG1, VH3 / L (WT) -IgG1-F698 and VH3 / L (WT) -IgG1-F699 to human FcRn at pH 7.0 was confirmed. Measured. Furthermore, using the method of Example 7, binding of VH3 / L (WT) -IgG1, VH3 / L (WT) -IgG1-F698 and VH3 / L (WT) -IgG1-F699 to human Fc ⁇ R at pH 7.4 Activity was measured. The results are also shown in Table 15 below.
- L (WT) -IgG1-F699 had binding activity to hFcgRI, although binding to hFcgRIIa (R), hFcgRIIa (H), hFcgRIIb, and hFcgRIIIa (F) was reduced.
- Example 7 Evaluation of immunogenicity of various A33-binding antibodies by in vitro T-cell assay
- the same method as in Example 7 was used to evaluate the immunogenicity of the prepared hA33-IgG1-F698 and hA33-IgG1-F699.
- the healthy volunteer who is a donor is not the same individual as the healthy volunteer from which PBMC used in Examples 7 and 8 were isolated. That is, donor A in Example 7 and Example 8 and donor A in this test are healthy volunteers of different individuals.
- hA33-IgG1-F698 having a strong binding activity to human FcRn under neutral pH conditions, and hA33 containing an Fc ⁇ R binding domain whose binding activity to human Fc ⁇ R is lower than that of the natural Fc ⁇ R domain.
- -IgG1-F699 results are compared.
- Donor G and I are donors that do not elicit an immune response against hA33-IgG1-F698 because no response to hA33-IgG1-F698 has been observed for PBMC isolated from donors G and I compared to the negative control. It is believed that there is.
- PBMCs isolated from the other seven donors we observed a higher immune response to hA33-IgG1-F698 compared to the negative control As in the case of hA33-IgG1-F21 described above, it showed high immunogenicity in vitro.
- isolated from 5 donors (donors A, B, C, D and F) against hA33-IgG1-F699 containing an Fc ⁇ R binding domain whose binding activity to human Fc ⁇ R is lower than that of the native Fc ⁇ R domain An effect is observed in which the immune response of the prepared PBMC is reduced compared to that against hA33-IgG1-F698.
- the immune response of PBMC isolated from donors C and F against hA33-IgG1-F699 was confirmed to be comparable to the negative control.
- the immunogenicity reducing effect was confirmed not only on hA33-IgG1-F21 but also on hA33-IgG1-F698, which has a stronger binding activity on human FcRn. It was shown that the immunogenicity can be reduced by inhibiting the formation of the quaternary complex by lowering the binding activity to human Fc ⁇ R to be lower than that of the natural Fc ⁇ R binding domain.
- position 235 represented by EU numbering of hA33H-IgG1-F698 (SEQ ID NO: 67) HA33H-IgG1-F763 (SEQ ID NO: 69) was prepared in which Leu was replaced with Arg and Ser at position 239 represented by EU numbering was replaced with Lys.
- hA33-IgG1-F698 having a strong binding activity to human FcRn under neutral pH conditions, and hA33 containing an Fc ⁇ R binding domain that has a lower binding activity to human Fc ⁇ R than that of the natural Fc ⁇ R domain.
- -Results for IgG1-F763 are compared.
- Donor B, E, F and K against hA33-IgG1-F698 because no response was observed for hBMC33-IgG1-F698 of PBMC isolated from donors B, E, F and K compared to the negative control It is considered that the donor does not cause an immune response.
- PBMCs isolated from the other seven donors the immune response to hA33-IgG1-F698 may be higher than the negative control Has been observed.
- An effect is observed in which the immune response of PBMC is reduced compared to that against hA33-IgG1-F698.
- the immune response of PBMCs isolated from donors C, D and H, in particular, to hA33-IgG1-F763 is similar to that of the negative control, and by reducing the binding to Fc ⁇ R, Of the 4 donors whose immune response had declined, as many as 3 were able to completely suppress the immune response of PBMC.
- an antigen-binding molecule containing an Fc ⁇ R binding domain with low binding activity to human Fc ⁇ R is a very effective molecule with reduced immunogenicity.
- Example 10 In vivo immunogenicity evaluation of humanized antibody having binding activity to human FcRn in the neutral pH range and not binding to mouse Fc ⁇ R
- the neutral pH range Antigen-binding molecules that have an Fc ⁇ R-binding domain that has a binding activity to human FcRn and a lower binding activity to Fc ⁇ R than the binding activity of the native Fc ⁇ R-binding domain compared to antigen-binding molecules that do not have reduced Fc ⁇ R-binding activity In vitro experiments have shown that immunogenicity is reduced. In order to confirm that this effect was also demonstrated in vivo, the following test was performed.
- a numerical value (X) obtained by adding 1.645 times the standard deviation (SD) of the numerical value measured in this manner was used as a positive criterion (Equation 3). On any blood collection day, an individual who showed a response exceeding the positive determination criterion at least once was determined to have a positive antibody production response to the test substance.
- FIG. 22 shows the antibodies of mouse antibodies produced against Fv4-IgG1-F11 3 days, 7 days, 14 days, 21 days and 28 days after Fv4-IgG1-F11 was administered to human FcRn transgenic mice. The value was shown. It was shown that mouse antibody production against Fv4-IgG1-F11 was positive in 1 out of 3 mice (# 3) on any blood collection day after administration (positive rate 1/3 ). On the other hand, FIG.
- the antibody titer of the antibody was shown. It was shown that the production of mouse antibodies against Fv4-IgG1-F821 was negative in all three mice on any blood collection day after administration (positive rate 0/3).
- FIG.24A and its enlarged view, FIG.24B show Fv4-IgG1-F890 at 3 days, 7 days, 14 days, 21 days and 28 days after Fv4-IgG1-F890 was administered to human FcRn transgenic mice.
- the antibody titer of the mouse antibody produced was shown.
- mouse antibodies against Fv4-IgG1-F890 were positive in 2 out of 3 mice (# 1, # 3) ( Positive rate 2/3).
- FIG.24B show Fv4-IgG1-F890 at 3 days, 7 days, 14 days, 21 days and 28 days after Fv4-IgG1-F890 was administered to human FcRn transgenic mice.
- the antibody titer of the mouse antibody produced was shown.
- mouse antibodies against Fv4-IgG1-F890 were positive in 2 out of 3 mice (# 1, # 3) ( Positive rate 2/3).
- mice 25 shows mice that were produced against Fv4-IgG1-F939 3 days, 7 days, 14 days, 21 days, and 28 days after Fv4-IgG1-F939 was administered to human FcRn transgenic mice.
- the antibody titer of the antibody was shown. It was shown that the production of mouse antibodies against Fv4-IgG1-F939 was negative in all three mice on any blood collection day after administration (positive rate 0/3).
- FIG. 26 shows the antibodies of mouse antibodies produced against Fv4-IgG1-F947 3 days, 7 days, 14 days, 21 days and 28 days after Fv4-IgG1-F947 was administered to human FcRn transgenic mice. The value was shown. At 14 days after administration, it was shown that mouse antibody production against Fv4-IgG1-F947 was positive in 2 out of 3 mice (# 1, # 3) (positive rate 2) / 3).
- FIG. 27 shows mice produced against Fv4-IgG1-F1009 3 days, 7 days, 14 days, 21 days and 28 days after Fv4-IgG1-F1009 was administered to human FcRn transgenic mice. The antibody titer of the antibody was shown. On the day of blood collection after 7 days after administration, production of mouse antibodies against Fv4-IgG1-F1009 was shown to be positive in 2 out of 3 mice (# 4, # 5) (positive) Rate 2/3).
- Fv4-IgG1-F821 has a reduced binding to various mouse Fc ⁇ Rs against Fv4-IgG1-F11, and similarly various mouse Fc ⁇ Rs against Fv4-IgG1-F890.
- Fv4-IgG1-F939 has a reduced binding to Fv4-IgG1-F939, and similarly Fv4-IgG1-F1009 has a decreased binding to various mouse Fc ⁇ Rs relative to Fv4-IgG1-F947.
- Fv4-IgG1-F947 did not show an immunogenicity suppressing effect by reducing the binding activity to Fc ⁇ R.
- the reason can be considered as follows. As shown in FIG. 16, the disappearance of Fv4-IgG1-F947 and Fv4-IgG1-F1009 from plasma is very rapid.
- Fv4-IgG1-F1009 has a reduced binding activity to mouse Fc ⁇ R, and it is considered that formation of a quaternary complex on antigen-presenting cells is inhibited.
- Fv4-IgG1-F1009 is considered to be taken into cells by binding only to FcRn expressed on cell membranes such as vascular endothelial cells and blood cells.
- FcRn is also expressed on the cell membrane of some antigen-presenting cells
- Fv4-IgG1-F1009 can be incorporated into antigen-presenting cells by binding only to FcRn. That is, some of the rapid disappearance of Fv4-IgG1-F1009 from plasma may have been taken up by antigen-presenting cells.
- Fv4-IgG1-F1009 is a human antibody and is a completely heterologous protein for mice. That is, it is considered that the mouse has many T cell populations that specifically respond to Fv4-IgG1-F1009. Fv4-IgG1-F1009, even taken up slightly by antigen-presenting cells, can be presented to T cells after being intracellularly processed, but mice specifically respond to Fv4-IgG1-F1009 Since it has many T cell populations, it is considered that an immune response against Fv4-IgG1-F1009 is likely to be induced.
- the antigen-binding molecule when the antigen-binding molecule is a heterologous protein (human protein is administered to a mouse), compared to the case where the antigen-binding molecule is a homologous protein (mouse protein is administered to a mouse), It may also be more difficult to suppress the immune response by inhibiting the formation of the quaternary complex.
- the antigen-binding molecule when the antigen-binding molecule is an antibody, the antibody administered to a human is a humanized antibody or a human antibody, so that an immune response against a homologous protein occurs. Therefore, in Example 11, whether or not inhibition of formation of the four-component complex leads to reduction in immunogenicity was evaluated by administering a mouse antibody to the mouse.
- Example 11 In vivo immunogenicity evaluation of a mouse antibody having binding activity to mouse FcRn under neutral pH conditions but not binding to mouse Fc ⁇ R (11-1) In vivo immunogenicity test in normal mice By inhibiting the formation of a quaternary complex on antigen-presenting cells when the antigen-binding molecule is a homologous protein (mouse antibody administered to the mouse) In order to verify the suppressive effect of immunogenicity, the following tests were conducted.
- Read Buffer T ( ⁇ 4) (Meso Scale Discovery) was dispensed and immediately measured with a SECTOR PR 2400 reader (Meso Scale Discovery).
- MEAN mean value
- SD standard deviation
- FIG. 28 shows the antibody titers of mouse antibodies produced against mPM1-mIgG1-mF14 14 days, 21 days and 28 days after mPM1-mIgG1-mF14 was administered to normal mice. At 21 days after administration, all three mice were shown to be positive in producing mouse antibodies against mPM1-mIgG1-mF14 (positive rate 3/3). On the other hand, FIG.
- FIG. 29 shows the antibody titers of mouse antibodies produced against mPM1-mIgG1-mF39, 14 days, 21 days and 28 days after mPM1-mIgG1-mF39 was administered to normal mice. Yes. It was shown that the production of mouse antibodies against mPM1-mIgG1-mF39 was negative in all three mice on any blood collection day after administration (positive rate 0/3).
- FIG. 30 shows antibody titers of mouse antibodies produced against mPM1-mIgG1-mF38 at 14 days, 21 days, and 28 days after mPM1-mIgG1-mF38 was administered to normal mice.
- FIG. 31 shows the antibody titers of mouse antibodies produced against mPM1-mIgG1-mF40, 14 days, 21 days and 28 days after mPM1-mIgG1-mF40 was administered to normal mice. It was shown that the production of mouse antibodies against mPM1-mIgG1-mF40 was negative in all three mice on any blood collection day after administration (positive rate 0/3).
- mPM1-mIgG1-mF40 has decreased binding to various mouse Fc ⁇ Rs against mPM1-mIgG1-mF38, and similarly various mouse Fc ⁇ Rs against mPM1-mIgG1-mF14.
- MPM1-mIgG1-mF39 has a decreased binding to. From these results, even when mPM1-mIgG1-mF38 and mPM1-mIgG1-mF14, which are mouse antibodies that are homologous proteins, were administered to normal mice, antibody production against the administered antibody was confirmed, and an immune response was confirmed.
- this enhances the binding activity to FcRn in the neutral pH range and forms a quaternary complex on the antigen-presenting cell, thereby allowing the antigen-presenting cell to be taken up. It is thought that it was because it was promoted.
- an antigen-binding molecule having binding activity to FcRn under neutral pH conditions and having a binding activity to active Fc ⁇ R lower than that of the natural Fc ⁇ R binding domain ie, the mode 1 described in Example 3
- the antigen-binding molecule having the same binding activity as that of the natural Fc ⁇ R-binding domain that is, the antigen-binding molecule capable of forming a quaternary complex described in Example 3
- Example 12 Production and evaluation of human antibody having binding activity to human FcRn in neutral pH range and lower binding activity to human Fc ⁇ R than that of natural Fc ⁇ R binding domain (12-1) pH neutrality Production and evaluation of human IgG1 antibody having binding activity to human FcRn in the region and lower binding activity to human Fc ⁇ R than that of natural Fc ⁇ R binding domain
- binding to active Fc ⁇ R Examples of the Fc region whose activity is lower than the binding activity to the active Fc ⁇ R of the natural Fc region are the 234, 235, 236, 237, 238, 239 represented by EU numbering among the amino acids of the Fc region.
- Preferred examples include Fc regions in which any one or more of amino acids at positions 270, 297, 298, 325, and 329 is modified to an amino acid different from the natural Fc region.
- deglycosylated chains described in Current Opinion in Biotechnology (2009) 20 (6), 685-691 N297A, N297Q
- IgG1-L234A / L235A IgG1-A325A / A330S / P331S
- IgG1- C226S / C229S IgG1-C226S / C229S / E233P / L234V / L235A
- IgG1-L234F / L235E / P331S IgG1-S267E / L328F
- IgG2-V234A / G237A IgG2-H268Q / V309L / A330S / A331S
- IgG4 deglycosyl
- Fv4-IgG1-F890 and Fv4-IgG1-F947 produced in Example 5 have binding activity to human FcRn under pH neutral conditions, and bind to human IL-6 receptor in a pH-dependent manner. It is an antibody.
- Various variants with reduced binding to human Fc ⁇ R were prepared by introducing amino acid substitutions into these amino acid sequences (Table 17).
- VH3-IgG1-F938 (SEQ ID NO: 156) in which Leu at position 235 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 was substituted with Lys, and Ser at position 239 was substituted with Lys
- VH3-IgG1-F1315 (SEQ ID NO: 157) in which Gly at position 237 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 was substituted with Lys, and Ser at position 239 was substituted with Lys
- VH3-IgG1-F1316 (SEQ ID NO: 158) in which Gly at position 237 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 is substituted with Arg
- Ser at position 239 is substituted with Lys
- VH3-IgG1-F1317 (SEQ ID NO: 159) in which Ser at position 239 represented by EU numbering of the amino acid sequence of
- VH3-IgG1-F1324 (SEQ ID NO: 161) in which Leu at position 234 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 was substituted with Ala, and Leu at position 235 was substituted with Ala
- VH3-IgG1-F1325 in which Leu at position 234 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 was substituted with Ala
- Leu at position 235 was substituted with Ala
- Asn at position 297 was substituted with Ala
- Leu at position 235 represented by EU numbering of the amino acid sequence of VH3-IgG1-F890 was substituted with Arg
- Gly at position 236 was substituted with Arg
- Ser at position 239 was substituted with Lys VH3-IgG1-F1333 (SEQ ID NO: 163), VH3-IgG1-F1356 (SEQ ID NO: 16
- the amino acid modifications introduced to reduce the binding activity to various human Fc ⁇ Rs compared to the binding activity of the natural Fc ⁇ R binding domain are not particularly limited, and can be achieved by various amino acid modifications. It has been shown.
- GpL16-k0 (SEQ ID NO: 75) of glypican 3 antibody with improved plasma kinetics disclosed in WO2009 / 041062 was commonly used as the antibody L chain.
- B3 (SEQ ID NO: 76) in which a mutation of K439E was introduced into G1d from which Gly and Lys at the C-terminus of IgG1 were deleted was used as the antibody H chain constant region.
- this H chain is called GpH7-B3 (SEQ ID NO: 77)
- the L chain is called GpL16-k0 (SEQ ID NO: 75).
- Biacore® T100 GE Healthcare
- Biacore® T200 GE Healthcare
- Biacore® A100 GE Healthcare
- Biacore® 4000 the interaction between each modified antibody and the Fc ⁇ receptor prepared above was analyzed. Measurement was performed at 25 ° C. using HBS-EP + (GE Healthcare) as a running buffer.
- Series S Sensor Chip CM5 GE Healthcare
- Series S sensor Chip CM4 GE Healthcare
- Antigen peptide ProteinA
- Protein A / G Thermo Scientific
- ProteinProL ACTIGEN
- a chip on which BioVision) was immobilized, or a chip obtained by interacting and immobilizing an antigen peptide previously biotinylated on Series ⁇ ⁇ S Sensor Chip SA (certified) (GE Healthcare) was used.
- the antibody of interest was captured by these sensor chips, and Fc ⁇ receptor diluted with a running buffer was allowed to interact, and the binding amount to the antibody was measured. The amount of binding was compared between antibodies. However, since the binding amount of Fc ⁇ receptor depends on the amount of captured antibody, a correction value obtained by dividing the binding amount of Fc ⁇ receptor by the capture amount of each antibody was compared.
- the sensor chip regenerated by washing the antibody captured on the sensor chip by reacting with 10 mM glycine-HCl, pH 1.5 was repeatedly used.
- the binding strength was evaluated according to the following method.
- the amount of binding to the Fc ⁇ R of the antibody derived from each B3 variant was compared with the antibody to which B3 mutation was not introduced (EU numbering from position 234 to position 239, position 265 to position 271, position 295 , 296, 298, 300, 324 to 337 are divided by the value of the amount of binding to Fc ⁇ R of the antibody having human natural IgG1 sequence).
- a value obtained by further multiplying the value by 100 was expressed as an index of relative binding activity to each Fc ⁇ R.
- Table 21 shows the modifications that reduce the binding to all FcgRs among the analyzed mutants.
- the 236 types of modifications shown in Table 20 are modifications that reduce the binding to at least one type of FcgR compared to the antibody (GpH7-B3 / GpL16-k0) before the modification was introduced, Similarly, even when introduced, it was considered that the modification had an effect of reducing the binding to at least one type of FcgR. Therefore, the amino acid modification introduced to reduce the binding activity to various human Fc ⁇ Rs compared to the binding activity of the natural Fc ⁇ R binding domain is not particularly limited, and at least one amino acid modification shown in Table 20 is introduced. This has been shown to be achievable. In addition, the amino acid modification introduced here may be one place or a combination of a plurality of places.
- an antibody containing VH3-IgG2 (SEQ ID NO: 166) as a heavy chain and L (WT) -CK (SEQ ID NO: 41) as a light chain It was produced by the method shown in Reference Example 2.
- VH3-IgG4 As a human IL-6 receptor binding antibody having human IgG4 as a constant region, VH3-IgG4 (SEQ ID NO: 167) is included as a heavy chain, and L (WT) -CK (SEQ ID NO: 41) is used as a light chain.
- the contained antibody was produced by the method shown in Reference Example 2.
- VH3-IgG2 and VH3-IgG4 were produced.
- Met at position 252 represented by EU numbering is replaced with Tyr
- Asn at position 434 is replaced with Tyr
- Tyr at position 436 is replaced with Val.
- VH3-IgG2-F890 (SEQ ID NO: 168) and VH3-IgG4-F890 (SEQ ID NO: 169) were produced.
- VH3-IgG2-F890 In order to reduce the binding to human Fc ⁇ R against VH3-IgG2-F890 and VH3-IgG4-F890, amino acid modifications were introduced into the respective constant regions. Specifically, with respect to VH3-IgG2-F890, VH3-IgG2-F939 (SEQ ID NO: 170) in which Ala at position 235 represented by EU numbering was substituted with Arg and Ser at position 239 was substituted with Lys. ) was produced.
- VH3-IgG4-F939 (SEQ ID NO: 171) in which Leu at position 235 represented by EU numbering was substituted with Arg and Ser at position 239 was substituted with Lys was prepared for VH3-IgG4-F890. It was done.
- Antibodies containing VH3-IgG2-F890, VH3-IgG4-F890, VH3-IgG2-F939 or VH3-IgG4-F939 as heavy chains and L (WT) -CK (SEQ ID NO: 41) as light chains was prepared by the method shown in Reference Example 2.
- the Fc region having a binding activity to human FcRn in the neutral pH range and having a binding activity to human Fc ⁇ R lower than that of the natural Fc ⁇ R binding domain is not particularly limited to human IgG1. It was also shown that this can be achieved by using human IgG2 or human IgG4.
- Example 13 Production and evaluation of an antigen-binding molecule having binding to FcRn under the condition of only one of the two polypeptides constituting the FcRn-binding domain under pH neutral conditions. Only one of the two polypeptides constituting the FcRn binding domain has binding to FcRn under pH neutral conditions, and the other has no binding activity to FcRn under pH neutral conditions.
- the preparation of the molecule was performed as follows.
- VH3-IgG1-F947 (SEQ ID NO: 70) is a reference example as a heavy chain of an anti-human IL-6R antibody having a binding to FcRn under neutral pH conditions. 1 was produced.
- an amino acid that substitutes Ala for Ile at position 253 represented by EU numbering for VH3-IgG1 was added to produce VH3-IgG1-F46 (SEQ ID NO: 71).
- Asp at position 356 in which one Fc region of the antibody is represented by EU numbering is Lys, and Glu at position 357 by EU numbering is used.
- VH3-IgG1-FA6a (SEQ ID NO: 72) in which Asp at position 356 represented by EU numbering of VH3-IgG1-F947 was replaced with Lys and Glu at position 357 represented by EU numbering was replaced with Lys (Hereinafter referred to as heavy chain A).
- Lys at position 370 represented by EU numbering of VH3-IgG1-F46 is Glu
- His at position 435 represented by EU numbering is Arg
- Lys at position 439 represented by EU numbering is Glu.
- a substituted VH3-IgG1-FB4a (SEQ ID NO: 73) was produced (hereinafter referred to as heavy chain B) (Table 23).
- VH3-IgG1-FA6a and VH3-IgG1-FB4a are added as heavy chains by adding equal amounts of VH3-IgG1-FA6a and VH3-IgG1-FB4a as heavy chain plasmids.
- Fv4-IgG1-FA6a / FB4a having VL3-CK as the light chain was produced.
- PK test for antigen-binding molecules not having Fk4-IgG1-F947 and Fv4-IgG1-FA6a / FB4a were administered to human FcRn transgenic mice by the following method.
- Anti-human IL-6 receptor subcutaneously in the back of human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg line 32 + / + mouse, Jackson Laboratories, Methods Mol. Biol. (2010) 602, 93-104) The antibody was administered once at 1 mg / kg.
- the anti-human IL-6 receptor antibody concentration in the mouse plasma was measured by ELISA as in the method of Example 4. The results are shown in FIG. Compared to Fv4-IgG1-F947, which can bind to two molecules of FcRn via two binding regions to human FcRn, to one molecule of FcRn via one binding region to human FcRn Fv4-IgG1-FA6a / FB4a, which can only bind, showed a high plasma concentration transition.
- IgG taken up into cells is recycled to plasma again by binding to FcRn in endosomes, but natural IgG is converted to two molecules of FcRn via two FcRn binding regions. Since it can bind, it binds to FcRn with high binding capacity, and many of them are thought to be recycled.
- IgG having only one FcRn-binding region has a low ability to bind to FcRn in endosomes and cannot be sufficiently recycled, so it was thought that disappearance from plasma is accelerated. Therefore, as shown in FIG. 32, in Fv4-IgG1-FA6a / FB4a having one FcRn binding region under pH neutral conditions, the phenomenon that plasma retention is improved is a natural type Since it was the opposite of IgG, it was completely unexpected.
- Example 1 when Fv4-IgG1-F1 was administered subcutaneously, rapid disappearance of Fv4-IgG1-F1 from the plasma was confirmed, and mice against Fv4-IgG1-F1 were confirmed. Antibody production was suggested. On the other hand, when administered intravenously, rapid disappearance of Fv4-IgG1-F1 from the plasma was not confirmed, suggesting that no mouse antibody against Fv4-IgG1-F1 was produced. That is, when an antibody administered subcutaneously is taken up by immune cells present in the lymphatic system in the absorption process, the bioavailability is reduced and it can also cause immunogenicity.
- any antigen binding molecule that does not form a quaternary complex on the cell membrane of immune cells may be used. That is, in any of the antigen-binding molecules of Embodiments 1, 2, and 3, the bioavailability when administered subcutaneously is higher than that of an antigen-binding molecule capable of forming a quaternary complex. It is considered possible to improve the plasma retention and further reduce the immunogenicity.
- the indication of the present invention is never limited to a specific route of administration, but an example that is expected to be particularly effective is given by the route of administration via the lymphatic system in the absorption process of antigen-binding molecules. It is conceivable that one example may be subcutaneous administration.
- Example 14 Production of an antibody having binding activity to human FcRn under neutral pH conditions and selective binding activity to inhibitory Fc ⁇ R. Also, enhanced binding to FcRn under neutral conditions. It is possible to produce the antigen-binding molecule of Embodiment 2 shown in Example 3 by using a modification that causes selective enhancement of binding activity to the inhibitory Fc ⁇ RIIb with respect to the antigen-binding molecule. In other words, an antigen-binding molecule that has a binding activity to FcRn under neutral conditions and further introduced a modification that selectively enhances the binding activity to inhibitory Fc ⁇ RIIb consists of two molecules of FcRn and one molecule of Fc ⁇ R. An intervening quaternary complex.
- GpL16-k0 (SEQ ID NO: 75) of glypican 3 antibody with improved plasma kinetics disclosed in WO2009 / 041062 was commonly used in combination with different H chains as the antibody L chain.
- B3 (SEQ ID NO: 76) in which a mutation of K439E was introduced into G1d from which Gly and Lys at the C-terminus of IgG1 were deleted was used as the antibody H chain constant region.
- this H chain is referred to as GpH7-B3 (SEQ ID NO: 77)
- the L chain is referred to as GpL16-k0 (SEQ ID NO: 75).
- a control antibody in which no mutation is introduced into B3, and the amount of binding between each B3 variant antibody and each Fc ⁇ R (EU numbering from position 234 to position 239, position 265 to position 271, position 295 , 296, 298, 300, 324 to 337 are divided by the value of human natural IgG1 sequence).
- a value obtained by multiplying the value by 100 was expressed as a binding value for each Fc ⁇ R.
- the horizontal axis represents the binding of each mutant to Fc ⁇ RIIb, and the vertical axis represents the values of Fc ⁇ RIa, Fc ⁇ RIIa (H), Fc ⁇ RIIa (R), and Fc ⁇ RIIIa, which are the active Fc ⁇ Rs of each mutant (FIGS. 33, 34, and 35) 36).
- mutation A a modification in which Pro at position 238 represented by EU numbering was replaced by Asp
- mutation B expressed by EU numbering
- variable region (SEQ ID NO: 78) of IL6R-H which is a variable region of an antibody against human interleukin-6 receptor disclosed in WO2009 / 125825 as an antibody H chain variable region, and the C-terminus of human IgG1 as an antibody H chain constant region IL6R-G1d (SEQ ID NO: 79) containing the G1d constant region from which Gly and Lys were removed was used as the heavy chain of IgG1.
- IL6R-G1d_v1 SEQ ID NO: 80 in which Pro at position 238 represented by EU numbering of IL6R-G1d was modified to Asp was prepared.
- IL6R-G1d_v2 SEQ ID NO: 81 in which Leu at position 328 represented by EU numbering of IL6R-G1d was modified to Glu was prepared.
- Ser 267 represented by EU numbering, which is a known mutation (Mol. Immunol.
- IL6R-G1d_v3 As the antibody L chain, IL6R-L (SEQ ID NO: 83), which is the L chain of tocilizumab, was commonly used in combination with these heavy chains. The antibody was expressed and purified according to the method of Reference Example 2.
- Antibodies containing IL6R-G1d, IL6R-G1d_v1, IL6R-G1d_v2, and IL6R-G1d_v3 as antibody heavy chains are hereinafter referred to as IgG1, IgG1-v1, IgG1-v2, and IgG1-v3, respectively.
- KD was not calculated by global fitting the measurement results using the above 1: 1 Langmuir binding model using Biacore Evaluation Software It was.
- KD was calculated using the following 1: 1 binding model formula described in Biacore T100 Software Handbook BR1006-48 Edition AE It was done.
- the behavior of molecules interacting with the 1: 1 binding model on Biacore can be expressed by the following equation 4.
- Equation 4 The meaning of each item in the above [Formula 4] is as follows: Req (RU): Steady state binding levels C (M): Analyte concentration C: concentration Rmax (RU): Analyte binding capacity of the surface RI (RU): Bulk refractive index contribution in the sample KD (M): Equilibrium dissociation constant It is represented by By transforming Equation 4, KD can be expressed as Equation 5 below.
- Rmax the analysis result of IgG1 interaction with each Fc ⁇ R is obtained by dividing the Rmax value obtained by global fitting with 1: 1 Langmuir binding model by the capture amount of IgG1, and capture of IgG1-v1 and IgG1-v2 It was set as the value obtained by multiplying by quantity.
- the binding of IgG1-v1 and IgG1-v2 to Fc ⁇ RIIa (H) was about 2.5 and 10 ⁇ RU, respectively, and the binding of IgG1-v1 and IgG1-v2 to Fc ⁇ RIIIa was about 2.5 and 5 ⁇ RU, respectively.
- the capture amounts of the IgG1-v1 and IgG1-v2 antibody sensor chips were 469.2 and 444.2 RU, and the IgG1 interaction analysis with IgG1 against Fc ⁇ RIIIa
- the capture amounts of -v1 and IgG1-v2 antibody sensor chips were 470.8 and 447.1 RU.
- Rmax obtained by global fitting with 1: 1 Langmuir binding model is 69.8 and 63.8 RU, respectively, and the amount of antibody captured on the sensor chip is 452, It was 454.5 RU.
- the Rmax of IgG1-v1 and IgG1-v2 for Fc ⁇ RIIa (H) was calculated to be 72.5 and 68.6 ⁇ RU, respectively, and the Rmax of IgG1-v1 and IgG1-v2 for Fc ⁇ RIIIa was calculated to be 66.0 and 62.7 ⁇ RU, respectively.
- the KD of IgG1-v1 and IgG1-v2 for Fc ⁇ RIIa (H) and Fc ⁇ RIIIa was calculated.
- the KD values for IgG1, IgG1-v1, IgG1-v2, and IgG1-v3 Fc ⁇ R are shown in Table 24 (KD values for each Fc ⁇ R of each antibody), and the KD values for each Fc ⁇ R of IgG1 are IgG1-v1, IgG1-v2
- the relative KD values of IgG1-v1, IgG1-v2, and IgG1-v3 divided by the KD value for each Fc ⁇ R of IgG1-v3 are shown in Table 25 (relative KD values for each Fc ⁇ R of each antibody).
- IgG1-v1 has a 0.047-fold lower affinity for Fc ⁇ RIa, a 0.10-fold lower affinity for Fc ⁇ R IIa (R), and an affinity for Fc ⁇ RIIa (H) Decreased to 0.014 times, and the affinity for Fc ⁇ RIIIa decreased to 0.061 times.
- the affinity for Fc ⁇ RIIb was improved by 4.8 times.
- IgG1-v2 has an affinity for Fc ⁇ RIa that is 0.74 times lower than IgG1, and affinity for Fc ⁇ R IIa (R) is reduced 0.41 times, and affinity for Fc ⁇ RIIa (H). And the affinity for Fc ⁇ RIIIa was reduced 0.14 times.
- the affinity for Fc ⁇ RIIb was improved 2.3 times.
- IgG1-v1 in which Pro at position 238 represented by EU numbering was replaced with Asp and IgG1-v2 in which Leu at position 328 represented by EU numbering was replaced by Glu were both Fc ⁇ RIIa. It was revealed that the binding to the active Fc ⁇ R including the gene polymorphism decreased while the binding to the inhibitory Fc ⁇ R, Fc ⁇ RIIb, increased. A variant having such properties has not been reported so far, and is extremely rare as shown in FIGS. A variant in which Pro at position 238 represented by EU numbering is replaced with Asp and a variant in which Leu at position 328 represented by EU numbering is replaced by Glu are used for the development of therapeutic drugs for immunoinflammatory diseases, etc. Very useful.
- IgG1-v3 certainly has a 408-fold increase in binding to Fc ⁇ RIIb and a 0.51-fold decrease in binding to Fc ⁇ RIIa (H), while binding to Fc ⁇ RIIa (R) is reduced. 522 times better. That is, IgG1-v1 and IgG1-v2 are more selective for Fc ⁇ RIIb than IgG1-v3 because binding to both Fc ⁇ RIIa (R) and Fc ⁇ RIIa (H) is suppressed and binding to Fc ⁇ RIIb is improved. It is considered to be a variant that binds to. In other words, a variant in which Pro at position 238 represented by EU numbering is replaced with Asp and a variant in which Leu at position 328 represented by EU numbering is replaced by Glu are development of therapeutic agents for immunoinflammatory diseases, etc. Very useful.
- amino acid substitution for substituting Pro at position 238 represented by EU numbering to Asp for VH3-IgG1-F11 was introduced by the method of Reference Example 1, and VH3-IgG1-F652 (SEQ ID NO: 85) was introduced. ) was produced.
- the IgG1-F652 obtained here is an antibody having a binding activity to FcRn under neutral pH conditions, and a selective enhancement of binding activity to inhibitory Fc ⁇ RIIb. That is, it corresponds to the antigen-binding molecule of embodiment 2 shown in Example 3.
- IgG1-F652 can form a quaternary complex via two molecules of FcRn and one molecule of Fc ⁇ R, but since it has enhanced selective binding activity to inhibitory Fc ⁇ R, active Fc ⁇ R The binding activity to is reduced. As a result, it is considered that a quaternary complex containing inhibitory Fc ⁇ R is formed predominantly on antigen-presenting cells.
- immunogenicity is thought to be caused by the formation of a quaternary complex containing active Fc ⁇ R.
- a quaternary complex containing inhibitory Fc ⁇ R the suppression of immune response is suppressed. It is considered possible.
- HEK293H strain derived from human fetal kidney cancer cells is suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and the dish (diameter) has a cell density of 5-6 ⁇ 10 5 cells / mL. 10 mL to each dish of 10 cm, CORNING) After incubating overnight in a CO 2 incubator (37 ° C, 5% CO 2 ), the medium is removed by suction and CHO-S-SFM-II (Invitrogen) medium 6.9 mL was added.
- the prepared plasmid was introduced into cells by the lipofection method. After collecting the obtained culture supernatant, the cells are removed by centrifugation (approximately 2000 g, 5 minutes, room temperature), and further sterilized through a 0.22 ⁇ m filter MILLEX (R) -GV (Millipore). Got.
- the obtained culture supernatant was purified by a method known to those skilled in the art using rProtein A Sepharose TM Fast Flow (Amersham Biosciences). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the method described in Protein Science 1995; 4: 2411-2423.
- soluble human IL-6 receptor Recombinant human IL-6 receptor of human IL-6 receptor as an antigen was prepared as follows.
- a soluble human IL-6 receptor (hereinafter also referred to as hsIL-6R) consisting of the amino acid sequence from the 1st to the 357th N-terminal side reported in J. Immunol. (1994) 152, 4958-4968
- a CHO strain that constantly expresses was constructed by methods known to those skilled in the art. By culturing the CHO strain, soluble human IL-6 receptor was expressed.
- soluble human IL-6 receptor was purified by two steps of Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the final step was used as the final purified product.
- PK test of soluble human IL-6 receptor and human antibody in normal mice Objective to evaluate plasma retention and immunogenicity of soluble human IL-6 receptor and human antibody in normal mice The following tests were conducted. (4-1) Evaluation of plasma retention and immunogenicity of soluble human IL-6 receptor in normal mice Purpose of evaluating plasma retention and immunogenicity of soluble human IL-6 receptor in normal mice The following tests were conducted. A soluble human IL-6 receptor (prepared in Reference Example 3) was administered at a single dose of 50 ⁇ g / kg to the tail vein of normal mice (C57BL / 6J mice, Charles River Japan).
- the concentration of soluble human IL-6 receptor in mouse plasma was measured by the electrochemiluminescence method.
- a soluble human IL-6 receptor calibration curve sample prepared at 2000, 1000, 500, 250, 125, 62.5, 31.25 pg / mL and a mouse plasma measurement sample diluted 50 times or more were used as SULFO-TAG NHS Ester ( The reaction was carried out overnight at 37 ° C. by mixing with Monoclonal Anti-human IL-6R Antibody (R & D) and Biotinylated Anti-human IL-6 R Antibody (R & D) and Tocilizumab ruthenated in Meso Scale Discovery. The final concentration of Tocilizumab was adjusted to 333 ⁇ g / mL.
- reaction solution was dispensed into MA400®PR®Streptavidin®Plate (Meso®Scale®Discovery). Further, after washing the reaction solution reacted at room temperature for 1 hour, Read Buffer T ( ⁇ 4) (Meso Scale Discovery) was dispensed. Immediately thereafter, measurements were taken with a SECTOR PR-400 reader (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).
- the antibody titer of mouse anti-human IL-6 receptor antibody in mouse plasma was measured by electrochemiluminescence method.
- human IL-6 receptor was dispensed into MA100 PR Uncoated Plate (Meso Discovery) and allowed to stand overnight at 4 ° C to prepare a human IL-6 receptor solid phase plate.
- a human IL-6 receptor-immobilized plate into which a 50-fold diluted mouse plasma measurement sample was dispensed was allowed to stand overnight at 4 ° C. Thereafter, the plate was washed with anti-mouse IgG (whole molecule) (Sigma-Aldrich) that was ruthenized with SULFO-TAG NHS Ester (Meso Scale Discovery) at room temperature for 1 hour.
- Read Buffer T ⁇ 4
- SECTOR PR 400 reader Meso Scale Discovery
- Monoclonal anti-mouse CD4 antibody (R & D) was administered to the tail vein at 20 mg / kg in order to suppress the production of mouse antibodies against soluble human IL-6 receptor in a group of mice with mimicking immune tolerance.
- a single dose was administered in the same manner once every 10 days (hereinafter referred to as an anti-mouse CD4 antibody administration group).
- the other group was used as a control group, ie, an anti-mouse CD4 antibody non-administered group to which no monoclonal anti-mouse CD4 antibody was administered.
- FIG. 38 shows changes in plasma soluble human IL-6 receptor concentration in individual normal mice measured by this method.
- Fv4-IgG1 an anti-human IL-6 receptor antibody
- Fv4-IgG1 an anti-human IL-6 receptor antibody
- Plasma was obtained by immediately centrifuging the collected blood at 4 ° C. and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer set to ⁇ 20 ° C. or lower until measurement was performed.
- the anti-human IL-6 receptor antibody concentration in mouse plasma was measured by ELISA.
- Anti-Human IgG ( ⁇ -chain specific) F (ab ') 2 Fragment of Antibody (SIGMA) is dispensed into Nunc-Immuno Plate and MaxiSoup (Nalge nunc International) and left at 4 °C overnight. Produced an anti-Human IgG immobilized plate.
- a calibration curve sample containing anti-human IL-6 receptor antibody having a plasma concentration of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 ⁇ g / mL and a mouse plasma measurement sample diluted 100 times or more were prepared.
- Substrate BioFX Laboratories was used as a substrate.
- the antibody concentration in the mouse plasma was calculated from the absorbance of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).
- mice have a large population of T cells that specifically respond to them.
- human soluble IL-6 receptor a heterologous protein
- human soluble IL-6 receptor disappears from plasma in a short time, and immune response to human soluble IL-6 receptor was confirmed.
- the rapid disappearance of human soluble IL-6 receptor from plasma means that many human soluble IL-6 receptors are taken up by antigen-presenting cells in a short time and processed in the cells. And then activate T cells that specifically respond to human soluble IL-6 receptor.
- an immune response to human soluble IL-6 receptor that is, production of mouse antibody to human soluble IL-6 receptor
- Table 27-2 is a continuation table of Table 27-1.
- Table 27-3 is a continuation table of Table 27-2.
- Table 27-4 is a continuation table of Table 27-3.
- Table 27-5 is a continuation table of Table 27-4.
- Table 27-6 is a continuation table of Table 27-5.
- Table 27-7 is a continuation table of Table 27-6.
- Table 27-8 is a continuation table of Table 27-7.
- Table 27-9 is a continuation table of Table 27-8.
- Table 27-10 is a continuation table of Table 27-9.
- Table 27-11 is a continuation table of Table 27-10.
- Table 27-12 is a continuation table of Table 27-11.
- Table 27-13 is a continuation table of Table 27-12.
- Table 27-14 is a continuation table of Table 27-13.
- Table 27-15 is a continuation table of Table 27-14.
- Table 27-16 is a continuation table of Table 27-15.
- Table 27-17 is a continuation table of Table 27-16.
- Table 27-18 is a continuation table of Table 27-17.
- Table 27-19 is a continuation table of Table 27-18.
- Table 27-20 is a continuation table of Table 27-19.
- Table 27-21 is a continuation table of Table 27-20.
- Table 27-22 is a continuation table of Table 27-21.
- Table 27-23 is a continuation table of Table 27-22.
- Table 27-24 is a continuation table of Table 27-23.
- Table 27-25 is a continuation table of Table 27-24.
- Table 27-26 is a continuation table of Table 27-25.
- Table 27-27 is a continuation table of Table 27-26.
- Table 27-28 is a continuation table of Table 27-27.
- Table 27-29 is a continuation table of Table 27-28.
- Table 27-30 is a continuation table of Table 27-29.
- Table 27-31 is a continuation table of Table 27-30.
- Table 27-32 is a continuation table of Table 27-31.
- Fv4-IgG1 comprising VH3-IgG1 (SEQ ID NO: 35) and VL3-CK (SEQ ID NO: 36)
- Fv4-IgG1-v2 comprising VH3-IgG1-F1 (SEQ ID NO: 37) and VL3-CK (SEQ ID NO: 36)
- Fv4-IgG1-F14 comprising VH3-IgG1-F14 (SEQ ID NO: 86) and VL3-CK (SEQ ID NO: 36)
- Fv4-IgG1-F20 comprising VH3-IgG1-F20 (SEQ ID NO: 39) and VL3-CK (SEQ ID NO: 36)
- Fv4-IgG1-F21 comprising VH3-IgG1-F21 (SEQ ID NO: 40) and VL3-CK (SEQ ID NO: 36)
- Fv4-IgG1-F25 comprising VH3-Ig
- human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg strain 276 + / + mice, Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104 .) was performed as follows: Human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg strain 276 + / + mice, Jackson Laboratories, Methods Mol Biol. 2010; 602: 93-104 .) was performed as follows: Human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg strain 276 + / + mice, Jackson Laboratories, Methods Mol Biol.
- HsIL-6R soluble human IL-6 receptor: prepared in Reference Example 3
- soluble human IL-6 receptor 10 mg
- a mixed solution of soluble human IL-6 receptor and anti-human IL-6 receptor antibody 5 ⁇ g / mL and 0.1 mg / mL, respectively
- Plasma soluble human IL-6 receptor concentration in mice was measured by electrochemiluminescence method.
- a soluble human IL-6 receptor calibration curve sample prepared at 2000, 1000, 500, 250, 125, 62.5, 31.25 pg / mL and a mouse plasma measurement sample diluted 50-fold or more were prepared using SULFO-TAG NHS Ester ( Monoclonal Anti-human IL-6R Antibody (R & D), Biotinylated Anti-human IL-6R Antibody (R & D) and Tocilizumab ruthenated by Meso Scale Discovery were reacted overnight at 37 ° C.
- the final concentration of Tocilizumab was adjusted to 333 ⁇ g / mL. Thereafter, the reaction solution was dispensed into MA400 PR Streptavidin Plate (Meso Scale Discovery). Further, after washing the reaction solution reacted at room temperature for 1 hour, Read Buffer T ( ⁇ 4) (Meso Scale Discovery) was dispensed. Immediately thereafter, measurements were taken with a SECTOR PR 400 reader (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated from the response of the calibration curve using the analysis software SOFTmax PRO (Molecular Devices).
- FIG. 40 shows changes in plasma soluble human IL-6 receptor concentration in the obtained human FcRn transgenic mice after intravenous administration.
- any pH-dependent human IL-6 receptor-binding antibody with enhanced binding to human FcRn under neutral conditions has no ability to bind to human FcRn under neutral conditions. It was shown that the plasma concentration of soluble human IL-6 receptor remained low.
- the plasma concentration of soluble human IL-6 receptor administered at the same time as Fv4-IgG1-F14 was administered at the same time as Fv4-IgG1. It was shown to be about 54 times lower than that.
- the plasma concentration of soluble human IL-6 receptor administered at the same time as Fv4-IgG1-F21 at 7 hours may be reduced by about 24 times compared to that administered at the same time as Fv4-IgG1. Indicated. Furthermore, the plasma concentration of soluble human IL-6 receptor administered at the same time as Fv4-IgG1-F25 after 7 hours is below the detection limit (1.56 ng / mL), and administered at the same time as Fv4-IgG1. Compared with that, it was considered that the antigen concentration can be significantly reduced by 200 times or more.
- the first selection from the constructed naive human antibody phage display library is the ability to bind to the antigen (IL-6 receptor). This was carried out by concentration of only antibody fragments having a phenotype or concentration of antibody fragments using the binding ability to an antigen (IL-6 receptor) dependent on Ca concentration as an index. Concentration of antibody fragments using the binding ability to Ca concentration-dependent antigen (IL-6 receptor) as an index is based on phage library bound to IL-6 receptor in the presence of Ca ion using EDTA chelating Ca ion. This was done by eluting the phage. IL-6 receptor labeled with biotin was used as an antigen.
- Phage production was performed from E. coli holding the constructed phagemid for phage display.
- a phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS.
- BSA and CaCl 2 were added to the phage library solution to a final concentration of 4% BSA and 1.2 mM calcium ion concentration.
- a panning method a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods.
- NeutrAvidin coated beads Sera-Mag SpeedBeads NeutrAvidin-coated
- Streptavidin coated beads Dynabeads M-280 Streptavidin
- the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. Beads were washed once with TBS) containing 1.2 mM CaCl 2 /TBS(1.2 mM CaCl 2 in 1 mL. Then, when concentrating antibody fragments that have the ability to bind to IL-6 receptor, the antibody fragments are concentrated by elution using a general method with the binding ability to IL-6 receptor dependent on Ca concentration as an index.
- phage solutions were recovered by elution from beads suspended in 2 mM EDTA / TBS (TBS containing 2 mM EDTA). The recovered phage solution was added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.7). E. coli was infected with phages by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates. Next, a phage library solution was prepared by recovering the phage from the seeded E. coli culture solution.
- phages were concentrated using Ca-dependent binding ability as an index.
- the phage library was contacted with the antigen at room temperature for 60 minutes by adding 40 pmol of biotin-labeled antigen to the prepared phage library solution. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed with 1 mL of 1.2 mM CaCl 2 / TBST and 1.2 mM CaCl 2 / TBS.
- the beads to which 0.1 mL of 2 mM EDTA / TBS was added were suspended at room temperature, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- the recovered phage solution was added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.7).
- E. coli was infected with phages by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates.
- the phage library solution was recovered by recovering the phage from the seeded E. coli culture solution. Panning with Ca-dependent binding ability as an index was repeated several times.
- Each well of the plate was washed with PBST to remove the antigen, and then the well was blocked with 250 ⁇ L of 4% BSA-TBS for 1 hour or longer.
- the antibody displaying the phage binds to the antigen present in each well. I let you.
- the antibody was purified from the culture supernatant obtained above using rProtein A Sepharose TM Fast Flow (Amersham Biosciences) using methods known to those skilled in the art. Absorbance at 280 nm of the purified antibody solution was measured using a spectrophotometer. By using the extinction coefficient calculated by the PACE method, the antibody concentration was calculated from the obtained measured value (Protein Science (1995) 4, 2411-2423).
- H54 / L28-IgG1 (heavy chain variable region (SEQ ID NO: 96), light chain variable region (sequence) described in WO2009 / 125825 No. 97)) was used.
- the kinetic analysis of the antigen-antibody reaction was carried out in a solution with a calcium ion concentration of 2 mM and 3 ⁇ M, respectively, as conditions for high calcium ion concentration and low calcium ion concentration.
- the antibody of interest was captured on Sensor chip CM4 (GE Healthcare) on which an appropriate amount of protein A (Invitrogen) had been immobilized by the amine coupling method.
- the running buffer is 10 mM ACES, 150 mM NaCl, 0.05% (w / v) Tween 20, 2 mM CaCl 2 (pH 7.4) or 10 mM ACES, 150 mM NaCl, 0.05% (w / v) Tween 20, 3 ⁇ mol
- Two types of buffer solutions were used: / L CaCl 2 (pH 7.4). Each buffer was also used to dilute the human IL-6 receptor. All measurements were performed at 37 ° C.
- the IL-6 receptor diluted solution and blank running buffer were injected onto the sensor chip by injecting at a flow rate of 20 ⁇ L / min for 3 minutes.
- IL-6 receptor was allowed to interact with the H54L28-IgG1 antibody.
- 10 ⁇ mM Glycine-HCl pH 1.5 was injected at a flow rate of 30 ⁇ L / min for 30 seconds. The chip has been regenerated.
- KD of H54 / L28-IgG1 antibody under the condition of Ca concentration of 3 ⁇ M can be calculated by the same method as in the presence of 2 ⁇ mM Ca concentration.
- FH4-IgG1 antibody and 6RL # 9-IgG1 antibody hardly observed binding to IL-6 receptor, and therefore it is difficult to calculate KD by the above method.
- Equation 5 described in Example 13 (Biacore T100 Software Handbook, BR-1006-48, AE 01/2007), the KD of these antibodies under the condition of a Ca concentration of 3 ⁇ M is predicted. Is possible.
- Table 29 shows the estimated approximate results of the dissociation constant KD between each antibody and IL-6 receptor when the Ca concentration is 3 ⁇ mol / L using Formula 3 described in Example 13. Req, Rmax, RI, and C in Table 29 are values assumed based on the measurement results.
- FH4-IgG1 antibody and 6RL # 9-IgG1 antibody decreased the concentration of CaCl 2 in the buffer from 2 mM to 3 ⁇ M, so that the KD for IL-6 receptor was about 60 times and about 120 times, respectively.
- a fold increase 60-fold, 120-fold or more decrease in affinity was predicted.
- Table 30 summarizes the KD values in the presence of 2 mM CaCl 2 and 3 ⁇ M CaCl 2 and the Ca dependence of the KD values for the three antibodies H54 / L28-IgG1, FH4-IgG1, and 6RL # 9-IgG1. It was.
- Tm value thermal denaturation intermediate temperature (Tm value) by differential scanning calorimetry (DSC) is Measured (MicroCal VP-Capillary DSC, MicroCal).
- Tm value is an indicator of stability. When protein is stabilized by the binding of calcium ions, the thermal denaturation intermediate temperature (Tm value) is higher than when calcium ions are not bound ( J. Biol. Chem. (2008) 283, 37, 25140-25149).
- the binding activity of the calcium ion to the antibody was evaluated by evaluating the change in the Tm value of the antibody according to the change in the calcium ion concentration in the antibody solution.
- the purified antibody solution is 20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2 (pH 7.4) or 20 mM Tris-HCl, 150 mM NaCl, 3 ⁇ M CaCl 2 (pH 7.4). It was subjected to dialysis (EasySEP, TOMY) treatment. DSC measurement was performed from 20 ° C. to 115 ° C.
- Table 31 shows the heat denaturation intermediate temperature (Tm value) of the Fab domain of each antibody calculated based on the obtained DSC denaturation curve.
- 6RL # 9 antibody expressed for use in X-ray crystal structure analysis was purified. Specifically, the 6RL # 9 antibody heavy chain (SEQ ID NO: 9 linked to a constant region sequence derived from IgG1) and the light chain (SEQ ID NO: 93) were prepared respectively. Animal expression plasmids were transiently introduced into animal cells. Prepared by lipofection method into 800 mL human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended in FreeStyle 293 Expression Medium medium (Invitrogen) to a final cell density of 1 x 10 6 cells / mL Plasmids were introduced.
- Cells into which the plasmid was introduced were cultured in a CO 2 incubator (37 ° C., 8% CO 2 , 90 rpm) for 5 days.
- the antibody was purified from the culture supernatant obtained as described above according to a method known to those skilled in the art using rProtein A Sepharose TM Fast Flow (Amersham Biosciences). Absorbance at 280 nm of the purified antibody solution was measured using a spectrophotometer. The antibody concentration was calculated from the measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).
- the purified fraction obtained was concentrated to about 0.8 mL using a 5000 MWCO ultrafiltration membrane.
- the concentrated solution was added to a gel filtration column Superdex 200 10/300 GL (GE Healthcare) equilibrated with 100 mM HEPES buffer (pH 8) containing 50 mM NaCl.
- the purified 6RL # 9 antibody Fab fragment for crystallization was eluted from the column using the same buffer. All the column operations described above were performed at a low temperature of 6 to 7.5 ° C.
- Crystallization drops were prepared by adding 0.2 ⁇ l of a dilution series solution in which crystals were diluted 100-10000 times. X-ray diffraction data of a thin plate crystal obtained by allowing the crystallization drop to stand at 20 ° C. for 2 to 3 days was measured.
- Crystallization drops were prepared by adding 0.2 ⁇ l of a dilution series solution of 6RL # 9 antibody Fab fragment crystals diluted 100-10000 fold. X-ray diffraction data of a thin plate crystal obtained by allowing the crystallization drop to stand at 20 ° C. for 2 to 3 days was measured.
- the frozen state was maintained by placing frozen crystals in a nitrogen stream at -178 ° C.
- a CCD detector Quantum 315r ADSC
- a total of 180 diffraction images were collected while rotating the crystal by 1 °.
- Lattice constant determination, diffraction spot indexing, and diffraction data processing were performed by the programs Xia2 (CCP4 Software Suite), XDS Package (Walfgang Kabsch) and Scala (CCP4 Software Suite). Finally, diffraction intensity data up to a resolution of 2.2 mm was obtained.
- the amino acid residue of light chain 3-147 extracted from the structural coordinates of PDB code 2A9M was used as a model molecule for searching the VL region.
- the initial structure model of the 6RL # 9 antibody Fab fragment was obtained by determining the orientation and position of each model molecule for search in the crystal lattice from the rotation function and the translation function.
- the crystallographic reliability factor R value for the reflection data of 25-3.0 mm is 46.9%, Free R value Was 48.6%.
- the structure refinement using the program Refmac5 (CCP4 Software Suite) and the coefficient of 2Fo-Fc and Fo-Fc calculated using the structure factor Fc and phase calculated from the experimentally determined structure factor Fo and model
- the model was refined by repeatedly modifying the model on the program Coot (Paul Emsley) while referring to the electron density map.
- refinement was performed using the program Refmac5 (CCP4 Software Suite).
- the crystallographic reliability factor R value for the 3440 atom model was finally 20.0% and the Free R value was 27.9%.
- the structure refinement using the program Refmac5 (CCP4 Software Suite) and the coefficient of 2Fo-Fc and Fo-Fc calculated using the structure factor Fc and phase calculated from the experimentally determined structure factor Fo and model
- the model was refined by repeatedly modifying the model on the program Coot (Paul Emsley) while referring to the electron density map.
- water molecules were incorporated into the model based on the electron density map with coefficients 2Fo-Fc and Fo-Fc, and refined using the program Refmac5 (CCP4 Software Suite).
- the crystallographic reliability factor R value for the 3351 atom model was finally 20.9% and the Free R value was 27.7%.
- the calcium binding motif present in the heavy chain CDR3 revealed from the structure of the Fab fragment of the 6RL # 9 antibody can also be a new element in the design of the Ca library.
- a library containing the heavy chain CDR3 of the 6RL # 9 antibody and a flexible residue in the other CDR containing the light chain can be considered.
- the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. After the beads was washed three times with TBST) containing 1.2 mM CaCl 2 /TBST(1.2 mM CaCl 2 , further washed 2 times with TBS) containing 1 mL of 1.2 mM CaCl 2 /TBS(1.2 mM CaCl 2 It was.
- the beads to which 0.5 mL of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- the recovered phage solution was added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.5).
- E. coli was infected with phages by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates.
- a phage library solution was prepared by recovering the phage from the seeded E. coli culture solution.
- phages were concentrated using Ca-dependent binding ability as an index.
- the phage library was contacted with the antigen at room temperature for 60 minutes by adding 40 pmol of biotin-labeled antigen to the prepared phage library solution. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed with 1 mL of 1.2 mM CaCl 2 / TBST and 1.2 mM CaCl 2 / TBS.
- the beads to which 0.1 mL of 2 mM EDTA / TBS was added were suspended at room temperature, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- the phage pIII protein that does not display Fab pIII protein derived from helper phage
- Phages recovered from the trypsinized phage solution were added to 10 mL of E. coli strain TG1 in the logarithmic growth phase (OD600 0.4-0.7).
- coli was infected with phages by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates. Next, the phage library solution was recovered by recovering the phage from the seeded E. coli culture solution. Panning with Ca-dependent binding ability as an index was repeated three times.
- Each well of the plate was washed with PBST to remove the antigen, and then the well was blocked with 250 ⁇ L of 4% BSA-TBS for 1 hour or longer.
- the antibody displaying the phage binds to the antigen present in each well. I let you.
Abstract
Description
〔1〕イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、およびpH中性域の条件下でFcRnに対する結合活性を有するFc領域を含む抗原結合分子のFc領域が、pH中性域の条件下で二分子のFcRnおよび一分子の活性型Fcγレセプターを含むヘテロ複合体を形成しないFc領域に改変することを含む、以下のいずれかの方法;
(a) 抗原結合分子の薬物動態を改善する方法、または
(b) 抗原結合分子の免疫原性を低減させる方法、
〔2〕前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域の活性型Fcγレセプターに対する結合活性が、天然型ヒトIgGのFc領域の当該活性型Fcγレセプターに対する結合活性よりも低いFc領域に改変することを含む、〔1〕に記載の方法、
〔3〕前記活性型FcγレセプターがヒトFcγRIa、ヒトFcγRIIa(R)、ヒトFcγRIIa(H)、ヒトFcγRIIIa(V)またはヒトFcγRIIIa(F)である、〔1〕または〔2〕に記載の方法、
〔4〕前記Fc領域のアミノ酸のうちEUナンバリングで表される235位、237位、238位、239位、270位、298位、325位および329位のいずれかひとつ以上のアミノ酸を置換することを含む、〔1〕から〔3〕のいずれか一項に記載の方法、
〔5〕前記Fc領域のEUナンバリングで表されるアミノ酸であって;
234位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Met、Phe、Pro、Ser、ThrまたはTrpのいずれか、
235位のアミノ酸をAla、Asn、Asp、Gln、Glu、Gly、His、Ile、Lys、Met、Pro、Ser、Thr、ValまたはArgのいずれか、
236位のアミノ酸をArg、Asn、Gln、His、Leu、Lys、Met、Phe、ProまたはTyrのいずれか、
237位のアミノ酸をAla、Asn、Asp、Gln、Glu、His、Ile、Leu、Lys、Met、Pro、Ser、Thr、Val、TyrまたはArgのいずれか、
238位のアミノ酸をAla、Asn、Gln、Glu、Gly、His、Ile、Lys、Thr、TrpまたはArgのいずれか、
239位のアミノ酸をGln、His、Lys、Phe、Pro、Trp、TyrまたはArgのいずれか、
265位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、TyrまたはValのいずれか、
266位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Phe、Pro、Ser、Thr、TrpまたはTyrのいずれか、
267位のアミノ酸をArg、His、Lys、Phe、Pro、TrpまたはTyrのいずれか、
269位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
270位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
271位のアミノ酸をArg、His、Phe、Ser、Thr、TrpまたはTyrのいずれか、
295位のアミノ酸をArg、Asn、Asp、Gly、His、Phe、Ser、TrpまたはTyrのいずれか、
296位のアミノ酸をArg、Gly、LysまたはProのいずれか、
297位のアミノ酸をAla、
298位のアミノ酸をArg、Gly、Lys、Pro、TrpまたはTyrのいずれか、
300位のアミノ酸をArg、LysまたはProのいずれか、
324位のアミノ酸をLysまたはProのいずれか、
325位のアミノ酸をAla、Arg、Gly、His、Ile、Lys、Phe、Pro、Thr、TrpTyr、もしくはValのいずれか、
327位のアミノ酸をArg、Gln、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
328位のアミノ酸をArg、Asn、Gly、His、LysまたはProのいずれか、
329位のアミノ酸をAsn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、Tyr、ValまたはArgのいずれか、
330位のアミノ酸をProまたはSerのいずれか、
331位のアミノ酸をArg、GlyまたはLysのいずれか、もしくは
332位のアミノ酸をArg、LysまたはProのいずれか、
のいずれかひとつ以上に置換することを含む、〔4〕に記載の方法、
〔6〕前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域の抑制型Fcγレセプターに対する結合活性が活性型Fcγレセプターに対する結合活性よりも高いFc領域に改変することを含む、〔1〕に記載の方法、
〔7〕前記抑制型FcγレセプターがヒトFcγRIIbである、〔6〕に記載の方法、
〔8〕前記活性型FcγレセプターがヒトFcγRIa、ヒトFcγRIIa(R)、ヒトFcγRIIa(H)、ヒトFcγRIIIa(V)またはヒトFcγRIIIa(F)である、〔6〕または〔7〕に記載の方法、
〔9〕EUナンバリングで表される238または328のアミノ酸を置換することを含む〔6〕から〔8〕のいずれか一項に記載の方法、
〔10〕EUナンバリングで表される238のアミノ酸をAsp、または328のアミノ酸をGluに置換することを含む、〔9〕に記載の方法、
〔11〕 EUナンバリングで表されるアミノ酸であって;
233位のアミノ酸をAsp、
234位のアミノ酸をTrp、またはTyrのいずれか、
237位のアミノ酸をAla、Asp、Glu、Leu、Met、Phe、TrpまたはTyrのいずれか、
239位のアミノ酸をAsp、
267位のアミノ酸をAla、GlnまたはValのいずれか、
268位のアミノ酸をAsn、Asp、またはGluのいずれか、
271位のアミノ酸をGly、
326位のアミノ酸をAla、Asn、Asp、Gln、Glu、Leu、Met、SerまたはThrのいずれか、
330位のアミノ酸をArg、Lys、またはMetのいずれか、
323位のアミノ酸をIle、Leu、またはMetのいずれか、
296位のアミノ酸をAsp、
のいずれかひとつ以上に置換することを含む〔9〕または〔10〕に記載の方法、
〔12〕前記Fc領域のアミノ酸のうちEUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、298、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸が天然型Fc領域のアミノ酸と異なるアミノ酸を含むFc領域である、〔1〕から〔11〕のいずれか一項に記載の方法、
〔13〕前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyrのいずれか、
252位のアミノ酸がPhe、Trp、またはTyrのいずれか、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsp、Asn、Glu、またはGlnのいずれか、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはValのいずれか、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGluのいずれか、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
298位のアミノ酸がGly、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyrのいずれか、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThrのいずれか、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArgのいずれか、
311位のアミノ酸がAla、His、またはIleのいずれか、
312位のアミノ酸がAlaまたはHisのいずれか、
314位のアミノ酸がLysまたはArgのいずれか、
315位のアミノ酸がAla、AspまたはHisのいずれか、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHisのいずれか、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSerのいずれか、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyrのいずれか、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyrのいずれか、もしくは
436位のアミノ酸がHis、Ile、Leu、Phe、ThrまたはVal 、
のいずれかひとつ以上の組合せである、〔12〕に記載の方法、
〔14〕前記抗原結合ドメインが、カルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔1〕から〔13〕のいずれか一項に記載の方法、
〔15〕前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔14〕に記載の方法、
〔16〕前記抗原結合ドメインが、pHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔1〕から〔13〕のいずれか一項に記載の方法、
〔17〕前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔16〕に記載の方法、
〔18〕前記抗原結合ドメインが抗体の可変領域である、〔1〕から〔17〕のいずれか一項に記載の方法、
〔19〕前記抗原結合分子が抗体である、〔1〕から〔18〕のいずれか一項に記載の方法、
〔20〕前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域を構成する二つのポリペプチドの一方がpH中性域の条件下でのFcRn結合活性を有し、他方がpH中性域の条件下でのFcRn結合活性を有しないFc領域に改変することを含む、〔1〕に記載の方法、
〔21〕前記Fc領域を構成する二つのポリペプチドの一方のアミノ酸配列のうち、EUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、298、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸を置換することを含む、〔20〕に記載の方法、
〔22〕前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸をMet、
248位のアミノ酸をIle、
250位のアミノ酸をAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyr、
252位のアミノ酸をPhe、Trp、またはTyr、
254位のアミノ酸をThr、
255位のアミノ酸をGlu、
256位のアミノ酸をAsp、Asn、Glu、またはGln、
257位のアミノ酸をAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはVal、
258位のアミノ酸をHis、
265位のアミノ酸をAla、
286位のアミノ酸をAlaまたはGlu、
289位のアミノ酸をHis、
297位のアミノ酸をAla、
298位のアミノ酸をGly、
303位のアミノ酸をAla、
305位のアミノ酸をAla、
307位のアミノ酸をAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyr、
308位のアミノ酸をAla、Phe、Ile、Leu、Met、Pro、Gln、またはThr、
309位のアミノ酸をAla、Asp、Glu、Pro、またはArg、
311位のアミノ酸をAla、His、またはIle、
312位のアミノ酸をAlaまたはHis、
314位のアミノ酸をLysまたはArg、
315位のアミノ酸をAla、AspまたはHis、
317位のアミノ酸をAla、
332位のアミノ酸をVal、
334位のアミノ酸をLeu、
360位のアミノ酸をHis、
376位のアミノ酸をAla、
380位のアミノ酸をAla、
382位のアミノ酸をAla、
384位のアミノ酸をAla、
385位のアミノ酸をAspまたはHis、
386位のアミノ酸をPro、
387位のアミノ酸をGlu、
389位のアミノ酸をAlaまたはSer、
424位のアミノ酸をAla、
428位のアミノ酸をAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyr、
433位のアミノ酸をLys、
434位のアミノ酸をAla、Phe、His、Ser、Trp、またはTyr、もしくは
436位のアミノ酸をHis 、Ile、Leu、Phe、Thr、またはVal
のいずれかひとつ以上に置換することを含む、〔21〕に記載の方法、
〔23〕前記抗原結合ドメインが、カルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔20〕から〔22〕のいずれか一項に記載の方法、
〔24〕前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔23〕に記載の方法、
〔25〕前記抗原結合ドメインが、pHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔20〕から〔22〕のいずれか一項に記載の方法、
〔26〕前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔25〕に記載の方法、
〔27〕前記抗原結合ドメインが抗体の可変領域である、〔20〕から〔26〕のいずれか一項に記載の方法、
〔28〕前記抗原結合分子が抗体である、〔20〕から〔27〕のいずれか一項に記載の方法、
〔29〕イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、ならびにpH中性域の条件下でFcRnに対する結合活性を有するFc領域のEUナンバリングで表されるアミノ酸であって;
234位のアミノ酸がAla、
235位のアミノ酸がAla、LysまたはArgのいずれか、
236位のアミノ酸がArg、238位のアミノ酸がArg、
239位のアミノ酸がLys、
270位のアミノ酸がPhe、
297位のアミノ酸がAla、
298位のアミノ酸がGly、
325位のアミノ酸がGly、
328位のアミノ酸がArg、もしくは329 位のアミノ酸がLys、またはArg、
の中から選択されるいずれかひとつ以上のアミノ酸を含むFc領域を含む抗原結合分子、
〔30〕前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がLysまたはArgのいずれか、
238位のアミノ酸がLys
239位のアミノ酸がArg、または
329位のアミノ酸がLysまたはArgのいずれか、
の中から選択されるいずれかひとつ以上のアミノ酸を含む、〔29〕に記載の抗原結合分子、
〔31〕イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、およびFc領域を構成する二つのポリペプチドの一方がpH中性域の条件下でのFcRnに対する結合活性を有し、他方がpH中性域の条件下でのFcRnに対する結合活性を有しないFc領域を含む抗原結合分子、
〔32〕前記Fc領域を構成する二つのポリペプチドの一方のアミノ酸配列のうち、EUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸が天然型Fc領域のアミノ酸と異なるFc領域である、〔29〕から〔31〕のいずれか一項に記載の抗原結合分子、
〔33〕前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyr、
252位のアミノ酸がPhe、Trp、またはTyr、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsp、Asn、Glu、またはGln、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはVal、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGlu、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyr、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThr、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArg、
311位のアミノ酸がAla、His、またはIle、
312位のアミノ酸がAlaまたはHis、
314位のアミノ酸がLysまたはArg、
315位のアミノ酸がAla、AspまたはHis、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHis、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSer、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyr、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyr、もしくは
436位のアミノ酸がHis 、Ile、Leu、Phe、Thr、またはVal、
のいずれかひとつ以上の組合せである、〔32〕に記載の抗原結合分子、
〔34〕前記抗原結合ドメインがカルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔29〕から〔33〕のいずれか一項に記載の抗原結合分子、
〔35〕前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔34〕に記載の抗原結合分子、
〔36〕前記抗原結合ドメインがpHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、〔29〕から〔33〕のいずれか一項に記載の抗原結合分子、
〔37〕前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、〔36〕に記載の抗原結合分子、
〔38〕前記抗原結合ドメインが抗体の可変領域である、〔29〕から〔37〕のいずれか一項に記載の抗原結合分子、
〔39〕前記抗原結合分子が抗体である、〔29〕から〔38〕のいずれか一項に記載の抗原結合分子、
〔40〕〔29〕から〔39〕のいずれか一項に記載の抗原結合分子をコードするポリヌクレオチド、
〔41〕〔40〕に記載のポリヌクレオチドが作用可能に連結されたベクター、
〔42〕〔41〕に記載のベクターが導入された細胞、
〔43〕〔42〕に記載の細胞の培養液から抗原結合分子を回収する工程を含む、〔29〕から〔39〕のいずれか一項に記載の抗原結合分子の製造方法、
〔44〕〔29〕から〔39〕のいずれか一項に記載の抗原結合分子、または〔43〕に記載の製造方法によって得られる抗原結合分子を有効成分として含む医薬組成物。
アミノ酸
本明細書において、たとえば、Ala/A、Leu/L、Arg/R、Lys/K、Asn/N、Met/M、Asp/D、Phe/F、Cys/C、Pro/P、Gln/Q、Ser/S、Glu/E、Thr/T、Gly/G、Trp/W、His/H、Tyr/Y、Ile/I、Val/Vと表されるように、アミノ酸は1文字コードまたは3文字コード、またはその両方で表記されている。
本明細書において「抗原」は抗原結合ドメインが結合するエピトープを含む限りその構造は特定の構造に限定されない。別の意味では、抗原は無機物でもあり得るし有機物でもあり得る。抗原としては下記のような分子;17-IA、4-1BB、4Dc、6-ケト-PGF1a、8-イソ-PGF2a、8-オキソ-dG、A1 アデノシン受容体、A33、ACE、ACE-2、アクチビン、アクチビンA、アクチビンAB、アクチビンB、アクチビンC、アクチビンRIA、アクチビンRIA ALK-2、アクチビンRIB ALK-4、アクチビンRIIA、アクチビンRIIB、ADAM、ADAM10、ADAM12、ADAM15、ADAM17/TACE、ADAM8、ADAM9、ADAMTS、ADAMTS4、ADAMTS5、アドレシン、aFGF、ALCAM、ALK、ALK-1、ALK-7、アルファ-1-アンチトリプシン、アルファ-V/ベータ-1アンタゴニスト、ANG、Ang、APAF-1、APE、APJ、APP、APRIL、AR、ARC、ART、アルテミン、抗Id、ASPARTIC、心房性ナトリウム利尿因子、av/b3インテグリン、Axl、b2M、B7-1、B7-2、B7-H、B-リンパ球刺激因子(BlyS)、BACE、BACE-1、Bad、BAFF、BAFF-R、Bag-1、BAK、Bax、BCA-1、BCAM、Bcl、BCMA、BDNF、b-ECGF、bFGF、BID、Bik、BIM、BLC、BL-CAM、BLK、BMP、BMP-2 BMP-2a、BMP-3 オステオゲニン(Osteogenin)、BMP-4 BMP-2b、BMP-5、BMP-6 Vgr-1、BMP-7(OP-1)、BMP-8(BMP-8a、OP-2)、BMPR、BMPR-IA(ALK-3)、BMPR-IB(ALK-6)、BRK-2、RPK-1、BMPR-II(BRK-3)、BMP、b-NGF、BOK、ボンベシン、骨由来神経栄養因子、BPDE、BPDE-DNA、BTC、補体因子3(C3)、C3a、C4、C5、C5a、C10、CA125、CAD-8、カルシトニン、cAMP、癌胎児性抗原(CEA)、癌関連抗原、カテプシンA、カテプシンB、カテプシンC/DPPI、カテプシンD、カテプシンE、カテプシンH、カテプシンL、カテプシンO、カテプシンS、カテプシンV、カテプシンX/Z/P、CBL、CCI、CCK2、CCL、CCL1、CCL11、CCL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL2、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27、CCL28、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9/10、CCR、CCR1、CCR10、CCR10、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CD1、CD2、CD3、CD3E、CD4、CD5、CD6、CD7、CD8、CD10、CD11a、CD11b、CD11c、CD13、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD27L、CD28、CD29、CD30、CD30L、CD32、CD33(p67タンパク質)、CD34、CD38、CD40、CD40L、CD44、CD45、CD46、CD49a、CD52、CD54、CD55、CD56、CD61、CD64、CD66e、CD74、CD80(B7-1)、CD89、CD95、CD123、CD137、CD138、CD140a、CD146、CD147、CD148、CD152、CD164、CEACAM5、CFTR、cGMP、CINC、ボツリヌス菌毒素、ウェルシュ菌毒素、CKb8-1、CLC、CMV、CMV UL、CNTF、CNTN-1、COX、C-Ret、CRG-2、CT-1、CTACK、CTGF、CTLA-4、CX3CL1、CX3CR1、CXCL、CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16、CXCR、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、サイトケラチン腫瘍関連抗原、DAN、DCC、DcR3、DC-SIGN、補体制御因子(Decay accelerating factor)、des(1-3)-IGF-I(脳IGF-1)、Dhh、ジゴキシン、DNAM-1、Dnase、Dpp、DPPIV/CD26、Dtk、ECAD、EDA、EDA-A1、EDA-A2、EDAR、EGF、EGFR(ErbB-1)、EMA、EMMPRIN、ENA、エンドセリン受容体、エンケファリナーゼ、eNOS、Eot、エオタキシン1、EpCAM、エフリンB2/EphB4、EPO、ERCC、E-セレクチン、ET-1、ファクターIIa、ファクターVII、ファクターVIIIc、ファクターIX、線維芽細胞活性化タンパク質(FAP)、Fas、FcR1、FEN-1、フェリチン、FGF、FGF-19、FGF-2、FGF3、FGF-8、FGFR、FGFR-3、フィブリン、FL、FLIP、Flt-3、Flt-4、卵胞刺激ホルモン、フラクタルカイン、FZD1、FZD2、FZD3、FZD4、FZD5、FZD6、FZD7、FZD8、FZD9、FZD10、G250、Gas6、GCP-2、GCSF、GD2、GD3、GDF、GDF-1、GDF-3(Vgr-2)、GDF-5(BMP-14、CDMP-1)、GDF-6(BMP-13、CDMP-2)、GDF-7(BMP-12、CDMP-3)、GDF-8(ミオスタチン)、GDF-9、GDF-15(MIC-1)、GDNF、GDNF、GFAP、GFRa-1、GFR-アルファ1、GFR-アルファ2、GFR-アルファ3、GITR、グルカゴン、Glut4、糖タンパク質IIb/IIIa(GPIIb/IIIa)、GM-CSF、gp130、gp72、GRO、成長ホルモン放出因子、ハプテン(NP-capまたはNIP-cap)、HB-EGF、HCC、HCMV gBエンベロープ糖タンパク質、HCMV gHエンベロープ糖タンパク質、HCMV UL、造血成長因子(HGF)、Hep B gp120、ヘパラナーゼ、Her2、Her2/neu(ErbB-2)、Her3(ErbB-3)、Her4(ErbB-4)、単純ヘルペスウイルス(HSV) gB糖タンパク質、HSV gD糖タンパク質、HGFA、高分子量黒色腫関連抗原(HMW-MAA)、HIV gp120、HIV IIIB gp 120 V3ループ、HLA、HLA-DR、HM1.24、HMFG PEM、HRG、Hrk、ヒト心臓ミオシン、ヒトサイトメガロウイルス(HCMV)、ヒト成長ホルモン(HGH)、HVEM、I-309、IAP、ICAM、ICAM-1、ICAM-3、ICE、ICOS、IFNg、Ig、IgA受容体、IgE、IGF、IGF結合タンパク質、IGF-1R、IGFBP、IGF-I、IGF-II、IL、IL-1、IL-1R、IL-2、IL-2R、IL-4、IL-4R、IL-5、IL-5R、IL-6、IL-6R、IL-8、IL-9、IL-10、IL-12、IL-13、IL-15、IL-18、IL-18R、IL-23、インターフェロン(INF)-アルファ、INF-ベータ、INF-ガンマ、インヒビン、iNOS、インスリンA鎖、インスリンB鎖、インスリン様増殖因子1、インテグリンアルファ2、インテグリンアルファ3、インテグリンアルファ4、インテグリンアルファ4/ベータ1、インテグリンアルファ4/ベータ7、インテグリンアルファ5(アルファV)、インテグリンアルファ5/ベータ1、インテグリンアルファ5/ベータ3、インテグリンアルファ6、インテグリンベータ1、インテグリンベータ2、インターフェロンガンマ、IP-10、I-TAC、JE、カリクレイン2、カリクレイン5、カリクレイン6、カリクレイン11、カリクレイン12、カリクレイン14、カリクレイン15、カリクレインL1、カリクレインL2、カリクレインL3、カリクレインL4、KC、KDR、ケラチノサイト増殖因子(KGF)、ラミニン5、LAMP、LAP、LAP(TGF-1)、潜在的TGF-1、潜在的TGF-1 bp1、LBP、LDGF、LECT2、レフティ、ルイス-Y抗原、ルイス-Y関連抗原、LFA-1、LFA-3、Lfo、LIF、LIGHT、リポタンパク質、LIX、LKN、Lptn、L-セレクチン、LT-a、LT-b、LTB4、LTBP-1、肺表面、黄体形成ホルモン、リンホトキシンベータ受容体、Mac-1、MAdCAM、MAG、MAP2、MARC、MCAM、MCAM、MCK-2、MCP、M-CSF、MDC、Mer、METALLOPROTEASES、MGDF受容体、MGMT、MHC(HLA-DR)、MIF、MIG、MIP、MIP-1-アルファ、MK、MMAC1、MMP、MMP-1、MMP-10、MMP-11、MMP-12、MMP-13、MMP-14、MMP-15、MMP-2、MMP-24、MMP-3、MMP-7、MMP-8、MMP-9、MPIF、Mpo、MSK、MSP、ムチン(Muc1)、MUC18、ミュラー管抑制物質、Mug、MuSK、NAIP、NAP、NCAD、N-Cアドヘリン、NCA 90、NCAM、NCAM、ネプリライシン、ニューロトロフィン-3、-4、または-6、ニュールツリン、神経成長因子(NGF)、NGFR、NGF-ベータ、nNOS、NO、NOS、Npn、NRG-3、NT、NTN、OB、OGG1、OPG、OPN、OSM、OX40L、OX40R、p150、p95、PADPr、副甲状腺ホルモン、PARC、PARP、PBR、PBSF、PCAD、P-カドヘリン、PCNA、PDGF、PDGF、PDK-1、PECAM、PEM、PF4、PGE、PGF、PGI2、PGJ2、PIN、PLA2、胎盤性アルカリホスファターゼ(PLAP)、PlGF、PLP、PP14、プロインスリン、プロレラキシン、プロテインC、PS、PSA、PSCA、前立腺特異的膜抗原(PSMA)、PTEN、PTHrp、Ptk、PTN、R51、RANK、RANKL、RANTES、RANTES、レラキシンA鎖、レラキシンB鎖、レニン、呼吸器多核体ウイルス(RSV)F、RSV Fgp、Ret、リウマイド因子、RLIP76、RPA2、RSK、S100、SCF/KL、SDF-1、SERINE、血清アルブミン、sFRP-3、Shh、SIGIRR、SK-1、SLAM、SLPI、SMAC、SMDF、SMOH、SOD、SPARC、Stat、STEAP、STEAP-II、TACE、TACI、TAG-72(腫瘍関連糖タンパク質-72)、TARC、TCA-3、T細胞受容体(例えば、T細胞受容体アルファ/ベータ)、TdT、TECK、TEM1、TEM5、TEM7、TEM8、TERT、睾丸PLAP様アルカリホスファターゼ、TfR、TGF、TGF-アルファ、TGF-ベータ、TGF-ベータ Pan Specific、TGF-ベータRI(ALK-5)、TGF-ベータRII、TGF-ベータRIIb、TGF-ベータRIII、TGF-ベータ1、TGF-ベータ2、TGF-ベータ3、TGF-ベータ4、TGF-ベータ5、トロンビン、胸腺Ck-1、甲状腺刺激ホルモン、Tie、TIMP、TIQ、組織因子、TMEFF2、Tmpo、TMPRSS2、TNF、TNF-アルファ、TNF-アルファベータ、TNF-ベータ2、TNFc、TNF-RI、TNF-RII、TNFRSF10A(TRAIL R1 Apo-2、DR4)、TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3 DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF RI CD120a、p55-60)、TNFRSF1B(TNF RII CD120b、p75-80)、TNFRSF26(TNFRH3)、TNFRSF3(LTbR TNF RIII、TNFC R)、TNFRSF4(OX40 ACT35、TXGP1 R)、TNFRSF5(CD40 p50)、TNFRSF6(Fas Apo-1、APT1、CD95)、TNFRSF6B(DcR3 M68、TR6)、TNFRSF7(CD27)、TNFRSF8(CD30)、TNFRSF9(4-1BB CD137、ILA)、TNFRSF21(DR6)、TNFRSF22(DcTRAIL R2 TNFRH2)、TNFRST23(DcTRAIL R1 TNFRH1)、TNFRSF25(DR3 Apo-3、LARD、TR-3、TRAMP、WSL-1)、TNFSF10(TRAIL Apo-2リガンド、TL2)、TNFSF11(TRANCE/RANKリガンド ODF、OPGリガンド)、TNFSF12(TWEAK Apo-3リガンド、DR3リガンド)、TNFSF13(APRIL TALL2)、TNFSF13B(BAFF BLYS、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT HVEMリガンド、LTg)、TNFSF15(TL1A/VEGI)、TNFSF18(GITRリガンド AITRリガンド、TL6)、TNFSF1A(TNF-a コネクチン(Conectin)、DIF、TNFSF2)、TNFSF1B(TNF-b LTa、TNFSF1)、TNFSF3(LTb TNFC、p33)、TNFSF4(OX40リガンド gp34、TXGP1)、TNFSF5(CD40リガンド CD154、gp39、HIGM1、IMD3、TRAP)、TNFSF6(Fasリガンド Apo-1リガンド、APT1リガンド)、TNFSF7(CD27リガンド CD70)、TNFSF8(CD30リガンド CD153)、TNFSF9(4-1BBリガンド CD137リガンド)、TP-1、t-PA、Tpo、TRAIL、TRAIL R、TRAIL-R1、TRAIL-R2、TRANCE、トランスフェリン受容体、TRF、Trk、TROP-2、TSG、TSLP、腫瘍関連抗原CA125、腫瘍関連抗原発現ルイスY関連炭水化物、TWEAK、TXB2、Ung、uPAR、uPAR-1、ウロキナーゼ、VCAM、VCAM-1、VECAD、VE-Cadherin、VE-cadherin-2、VEFGR-1(flt-1)、VEGF、VEGFR、VEGFR-3(flt-4)、VEGI、VIM、ウイルス抗原、VLA、VLA-1、VLA-4、VNRインテグリン、フォン・ヴィレブランド因子、WIF-1、WNT1、WNT2、WNT2B/13、WNT3、WNT3A、WNT4、WNT5A、WNT5B、WNT6、WNT7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9A、WNT9B、WNT10A、WNT10B、WNT11、WNT16、XCL1、XCL2、XCR1、XCR1、XEDAR、XIAP、XPD、HMGB1、IgA、Aβ、CD81, CD97, CD98, DDR1, DKK1, EREG、Hsp90, IL-17/IL-17R、IL-20/IL-20R、酸化LDL, PCSK9, prekallikrein , RON, TMEM16F、SOD1, Chromogranin A, Chromogranin B、tau, VAP1、高分子キニノーゲン、IL-31、IL-31R、Nav1.1、Nav1.2、Nav1.3、Nav1.4、Nav1.5、Nav1.6、Nav1.7、Nav1.8、Nav1.9、EPCR、C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin、sclerostin、fibrinogen, fibrin, prothrombin, thrombin, 組織因子, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, トロンボモデュリン、TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2、GPC3、Syndecan-1、Syndecan-2、Syndecan-3、Syndecan-4、LPA、S1Pならびにホルモンおよび成長因子のための受容体が例示され得る。
下記にIL-6Rに対する抗原結合ドメインを含む被験抗原結合分子によるエピトープへの結合の確認方法が例示されるが、IL-6R以外の抗原に対する抗原結合ドメインを含む被験抗原結合分子によるエピトープへの結合の確認方法も下記の例示に準じて適宜実施され得る。
FACSCantoTM II
FACSAriaTM
FACSArrayTM
FACSVantageTM SE
FACSCaliburTM (いずれもBD Biosciences社の商品名)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
Cell Lab Quanta / Cell Lab Quanta SC(いずれもBeckman Coulter社の商品名)
本明細書において、「抗原結合ドメイン」は目的とする抗原に結合するかぎりどのような構造のドメインも使用され得る。そのようなドメインの例として、例えば、抗体の重鎖および軽鎖の可変領域、生体内に存在する細胞膜タンパクであるAvimerに含まれる35アミノ酸程度のAドメインと呼ばれるモジュール(WO2004/044011、WO2005/040229)、細胞膜に発現する糖たんぱく質であるfibronectin中のタンパク質に結合するドメインである10Fn3ドメインを含むAdnectin(WO2002/032925)、ProteinAの58アミノ酸からなる3つのヘリックスの束(bundle)を構成するIgG結合ドメインをscaffoldとするAffibody(WO1995/001937)、33アミノ酸残基を含むターンと2つの逆並行ヘリックスおよびループのサブユニットが繰り返し積み重なった構造を有するアンキリン反復(ankyrin repeat:AR)の分子表面に露出する領域であるDARPins(Designed Ankyrin Repeat proteins)(WO2002/020565)、好中球ゲラチナーゼ結合リポカリン(neutrophil gelatinase-associated lipocalin(NGAL))等のリポカリン分子において高度に保存された8つの逆並行ストランドが中央方向にねじれたバレル構造の片側を支える4つのループ領域であるAnticalin等(WO2003/029462)、ヤツメウナギ、ヌタウナギなど無顎類の獲得免疫システムとしてイムノグロブリンの構造を有さない可変性リンパ球受容体(variable lymphocyte receptor(VLR))のロイシン残基に富んだリピート(leucine-rich-repeat(LRR))モジュールが繰り返し積み重なった馬てい形の構造の内部の並行型シート構造のくぼんだ領域(WO2008/016854)が好適に挙げられる。本発明の抗原結合ドメインの好適な例として、抗体の重鎖および軽鎖の可変領域を含む抗原結合ドメインが挙げられる。こうした抗原結合ドメインの例としては、「scFv(single chain Fv)」、「単鎖抗体(single chain antibody)」、「Fv」、「scFv2(single chain Fv 2)」、「Fab」または「F(ab')2」等が好適に挙げられる。
特異的とは、特異的に結合する分子の一方の分子がその一または複数の結合する相手方の分子以外の分子に対しては何ら有意な結合を示さない状態をいう。また、抗原結合ドメインが、ある抗原中に含まれる複数のエピトープのうち特定のエピトープに対して特異的である場合にも用いられる。また、抗原結合ドメインが結合するエピトープが複数の異なる抗原に含まれる場合には、当該抗原結合ドメインを有する抗原結合分子は当該エピトープを含む様々な抗原と結合することができる。
本明細書において、抗体とは、天然のものであるかまたは部分的もしくは完全合成により製造された免疫グロブリンをいう。抗体はそれが天然に存在する血漿や血清等の天然資源や抗体を産生するハイブリドーマ細胞の培養上清から単離され得るし、または遺伝子組換え等の手法を用いることによって部分的にもしくは完全に合成され得る。抗体の例としては免疫グロブリンのアイソタイプおよびそれらのアイソタイプのサブクラスが好適に挙げられる。ヒトの免疫グロブリンとして、IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、IgE、IgMの9種類のクラス(アイソタイプ)が知られている。本発明の抗体には、これらのアイソタイプのうちIgG1、IgG2、IgG3、IgG4が含まれ得る。
-IL-6Rのような膜蛋白質の構造を維持して免疫刺激が与えられ得る
-免疫抗原を精製する必要が無い
より具体的には、例えば細胞融合促進剤の存在下で通常の栄養培養液中で、前記細胞融合が実施され得る。融合促進剤としては、例えばポリエチレングリコール(PEG)、センダイウイルス(HVJ)等が使用され、更に融合効率を高めるために所望によりジメチルスルホキシド等の補助剤が添加されて使用される。
-グアニジン超遠心法(Biochemistry (1979) 18 (24), 5294-5299)
-AGPC法(Anal. Biochem. (1987) 162 (1), 156-159)
(1)ハイブリドーマから得られたcDNAによってコードされるV領域を含む抗体をIL-6R発現細胞に接触させる工程、
(2)IL-6R発現細胞と抗体との結合を検出する工程、および
(3)IL-6R発現細胞に結合する抗体を選択する工程。
(1)哺乳類細胞、:CHO、COS、ミエローマ、BHK (baby hamster kidney )、Hela、Vero、HEK(human embryonic kidney)293など
(2)両生類細胞:アフリカツメガエル卵母細胞など
(3)昆虫細胞:sf9、sf21、Tn5など
-酵母:サッカロミセス・セレビシエ(Saccharomyces serevisiae)などのサッカロミセス(Saccharomyces )属、メタノール資化酵母(Pichia pastoris)などのPichia属
-糸状菌:アスペスギルス・ニガー(Aspergillus niger)などのアスペルギルス(Aspergillus )属
本発明で使用されている方法によると、抗体のCDRとFRに割り当てられるアミノ酸位置はKabatにしたがって規定される(Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md., 1987年および1991年)。本明細書において、抗原結合分子が抗体または抗原結合断片である場合、可変領域のアミノ酸はKabatナンバリングにしたがい、定常領域のアミノ酸はKabatのアミノ酸位置に準じたEUナンバリングにしたがって表される。
金属イオン濃度の条件
本発明の一つの態様では、イオン濃度とは金属イオン濃度のことをいう。「金属イオン」とは、水素を除くアルカリ金属および銅族等の第I族、アルカリ土類金属および亜鉛族等の第II族、ホウ素を除く第III族、炭素とケイ素を除く第IV族、鉄族および白金族等の第VIII族、V、VIおよびVII族の各A亜族に属する元素と、アンチモン、ビスマス、ポロニウム等の金属元素のイオンをいう。金属原子は原子価電子を放出して陽イオンになる性質を有しており、これをイオン化傾向という。イオン化傾向の大きい金属は、化学的に活性に富むとされる。
(a) 低カルシウム濃度の条件における抗原結合ドメインまたは抗体の抗原結合活性を得る工程、
(b) 高カルシウム濃度の条件における抗原結合ドメインまたは抗体の抗原結合活性を得る工程、
(c) 低カルシウム濃度の条件における抗原結合活性が、高カルシウム濃度の条件における抗原結合活性より低い抗原結合ドメインまたは抗体を選択する工程。
(a) 高カルシウム濃度の条件における抗原結合ドメインまたは抗体もしくはそれらのライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合した抗原結合ドメインまたは抗体を低カルシウム濃度条件下に置く工程、
(c) 前記工程(b)で解離した抗原結合ドメインまたは抗体を単離する工程。
(a) 低カルシウム濃度条件下で抗原結合ドメイン又は抗体のライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合しない抗原結合ドメイン又は抗体を選択する工程、
(c) 前記工程(b)で選択された抗原結合ドメイン又は抗体を高カルシウム濃度条件下で抗原に結合させる工程、
(d) 前記工程(c)で抗原に結合した抗原結合ドメイン又は抗体を単離する工程。
(a) 抗原を固定したカラムに高カルシウム濃度条件下で抗原結合ドメイン又は抗体のライブラリを接触させる工程、
(b) 前記工程(a)でカラムに結合した抗原結合ドメイン又は抗体を低カルシウム濃度条件下でカラムから溶出する工程、
(c) 前記工程(b)で溶出された抗原結合ドメイン又は抗体を単離する工程。
(a) 抗原を固定したカラムに低カルシウム濃度条件下で抗原結合ドメイン又は抗体のライブラリを通過させる工程、
(b) 前記工程(a)でカラムに結合せずに溶出した抗原結合ドメイン又は抗体を回収する工程、
(c) 前記工程(b)で回収された抗原結合ドメイン又は抗体を高カルシウム濃度条件下で抗原に結合させる工程、
(d) 前記工程(c)で抗原に結合した抗原結合ドメイン又は抗体を単離する工程。
(a) 高カルシウム濃度条件下で抗原結合ドメイン又は抗体のライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合した抗原結合ドメイン又は抗体を取得する工程、
(c) 前記工程(b)で取得した抗原結合ドメイン又は抗体を低カルシウム濃度条件下に置く工程、
(d) 前記工程(c)で抗原結合活性が、前記工程(b)で選択した基準より弱い抗原結合ドメイン又は抗体を単離する工程。
ある一態様によれば、本発明の抗原結合ドメイン又は抗体は、イオン濃度の条件によって抗原に対する抗原結合分子の結合活性を変化させる少なくとも一つのアミノ酸残基が抗原結合ドメインに含まれている互いに配列の異なる複数の抗原結合分子から主としてなるライブラリから取得され得る。イオン濃度の例としては金属イオン濃度や水素イオン濃度が好適に挙げられる。
前記のスクリーニング方法によってスクリーニングされる本発明の抗原結合ドメイン又は抗体はどのように調製されてもよく、例えば、金属イオンがカルシウムイオン濃度である場合には、あらかじめ存在している抗体、あらかじめ存在しているライブラリ(ファージライブラリ等)、動物への免疫から得られたハイブリドーマや免疫動物からのB細胞から作製された抗体又はライブラリ、これらの抗体やライブラリにカルシウムをキレート可能なアミノ酸(例えばアスパラギン酸やグルタミン酸)や非天然アミノ酸変異を導入した抗体又はライブラリ(カルシウムをキレート可能なアミノ酸(例えばアスパラギン酸やグルタミン酸)又は非天然アミノ酸の含有率を高くしたライブラリや特定箇所にカルシウムをキレート可能なアミノ酸(例えばアスパラギン酸やグルタミン酸)又は非天然アミノ酸変異を導入したライブラリ等)などを用いることが可能である 。
また、本発明の一つの態様では、イオン濃度の条件とは水素イオン濃度の条件またはpHの条件をいう。本発明で、プロトンすなわち水素原子の原子核の濃度の条件は、水素指数(pH)の条件とも同義に取り扱われる。水溶液中の水素イオンの活動量をaH+で表すと、pHは-log10aH+と定義される。水溶液中のイオン強度が(例えば10-3より)低ければ、aH+は水素イオン強度にほぼ等しい。例えば25℃、1気圧における水のイオン積はKw=aH+aOH=10-14であるため、純水ではaH+=aOH=10-7である。この場合のpH=7が中性であり、pHが7より小さい水溶液は酸性、pHが7より大きい水溶液はアルカリ性である。
(a) pH酸性域の条件における抗原結合ドメインまたは抗体の抗原結合活性を得る工程、
(b) pH中性域の条件における抗原結合ドメインまたは抗体の抗原結合活性を得る工程、
(c) pH酸性域の条件における抗原結合活性が、pH中性域の条件における抗原結合活性より低い抗原結合ドメインまたは抗体を選択する工程。
(a) pH中性域の条件における抗原結合ドメインまたは抗体もしくはそれらのライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合した抗原結合ドメインまたは抗体をpH酸性域の条件に置く工程、
(c) 前記工程(b)で解離した抗原結合ドメインまたは抗体を単離する工程。
(a) pH酸性域の条件で抗原結合ドメイン又は抗体のライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合しない抗原結合ドメイン又は抗体を選択する工程、
(c) 前記工程(b)で選択された抗原結合ドメイン又は抗体をpH中性域の条件で抗原に結合させる工程、
(d) 前記工程(c)で抗原に結合した抗原結合ドメイン又は抗体を単離する工程。
(a) 抗原を固定したカラムにpH中性域の条件で抗原結合ドメイン又は抗体のライブラリを接触させる工程、
(b) 前記工程(a)でカラムに結合した抗原結合ドメイン又は抗体をpH酸性域の条件でカラムから溶出する工程、
(c) 前記工程(b)で溶出された抗原結合ドメイン又は抗体を単離する工程。
(a) 抗原を固定したカラムにpH酸性域の条件で抗原結合ドメイン又は抗体のライブラリを通過させる工程、
(b) 前記工程(a)でカラムに結合せずに溶出した抗原結合ドメイン又は抗体を回収する工程、
(c) 前記工程(b)で回収された抗原結合ドメイン又は抗体をpH中性域の条件で抗原に結合させる工程、
(d) 前記工程(c)で抗原に結合した抗原結合ドメイン又は抗体を単離する工程。
(a) pH中性域の条件で抗原結合ドメイン又は抗体のライブラリを抗原に接触させる工程、
(b) 前記工程(a)で抗原に結合した抗原結合ドメイン又は抗体を取得する工程、
(c) 前記工程(b)で取得した抗原結合ドメイン又は抗体をpH酸性域の条件に置く工程、
(d) 前記工程(c)で抗原結合活性が、前記工程(b)で選択した基準より弱い抗原結合ドメイン又は抗体を単離する工程。
前記のスクリーニング方法によってスクリーニングされる本発明の抗原結合ドメイン又は抗体はどのように調製されてもよく、例えば、イオン濃度の条件が水素イオン濃度の条件もしくはpHの条件である場合には、あらかじめ存在している抗体、あらかじめ存在しているライブラリ(ファージライブラリ等)、動物への免疫から得られたハイブリドーマや免疫動物からのB細胞から作製された抗体又はライブラリ、これらの抗体やライブラリに側鎖のpKaが4.0-8.0であるアミノ酸(例えばヒスチジンやグルタミン酸)や非天然アミノ酸の変異を導入した抗体又はライブラリ(側鎖のpKaが4.0-8.0であるアミノ酸(例えばヒスチジンやグルタミン酸)や非天然アミノ酸の含有率を高くしたライブラリや特定箇所に側鎖のpKaが4.0-8.0であるアミノ酸(例えばヒスチジンやグルタミン酸)や非天然アミノ酸の変異を導入したライブラリ等)などを用いることが可能である 。
免疫グロブリンスーパーファミリーに属するFcγレセプターと異なり、ヒトFcRnは構造的には主要組織不適合性複合体(MHC)クラスIのポリペプチドに構造的に類似しクラスIのMHC分子と22から29%の配列同一性を有する(Ghetieら,Immunol. Today (1997) 18 (12), 592-598)。FcRnは、可溶性βまたは軽鎖(β2マイクログロブリン)と複合体化された膜貫通αまたは重鎖よりなるヘテロダイマーとして発現される。MHCのように、FcRnのα鎖は3つの細胞外ドメイン(α1,α2,α3)よりなり、短い細胞質ドメインはタンパク質を細胞表面に繋留する。α1およびα2ドメインが抗体のFc領域中のFcRn結合ドメインと相互作用する(Raghavanら(Immunity (1994) 1, 303-315)。
Fc領域は、抗体重鎖の定常領域に由来するアミノ酸配列を含む。Fc領域は、EUナンバリングで表されるおよそ216のアミノ酸における、パパイン切断部位のヒンジ領域のN末端から、当該ヒンジ、CH2およびCH3ドメインを含める抗体の重鎖定常領域の部分である。
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyrのいずれか、
252位のアミノ酸がPhe、Trp、またはTyrのいずれか、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsp、Asn、Glu、またはGlnのいずれか、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはValのいずれか、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGluのいずれか、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyrのいずれか、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThrのいずれか、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArgのいずれか、
311位のアミノ酸がAla、His、またはIleのいずれか、
312位のアミノ酸がAlaまたはHisのいずれか、
314位のアミノ酸がLysまたはArgのいずれか、
315位のアミノ酸がAla、AspまたはHisのいずれか、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHisのいずれか、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSerのいずれか、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyrのいずれか、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyrのいずれか、および
436位のアミノ酸がHis 、Ile、Leu、Phe、Thr、またはVal、
が挙げられる。また、改変されるアミノ酸の数は特に限定されず、一箇所のみのアミノ酸が改変され得るし、二箇所以上のアミノ酸が改変され得る。二箇所以上のアミノ酸の改変の組合せとしては、例えば表6に記載されるような組合せが挙げられる。
本発明において、抗原結合分子は抗原結合ドメインおよびFc領域を含む分子を表す最も広義な意味として使用されており、具体的には、それらが抗原に対する結合活性を示す限り、様々な分子型が含まれる。例えば、抗原結合ドメインがFc領域と結合した分子の例として、抗体が挙げられる。抗体には、単一のモノクローナル抗体(アゴニストおよびアンタゴニスト抗体を含む)、ヒト抗体、ヒト化抗体、キメラ抗体等が含まれ得る。また抗体の断片として使用される場合としては、抗原結合ドメインおよび抗原結合断片(例えば、Fab、F(ab')2、scFvおよびFv)が好適に挙げられ得る。既存の安定なα/βバレルタンパク質構造等の立体構造が scaffold(土台)として用いられ、その一部分の構造のみが抗原結合ドメインの構築のためにライブラリ化されたスキャフォールド分子も、本発明の抗原結合分子に含まれ得る 。
Ser
Gly・Ser
Gly・Gly・Ser
Ser・Gly・Gly
Gly・Gly・Gly・Ser(配列番号:17)
Ser・Gly・Gly・Gly(配列番号:18)
Gly・Gly・Gly・Gly・Ser(配列番号:19)
Ser・Gly・Gly・Gly・Gly(配列番号:20)
Gly・Gly・Gly・Gly・Gly・Ser(配列番号:21)
Ser・Gly・Gly・Gly・Gly・Gly(配列番号:22)
Gly・Gly・Gly・Gly・Gly・Gly・Ser(配列番号:23)
Ser・Gly・Gly・Gly・Gly・Gly・Gly(配列番号:24)
(Gly・Gly・Gly・Gly・Ser(配列番号:19))n
(Ser・Gly・Gly・Gly・Gly(配列番号:20))n
[nは1以上の整数である]等が好適に挙げられる。但し、ペプチドリンカーの長さや配列は目的に応じて当業者が適宜選択することができる。
Fcγレセプター(FcγRとも記載される)とは、IgG1、IgG2、IgG3、IgG4モノクローナル抗体のFc領域に結合し得るレセプターをいい、実質的にFcγレセプター遺伝子にコードされるタンパク質のファミリーのいかなるメンバーをも意味する。ヒトでは、このファミリーには、アイソフォームFcγRIa、FcγRIbおよびFcγRIcを含むFcγRI(CD64);アイソフォームFcγRIIa(アロタイプH131およびR131を含む)、FcγRIIb(FcγRIIb-1およびFcγRIIb-2を含む)およびFcγRIIcを含むFcγRII(CD32);およびアイソフォームFcγRIIIa(アロタイプV158およびF158を含む)およびFcγRIIIb(アロタイプFcγRIIIb-NA1およびFcγRIIIb-NA2を含む)を含むFcγRIII(CD16)、並びにいかなる未発見のヒトFcγR類またはFcγRアイソフォームまたはアロタイプも含まれるが、これらに限定されるものではない。FcγRは、ヒト、マウス、ラット、ウサギおよびサルを含むが、これらに限定されるものではない、いかなる生物由来でもよい。マウスFcγR類には、FcγRI(CD64)、FcγRII(CD32)、FcγRIII(CD16)およびFcγRIII-2(FcγRIV、CD16-2)、並びにいかなる未発見のマウスFcγR類またはFcγRアイソフォームまたはアロタイプも含まれるが、これらに限定されない。こうしたFcγレセプターの好適な例としてはヒトFcγRI(CD64)、FcγRIIa(CD32)、FcγRIIb(CD32)、FcγRIIIa(CD16)及び/又はFcγRIIIb(CD16)が挙げられる。ヒトFcγRIのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:25(NM_000566.3)及び26(NP_000557.1)に、ヒトFcγRIIa(アロタイプH131)のポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:27(BC020823.1)及び28(AAH20823.1)に(アロタイプR131は配列番号:28の166番目のアミノ酸がArgに置換されている配列である)、FcγRIIbのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:29(BC146678.1)及び30(AAI46679.1)に、FcγRIIIaのポリヌクレオチド配列及びアミノ酸配列はそれぞれ配列番号:31(BC033678.1)及び32(AAH33678.1)に、及びFcγRIIIbのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:33(BC128562.1)及び34(AAI28563.1)に記載されている(カッコ内はRefSeq登録番号を示す)。例えば参考実施例27等でアロタイプV158が用いられている場合にFcγRIIIaVと表記されているように、特記されないかぎり、アロタイプF158が用いられているが、本願で記載されるアイソフォームFcγRIIIaのアロタイプが特に限定して解釈されるものではない。 Fcγレセプターが、IgG1、IgG2、IgG3、IgG4モノクローナル抗体のFc領域に結合活性を有するか否かは、上記に記載されるFACSやELISAフォーマットのほか、ALPHAスクリーン(Amplified Luminescent Proximity Homogeneous Assay)や表面プラズモン共鳴(SPR)現象を利用したBIACORE法等によって確認され得る(Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010)。
FcRnとIgG抗体との結晶学的研究によって、FcRn-IgG複合体は、二分子のFcRnに対して一分子のIgGから構成され、IgGのFc領域の両側に位置するCH2およびCH3ドメインの接触面付近において、二分子の結合が起こると考えられている(Burmeisterら(Nature (1994) 372, 336-343)。一方、後述する実施例3において確認されたように、抗体のFc領域が二分子のFcRnおよび一分子の活性型Fcγレセプターの四者を含む複合体を形成できることが明らかとなった(図48)。このヘテロ複合体の形成は、pH中性域の条件下でFcRnに対する結合活性を有するFc領域を含む抗原結合分子の性質について解析を進めた結果明らかとなった現象である。
前記Fc領域のアミノ酸のうちEUナンバリングで表される234、235、236、237、238、239、270、297、298、325、328、および329のいずれかひとつ以上のアミノ酸が天然型Fc領域と異なるアミノ酸に改変されているFc領域が好適に挙げられるが、Fc領域の改変は上記改変に限定されず、例えばCurrent Opinion in Biotechnology (2009) 20 (6), 685-691に記載されている脱糖鎖 (N297A, N297Q)、IgG1-L234A/L235A、IgG1-A325A/A330S/P331S、IgG1-C226S/C229S、IgG1-C226S/C229S/E233P/L234V/L235A、IgG1-L234F/L235E/P331S、IgG1-S267E/L328F、IgG2-V234A/G237A、IgG2-H268Q/V309L/A330S/A331S、IgG4-L235A/G237A/E318A、IgG4-L236E等の改変、および、WO 2008/092117に記載されているG236R/L328R、L235G/G236R、N325A/L328R、N325LL328R等の改変、および、EUナンバリング233位、234位、235位、237位におけるアミノ酸の挿入、WO 2000/042072に記載されている個所の改変であってもよい。
234位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Met、Phe、Pro、Ser、ThrまたはTrpのいずれか、
235位のアミノ酸をAla、Asn、Asp、Gln、Glu、Gly、His、Ile、Lys、Met、Pro、Ser、Thr、ValまたはArgのいずれか、
236位のアミノ酸をArg、Asn、Gln、His、Leu、Lys、Met、Phe、ProまたはTyrのいずれか、
237位のアミノ酸をAla、Asn、Asp、Gln、Glu、His、Ile、Leu、Lys、Met、Pro、Ser、Thr、Val、TyrまたはArgのいずれか、
238位のアミノ酸をAla、Asn、Gln、Glu、Gly、His、Ile、Lys、Thr、TrpまたはArgのいずれか、
239位のアミノ酸をGln、His、Lys、Phe、Pro、Trp、TyrまたはArgのいずれか、
265位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、TyrまたはValのいずれか、
266位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Phe、Pro、Ser、Thr、TrpまたはTyrのいずれか、
267位のアミノ酸をArg、His、Lys、Phe、Pro、TrpまたはTyrのいずれか、
269位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
270位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
271位のアミノ酸をArg、His、Phe、Ser、Thr、TrpまたはTyrのいずれか、
295位のアミノ酸をArg、Asn、Asp、Gly、His、Phe、Ser、TrpまたはTyrのいずれか、
296位のアミノ酸をArg、Gly、LysまたはProのいずれか、
297位のアミノ酸をAla、
298位のアミノ酸をArg、Gly、Lys、Pro、TrpまたはTyrのいずれか、
300位のアミノ酸をArg、LysまたはProのいずれか、
324位のアミノ酸をLysまたはProのいずれか、
325位のアミノ酸をAla、Arg、Gly、His、Ile、Lys、Phe、Pro、Thr、TrpTyr、もしくはValのいずれか、
327位のアミノ酸をArg、Gln、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
328位のアミノ酸をArg、Asn、Gly、His、LysまたはProのいずれか、
329位のアミノ酸をAsn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、Tyr、ValまたはArgのいずれか、
330位のアミノ酸をProまたはSerのいずれか、
331位のアミノ酸をArg、GlyまたはLysのいずれか、もしくは
332位のアミノ酸をArg、LysまたはProのいずれか、
のいずれかひとつ以上に改変されているFc領域が好適に挙げられる。
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409のアミノ酸がAsp、370のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399のアミノ酸がLys、357のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される370のアミノ酸のGluに代えてAspであってもよく、EUナンバリングで表される370のアミノ酸のGluに代えて392のアミノ酸のAspであってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409のアミノ酸がAsp、439のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399のアミノ酸がLys、356のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される439のアミノ酸のGluに代えて360のアミノ酸のAsp、EUナンバリングで表される392のアミノ酸のAsp又は439のアミノ酸のAspであってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される370のアミノ酸がGlu、439のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される357のアミノ酸がLys、356のアミノ酸がLysであることを特徴とする二つのポリペプチド、または、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409のアミノ酸がAsp、370のアミノ酸がGlu、439のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399のアミノ酸がLys、357のアミノ酸がLys、356のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される370のアミノ酸をGluに置換しなくてもよく、更に、370のアミノ酸をGluに置換しない上で、439のアミノ酸のGluに代えてAsp又は439のアミノ酸のGluに代えて392のアミノ酸のAspであってもよい)、
が好適に用いられる。
本発明の抗原結合分子に対する免疫応答が改変されたか否かは、抗原結合分子を有効成分として含む医薬組成物が投与された生体の応答反応を測定することによって評価され得る。生体の応答反応としては、主として細胞性免疫(MHCクラスIに結合した抗原結合分子のペプチド断片を認識する細胞障害性T細胞の誘導)と液性免疫(抗原結合分子に結合する抗体産生の誘導)の二つの免疫応答が挙げられるが、特にタンパク質医薬品の場合は、投与された抗原結合分子に対する抗体産生が免疫原性と呼ばれる。免疫原性を評価する方法としては、in vivoで抗体産生を評価する方法と、in vitroで免疫細胞の反応を評価する方法の2種類がある。
本発明は特定の理論により拘束されるものではないが、例えば、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように、イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、およびpH中性域の条件下でヒトFcRnに対する結合活性を有するFc領域を含む抗原結合分子が生体に投与されたときに生体中の細胞への取込みが促進されることによって、一分子の抗原結合分子が結合可能な抗原の数が増加する理由、および、血漿中抗原濃度の消失が促進される理由はたとえば以下のように説明することが可能である。
A値=各時点での抗原のモル濃度
B値=各時点での抗原結合分子のモル濃度
C値=各時点での抗原結合分子のモル濃度あたりの抗原のモル濃度(抗原/抗原結合分子モル比)
C=A/B。
本発明の非限定の一態様では、前記のように選択された条件によって結合活性が変化する抗原結合ドメインをコードするポリヌクレオチドが単離された後に、当該ポリヌクレオチドが適切な発現ベクターに挿入される。例えば、抗原結合ドメインが抗体の可変領域である場合には、当該可変領域をコードするcDNAが得られた後に、当該cDNAの両末端に挿入された制限酵素サイトを認識する制限酵素によって該cDNAが消化される。好ましい制限酵素は、抗原結合分子の遺伝子を構成する塩基配列に出現する頻度が低い塩基配列を認識して消化する。更に1コピーの消化断片をベクターに正しい方向で挿入するためには、付着末端を与える制限酵素の挿入が好ましい。上記のように消化された抗原結合分子の可変領域をコードするcDNAを適当な発現ベクターに挿入することによって、本発明の抗原結合分子の発現ベクターが取得され得る。このとき、抗体定常領域(C領域)をコードする遺伝子と、前記可変領域をコードする遺伝子とがインフレームで融合され得る。
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyrのいずれか、
252位のアミノ酸がPhe、Trp、またはTyrのいずれか、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsp、Asn、Glu、またはGlnのいずれか、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはValのいずれか、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGluのいずれか、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyrのいずれか、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThrのいずれか、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArgのいずれか、
311位のアミノ酸がAla、His、またはIleのいずれか、
312位のアミノ酸がAlaまたはHisのいずれか、
314位のアミノ酸がLysまたはArgのいずれか、
315位のアミノ酸がAla、AspまたはHisのいずれか、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHisのいずれか、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSerのいずれか、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyrのいずれか、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyrのいずれか、および
436位のアミノ酸がHis 、Ile、Leu、Phe、Thr、またはVal、
が挙げられる。また、改変されるアミノ酸の数は特に限定されず、一箇所のみのアミノ酸が改変され得るし、二箇所以上のアミノ酸が改変され得る。二箇所以上のアミノ酸の改変の組合せとしては、例えば表6に記載されるような組合せが挙げられる。
様態1の抗原結合分子は、二分子のFcRnに結合することによって三者複合体を形成するが、活性型FcγRを含めた複合体は形成しない(図49)。活性型FcγRに対する結合活性が天然型Fc領域の活性型FcγRに対する結合活性より低いFc領域は、前記のように天然型Fc領域のアミノ酸を改変することによって作製され得る。改変Fc領域の活性型FcγRに対する結合活性が、天然型Fc領域の活性型FcγRに対する結合活性より低いか否かは、前記の結合活性の項で記載された方法を用いて適宜実施され得る。
234位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Met、Phe、Pro、Ser、ThrまたはTrpのいずれか、
235位のアミノ酸をAla、Asn、Asp、Gln、Glu、Gly、His、Ile、Lys、Met、Pro、Ser、Thr、ValまたはArgのいずれか、
236位のアミノ酸をArg、Asn、Gln、His、Leu、Lys、Met、Phe、ProまたはTyrのいずれか、
237位のアミノ酸をAla、Asn、Asp、Gln、Glu、His、Ile、Leu、Lys、Met、Pro、Ser、Thr、Val、TyrまたはArgのいずれか、
238位のアミノ酸をAla、Asn、Gln、Glu、Gly、His、Ile、Lys、Thr、TrpまたはArgのいずれか、
239位のアミノ酸をGln、His、Lys、Phe、Pro、Trp、TyrまたはArgのいずれか、
265位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、TyrまたはValのいずれか、
266位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Phe、Pro、Ser、Thr、TrpまたはTyrのいずれか、
267位のアミノ酸をArg、His、Lys、Phe、Pro、TrpまたはTyrのいずれか、
269位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
270位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか
271位のアミノ酸をArg、His、Phe、Ser、Thr、TrpまたはTyrのいずれか、
295位のアミノ酸をArg、Asn、Asp、Gly、His、Phe、Ser、TrpまたはTyrのいずれか、
296位のアミノ酸をArg、Gly、LysまたはProのいずれか、
297位のアミノ酸をAla、
298位のアミノ酸をArg、Gly、Lys、Pro、TrpまたはTyrのいずれか、
300位のアミノ酸をArg、LysまたはProのいずれか、
324位のアミノ酸をLysまたはProのいずれか、
325位のアミノ酸をAla、Arg、Gly、His、Ile、Lys、Phe、Pro、Thr、TrpTyr、もしくはValのいずれか、
327位のアミノ酸をArg、Gln、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
328位のアミノ酸をArg、Asn、Gly、His、LysまたはProのいずれか、
329位のアミノ酸をAsn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、Tyr、ValまたはArgのいずれか、
330位のアミノ酸をProまたはSerのいずれか、
331位のアミノ酸をArg、GlyまたはLysのいずれか、もしくは
332位のアミノ酸をArg、LysまたはProのいずれか、
のいずれかひとつ以上に改変されているFc領域が好適に挙げられる。
様態2の抗原結合分子は、二分子のFcRnと一分子の抑制型FcγRに結合することによってこれら四者を含む複合体を形成し得る。しかしながら、一分子の抗原結合分子は一分子のFcγRとしか結合できないため、一分子の抗原結合分子は抑制型FcγRに結合した状態で他の活性型FcγRに結合することはできない(図50)。さらに、抑制型FcγRに結合した状態で細胞内へと取り込まれた抗原結合分子は、細胞膜上へとリサイクルされ、細胞内での分解を回避することが報告されている(Immunity (2005) 23, 503-514)。すなわち、抑制型FcγRに対する選択的結合活性を有する抗原結合分子は、免疫応答の原因となる活性型FcγRおよび二分子のFcRnを含めたヘテロ複合体を形成することができないと考えられる。
様態3の抗原結合分子は、一分子のFcRnと一分子のFcγRに結合することによって三者複合体を形成しうるが、二分子のFcRnと一分子のFcγRの四者を含むヘテロ複合体は形成しない(図51)。本様態3の抗原結合分子に含まれる、Fc領域を構成する二つのポリペプチドの一方がpH中性域の条件下でのFcRnに対する結合活性を有し、他方のポリペプチドがpH中性域の条件下でのFcRnに対する結合能活性を有しないFc領域として、二重特異性抗体(bispecific抗体)を起源とするFc領域も適宜使用され得る。二重特異性抗体とは、異なる抗原に対して特異性を有する二種類の抗体である。IgG型の二重特異性抗体はIgG抗体を産生するハイブリドーマ二種を融合することによって生じるhybrid hybridoma(quadroma)によって分泌させることが可能である(Milsteinら(Nature (1983) 305, 537-540)。
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409位のアミノ酸がAsp、370位のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399位のアミノ酸がLys、357位のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される370位のアミノ酸のGluに代えてAspであってもよく、EUナンバリングで表される370位のアミノ酸のGluに代えて392位のアミノ酸のAspであってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409位のアミノ酸がAsp、439位のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399位のアミノ酸がLys、356位のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される439位のアミノ酸のGluに代えて360位のアミノ酸のAsp、EUナンバリングで表される392位のアミノ酸のAsp又は439位のアミノ酸のAspであってもよい)、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される370位のアミノ酸がGlu、439位のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される357位のアミノ酸がLys、356位のアミノ酸がLysであることを特徴とする二つのポリペプチド、または、
Fc領域を構成する二つのポリペプチドであって、その一方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される409位のアミノ酸がAsp、370位のアミノ酸がGlu、439位のアミノ酸がGluであり、他方のポリペプチドのアミノ酸配列のうちEUナンバリングで表される399位のアミノ酸がLys、357位のアミノ酸がLys、356位のアミノ酸がLysであることを特徴とする二つのポリペプチド(本態様では、EUナンバリングで表される370位のアミノ酸をGluに置換しなくてもよく、更に、370位のアミノ酸をGluに置換しない上で、439位のアミノ酸のGluに代えてAsp又は439位のアミノ酸のGluに代えて392位のアミノ酸のAspであってもよい)、
が好適に用いられる。
可溶型抗原に対する既存の中和抗体を投与すると、抗原が抗体に結合することで血漿中での持続性が高まることが予想される。抗体は一般的に長い半減期(1週間~3週間)を有するが、一方で抗原は一般的に短い半減期(1日以下)を有する。そのため、血漿中で抗体に結合した抗原は、抗原単独で存在する場合に比べて顕著に長い半減期を有するようになる。その結果として、既存の中和抗体を投与することにより、血漿中の抗原濃度の上昇が起こる。このような事例は様々な可溶型抗原を標的とした中和抗体において報告されており、一例を挙げるとIL-6(J. Immunotoxicol. (2005) 3, 131-139)、amyloid beta(mAbs (2010) 2 (5), 1-13)、MCP-1(ARTHRITIS & RHEUMATISM (2006) 54,2387-2392)、hepcidin(AAPS J. (2010) 4, 646-657) 、sIL-6 receptor(Blood (2008) 112 (10), 3959-64)などがある。既存の中和抗体の投与により、ベースラインからおよそ10倍~1000倍程度(上昇の程度は、抗原によって異なる)の血漿中総抗原濃度の上昇が報告されている。ここで、血漿中総抗原濃度とは、血漿中に存在する抗原の総量としての濃度を意味しており、すなわち抗体結合型と抗体非結合型の抗原濃度の和として表される。このような可溶型抗原を標的とした抗体医薬にとっては、血漿中総抗原濃度の上昇が起こることは好ましくない。なぜなら、可溶型抗原を中和するためには、少なくとも血漿中総抗原濃度を上回る血漿中抗体濃度が必要なためである。つまり、血漿中総抗原濃度が10倍~1000倍上昇するということは、それを中和するための血漿中抗体濃度(すなわち抗体投与量)としても、血漿中総抗原濃度の上昇が起こらない場合に比べて10倍~1000倍が必要になることを意味する。一方で、既存の中和抗体に比較して血漿中総抗原濃度を10倍~1000倍低下することができれば、抗体の投与量を同じだけ減らすことが可能である。このように、血漿中から可溶型抗原を消失させて、血漿中総抗原濃度を低下させることができる抗体は、既存の中和抗体に比較して顕著に有用性が高い。
〔実施例1〕中性条件下におけるヒトFcRnへの結合を増強することによるpH依存的ヒトIL-6レセプター結合ヒト抗体の血漿中滞留性および免疫原性への影響
血漿中からの可溶型抗原を消失させるために、FcRnと相互作用する抗体等の抗原結合分子のFc領域(Nat. Rev. Immunol. (2007) 7 (9), 715-25)等のFcRn結合ドメインにがpH中性域においてFcRnに対する結合活性を有することが重要である。参考実施例5で示したように、FcRn結合ドメインのpH中性域でのFcRnに対する結合活性を有するFcRn結合ドメイン変異(アミノ酸置換)体が研究されている。Fc変異体として創生されたF1~F600のpH中性域におけるFcRnに対する結合活性が評価され、pH中性域においてFcRnに対する結合活性を増強することにより血漿中からの抗原の消失を加速することが確認された。こういったFc変異体を医薬品として開発するためには、薬理的な性質(FcRnの結合増強による血漿中からの抗原の消失の加速など)のみならず、抗原結合分子の安定性や純度、抗原結合分子の生体内における血漿中滞留性が優れ、免疫原性が低いことが好ましい。
そこで、pH中性域の条件下におけるヒトFcRnに対する結合を有するFcRn結合ドメインを含む抗原結合分子の血漿中滞留性の評価、および、その抗原結合分子の免疫原性の評価を行うために、pH中性域の条件下におけるヒトFcRnに対する結合活性を有するヒトIL-6レセプター結合ヒト抗体として、VH3-IgG1(配列番号:35)とVL3-CK(配列番号:36)からなるFv4-IgG1、VH3-IgG1-F1(配列番号:37)とVL3-CK からなるFv4-IgG1-F1、VH3-IgG1-F157(配列番号:38)とVL3-CK からなるFv4-IgG1-F157、VH3-IgG1-F20(配列番号:39)とVL3-CK からなるFv4-IgG1-F20、VH3-IgG1-F21(配列番号:40)とVL3-CK からなるFv4-IgG1-F21が参考実施例1および参考実施例2に示した方法によって作製された。
重鎖としてVH3-IgG1あるいはVH3-IgG1-F1を含み、軽鎖としてL(WT)-CK(配列番号:41)を含む抗体が参考実施例2に示した方法で作製され、下記のようにマウスFcRnに対する結合活性が評価された。
Biacore T100(GE Healthcare)を用いて、マウスFcRnと抗体との速度論的解析を行った。センサーチップCM4(GE Healthcare)上にアミンカップリング法でプロテインL(ACTIGEN)を適当量固定化し、そこへ目的の抗体を捕捉させた。次に、FcRn希釈液とランニングバッファー(参照溶液として)とをインジェクトし、センサーチップ上に捕捉させた抗体にマウスFcRnを相互作用させた。ランニングバッファーには50 mmol/Lリン酸ナトリウム、150 mmol/L NaCl、0.05% (w/v) Tween20、pH7.4を用い、FcRnの希釈にもそれぞれのバッファーが使用された。センサーチップの再生には10 mmol/Lグリシン-HCl, pH1.5が用いられた。測定は全て25 ℃で実施された。測定で得られたセンサーグラムから算出されたカイネティクスパラメーターである結合速度定数 ka (1/Ms)、および解離速度定数 kd (1/s)をもとに各抗体のマウスFcRnに対する KD (M) が算出された。各パラメーターの算出には Biacore T100 Evaluation Software(GE Healthcare)が用いられた。
その結果、IgG1のKD (M)は検出されなかった一方で、作製されたIgG1-F1のKD (M)は1.06E-06 (M)であった。作製されたIgG1-F1は、pH中性域(pH7.4)の条件下において、マウスFcRnに対する結合活性が増強されていることが示された。
作製されたpH依存的ヒトIL-6レセプター結合ヒト抗体であるFv4-IgG1およびFv4-IgG1-F1のノーマルマウスを用いたPK試験が下記の方法で実施された。ノーマルマウス(C57BL/6J mouse、Charles River Japan)の尾静脈あるいは背部皮下に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後5分、7時間、1日、2日、4日、7日、14日、21日、28日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
マウス血漿中の抗ヒトIL-6レセプター抗体濃度はELISA法にて測定された。まず、Anti-Human IgG(γ-chain specific)F(ab')2 Fragment of Antibody(SIGMA)をNunc-Immuno Plate, MaxiSoup(Nalge nunc International)に分注し、4℃で1晩静置することによってAnti-Human IgG固相化プレートが作成された。血漿中濃度として0.8、0.4、0.2、0.1、0.05、0.025、0.0125μg/mLの抗ヒトIL-6レセプター抗体を含む検量線試料と100倍以上希釈されたマウス血漿測定試料が調製された。これらの検量線試料および血漿測定試料100μLに20 ng/mLの可溶型ヒトIL-6レセプターが200μL加えられた混合液を、室温で1時間静置させた。その後当該混合液が各ウェルに分注されたAnti-Human IgG固相化プレートをさらに室温で1時間静置させた。その後Biotinylated Anti-human IL-6 R Antibody(R&D)と室温で1時間反応させ、さらにStreptavidin-PolyHRP80(Stereospecific Detection Technologies)を室温で1時間反応させた反応液の発色反応が、TMB One Component HRP Microwell Substrate(BioFX Laboratories)を基質として用いて行われた。1N-Sulfuric acid(Showa Chemical)を添加することによって反応が停止された各ウェルの反応液の450 nmの吸光度が、マイクロプレートリーダーにて測定された。マウス血漿中の抗体濃度は検量線の吸光度から解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。
以下の方法により、pH中性域の条件下におけるマウスFcRnに対する結合活性を有するマウス抗体が作製された。
マウス抗体の可変領域として、ヒトIL-6Rへの結合能を有するマウス抗体である、mouse PM-1(Sato K, et al. Cancer Res. (1993) 53(4), 851-856)のアミノ酸配列が用いられた。これ以降、mouse PM-1の重鎖可変領域はmPM1H(配列番号:42)、軽鎖可変領域はmPM1L(配列番号:43)と表記される。
また、重鎖定常領域として天然型マウスIgG1(配列番号:44、以降はmIgG1と表記される)、軽鎖定常領域として天然型マウスkappa(配列番号:45、以降はmk1と表記される)が用いられた。
参考実施例1の方法に従い、重鎖mPM1H-mIgG1(配列番号:46)および軽鎖mPM1L-mk1(配列番号:47)の塩基配列を有する発現ベクターが作製された。また、参考実施例2の方法に従い、mPM1H-mIgG1とmPM1L-mk1からなる、ヒトIL-6R結合マウス抗体であるmPM1-mIgG1が作製された。
作製されたmPM1-mIgG1は、天然型マウスFc領域を含むマウス抗体であり、pH中性域の条件下でのマウスFcRnに対する結合活性を有しない。そこで、pH中性域の条件下でのマウスFcRnに対する結合活性を付与するために、mPM1-mIgG1の重鎖定常領域にアミノ酸改変が導入された。
同様に、mPM1H-mIgG1のEUナンバリングで表される252位のThrがTyrに置換されたアミノ酸置換、EUナンバリングで表される256位のThrがGluに置換されたアミノ酸置換、EUナンバリングで表される433位のHisがLysに置換されたアミノ酸置換が加えられたmPM1H-mIgG1-mF14(配列番号:49)が作製された。
更に、mPM1H-mIgG1のEUナンバリングで表される252位のThrがTyrに置換されたアミノ酸置換、EUナンバリングで表される256位のThrがGluに置換されたアミノ酸置換、EUナンバリングで表される434位のAsnがTrpに置換されたアミノ酸置換が加えられたmPM1H-mIgG1-mF38(配列番号:50)が作製された。
参考実施例2の方法を用いて、pH中性域の条件下でのマウスFcRnに対する結合を有するマウスIgG1抗体として、mPM1H-mIgG1-mF3とmPM1L-mk1からなるmPM1-mIgG1-mF3が作製された。
mPM1-mIgG1またはmPM1-mIgG1-mF3の重鎖および、L(WT)-CK(配列番号:41)の軽鎖を含む抗体が作製され、これらの抗体のpH7.0におけるマウスFcRnに対する結合活性(解離定数KD)が測定された。結果を以下の表5に示した。
実施例1において、抗原結合分子の中性条件下におけるFcRnへの結合を増強することによって、血漿中滞留性および免疫原性が悪化することが確認された。天然型IgG1は中性領域でヒトFcRnに対して結合活性を有さないため、中性条件下におけるFcRnへの結合を付与したことで血漿中滞留性および免疫原性が悪化したと考えられた。
抗体のFc領域にはFcRnに対する結合ドメインとFcγRに対する結合ドメインが存在する。FcRnに対する結合ドメインはFc領域の2箇所に存在し、抗体1分子のFc領域に対して2分子のFcRnが同時に結合できることが既に報告されている(Nature (1994) 372 (6504), 379-383)。一方で、FcγRに対する結合ドメインもFc領域の2箇所に存在するが、2分子のFcγRが同時に結合することはできないと考えられている。これは、1分子目のFcγRがFc領域に結合することによって生じたFc領域の構造変化によって、2分子目のFcγRが結合できないためである(J. Biol. Chem. (2001) 276 (19), 16469-16477)。
FcγR/二分子のFcRn/IgGの四者複合体の形成できるかどうかは、pH中性域の条件下でのFcRnに対する結合活性を有するFc領域を含む抗原結合分子がFcγRとFcRnに対して同時に結合できるか否かで判断することが可能である。そこで、下記の方法にしたがい、抗原結合分子が含むFc領域のFcRnとFcγRに対する同時結合実験が実施された。
Biacore T100又はT200(GE Healthcare)を用いて、ヒト又はマウスFcRnとヒト又はマウスFcγRsとが抗原結合分子に同時に結合するかが評価された。Sensor chip CM4 (GE Healthcare) 上にアミンカップリング法によって固定化されたヒト又はマウスFcRnに、被験対象の抗原結合分子をキャプチャーさせた。次に、ヒト又はマウスFcγRsの希釈液とブランクとして使用されたランニングバッファーが注入され、センサーチップ上のFcRnに結合した抗原結合分子にヒト又はマウスFcγRsを相互作用させた。ランニングバッファーとして50 mmol/L sodium phosphate、150 mmol/L NaCl、0.05% (w/v) Tween20、pH7.4が用いられ、FcγRsの希釈にもこのバッファーが使用された。センサーチップの再生には10 mmol/L Trsi-HCl、pH9.5が用いられた。結合の測定は全て25℃で実施された。
pH中性域における条件下でヒトFcRnに対する結合能を有するヒト抗体である、実施例1で作製されたFv4-IgG1-F157が、ヒトFcRnに結合するのと同時に、各種ヒトFcγRまたは各種マウスFcγRに結合するか否かが評価された。
その結果、Fv4-IgG1-F157が、 ヒトFcRnに結合するのと同時に、ヒトFcγRIa、FcγRIIa(R)、FcγRIIa(H)、FcγRIIb、FcγRIIIa(F)に対して結合できることが示された(図3、4、5、6、7)。また、Fv4-IgG1-F157は同様に、ヒトFcRnに結合するのと同時に、マウスFcγRI、FcγRIIb、FcγRIII、FcγRIVに対しても結合できることが示された。(図8、9、10、11)
以上のことから、pH中性域の条件下におけるヒトFcRnに対する結合活性を有するヒト抗体が、 ヒトFcRnに結合するのと同時に、ヒトFcγRIa、FcγRIIa(R)、FcγRIIa(H)、FcγRIIb、FcγRIIIa(F)やマウスFcγRI、FcγRIIb、FcγRIII、FcγRIV等の各種ヒトFcγRおよび各種マウスFcγRに対しても結合できることが示された。
pH中性域における条件下でマウスFcRnに対する結合活性を有するヒト抗体である、実施例1で作製されたFv4-IgG1-F20が、マウスFcRnに結合するのと同時に、各種マウスFcγRに結合するか否かが評価された。
その結果、Fv4-IgG1-F20が、マウスFcRnに結合するのと同時に、マウスFcγRI、FcγRIIb、FcγRIII、FcγRIVに対して結合できることが示された(図12)。
pH中性域における条件下でマウスFcRnに対する結合能を有するマウス抗体である、実施例2で作製されたmPM1-mIgG1-mF3が、マウスFcRnに結合するのと同時に、各種マウスFcγRに結合するか否かが評価された。
その結果、mPM1-mIgG1-mF3は、マウスFcRnに結合するのと同時に、マウスFcγRIIbおよびFcγRIIIに対して結合できることが示された(図13)。マウスFcγRIおよびIVに対して結合が確認されなかった結果は、マウスIgG1抗体はマウスFcγRIおよびIVに対して結合能を持たないとの報告(J. Immunol. (2011) 187 (4), 1754-1763))から判断すると、妥当な結果であると考えられる。
これらのことから、pH中性域の条件下におけるマウスFcRnに対する結合活性を有するヒト抗体およびマウス抗体は、マウスFcRnに結合するのと同時に各種マウスFcγRに対しても結合できることが示された。
以上のことから、ヒトおよびマウスIgGのFc領域にはFcRnへの結合領域とFcγRへの結合領域が存在するが、それらは互いに干渉することはなく、一分子のFcと二分子のFcRn、一分子のFcγRの四者を含むヘテロ複合体を形成することが可能であることが示された。
すなわち、pH中性域におけるFcRnに対する結合活性を有する抗原結合分子は、一分子の活性型FcγRと二分子のFcRnの四者を含むヘテロ複合体を形成することで、抗原提示細胞への取り込みが増大し、血漿中滞留性が悪化し、さらに免疫原性が悪化したと考えられた。
様態1の抗原結合分子は、2分子のFcRnに結合することによって三者を含む複合体を形成するが、活性型FcγRを含めた複合体は形成しない。
様態2の抗原結合分子は、二分子のFcRnと一分子の抑制性FcγRに結合することによってこれら四者を含む複合体を形成し得る。しかしながら、一分子の抗原結合分子は一分子のFcγRとしか結合できないため、一分子の抗原結合分子は抑制性FcγRに結合した状態で他の活性型FcγRに結合することはできない。さらに、抑制性FcγRに結合した状態で細胞内へと取り込まれた抗原結合分子は、細胞膜上へとリサイクルされ、細胞内での分解を回避することが報告されている(Immunity (2005) 23, 503-514)。すなわち、抑制性FcγRに対する選択的結合活性を有する抗原結合分子は、免疫応答の原因となる活性型FcγRを含めた複合体を形成することができないと考えられる。
様態3の抗原結合分子は、一分子のFcRnと一分子のFcγRに結合することによって三者複合体を形成しうるが、二分子のFcRnと一分子のFcγRの四者を含むヘテロ複合体は形成しない。
(4-1)ヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性より低く、pH依存的にヒトIL-6レセプターに結合する抗体の作製
実施例3で示された三つの態様のうち、様態1の抗原結合分子、つまり、pH中性域の条件下でのFcRnに対する結合活性を有し、活性型FcγRに対する結合活性が天然型FcγR結合ドメインの結合活性より低い抗原結合分子が、以下のように作製された。
実施例1で作製された、Fv4-IgG1-F21およびFv4-IgG1-F157は、pH中性域の条件下におけるヒトFcRnに対する結合活性を有し、pH依存的にヒトIL-6レセプターに結合する抗体である。これらのアミノ酸配列の、EUナンバリングで表される239位のSerがLysに置換されたアミノ酸置換によって、マウスFcγRに対する結合を低下させた改変体が作製された。具体的には、VH3-IgG1-F21のアミノ酸配列のEUナンバリングで表される239位のSerがLysに置換されたVH3-IgG1-F140(配列番号:51)が作製された。また、VH3-IgG1-F157のアミノ酸配列のEUナンバリングで表される239位のSerがLysに置換されたVH3-IgG1-F424(配列番号:52)が作製された。
参考実施例2の方法を用いて、これらの重鎖およびVL3-CKの軽鎖を含む、Fv4-IgG1-F140およびFv4-IgG1-F424が作製された。
作製されたVH3-IgG1-F21、VH3-IgG1-F140、VH3-IgG1-F157、またはVH3-IgG1-F424を重鎖として含み、L(WT)-CKを軽鎖として含む抗体の、pH7.0におけるヒトFcRnに対する結合活性(解離定数KD)およびpH7.4におけるマウスFcγRに対する結合活性が、下記の方法を用いて測定された。
Biacore T100 又はT200(GE Healthcare)を用いて、ヒトFcRnと前記の抗体との結合の速度論的解析を行った。上にアミンカップリング法でprotein L(ACTIGEN)が適当量固定化されたSensor chip CM4(GE Healthcare)に、被験対象の抗体をキャプチャーさせた。次に、ヒトFcRnの希釈液とブランクとして使用されたランニングバッファーが注入され、センサーチップ上にキャプチャーさした抗体にヒトFcRnを相互作用させた。ランニングバッファーとして50 mmol/L sodium phosphate、150 mmol/L NaCl、0.05%(w/v)Tween20、pH7.0またはpH7.4が用られ、ヒトFcRnの希釈にもそれぞれのバッファーが使用された。センサーチップの再生には10 mmol/L Glycine-HCl, pH1.5が用いられた。結合の測定は全て25℃で実施された。測定で得られたセンサーグラムから算出された、カイネティクスパラメーターである結合速度定数 ka(1/Ms)、および解離速度定数 kd(1/s)をもとに各抗体のヒトFcRnに対する KD(M)が算出された。各パラメーターの算出にはBiacore T100 又はT200 Evaluation Software(GE Healthcare)が用いられた。
その結果を以下の表6に示した。
Biacore T100 又はT200(GE Healthcare)を用いて、マウスFcγRI、FcγRII、FcγRIII、FcγRIV(R&D sytems、Sino Biological)(以下、マウスFcγRsと呼ばれる)と抗体との結合活性が評価された。Sensor chip CM4(GE Healthcare)上にアミンカップリング法で適当量固定化されたprotein L(ACTIGEN)に、被験対象の抗体をキャプチャーさせた。次に、マウスFcγRsの希釈液とブランクとして使用されたランニングバッファーが注入され、センサーチップ上にキャプチャーされた抗体に相互作用させた。ランニングバッファーとして20 mmol/L ACES、150 mmol/L NaCl、0.05%(w/v)Tween20、pH7.4が用いられ、マウスFcγRsの希釈にもこのバッファーが使用された。センサーチップの再生には10 mmol/L Glycine-HCl、pH1.5が用いられた。測定は全て25℃で実施された。
マウスFcγRsの結合活性(Y)=(ΔA1 - ΔA2)/X x 1500
作製されたFv4-IgG1-F140、Fv4-IgG1-F424、Fv4-IgG1-F21およびFv4-IgG1-F157がヒトFcRnトランスジェニックマウスに投与された際のPK試験が下記の方法で実施された。
ヒトFcRnトランスジェニックマウス(B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse、Jackson Laboratories、Methods Mol. Biol. (2010) 602, 93-104)の尾静脈に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後15分、7時間、1日、2日、3日、4日、7日、14日、21日、28日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
マウス血漿中の抗ヒトIL-6レセプター抗体濃度はELISA法にて測定された。まず、Anti-Human IgG(γ-chain specific)F(ab')2 Fragment of Antibody(SIGMA)をNunc-Immuno Plate, MaxiSoup (Nalge nunc International)に分注し、4℃で1晩静置することによってAnti-Human IgG固相化プレートが作成された。血漿中濃度として0.8、0.4、0.2、0.1、0.05、0.025、0.0125μg/mLの抗ヒトIL-6レセプター抗体を含む検量線試料と100倍以上希釈されたマウス血漿測定試料が調製された。これらの検量線試料および血漿測定試料100μLに20 ng/mLの可溶型ヒトIL-6レセプターが200μL加えられた混合液を、室温で1時間静置させた。その後当該混合液が各ウェルに分注されたAnti-Human IgG固相化プレートをさらに室温で1時間静置させた。その後Biotinylated Anti-human IL-6 R Antibody(R&D)と室温で1時間反応させ、さらにStreptavidin-PolyHRP80(Stereospecific Detection Technologies)を室温で1時間反応させた反応液の発色反応が、TMB One Component HRP Microwell Substrate(BioFX Laboratories)を基質として用いて行われた。1N-Sulfuric acid(Showa Chemical)を添加することによって反応が停止された各ウェルの反応液の450 nmの吸光度が、マイクロプレートリーダーにて測定された。マウス血漿中の抗体濃度は検量線の吸光度から解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。
図14の結果から、Fv4-IgG1-F21に比較してマウスFcγRへの結合が低いFv4-IgG1-F140は、Fv4-IgG1-F21に比べて血漿中滞留性の向上が認められた。同様に、Fv4-IgG1-F157に比較してマウスFcγRへの結合が低いFv4-IgG1-F424は、Fv4-IgG1-F157に比べて血漿中滞留性の延長が認められた。
このことから、pH中性域の条件下でのヒトFcRnに対する結合を有し、FcγRに対する結合が通常のFcγR結合ドメインよりも低いFcγR結合ドメインを有する抗体は、通常のFcγR結合ドメインを有する抗体よりも血漿中滞留性が高いことが示された。
(5-1)ヒトおよびマウスFcγRに対する結合活性を有しないpH依存的にヒトIL-6レセプターに結合するヒト抗体の作製
ヒトおよびマウスFcγRに対する結合活性を有しないpH依存的にヒトIL-6レセプターに結合するヒト抗体を作製するため、以下のように抗体作製が行われた。
VH3-IgG1のアミノ酸配列のEUナンバリングで表される235位のLeuがArgに置換されたアミノ酸置換および239位のSerがLysに置換されたアミノ酸置換によって、ヒトおよびマウスFcγRに対する結合活性を有しないVH3-IgG1-F760(配列番号:53)が作製された。
同様に、VH3-IgG1-F11(配列番号:54)、VH3-IgG1-F890(配列番号:55)およびVH3-IgG1-F947(配列番号:56)のそれぞれのアミノ酸配列の、EUナンバリングで表される235位のLeuがArgに置換されたアミノ酸置換および239位のSerがLysに置換されたアミノ酸置換によって、ヒトおよびマウスFcγRに対する結合活性を有しないVH3-IgG1-F821(配列番号:57)、VH3-IgG1-F939(配列番号:58)およびVH3-IgG1-F1009(配列番号:59)が作製された。
参考実施例2の方法を用いて、これらの重鎖およびVL3-CKの軽鎖を含むFv4-IgG1、Fv4-IgG1-F11、Fv4-IgG1-F890、Fv4-IgG1-F947、Fv4-IgG1-F760、Fv4-IgG1-F821、Fv4-IgG1-F939およびFv4-IgG1-F1009が作製された。
参考実施例2の方法で作製されたVH3-IgG1、VH3-IgG1-F11、VH3-IgG1-F890、VH3-IgG1-F947、VH3-IgG1-F760、VH3-IgG1-F821、VH3-IgG1-F939またはVH3-IgG1-F1009を重鎖として含み、L(WT)-CKを軽鎖として含む抗体のpH7.0におけるヒトFcRnに対する結合活性(解離定数KD)が、実施例4の方法を用いて測定された。測定した結果を以下の表8に示した。
作製されたFv4-IgG1およびFv4-IgG1-F760がヒトFcRnトランスジェニックマウスに投与された際のPK試験が下記の方法で実施された。
ヒトFcRnトランスジェニックマウス(B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse、Jackson Laboratories、Methods Mol. Biol. (2010) 602, 93-104)の尾静脈に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後15分、7時間、1日、2日、3日、4日、7日、14日、21日、28日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
マウス血漿中の抗ヒトIL-6レセプター抗体濃度は、実施例4の方法と同様にELISA法にて測定された。その結果を図15に示した。Fv4-IgG1のマウスFcγRに対する結合活性を低下させたFv4-IgG1-F760は、Fv4-IgG1-F11に比べてほぼ同等の血漿中滞留性を示し、FcγRに対する結合活性を低下させることによる血漿滞留性の向上効果は見られなかった。
作製されたFv4-IgG1-F11、Fv4-IgG1-F890、Fv4-IgG1-F947、Fv4-IgG1-F821、Fv4-IgG1-F939およびFv4-IgG1-F1009がヒトFcRnトランスジェニックマウスに投与された際のPK試験が下記の方法で実施された。
ヒトFcRnトランスジェニックマウス(B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse、Jackson Laboratories、Methods Mol. Biol. (2010) 602, 93-104)の背部皮下に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後15分、7時間、1日、2日、3日、4日、7日、14日、21日、28日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
一方、Fv4-IgG1とIgG1-F760の両者においては血漿中滞留性の違いは認められず、pH中性域におけるFcRn結合活性を有さないFv4-IgG1は、免疫細胞上でFcγRとの二者複合体を形成し、四者複合体を形成することができないことから、FcγRへの結合活性の低下により血漿中滞留性の向上が認められなかったと考えられた。すなわち、pH中性域におけるFcRn結合活性を有する抗原結合分子に対して、FcγRへの結合活性を低下させ四者複合体の形成を阻害することで初めて血漿中滞留性の向上が認められたと言える。このことからも、四者複合体の形成が血漿中滞留性の悪化に重要な役割を果たしていると考えられる。
VH3-IgG1-F947(配列番号:56)のアミノ酸配列の、EUナンバリングで表される234位のLeuがAlaに置換されたアミノ酸置換および235位のLeuがAlaに置換されたアミノ酸置換によって、ヒトおよびマウスFcγRに対する結合活性が低下されたVH3-IgG1-F1326(配列番号:155)が作製された。
参考実施例2の方法を用いて、VH3-IgG1-F1326の重鎖およびVL3-CKの軽鎖を含むFv4-IgG1-F1326が作製された。
参考実施例2の方法で作製されたVH3-IgG1-F1326を重鎖として含み、L(WT)-CKを軽鎖として含む抗体のpH7.0におけるヒトFcRnに対する結合活性(解離定数KD)が、実施例4の方法を用いて測定された。また、実施例4の方法と同様に、pH7.4におけるマウスFcγRに対する結合活性が測定された。測定した結果を以下の表10に示した。
作製されたFv4-IgG1-F1326がヒトFcRnトランスジェニックマウスに投与された際のPK試験が実施例5-4の方法と同様に実施された。 マウス血漿中の抗ヒトIL-6レセプター抗体濃度は、実施例4の方法と同様にELISA法にて測定された。その結果を、実施例5-4で得られたFv4-IgG1-F947の結果とあわせて図54に示した。Fv4-IgG1-F947のマウスFcγRに対する結合活性を低下させたFv4-IgG1-F1326は、Fv4-IgG1-F947に比べて血漿中滞留性の向上が認められた。
以上のことから、中性条件下におけるヒトFcRnへの結合を増強したヒト抗体において、マウスFcγRへの結合活性を低下させ四者複合体の形成を阻害することにより、ヒトFcRnトランスジェニックマウスにおける血漿中滞留性の向上が可能であることが示された。ここで、マウスFcγRへの結合活性を低下させることにより血漿中滞留性向上の効果が示されるためには、好ましくはヒトFcRnへのpH7.0でのアフィニティー(KD)が310 nMよりも強く、更に好ましくは110 nM以下である。
結果として、実施例4と同様に、抗原結合分子に対して様態1の性質を付与することにより血漿中滞留性の向上が確認された。ここで見られている血漿中滞留性の向上は、抗原提示細胞を含む免疫細胞への取り込みを選択的に阻害したためであると考えられ、その結果として免疫応答の誘起を阻害することも可能であると期待される。
(6-1)マウスFcγRに対する結合活性を有しないヒトIL-6レセプターに結合するマウス抗体の作製
実施例4および5において、pH中性域の条件下においてヒトFcRnに対する結合活性を有し、マウスFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFcγR結合ドメインを含む抗原結合分子は、ヒトFcRnトランスジェニックマウスにおける血漿中滞留性が向上していることが示された。同様に、pH中性域の条件下においてマウスFcRnに対する結合活性を有し、マウスFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFcγR結合ドメインを含む抗原結合分子の、ノーマルマウスにおける血漿中滞留性が向上されているかどうかが検証された。
実施例2で作製されたmPM1H-mIgG1-mF38のアミノ酸配列の、EUナンバリングで表される235位のProがLysに置換されたアミノ酸置換および239位のSerがLysに置換されたアミノ酸置換によって、mPM1H-mIgG1-mF40(配列番号:60)が、mPM1H-mIgG1-mF14のアミノ酸配列の、EUナンバリングで表される235位のProがLysに置換されたアミノ酸置換および239位のSerがLysに置換されたアミノ酸置換によって、mPM1H-mIgG1-mF39(配列番号:61)が作製された。
実施例2の方法を用いて、pH7.0におけるマウスFcRnに対する結合活性(解離定数KD)が測定された。その結果を以下の表11に示した。
作製されたmPM1-mIgG1-mF14、mPM1-mIgG1-mF38、mPM1-mIgG1-mF39、mPM1-mIgG1-mF40が、ノーマルマウスに投与された際のPK試験が下記の方法で実施された。
ノーマルマウス(C57BL/6J mouse、Charles River Japan)の背部皮下に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後5分、7時間、1日、2日、4日、7日、14日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
マウス血漿中の抗ヒトIL-6レセプターマウス抗体濃度はELISA法にて測定された。まず、可溶型ヒトIL-6レセプター をNunc-Immuno Plate, MaxiSoup(Nalge nunc International)に分注し、4℃で1晩静置することによって可溶型ヒトIL-6レセプター固相化プレートが作成された。血漿中濃度として1.25、0.625、 0.313、0.156、0.078、0.039、0.020μg/mLの抗ヒトIL-6レセプターマウス抗体を含む検量線試料と100倍以上希釈されたマウス血漿測定試料が調製された。これらの検量線試料および血漿測定試料100μLが各ウェルに分注された可溶型ヒトIL-6レセプター固相化プレートを室温で2時間静置させた。その後Anti-Mouse IgG-Peroxidase antibody(SIGMA)と室温で1時間反応させ、さらにStreptavidin-PolyHRP80(Stereospecific Detection Technologies)を室温で1時間反応させた反応液の発色反応が、TMB One Component HRP Microwell Substrate(BioFX Laboratories)を基質として用いて行われた。1N-Sulfuric acid(Showa Chemical)を添加することによって反応が停止された各ウェルの反応液の450 nmの吸光度が、マイクロプレートリーダーにて測定された。マウス血漿中の抗体濃度は検量線の吸光度から解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。この方法で測定された静脈内投与後のノーマルマウスにおける血漿中の抗体濃度の推移を図17に示した。
以上のことから、pH中性域の条件下におけるマウスFcRnに対する結合を有し、マウスFcγRに対する結合活性を有しないFcγR結合ドメインを有する抗体は、通常のFcγR結合ドメインを有する抗体よりもノーマルマウスにおける血漿中滞留性が高いことが示された。
結果として、実施例4および5と同様に、抗原結合分子に対して様態1の性質を有する抗原結合分子は血漿中滞留性が高いことが確認された。本発明は特定の理論に拘束されるものではないが、ここで観察された血漿中滞留性の向上は、抗原提示細胞等の免疫細胞への取り込みを選択的に阻害したためであると考えられ、その結果として免疫応答の誘起を阻害することも可能であると期待される。
様態1の抗原結合分子、つまり、pH中性域の条件下でのFcRnに対する結合活性を有し、活性型FcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低い抗原結合ドメインを含む抗原結合分子の、ヒトにおける免疫原性を評価するために、下記の方法によって当該抗原結合分子に対するin vitroにおけるT細胞応答が評価された。
実施例4で測定された、VH3/L(WT)-IgG1、VH3/L(WT)-IgG1-F21およびVH3/L(WT)-IgG1-F140のpH中性域の条件下(pH7.0)におけるヒトFcRnに対する結合定数(KD)を以下の表13に示した。
以下の方法を用いて、VH3/L(WT)-IgG1、VH3/L(WT)-IgG1-F21、VH3/L(WT)-IgG1-F140のpH7.4におけるヒトFcγRに対する結合活性が測定された。
Biacore T100 又はT200 (GE Healthcare) を用いて、ヒトFcγRIa、FcγRIIa(H)、FcγRIIa(R)、FcγRIIb、FcγRIIIa(F)(以下、ヒトFcγRsと呼ばれる)と抗体との結合活性が評価された。Sensor chip CM4(GE Healthcare)上にアミンカップリング法で適切な量固定化されたprotein L(ACTIGEN)に、被験対象の抗体をキャプチャーさせた。次に、ヒトFcγRsの希釈液とブランクとして使用されたランニングバッファーが注入され、センサーチップ上にキャプチャーされた抗体に相互作用させた。ランニングバッファーとして20 mmol/L ACES、150 mmol/L NaCl、0.05%(w/v)Tween20、pH7.4が用いられ、ヒトFcγRsの希釈にもこのバッファーが使用された。センサーチップの再生には10 mmol/L Glycine-HCl、pH1.5が用いられた。測定は全て25℃で実施された。
ヒトFcγRsの結合活性(Y)=(ΔA1 - ΔA2)/X x 1500
実施例1で作製されたFv4-IgG1-F21、Fv4-IgG1-F140を用いて、下記の通りin vitro免疫原性試験が実施された。
末梢血単核球細胞(PBMC)が、健常人ボランティアから採取した血液より単離された。Ficoll(GE Healthcare)密度遠心分離によって血液から分離されたPBMCから、Dynabeads CD8(invitrogen)を用いて付属の標準プロトコールに従い、マグネットによってCD8+T細胞が除去された。次いでDynabeads CD25(invitrogen)を用いて付属の標準プロトコールに従い、CD25hiT細胞がマグネットによって除去された。
増殖アッセイが以下のように実施された。CD8+ 及び CD25hiT 細胞が除去され、2×106 /mL となるように 3% 不活性化ヒト血清を含む AIMV 培地 (Invitrogen) に再懸濁された各ドナーのPBMCが、平底の24ウェルプレートに1ウェルあたり 2×106細胞加えられた。37℃、5%CO2の条件下で 2 時間の培養後、各被験物質が終濃度 10、30、100、300μg/mLとなるように添加された細胞が8日間培養された。培養 6、7及び8日の時点で、丸底 96 ウェルプレートに移された培養中の細胞懸濁液 150μLに対してBrdU(Bromodeoxyuridine)が加えられ、さらに当該細胞が24 時間培養された。BrdUとともに培養された細胞の核内に取り込まれた BrdUが、BrdU Flow Kit(BD bioscience)を用いて付属の標準プロトコールに従い染色されるのと同時に抗CD3、CD4 及び CD19 抗体(BD bioscience)によって表面抗原(CD3、CD4 及び CD19)が染色された。次いで BD FACS Calibur 又は BD FACS CantII(BD)によってBrdU陽性CD4+T細胞の割合が検出された。培養6、7及び 8日において、被検物質の10、30、100、300μg/mLの各終濃度でのBrdU陽性CD4+T細胞の割合が算出され、それらの平均値が算出された。
(8-1)hA33-IgG1の作製
実施例7で示されたように、Fv4-IgG1-F21に対するヒトPBMCの免疫応答性が元来低いため、FcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低い抗原結合ドメインを含むFv4-IgG1-F140に対する免疫応答の抑制を評価するためには、適さないことが示唆された。そこで、in vitro免疫原性評価系において、免疫原性低減効果の検出力を高めるために、A33 抗原に対するヒト化IgG1抗体であるヒト化A33抗体(hA33-IgG1)が作製された。
hA33-IgG1は、臨床試験において 33-73% の被験者で抗抗体の産生が確認されている(Hwangら(Methods (2005) 36, 3-10)およびWalleら(Expert Opin. Bio. Ther. (2007) 7 (3), 405-418))。hA33-IgG1はこの高い免疫原性は可変領域配列によるものであることから、hA33-IgG1に対してpH中性域におけるFcRnに対する結合活性を増強させた分子に対して、FcγRに対する結合活性を低下させて四者複合体形成を阻害することによる免疫原性低減効果を検出しやすいと考えられた。
参考実施例1の方法に従い、重鎖hA33H-IgG1および軽鎖hA33L-k0の塩基配列を含む発現ベクターが作製された。また、参考実施例2の方法に従い、重鎖hA33H-IgG1および軽鎖hA33L-k0を含む、ヒト化A33抗体であるhA33-IgG1が作製された。
作製されたhA33-IgG1は、天然型ヒトFc領域を有するヒト抗体であるため、pH中性域の条件下でのヒトFcRnに対する結合活性を有しない。そこで、pH中性域の条件下でのヒトFcRnに対する結合能を付与するために、hA33-IgG1の重鎖定常領域にアミノ酸改変が導入された。
具体的には、hA33-IgG1の重鎖定常領域であるhA33H-IgG1のEUナンバリングで表される252位のアミノ酸がMetからTyrに置換され、EUナンバリングで表される308位のアミノ酸がValからProに置換され、EUナンバリングで表される434位のアミノ酸がAsnからTyrに置換されたことにより、hA33H-IgG1-F21(配列番号:65)が作製された。参考実施例2の方法を用いて、pH中性域の条件下におけるヒトFcRnに対する結合活性を有するA33結合抗体として、hA33H-IgG1-F21を重鎖として含み、hA33L-k0を軽鎖として含むhA33-IgG1-F21が作製された。
hA33-IgG1-F21のヒトFcγRに対する結合活性を低下させるため、hA33H-IgG1-F21のアミノ酸配列のEUナンバリングで表される239位のSerがLysに置換された、hA33H-IgG1-F140(配列番号:66)が作製された。
実施例7と同様の方法を用いて、作製されたhA33-IgG1-F21、hA33-IgG1-F140に対する免疫原性の評価が行われた。なお、ドナーである健常人ボランティアは実施例7で用いられたPBMCが単離された健常人ボランティアとは同一の個体ではない。つまり、実施例7におけるドナーAと当試験におけるドナーAは別の個体の健常人ボランティアである。
試験の結果を図19に示した。図19では、pH中性域におけるヒトFcRnに対する結合を有するhA33-IgG1-F21と、さらにヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFcγR結合ドメインを含むhA33-IgG1-F140の結果が比較されている。陰性対照に比べて、ドナーC、DおよびFから単離されたPBMCのhA33-IgG1-F21に対する反応は観察されていないため、ドナーC、DおよびFはhA33-IgG1-F21に対して免疫応答を起こさないドナーであると考えられる。それ以外の7名のドナー(ドナーA、B、E、G、H、IおよびJ)から単離されたPBMCにおいては、hA33-IgG1-F21に対する免疫応答が陰性対照に比べて高いことが観察されており、hA33-IgG1-F21はin vitroにおいて期待通り高い免疫原性を示した。一方で、ヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFcγR結合ドメインを含むhA33-IgG1-F140に対する、これら7名の全ドナー(ドナーA、B、E、G、H、IおよびJ)から単離されたPBMCの免疫応答が、hA33-IgG1-F21に対するそれと比較して、低下している効果が観察される。また、hA33-IgG1-F140に対する、ドナーEおよびJから単離されたPBMCの免疫応答は陰性対照と同程度あることからも、pH中性域におけるヒトFcRnに対する結合活性を有する抗原結合分子において、ヒトFcγRに対する結合活性を天然型FcγR結合ドメインの結合活性よりも低くして四者複合体の形成を阻害することで免疫原性を低減することできると考えられた。
(9-1)pH中性域の条件下でのヒトFcRnに対する強い結合活性を有するA33結合抗体の作製
hA33H-IgG1に対してEUナンバリングで表される252位のアミノ酸がMetからTyrに置換され、EUナンバリングで表される286位のアミノ酸がAsnからGluに置換され、EUナンバリングで表される307位のアミノ酸がThrからGlnに置換され、EUナンバリングで表される311位のアミノ酸がGlnからAlaに置換され、EUナンバリングで表される434位のアミノ酸がAsnからTyrに置換されたことにより、hA33H-IgG1-F698(配列番号:67)が参考実施例1の方法で作製された。pH中性域の条件下におけるヒトFcRnに対する強い結合活性を有するヒトA33結合抗体として、hA33H-IgG1-F698を重鎖として含み、hA33L-k0を軽鎖として含むhA33-IgG1-F698が作製された。
hA33H-F698のEUナンバリングで表される239番目のSerがLysに置換され、ヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低い抗原結合ドメインを含む、hA33H-IgG1-F699(配列番号:68)が作製された。
実施例4の方法を用いて、VH3/L(WT)-IgG1、VH3/L(WT)-IgG1-F698およびVH3/L(WT)-IgG1-F699のpH7.0におけるヒトFcRnに対する結合活性が測定された。更に、実施例7の方法を用いて、pH7.4におけるヒトFcγRに対するVH3/L(WT)-IgG1、VH3/L(WT)-IgG1-F698およびVH3/L(WT)-IgG1-F699の結合活性が測定された。その結果を併せて以下の表15に示した。
作製されたhA33-IgG1-F698、hA33-IgG1-F699に対する免疫原性の評価が、実施例7と同様の方法で行われた。なお、ドナーである健常人ボランティアは実施例7および8で用いられたPBMCが単離された健常人ボランティアとは同一の個体ではない。つまり、実施例7および実施例8におけるドナーAと、当試験におけるドナーAは別の個体の健常人ボランティアである。
(9-3)に記載したとおり、hA33-IgG1-F698のEUナンバリングで表される239番のSerをLysに置換することによって各種ヒトFcγRに対する結合活性が低下しているhA33-IgG1-F699は、hFcgRIIa(R)、hFcgRIIa(H)、hFcgRIIb、hFcgRIIIa(F)に対する結合は顕著に低下しているものの、hFcgRIに対する結合は残存していた。
そこで、hFcgRIaも含めた全てのヒトFcγRに対する結合を有しないFcγR結合ドメインを含む、A33結合抗体を作製するために、hA33H-IgG1-F698(配列番号:67)のEUナンバリングで表される235位のLeuがArgに置換され、EUナンバリングで表される239位のSerがLysに置換された、hA33H-IgG1-F763(配列番号:69)が作製された。
実施例7と同様の方法を用いて、作製されたhA33-IgG1-F698、hA33-IgG1-F763の免疫原性が評価された。なお、これまでと同様に、ドナーである健常人ボランティアは前記実施例で用いられたPBMCが単離された健常人ボランティアとは同一の個体ではない。つまり、前記実施例におけるドナーAと、当試験におけるドナーAは別の個体の健常人ボランティアである。
実施例7、8および9において、pH中性域におけるヒトFcRnに対する結合活性を有し、FcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFcγR結合ドメインを含む抗原結合分子は、FcγR結合活性が低下されていない抗原結合分子に比較して、免疫原性が低減していることがin vitroの実験において示された。この効果がin vivoにおいても示されることを確認するために、下記の試験が実施された。
実施例5で得られたマウス血漿を用いて、Fv4-IgG1-F11、Fv4-IgG1-F890、Fv4-IgG1-F947、Fv4-IgG1-F821、Fv4-IgG1-F939、Fv4-IgG1-F1009に対する抗体産生が下記の方法で評価された。
マウスの血漿中抗投与検体抗体は電気化学発光法にて測定された。まず投与検体がUncoated MULTI-ARRAY Plate(Meso Scale Discovery)に分注され、4℃で1晩静置することにより投与検体固相化プレートが作成された。50倍に希釈されたマウス血漿測定試料が調製され、投与検体固相化プレートに分注し4℃で1晩反応された。その後SULFO-TAG NHS Ester(Meso Scale Discovery)でルテニウム化したAnti-Mouse IgG(whole molecule)(SIGMA)を室温で1時間反応させ、Read Buffer T(×4)(Meso Scale Discovery)を分注し、ただちにSECTOR PR 400(Meso Scale Discovery)で測定が行われた。測定系毎に、抗体が投与されていない個体5個体の血漿が陰性対照サンプルとして測定され、その5個体の血漿を用いて測定された数値の平均値(MEAN)に、5個体の血漿を用いて測定された数値の標準偏差(SD)の1.645倍を加えた数値(X)が、陽性判定基準として用いられた(式3)。いずれかの採血日において、1度でも陽性判定基準を上回る反応が示された個体が、被検物質に対する抗体産生応答が陽性であると判定された。
抗体産生の陽性判定基準(X)= MEAN + 1.645 x SD
その結果を図22から図27に示した。図22にFv4-IgG1-F11がヒトFcRnトランスジェニックマウスに投与されてから3日後、7日後、14日後、21日後および28日後の、Fv4-IgG1-F11に対して産生されたマウス抗体の抗体価を示した。投与後いずれの採血日においても、3匹のマウスの内1匹のマウス(#3)において、Fv4-IgG1-F11に対するマウス抗体の産生が陽性であることが示された(陽性率1/3)。一方で、図23にFv4-IgG1-F821がヒトFcRnトランスジェニックマウスに投与されてから3日後、7日後、14日後、21日後および28日後の、Fv4-IgG1-F821に対して産生されたマウス抗体の抗体価を示した。投与後いずれの採血日においても、3匹のマウス全てにおいて、Fv4-IgG1-F821に対するマウス抗体の産生は陰性であることが示された(陽性率0/3)。
Fv4-IgG1-F11およびFv4-IgG1-F890に対しては、各種マウスFcγRへの結合を低下させることにより、生体内における免疫原性を顕著に低減させることが可能であることが示された。一方、Fv4-IgG1-F947に対しては、各種マウスFcγRへの結合を低下させることによる、生体内における免疫原性を低下させる効果は示されなかった。
実施例3に記載されたように、pH中性域の条件下においてFcRnに対する結合活性を有する抗原結合分子に対して、FcγRへの結合活性を低下させることにより、抗原提示細胞の細胞膜上における四者複合体の形成を阻害することが可能であると考えられる。四者複合体の形成が阻害されることによって、抗原提示細胞への抗原結合分子の取り込みも抑制され、結果的に抗原結合分子に対する免疫原性の誘導が抑制されると考えられる。Fv4-IgG1-F11およびFv4-IgG1-F890については、FcγRへの結合活性を低下させることにより、このようにして免疫原性の誘導が抑制されたとも考えられる。
図16に示されたように、Fv4-IgG1-F947およびFv4-IgG1-F1009の血漿中からの消失は非常に速い。ここで、Fv4-IgG1-F1009はマウスFcγRへの結合活性が低下しており、抗原提示細胞上での四者複合体の形成は阻害されていると考えられる。そのため、Fv4-IgG1-F1009は血管内皮細胞や血球系細胞等の細胞膜上に発現しているFcRnのみに結合することにより、細胞内へと取り込まれていると考えられる。ここで、一部の抗原提示細胞の細胞膜上にもFcRnが発現していることから、Fv4-IgG1-F1009はFcRnのみに結合することによっても、抗原提示細胞に取り込まれ得る。つまり、Fv4-IgG1-F1009の血漿中からの急速な消失のうち、一部は抗原提示細胞への取り込みが起きている可能性がある。
実際、抗原結合分子が抗体である場合、ヒトに投与される抗体は、ヒト化抗体あるいはヒト抗体であることから、同種タンパク質に対する免疫応答が起こることになる。そこで、実施例11において、四者複合体の形成阻害が免疫原性の低減につながるか否かについて、マウス抗体をマウスに投与することで評価された。
(11-1)ノーマルマウスにおけるin vivo免疫原性試験
抗原結合分子が同種タンパク質(マウス抗体をマウスに投与)である場合の、抗原提示細胞上での四者複合体の形成を阻害することによる免疫原性の抑制効果を検証する目的で、以下のような試験が実施された。
実施例6で得られたマウス血漿を用いて、以下の方法を用いて、mPM1-mIgG1-mF38、mPM1-mIgG1-mF40、mPM1-mIgG1-mF14、mPM1-mIgG1-mF39に対する抗体産生が評価された。
マウスの血漿中抗投与検体抗体は電気化学発光法にて測定された。MULTI-ARRAY 96 well plate に投与検体が分注され、室温で1hr反応させた。plateを洗浄後に、50倍希釈されたマウス血漿測定試料が調製され、室温で2hr反応させて洗浄された後、SULFO-TAG NHS Ester(Meso Scale Discovery)でルテニウム化した投与検体を分注して4℃で一晩反応させた。翌日plateを洗浄後にRead Buffer T(×4)(Meso Scale Discovery)が分注され、ただちにSECTOR PR 2400 reader(Meso Scale Discovery)で測定が行われた。測定系毎に、抗体が投与されていない個体5個体の血漿が陰性対照サンプルとして測定され、その5個体の血漿を用いて測定された数値の平均値(MEAN)に、5個体の血漿を用いて測定された数値の標準偏差(SD)の1.645倍を加えた数値(X)が、陽性判定基準として用いられた(式3)。いずれかの採血日において、1度でも陽性判定基準を上回る反応が示された個体が、被検物質に対する抗体産生応答が陽性であると判定された。
抗体産生の陽性判定基準(X)= MEAN + 1.645 x SD
その結果を図28から図31に示した。図28にmPM1-mIgG1-mF14がノーマルマウスに投与されてから14日後、21日後および28日後の、mPM1-mIgG1-mF14に対して産生されたマウス抗体の抗体価が示されている。投与から21日後の時点で、3匹のマウス全てにおいて、mPM1-mIgG1-mF14に対するマウス抗体の産生が陽性であることが示された(陽性率3/3)。一方で、図29にはmPM1-mIgG1-mF39がノーマルマウスに投与されてから14日後、21日後および28日後の、mPM1-mIgG1-mF39に対して産生されたマウス抗体の抗体価が示されている。投与後いずれの採血日においても、3匹のマウス全てにおいて、mPM1-mIgG1-mF39に対するマウス抗体の産生は陰性であることが示された(陽性率0/3)。
図30にmPM1-mIgG1-mF38がノーマルマウスに投与されてから14日後、21日後および28日後の、mPM1-mIgG1-mF38に対して産生されたマウス抗体の抗体価を示した。投与から28日後の時点で、3匹のマウスの内2匹のマウス(#1、#2)において、mPM1-mIgG1-mF38に対するマウス抗体の産生が陽性であることが示された(陽性率2/3)。一方で、図31にmPM1-mIgG1-mF40がノーマルマウスに投与されてから14日後、21日後および28日後の、mPM1-mIgG1-mF40に対して産生されたマウス抗体の抗体価を示した。投与後いずれの採血日においても、3匹のマウス全てにおいて、mPM1-mIgG1-mF40に対するマウス抗体の産生は陰性であることが示された(陽性率0/3)。
これらの結果から、同種タンパク質であるマウス抗体であるmPM1-mIgG1-mF38およびmPM1-mIgG1-mF14をノーマルマウスに投与しても、投与抗体に対する抗体産生が確認され、免疫応答が確認された。これは、実施例1、2で示したように、pH中性域においてFcRnへの結合活性を増強させ、抗原提示細胞上で四者複合体を形成することにより、抗原提示細胞への取り込みが促進されたためであると考えられる。
以上のことから、in vitroおよびin vivoの両方において、pH中性域の条件下におけるFcRnに対する結合活性を有する抗原結合分子に対して、FcγRへの結合活性を低下させることにより、当該抗原結合分子の免疫原性を極めて有効に低下させることが可能であることが示された。言い換えれば、pH中性域の条件下におけるFcRnに対する結合活性を有し、活性型FcγRに対する結合活性が天然型FcγR結合ドメインの結合活性より低い抗原結合分子(すなわち、実施例3で記載した様態1の抗原結合分子)は、天然型FcγR結合ドメインと同程度の結合活性を有する抗原結合分子(すなわち、実施例3で記載した四者複合体を形成し得る抗原結合分子)に比較して、免疫原性が顕著に低下されていることが示された。
(12-1)pH中性域におけるヒトFcRnに対する結合活性を有し、ヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いヒトIgG1抗体の作製および評価
本発明の非限定の一態様では、活性型FcγRに対する結合活性が天然型Fc領域の活性型FcγRに対する結合活性より低いFc領域の例として、前記Fc領域のアミノ酸のうちEUナンバリングで表される234位、235位、236位、237位、238位、239位、270位、297位、298位、325位および329位のいずれかひとつ以上のアミノ酸が天然型Fc領域と異なるアミノ酸に改変されているFc領域が好適に挙げられるが、Fc領域の改変は上記改変に限定されず、例えばCurrent Opinion in Biotechnology (2009) 20 (6), 685-691に記載されている脱糖鎖 (N297A, N297Q)、IgG1-L234A/L235A、IgG1-A325A/A330S/P331S、IgG1-C226S/C229S、IgG1-C226S/C229S/E233P/L234V/L235A、IgG1-L234F/L235E/P331S、IgG1-S267E/L328F、IgG2-V234A/G237A、IgG2-H268Q/V309L/A330S/A331S、IgG4-L235A/G237A/E318A、IgG4-L236E等の改変、および、WO 2008/092117に記載されているG236R/L328R、L235G/G236R、N325A/L328R、N325LL328R等の改変、および、EUナンバリング233位、234位、235位、237位におけるアミノ酸の挿入、WO 2000/042072に記載されている個所の改変であってもよい。
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される235位のLeuがLysに置換され、239位のSerがLysに置換されたVH3-IgG1-F938(配列番号:156)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される237位のGlyがLysに置換され、239位のSerがLysに置換されたVH3-IgG1-F1315(配列番号:157)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される237位のGlyがArgに置換され、239位のSerがLysに置換されたVH3-IgG1-F1316(配列番号:158)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される239位のSerがLysに置換され、329位のProがLysに置換されたVH3-IgG1-F1317(配列番号:159)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される239位のSerがLysに置換され、329位のProがArgに置換されたVH3-IgG1-F1318(配列番号:160)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される234位のLeuがAlaに置換され、235位のLeuがAlaに置換されたVH3-IgG1-F1324(配列番号:161)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される234位のLeuがAlaに置換され、235位のLeuがAlaに置換され、297位のAsnがAlaに置換されたVH3-IgG1-F1325(配列番号:162)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される235位のLeuがArgに置換され、236位のGlyがArgに置換され、239位のSerがLysに置換されたVH3-IgG1-F1333(配列番号:163)、
VH3-IgG1-F890のアミノ酸配列のEUナンバリングで表される236位のGlyがArgに置換され、328位のLeuがArgに置換されたVH3-IgG1-F1356(配列番号:164)、
VH3-IgG1-F947のアミノ酸配列のEUナンバリングで表される234位のLeuがAlaに置換され、235位のLeuがAlaに置換されたVH3-IgG1-F1326(配列番号:155)、
VH3-IgG1-F947のアミノ酸配列のEUナンバリングで表される234位のLeuがAlaに置換され、235位のLeuがAlaに置換され、297位のAsnがAlaに置換されたVH3-IgG1-F1327(配列番号:165)が作製された。
(12-1)で作製されたそれぞれのアミノ酸配列を重鎖として含み、L(WT)-CKを軽鎖として含む抗体のpH7.0におけるヒトFcRnに対する結合活性(解離定数KD)が、実施例4の方法を用いて測定された。また、pH7.4におけるヒトFcγRに対する結合活性が実施例7の方法を用いて測定された。測定した結果を以下の表18に示した。
天然型IgG1と比較してFcgRへの結合が低下している改変を見出すため、IgG1のFc領域においてFcγRに対する結合部位と考えられるアミノ酸残基の改変体の各FcγRに対する結合が網羅的に解析された。 抗体H鎖として、WO2009/041062に開示されている血漿中動態が改善した抗グリピカン3抗体であるGpH7のCDRを含むグリピカン3抗体の可変領域(配列番号:74)が使用された。同様に、抗体L鎖として、 WO2009/041062に開示される血漿中動態が改善したグリピカン3抗体のGpL16-k0(配列番号:75)が共通に使用された。また、抗体H鎖定常領域として、IgG1のC末端のGlyおよびLysが欠失されたG1dに、K439Eの変異が導入されたB3(配列番号:76)が使用された。以後、このH鎖はGpH7-B3(配列番号:77)、L鎖はGpL16-k0(配列番号:75)と呼ばれる。
まずGpH7-B3/GpL16-k0をコントロールとして網羅的に解析することの妥当性を検証するため、GpH7-B3/GpL16-k0と、GpH7-G1d/GpL16-k0の各FcgRに対する結合能が比較された(表19)。参考実施例2の方法により発現、精製された両抗体の各ヒトFcγR(FcγRIa、FcγRIIa H型、FcγRIIa R型、FcγRIIb、FcγRIIIaF型)に対する結合が以下の方法により評価された。
次に、GpH7-B3のアミノ酸配列において、FcγRの結合に関与すると考えられるアミノ酸とその周辺のアミノ酸(EUナンバリングで表される234位から239位、265位から271位、295位、296位、298位、300位、324位から337位)が元のアミノ酸とCysを除く18種類のアミノ酸にそれぞれ置換された。これらのFc変異体はB3 variantと呼ばれる。参考実施例2の方法により発現、精製されたB3 variantの各FcγR(FcγRIa、FcγRIIa H型、FcγRIIa R型、FcγRIIb、FcγRIIIaF型)に対する結合が、(12-4)の方法により網羅的に評価された。
そのため、各種ヒトFcγRに対する結合活性を天然型FcγR結合ドメインの結合活性に比べて低下させるために導入されるアミノ酸改変は、特に限定されず、表20に示されたアミノ酸改変を少なくとも1箇所導入することによって、達成可能であることが示された。また、ここで導入されるアミノ酸改変は、1箇所であってもよいし、複数箇所の組合せであってもよい。
ヒトIgG2あるいはヒトIgG4を用いて、pH中性域におけるヒトFcRnに対する結合活性を有し、ヒトFcγRに対する結合活性が天然型FcγR結合ドメインの結合活性よりも低いFc領域を以下のように作製した。
(12-6)で作製された抗体(表21)のpH7.0におけるヒトFcRnに対する結合活性(解離定数KD)が、実施例4の方法を用いて測定された。また、pH7.4におけるヒトFcγRに対する結合活性が実施例7の方法を用いて測定された。測定した結果を以下の表22に示した。
実施例3において様態3として示された、FcRn結合ドメインを構成する二つのポリペプチドの一方のみpH中性域の条件下でのFcRnに対する結合を有し、もう一方はpH中性域の条件下でのFcRnに対する結合活性を有しない抗原結合分子の作製が、以下のように行われた。
まず、pH中性域の条件下におけるFcRnに対する結合を有する抗ヒトIL-6R抗体の重鎖として、VH3-IgG1-F947(配列番号:70)が参考実施例1の方法で作製された。また、pH酸性域およびpH中性域の両方の条件下においてFcRnに対する結合活性を有しない抗原結合分子として、VH3-IgG1に対してEUナンバリングで表される253位のIleをAlaに置換するアミノ酸を加えてVH3-IgG1-F46(配列番号:71)が作製された。
抗体のヘテロ二量体を高純度で得るための方法として、抗体のうちの一方のFc領域がEUナンバリングで表される356位のAspがLysに、およびEUナンバリングで表される357位のGluがLysに置換されており、もう一方のFc領域がEUナンバリングで表される370位のLysがGluに、EUナンバリングで表される435位のHisがArgに、およびEUナンバリングで表される439位のLysがGluに置換されたFc領域を用いることが知られている(WO2006/106905)。
Fv4-IgG1-F947およびFv4-IgG1-FA6a/FB4aが、ヒトFcRnトランスジェニックマウスに投与された際のPK試験が下記の方法で実施された。
ヒトFcRnトランスジェニックマウス(B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse、Jackson Laboratories、Methods Mol. Biol. (2010) 602, 93-104)の背部皮下に、抗ヒトIL-6レセプター抗体が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後15分、7時間、1日、2日、3日、4日、7日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
そのため、図32で示したような、pH中性域の条件下において1箇所のFcRn結合領域を有するFv4-IgG1-FA6a/FB4aにおいて、血漿中滞留性の向上が見られるという現象は、天然型IgGの場合とは逆であることから、完全に予想外のものであった。
一般的に、皮下に投与された抗体はリンパ系を介して吸収され、血漿中へと移行すると考えられている(J. Pharm. Sci. (2000) 89 (3), 297-310.)。リンパ系には多量の免疫細胞が存在するため、皮下に投与された抗体は多量の免疫細胞に曝露され、その後血漿中へと移行すると考えられる。一般的に、抗体医薬品が皮下投与された場合、静脈内投与された場合と比べて免疫原性が高まることが知られているが、その一因として、皮下に投与された抗体がリンパ系において多量の免疫細胞に曝露されることが考えられる。実際に、実施例1において示されたように、Fv4-IgG1-F1は皮下投与された場合においてはFv4-IgG1-F1の血漿中からの急速な消失が確認され、Fv4-IgG1-F1に対するマウス抗体の産生が示唆された。一方で、静脈内投与された場合においてはFv4-IgG1-F1の血漿中からの急速な消失は確認されず、Fv4-IgG1-F1に対するマウス抗体は産生されていないことが示唆された。
すなわち、皮下に投与された抗体がその吸収過程において、リンパ系に存在する免疫細胞に取り込まれると、生物学的利用率(Bioavailablity)の低下が起こるとともに、免疫原性の原因にもなり得る。
このような、皮下に投与された抗体の生物学的利用率(Bioavailablity)を上昇させることで血漿中濃度を上昇させる、あるいは免疫原性を低下させる方法は、実施例3において様態3として示された抗原結合分子には限らず、免疫細胞の細胞膜上で四者複合体を形成しない抗原結合分子であれば、いずれを用いても良いと考えられる。すなわち、様態1、2、3のいずれの抗原結合分子においても、四者複合体を形成し得る抗原結合分子に比べ、皮下に投与された際の生物学的利用率(Bioavailablity)を上昇させるとともに血漿中滞留性を向上させ、更に免疫原性を低下させることが可能であると考えられる。
また、中性条件下でのFcRnへの結合を増強させた抗原結合分子に対して、抑制型FcγRIIbに対する選択的な結合活性の増強をもたらす改変を用いることにより、実施例3で示された様態2の抗原結合分子を作製することが可能である。つまり、中性条件下でのFcRnに対する結合活性を有し、さらに抑制型FcγRIIbに対する選択的な結合活性の増強をもたらす改変が導入された抗原結合分子は、2分子のFcRnと1分子のFcγRを介した四者複合体を形成し得る。しかし、当該改変の効果により、抑制性FcγRに対する選択的結合がもたらされているため、活性型FcγRに対する結合活性は低下している。その結果、抗原提示細胞上では抑制型FcγRを含む四者複合体が優位に形成されると考えられる。先述したとおり、免疫原性には活性型FcγRを含む四者複合体の形成が原因となると考えられ、このように抑制型FcγRを含む四者複合体を形成させることにより、免疫応答の抑制が可能であると考えられる。
そこで、抑制型FcγRIIbに対する選択的な結合活性の増強をもたらすアミノ酸変異を見出すために、以下に示す検討が実施された。
天然型IgG1と比較して、活性型FcγR、特にFcγRIIaのH型およびR型のいずれの遺伝子多型に対してもFcを介した結合が減少し、かつFcγRIIbに対する結合が増強する変異が導入された複数のIgG1抗体改変体の、各FcγRに対する結合活性が網羅的に解析された。
抗体H鎖には、WO2009/041062に開示されている血漿中動態が改善した抗グリピカン3抗体であるGpH7のCDRを含むグリピカン3抗体の可変領域(配列番号:74)が使用された。同様に、抗体L鎖には WO2009/041062に開示される血漿中動態が改善したグリピカン3抗体のGpL16-k0(配列番号:75)が異なるH鎖との組合せにおいて共通に使用された。また、抗体H鎖定常領域として、IgG1のC末端のGlyおよびLysが欠失されたG1dに、K439Eの変異が導入されたB3(配列番号:76)が使用された。以後、このH鎖はGpH7-B3(配列番号:77)、L鎖はGpL16-k0(配列番号:75)とそれぞれ呼ばれる。
それぞれのFcγRについて、以下の方法に従って図が作製された。各B3 variantに由来する抗体と各FcγRに対する結合量の値が、B3に何も変異が導入されていない対照抗体(EUナンバリングで表される234位から239位、265位から271位、295位、296位、298位、300位、324位から337位がヒト天然型IgG1の配列を有する抗体)の値で除された。その値に更に100を乗じた値が、各FcγRに対する結合の値として表された。横軸に各変異体のFcγRIIbに対する結合、縦軸に各変異体の各活性型FcγRであるFcγRIa、FcγRIIa(H)、FcγRIIa(R)、FcγRIIIaの値をそれぞれ表示した(図33、34、35、36)。
その結果、図33~36にラベルで表示したように、全改変のうち、mutation A(EUナンバリングで表される238位のProがAspに置換された改変)およびmutation B(EUナンバリングで表される328位のLeuがGluに置換された改変)天然型IgG1と比べてFcγRIIbに対する結合が顕著に増強され、FcγRIIaの両タイプに対する結合を顕著に抑制する効果があることを見出した。
(14-1)で見出されたEUナンバリングで表される238位のProがAspに置換された改変体の各FcγRに対する結合がより詳細に解析された。
抗体H鎖可変領域としてWO2009/125825に開示されるヒトインターロイキン6レセプターに対する抗体の可変領域であるIL6R-Hの可変領域(配列番号:78)と、抗体H鎖定常領域としてヒトIgG1のC末端のGlyおよびLysが除去されたG1d定常領域を含むIL6R-G1d(配列番号:79)がIgG1のH鎖として用いられた。IL6R-G1dのEUナンバリングで表される238位のProがAspに改変されたIL6R-G1d_v1(配列番号:80)が作製された。次に、IL6R-G1dのEUナンバリングで表される328位のLeuがGluに改変されたIL6R-G1d_v2(配列番号:81)が作製された。また、比較のために公知の変異(Mol. Immunol. (2008) 45, 3926-3933)であるEUナンバリングで表される267位のSerがGluに置換され、およびEUナンバリングで表される328位のLeuがPheに置換されたIL6R-G1dの改変体であるIL6R-G1d_v3(配列番号:82)が作製された。抗体L鎖としてはトシリズマブのL鎖であるIL6R-L(配列番号:83)がこれらの重鎖との組合せにおいて共通に用いられた。参考実施例2の方法に従い、抗体が発現、精製された。抗体H鎖としてIL6R-G1d、IL6R-G1d_v1、IL6R-G1d_v2、IL6R-G1d_v3を含む抗体は、以下それぞれIgG1、IgG1-v1、IgG1-v2、IgG1-v3と呼ばれる。
1:1 binding modelで相互作用する分子のBiacore上での挙動は以下の式4によって表わされ得る。
Req=C x Rmax / (KD + C) + RI
Req(RU): 定常状態結合レベル(Steady state binding levels)
C(M): アナライト濃度(Analyte concentration)
C: concentration
Rmax(RU):アナライトの表面結合能(Analyte binding capacity of the surface)
RI(RU): 試料中の容積屈折率寄与(Bulk refractive index contribution in the sample)
KD(M): 平衡解離定数(Equilibrium dissociation constant)
で表される。
この式4を変形すると、KDは以下の式5のように表わすことができる。
KD =C x Rmax / (Req - RI) - C
KD =C x Rmax / (Req - RI) - C
〔式5〕
KD =C x Rmax / (Req - RI) - C
また、表25に示したように、IgG1と比べてIgG1-v2はFcγRIaに対する親和性が0.74倍に低下し、FcγR IIa(R)に対する親和性は0.41倍に低下し、FcγRIIa(H)に対する親和性が0.064倍に低下し、FcγRIIIaに対する親和性は0.14倍に低下していた。一方で、FcγRIIbに対する親和性は2.3倍向上していた。
(14-2)において、ヒト天然型IgG1のEUナンバリングで表される238位のProがAspに置換された改変体又はEUナンバリングで表される328位のLeuがGluに置換された改変体は、FcγRIa、FcγRIIIaおよびFcγRIIaのいずれの遺伝子多型に対してもFcを介した結合が減少し、かつFcγRIIbに対する結合が向上することが見出された。そこで、EUナンバリングで表される238位のProがAspに置換された改変体又はEUナンバリングで表される328位のLeuがGluに置換された改変体に対して、さらにアミノ酸置換を導入することによって、FcγRI、FcγRIIa(H)、FcγRIIa(R)、FcγRIIIaのいずれかに対する結合がさらに低減された、あるいは、FcγRIIbに対する結合がさらに向上されたFc改変体の創出が行われる。
VH3-IgG1およびVH3-IgG1-F11に対して、ヒトFcγRIIbに対して選択的に結合活性を増強するため、以下の方法で抗体を作製した。VH3-IgG1に対してEUナンバリングで表される238位のProをAspに置換するためのアミノ酸置換が参考実施例1の方法で導入され、VH3-IgG1-F648(配列番号:84)が作製された。同様にVH3-IgG1-F11に対してEUナンバリングで表される238位のProをAspに置換するためのアミノ酸置換が参考実施例1の方法で導入され、VH3-IgG1-F652(配列番号:85)が作製された。
VH3-IgG1、VH3-IgG1-F648、VH3-IgG1-F11、あるいはVH3-IgG1-F652を重鎖として含み、L(WT)-CKを軽鎖として含む抗体が、参考実施例2の方法で作製された。
これらの抗体とFcγRIIa(R)およびFcγRIIbとの相互作用をBiacore T100(GE Healthcare)を用いて解析した。ランニングバッファーとして20 mM ACES, 150 mM NaCl, 0.05% Tween20, pH7.4を用いて25℃の温度で測定した。アミンカップリング法によりProtein Lが固定化されたSeries S Sencor Chip CM4(GE Healthcare)を用いた。目的の抗体をキャプチャーさせたチップに対して、ランニングバッファーで希釈された各FcγRを作用させることによって、各FcγRの抗体に対する相互作用を測定した。測定後は10 mM glycine-HCl、pH1.5を反応させることによって、チップにキャプチャーされた抗体を洗浄し、このように再生したチップは繰り返し用いられた。
マウスFcγRsの結合活性(Y)=(ΔA1 - ΔA2)/X x 1500
QuikChange Site-Directed Mutagenesis Kit(Stratagene)を用いて、添付説明書記載の方法で作製された変異体を含むプラスミド断片を動物細胞発現ベクターに挿入することによって、目的のH鎖発現ベクターおよびL鎖発現ベクターが作製された。得られた発現ベクターの塩基配列は当業者公知の方法で決定された。
抗体の発現は以下の方法を用いて行われた。ヒト胎児腎癌細胞由来HEK293H株(Invitrogen)を10 % Fetal Bovine Serum(Invitrogen)を含むDMEM培地(Invitrogen)へ懸濁し、5~6 × 105細胞/mLの細胞密度で接着細胞用ディッシュ(直径10 cm, CORNING)の各ディッシュへ10 mLずつ蒔きこみCO2インキュベーター(37℃、5 % CO2)内で一昼夜培養した後に、培地を吸引除去し、CHO-S-SFM-II(Invitrogen)培地6.9 mLを添加した。調製したプラスミドをlipofection法により細胞へ導入した。得られた培養上清を回収した後、遠心分離(約2000 g、5分間、室温)して細胞を除去し、さらに0.22μmフィルターMILLEX(R)-GV(Millipore)を通して滅菌して培養上清を得た。得られた培養上清にrProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いて当業者公知の方法で精製した。精製抗体濃度は、分光光度計を用いて280 nmでの吸光度を測定した。得られた値からProtein Science 1995 ; 4 : 2411-2423に記された方法により算出された吸光係数を用いて抗体濃度を算出した。
抗原であるヒトIL-6レセプターの組み換えヒトIL-6レセプターは以下のように調製された。J. Immunol. (1994) 152, 4958-4968で報告されているN末端側1番目から357番目のアミノ酸配列からなる可溶型ヒトIL-6レセプター(以下、hsIL-6Rとも表記される)を定常的に発現するCHO株が当業者公知の方法で構築された。当該CHO株を培養することによって、可溶型ヒトIL-6レセプターを発現させた。得られた当該CHO株の培養上清から、Blue Sepharose 6 FFカラムクロマトグラフィー、ゲルろ過カラムクロマトグラフィーの二工程によって可溶型ヒトIL-6レセプターが精製された。最終工程においてメインピークとして溶出された画分が最終精製品として用いられた。
ノーマルマウスにおける可溶型ヒトIL-6レセプターおよびヒト抗体の血漿中滞留性と免疫原性を評価する目的で、以下のような試験が実施された。
(4-1)ノーマルマウスにおける可溶型ヒトIL-6レセプターの血漿中滞留性および免疫原性評価
ノーマルマウスにおける可溶型ヒトIL-6レセプターの血漿中滞留性および免疫原性を評価する目的で、以下の試験が実施された。
ノーマルマウス(C57BL/6J mouse、Charles River Japan)の尾静脈に、可溶型ヒトIL-6レセプター(参考実施例3にて作製)が50μg/kgで単回投与された。可溶型ヒトIL-6レセプターの投与後15分、7時間、1日、2日、3日、4日、7日、14日、21日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。可溶型ヒトIL-6レセプターの血漿中濃度およびマウス抗可溶型ヒトIL-6レセプター抗体の抗体価は以下の方法で測定された。
可溶型ヒトIL-6レセプターに対するマウス抗体が産生されることによる、可溶型ヒトIL-6レセプターの血漿中濃度への影響を評価する目的で、以下の試験が実施された。
可溶型ヒトIL-6レセプターの血漿中濃度を定常状態(約20 ng/mL)に維持するモデルとして以下の試験モデルが構築された。ノーマルマウス(C57BL/6J mouse、Charles River Japan)の背部皮下に可溶型ヒトIL-6レセプターを充填したinfusion pump(MINI-OSMOTIC PUMP MODEL2004、alzet)を埋め込むことで、血漿中可溶型ヒトIL-6レセプター濃度が定常状態に維持される動物モデルが作製された。
この方法で測定したノーマルマウスにおける個体別の血漿中可溶型ヒトIL-6レセプター濃度推移を図38に示した。
(1)可溶型ヒトIL-6レセプターがマウスに投与された後の、血漿中からの消失が非常に早いこと、
(2)マウスにとって異種タンパク質である可溶型ヒトIL-6レセプターは、マウスに投与された場合に免疫原性を有し、可溶型ヒトIL-6レセプターに対するマウス抗体の産生が起こること、
(3)可溶型ヒトIL-6レセプターに対するマウス抗体の産生が起こると、可溶型ヒトIL-6レセプターの消失が更に早まり、可溶型ヒトIL-6レセプターの血漿中濃度を一定に維持するモデルにおいても、血漿中濃度の低下が起こること、
の3点が示された。
ノーマルマウスにおけるヒト抗体の血漿中滞留性および免疫原性を評価する目的で、以下の試験が実施された。
ノーマルマウス(C57BL/6J mouse、Charles River Japan)の尾静脈に、抗ヒトIL-6レセプター抗体であるFv4-IgG1が1 mg/kgで単回投与された。抗ヒトIL-6レセプター抗体の投与後15分、7時間、1日、2日、3日、4日、7日、14日、21日の時点で採血が行われた。採取された血液を直ちに4℃、15,000 rpmで15分間遠心分離することによって、血漿が得られた。分離された血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存された。
さらに、ヒト抗体が投与された3匹のマウスの全例において、可溶型ヒトIL-6レセプターの定常状態モデル(図38)で見られたような血漿中濃度の低下は認められなかった。すなわち、ヒトIL-6レセプターとは異なり、ヒト抗体に対してはマウス抗体が産生されていないことが示唆された。
マウスに異種タンパク質であるヒト可溶型IL-6レセプターが投与された場合、ヒト可溶型IL-6レセプターは血漿中から短時間で消失し、またヒト可溶型IL-6レセプターに対する免疫応答が確認された。ここで、血漿中からのヒト可溶型IL-6レセプターの消失が早いということは、多くのヒト可溶型IL-6レセプターが短時間のうちに抗原提示細胞に取り込まれ、細胞内でプロセシングを受けた後、ヒト可溶型IL-6レセプターに特異的に応答するT細胞を活性化すると考えられる。その結果として、ヒト可溶型IL-6レセプターに対する免疫応答(つまりヒト可溶型IL-6レセプターに対するマウス抗体の産生)が起こったと考えられる。
(5-1)中性pHにおけるヒトFcRnに対する結合アフィニティーが増大した様々な抗体Fc改変体の作製とその結合活性評価
pH中性域におけるヒトFcRnに対する結合アフィニティーを増大させることを目的として、様々な変異がVH3-IgG1(配列番号:35)に導入され、評価がなされた。作製された重鎖と軽鎖であるL(WT)-CK(配列番号:41)とを各々含む改変体(IgG1-F1からIgG1-F1052)が、参考実施例2に記載された方法に準じて発現および精製された。
抗体とヒトFcRnとの結合が、実施例4に記載される方法に準じて解析された。すなわち、Biacoreを用いた中性条件下(pH7.0)におけるヒトFcRnに対する改変体の結合活性を表27-1~27-32に示した。
実施例5-1で調製した中性条件下でのヒトFcRnへの結合能を付与した重鎖を用いて、中性条件下でのヒトFcRnへの結合能を有するpH依存的ヒトIL-6レセプター結合抗体を作製し、in vivoでの抗原消失効果の検証を行った。具体的には、
VH3-IgG1(配列番号:35)とVL3-CK(配列番号:36)とを含むFv4-IgG1、
VH3-IgG1-F1(配列番号:37)とVL3-CK(配列番号:36)とを含むFv4-IgG1-v2、
VH3-IgG1-F14(配列番号:86)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F14、
VH3-IgG1-F20(配列番号:39)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F20、
VH3-IgG1-F21(配列番号:40)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F21、
VH3-IgG1-F25(配列番号:87)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F25、
VH3-IgG1-F29(配列番号:88)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F29、
VH3-IgG1-F35(配列番号:89)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F35、
VH3-IgG1-F48(配列番号:90)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F48、
VH3-IgG1-F93(配列番号:91)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F93、
VH3-IgG1-F94(配列番号:92)とVL3-CK(配列番号:36)とを含むFv4-IgG1-F94
を、参考実施例2に記載の当業者公知の方法によって発現させて精製した。
ヒトFcRnトランスジェニックマウス(B6.mFcRn-/-.hFcRn Tg 系統276 +/+マウス、Jackson Laboratories、Methods Mol Biol. 2010;602:93-104.)および正常マウス(C57BL/6Jマウス、Charles River Japan)にhsIL-6R(可溶型ヒトIL-6レセプター:参考例3にて調製)を単独投与もしくは可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体を同時投与した後の可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体の生体内における薬物動態を評価した。可溶型ヒトIL-6レセプター溶液(5μg/mL)もしくは可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体の混合溶液(それぞれ5μg/mL、0.1 mg/mL)を尾静脈に10 mL/kgで単回投与した。このとき、可溶型ヒトIL-6レセプターに対して抗ヒトIL-6レセプター抗体は十分量過剰に存在することから、可溶型ヒトIL-6レセプターはほぼ全て抗体に結合していると考えられる。投与15分後、7時間後、1日後、2日後、3日後、4日後、7日後、14日後、21日後、28日後に血液を採取した。採取した血液は直ちに4℃、15,000 rpmで15分間遠心分離し、血漿を得た。分離した血漿は、測定を実施するまで-20℃以下に設定された冷凍庫に保存した。
マウスの血漿中可溶型ヒトIL-6レセプター濃度は電気化学発光法にて測定された。2000、1000、500、250、125、62.5、31.25 pg/mLに調製された可溶型ヒトIL-6レセプター検量線試料および50倍以上希釈されたマウス血漿測定試料を、SULFO-TAG NHS Ester(Meso Scale Discovery)でルテニウム化したMonoclonal Anti-human IL-6R Antibody(R&D)およびBiotinylated Anti-human IL-6 R Antibody (R&D)およびTocilizumabと混合することによって37℃で1晩反応させた。Tocilizumabの終濃度は333μg/mLとなるように調製された。その後、反応液がMA400 PR Streptavidin Plate(Meso Scale Discovery)に分注された。さらに室温で1時間反応させた反応液を洗浄後、Read Buffer T(×4)(Meso Scale Discovery)が分注された。その後ただちにSECTOR PR 400 reader(Meso Scale Discovery)で測定が行われた。可溶型ヒトIL-6レセプター濃度は検量線のレスポンスから解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。
(6-1)ナイーブヒト抗体ファージディスプレイライブラリの作製
ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型として当業者に公知な方法にしたがい、互いに異なるヒト抗体配列のFabドメインを提示する複数のファージからなるヒト抗体ファージディスプレイライブラリが構築された。
構築されたナイーブヒト抗体ファージディスプレイライブラリからの最初の選抜は、抗原(IL-6レセプター)への結合能をもつ抗体断片のみの濃縮、または、Ca濃度依存的な抗原(IL-6レセプター)への結合能を指標とした抗体断片の濃縮によって実施された。Ca濃度依存的な抗原(IL-6レセプター)への結合能を指標として抗体断片の濃縮は、CaイオンをキレートするEDTAを用いてCaイオン存在下でIL-6レセプターと結合させたファージライブラリからファージを溶出することによって実施された。抗原としてビオチン標識されたIL-6レセプターが用いられた。
上記の方法によって得られた大腸菌のシングルコロニーから、常法(Methods Mol. Biol. (2002) 178, 133-145)に習い、ファージ含有培養上清が回収された。
終濃度4%BSAおよび1.2 mMカルシウムイオン濃度となるようにBSAおよびCaCl2が加えられたファージを含有する培養上清が以下の手順でELISAに供された。StreptaWell 96マイクロタイタープレート(Roche)がビオチン標識抗原を含む100μLのPBSにて一晩コートされた。当該プレートの各ウェルをPBSTにて洗浄することによって抗原が除かれた後、当該ウェルが1時間以上250μLの4%BSA-TBSにてブロッキングされた。4%BSA-TBSが除かれた各ウェルに調製された培養上清が加えられた当該プレートを37℃で1時間静置することによって、ファージを提示する抗体を各ウェルに存在する抗原に結合させた。1.2 mM CaCl2/TBSTにて洗浄された各ウェルに、1.2 mM CaCl2/TBSもしくは1 mM EDTA/TBSが加えられ、当該プレートは37℃で30分間静置しインキュベートされた。1.2 mM CaCl2/TBSTにて洗浄された後に、終濃度4%のBSAおよび1.2 mMのイオン化カルシウム濃度としたTBSによって希釈されたHRP結合抗M13抗体(Amersham Pharmacia Biotech)が各ウェルに添加されたプレートを1時間インキュベートさせた。1.2 mM CaCl2/TBSTにて洗浄後、TMB single溶液(ZYMED)が添加された各ウェル中の溶液の発色反応が、硫酸の添加により停止された後、450 nmの吸光度によって当該発色が測定された。
上記のファージELISAの結果、Ca依存的な抗原に対する結合能があると判断される抗体断片を鋳型として特異的なプライマーによって増幅された遺伝子の塩基配列解析が行われた。
ファージELISAの結果、Ca依存的な抗原に対する結合能があると判断されたクローンが、動物細胞発現用プラスミドへ導入された。抗体の発現は以下の方法を用いて行われた。ヒト胎児腎細胞由来FreeStyle 293-F株(Invitrogen)がFreeStyle 293 Expression Medium培地(Invitrogen)に懸濁され、1.33 x 106細胞/mLの細胞密度で6ウェルプレートの各ウェルへ3 mLずつ蒔きこまれた。調製されたプラスミドは、リポフェクション法によって細胞へ導入された。CO2インキュベーター(37度、8%CO2、90 rpm)中で4日間培養が行われる。rProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いて当業者公知の方法を用いて、上記で得られた培養上清から抗体が精製された。分光光度計を用いて精製された抗体溶液の280 nmでの吸光度が測定された。PACE法により算出された吸光係数を用いることによって、得られた測定値から抗体濃度が算出された(Protein Science (1995) 4, 2411-2423)。
参考実施例6で取得された抗体6RL#9-IgG1(重鎖(配列番号:9にIgG1由来の定常領域配列が連結されたもの)、軽鎖(配列番号:93))、および、FH4-IgG1(重鎖(配列番号:94)、軽鎖(配列番号:95))のヒトIL-6レセプターに対する結合活性がCa依存的であるかどうかを判断するため、これらの抗体とヒトIL-6レセプターとの抗原抗体反応の速度論的解析がBiacore T100(GE Healthcare)を用いて行われた。ヒトIL-6レセプターに対するCa依存性の結合活性を有しない対照抗体として、WO2009/125825に記載されているH54/L28-IgG1(重鎖可変領域(配列番号:96)、軽鎖可変領域(配列番号:97))が用いられた。高カルシウムイオン濃度および低カルシウムイオン濃度の条件として、それぞれ2 mMおよび3 μMのカルシウムイオン濃度の溶液中で抗原抗体反応の速度論的解析が行われた。アミンカップリング法でprotein A(Invitrogen)が適当量固定化されたSensor chip CM4(GE Healthcare)上に、目的の抗体がキャプチャーされた。ランニングバッファーには10 mM ACES、150 mM NaCl、0.05% (w/v) Tween20、2 mM CaCl2(pH7.4)または10 mM ACES、150 mM NaCl、0.05% (w/v) Tween20、3 μmol/L CaCl2(pH7.4)の2種類の緩衝液が用いられた。ヒトIL-6レセプターの希釈にもそれぞれのバッファーが使用された。測定は全て37℃で実施された。
この方法により決定された2 mM CaCl2存在下における各抗体とIL-6レセプターとの解離定数KDを表28に示した。
表30にH54/L28-IgG1、FH4-IgG1、6RL#9-IgG1の3種類の抗体の2 mM CaCl2存在下および3μM CaCl2存在下におけるKD値、および、KD値のCa依存性についてまとめた。
次に、抗体へのカルシウムイオンの結合の評価の指標として、示差走査型熱量測定(DSC)による熱変性中間温度(Tm値)が測定された(MicroCal VP-Capillary DSC、MicroCal)。熱変性中間温度(Tm値)は安定性の指標であり、カルシウムイオンの結合によってタンパク質が安定化すると、熱変性中間温度(Tm値)はカルシウムイオンが結合していない場合に比べて高くなる(J. Biol. Chem. (2008) 283, 37, 25140-25149)。抗体溶液中のカルシウムイオン濃度の変化に応じた抗体のTm値の変化を評価することによって、抗体へのカルシウムイオンの結合活性が評価された。精製された抗体が20 mM Tris-HCl、150 mM NaCl、2 mM CaCl2(pH7.4)または20 mM Tris-HCl、150 mM NaCl, 3μM CaCl2(pH7.4)の溶液を外液とする透析(EasySEP、TOMY)処理に供された。透析に用いられた溶液を用いておよそ0.1 mg/mLに調製された抗体溶液を被験物質として、20℃から115℃まで240℃/hrの昇温速度でDSC測定が行われた。得られたDSCの変性曲線にもとづいて算出された各抗体のFabドメインの熱変性中間温度(Tm値)を表31に示した。
(9-1)X線結晶構造解析
参考実施例8に示されたように、6RL#9抗体はカルシウムイオンと結合することが熱変性温度Tm値の測定から示唆された。しかし、6RL#9抗体のどの部位がカルシウムイオンと結合しているか予想できなかったため、X線結晶構造解析の手法を用いることによって、カルシウムイオンが相互作用する6RL#9抗体の配列中の残基が特定された。
X線結晶構造解析に用いるために発現させた6RL#9抗体が精製された。具体的には、6RL#9抗体の重鎖(配列番号:9にIgG1由来の定常領域配列が連結されたもの)と軽鎖(配列番号:93)をそれぞれ発現させることが出来るように調製された動物発現用プラスミドが動物細胞に一過的に導入された。最終細胞密度1 x 106細胞/mLとなるようにFreeStyle 293 Expression Medium培地(Invitrogen)へ懸濁された800 mLのヒト胎児腎細胞由来FreeStyle 293-F株(Invitrogen)に、リポフェクション法により調製されたプラスミドが導入された。プラスミドが導入された細胞はCO2インキュベーター(37℃、8%CO2、90 rpm)中で5日間培養された。rProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いた当業者公知の方法にしたがって、上記のように得られた培養上清から抗体が精製された。分光光度計を用いて精製された抗体溶液の280 nmでの吸光度が測定された。PACE法により算出された吸光係数を用いて測定値から抗体濃度が算出された(Protein Science (1995) 4, 2411-2423)。
分子量分画サイズ10000MWCOの限外ろ過膜 を用いて6RL#9抗体が21 mg/mLまで濃縮された。L-Cystein 4 mM、EDTA 5 mM、20 mMリン酸ナトリウム緩衝液(pH 6.5)を用いて5 mg/mLによって希釈された2.5 mLの当該抗体の試料が調製された。0.125 mgのPapain(Roche Applied Science)を加えて攪拌された当該試料が35℃にて2時間静置された。静置後、プロテアーゼインヒビターカクテルミニ、EDTAフリー(Roche Applied Science)1錠を溶かした10 mLの25 mM MES 緩衝液(pH6)をさらに当該試料に加え、氷中に静置することによって、Papainによるプロテアーゼ反応が停止された。次に、当該試料が、下流に1 mLサイズのProteinA担体カラムHiTrap MabSelect Sure(GE Healthcare)がタンデムにつながれた25 mM MES 緩衝液pH6で平衡化された1 mLサイズの陽イオン交換カラムHiTrap SP HP(GE Healthcare)に添加された。同緩衝液中NaCl濃度を300 mMまで直線的に上げて溶出をおこなうことで6RL#9抗体のFabフラグメントの精製画分が得られた。次に、得られた精製画分が5000MWCOの限外ろ過膜 により0.8 mL程度まで濃縮された。50 mM NaCl を含む100 mM HEPES緩衝液(pH 8)で平衡化されたゲルろ過カラムSuperdex 200 10/300 GL(GE Healthcare)に濃縮液が添加された。結晶化用の精製6RL#9抗体のFabフラグメントが同緩衝液を用いてカラムから溶出された。なお、上記のすべてのカラム操作は6から7.5℃の低温下にて実施された。
予め一般的な条件設定で6RL#9 Fabフラグメントの種結晶が得られた。つぎに5 mM となるようにCaCl2が加えられた精製6RL#9抗体のFabフラグメントが5000MWCOの限外ろ過膜を用いて12 mg/mLに濃縮された。つぎに、ハンギングドロップ蒸気拡散法によって、前記のように濃縮された試料の結晶化が実施された。リザーバー溶液として20-29%のPEG4000を含む100 mM HEPES緩衝液(pH7.5)が用いられた。カバーグラス上で0.8μlのリザーバー溶液および0.8μlの前記濃縮試料の混合液に対して、29% PEG4000および5 mM CaCl2を含む100 mM HEPES緩衝液(pH7.5)中で破砕された前記種結晶が100-10000倍に希釈された希釈系列の溶液0.2μlを加えることによって結晶化ドロップが調製された。当該結晶化ドロップを20℃に2日から3日静置することによって得られた薄い板状の結晶のX線回折データが測定された。
精製6RL#9抗体のFabフラグメントが5000MWCOの限外ろ過膜 を用いて15 mg/mlに濃縮された。つぎに、ハンギングドロップ蒸気拡散法によって、前記のように濃縮された試料の結晶化が実施された。リザーバー溶液として18-25%のPEG4000を含む100 mM HEPES緩衝液(pH7.5)が用いられた。カバーグラス上で0.8μlのリザーバー溶液および0.8μlの前記濃縮試料の混合液に対して、25% PEG4000を含む100 mM HEPES緩衝液(pH7.5)中で破砕されたCa存在下で得られた6RL#9抗体のFabフラグメントの結晶が100-10000倍に希釈された希釈系列の溶液0.2μlを加えることによって結晶化ドロップが調製された。当該結晶化ドロップを20℃に2日から3日静置することによって得られた薄い板状の結晶のX線回折データが測定された。
35% PEG4000および5 mM CaCl2を含む100mM HEPES緩衝液(pH7.5)の溶液に浸された6RL#9抗体のFabフラグメントのCa存在下で得られた単結晶一つを、微小なナイロンループ付きのピンを用いて外液ごとすくいとることによって、当該単結晶が液体窒素中で凍結された。高エネルギー加速器研究機構の放射光施設フォトンファクトリーのビームラインBL-17Aを用いて、前記の凍結結晶のX線回折データが測定された。なお、測定中は常に-178℃の窒素気流中に凍結結晶を置くことで凍結状態が維持された。ビームラインに備え付けられたCCDディテクタQuantum315r(ADSC)を用い、結晶を1°ずつ回転させながらトータル180枚の回折画像が収集された。格子定数の決定、回折斑点の指数付け、および回折データの処理がプログラムXia2(CCP4 Software Suite)、XDS Package(Walfgang Kabsch)ならびにScala(CCP4 Software Suite)によって行われた。最終的に分解能2.2Åまでの回折強度データが得られた。本結晶は、空間群P212121に属し、格子定数a=45.47Å、b=79.86Å、c=116.25Å、α=90°、β=90°、γ=90°であった。
35% PEG4000を含む100 mM HEPES緩衝液(pH7.5)の溶液に浸された6RL#9抗体のFabフラグメントのCa非存在下で得られた単結晶一つを、微小なナイロンループ付きのピンを用いて外液ごとすくいとることによって、当該単結晶が液体窒素中で凍結された。高エネルギー加速器研究機構の放射光施設フォトンファクトリーのビームラインBL-5Aを用いて、前記の凍結結晶のX線回折データが測定された。なお、測定中は常に-178℃の窒素気流中に凍結結晶を置くことで凍結状態が維持された。ビームラインに備え付けられたCCDディテクタQuantum210r(ADSC)を用い、結晶を1°ずつ回転させながらトータル180枚の回折画像が収集された。格子定数の決定、回折斑点の指数付け、および回折データの処理がプログラムXia2(CCP4 Software Suite)、XDS Package(Walfgang Kabsch)ならびにScala(CCP4 Software Suite)によって行われた。最終的に分解能2.3Åまでの回折強度データが得られた。本結晶は、空間群P212121に属し、格子定数a=45.40Å、b=79.63Å、c=116.07Å、α=90°、β=90°、γ=90°であり、Ca存在下の結晶と同型であった。
プログラムPhaser(CCP4 Software Suite)を用いた分子置換法によって、6RL#9抗体のFabフラグメントのCa存在下での結晶の構造が決定された。得られた結晶格子の大きさと6RL#9抗体のFabフラグメントの分子量から、非対称単位中の分子数が一個であると予想された。一次配列上の相同性をもとにPDB code: 1ZA6の構造座標から取り出されたA鎖112-220番およびB鎖116-218番のアミノ酸残基部分が、CLおよびCH1領域の探索用モデル分子とされた。次にPDB code: 1ZA6の構造座標から取り出されたB鎖1-115番のアミノ酸残基部分が、VH領域の探索用モデル分子とされた。最後にPDB code 2A9Mの構造座標から取り出された軽鎖3-147番のアミノ酸残基が、VL領域の探索用モデル分子とされた。この順番にしたがい各探索用モデル分子の結晶格子内での向きと位置を回転関数および並進関数から決定することによって、6RL#9抗体のFabフラグメントの初期構造モデルが得られた。当該初期構造モデルに対してVH、VL、CH1、CLの各ドメインを動かす剛体精密化をおこなうことにより、25-3.0Åの反射データに対する結晶学的信頼度因子R値は46.9%、Free R値は48.6%となった。さらにプログラムRefmac5(CCP4 Software Suite)を用いた構造精密化と、実験的に決定された構造因子Foとモデルから計算された構造因子Fcおよび位相を用い計算された2Fo-Fc、Fo-Fcを係数とする電子密度マップを参照しながらモデル修正を繰り返しプログラムCoot(Paul Emsley)上でおこなうことによってモデルの精密化がおこなわれた。最後に2Fo-Fc、Fo-Fcを係数とする電子密度マップをもとにCaイオンおよび水分子をモデルに組み込むことによって、プログラムRefmac5(CCP4 Software Suite)を用いて精密化がおこなわれた。分解能25-2.2Åの21020個の反射データを用いることによって、最終的に3440原子のモデルに対する結晶学的信頼度因子R値は20.0%、Free R値は27.9%となった。
6RL#9抗体のFabフラグメントのCa非存在下での結晶の構造は、同型であるCa存在下結晶の構造を使って決定された。6RL#9抗体のFabフラグメントのCa存在下での結晶の構造座標から水分子とCaイオン分子がのぞかれ、VH、VL、CH1、CLの各ドメインを動かす剛体精密化がおこなわれた。25-3.0Åの反射データに対する結晶学的信頼度因子R値は30.3%、Free R値は31.7%となった。さらにプログラムRefmac5(CCP4 Software Suite)を用いた構造精密化と、実験的に決定された構造因子Foとモデルから計算された構造因子Fcおよび位相を用い計算された2Fo-Fc、Fo-Fcを係数とする電子密度マップを参照しながらモデル修正を繰り返しプログラムCoot(Paul Emsley)上でおこなうことによってモデルの精密化がおこなわれた。最後に2Fo-Fc、Fo-Fcを係数とする電子密度マップをもとに水分子をモデルに組み込むことによって、プログラムRefmac5(CCP4 Software Suite)を用いて精密化がおこなわれた。分解能25-2.3Åの18357個の反射データを用いることによって、最終的に3351原子のモデルに対する結晶学的信頼度因子R値は20.9%、Free R値は27.7%となった。
6RL#9抗体のFabフラグメントのCa存在下での結晶およびCa非存在下での結晶の構造を比較すると、重鎖CDR3に大きな変化がみられた。X線結晶構造解析で決定された6RL#9抗体のFabフラグメントの重鎖CDR3の構造を図41に示した。具体的には、Ca存在下での6RL#9抗体のFabフラグメントの結晶では、重鎖CDR3ループ部分の中心部分にカルシウムイオンが存在していた。カルシウムイオンは、重鎖CDR3の95位、96位および100a番目位(Kabatナンバリング)と相互作用していると考えられた。Ca存在下では、抗原との結合に重要である重鎖CDR3ループがカルシウムと結合することによって安定化し、抗原との結合に最適な構造となっていることが考えられた。抗体の重鎖CDR3にカルシウムが結合する例は今までに報告されておらず、抗体の重鎖CDR3にカルシウムが結合した構造は新規な構造である。
6RL#9抗体のFabフラグメントの構造から明らかになった重鎖CDR3に存在するカルシウム結合モチーフも、Caライブラリのデザインの新たな要素となりうる。たとえば6RL#9抗体の重鎖CDR3を含み、軽鎖を含むそれ以外のCDRにフレキシブル残基を含むライブラリが考えられる。
(10-1)ナイーブヒト抗体ファージディスプレイライブラリの作製
ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型として当業者に公知な方法にしたがい、互いに異なるヒト抗体配列のFabドメインを提示する複数のファージからなるヒト抗体ファージディスプレイライブラリが構築された。
構築されたナイーブヒト抗体ファージディスプレイライブラリからの最初の選抜は、抗原(IL-6)への結合能をもつ抗体断片のみの濃縮によって実施された。抗原としてビオチン標識されたIL-6が用いられた。
構築されたファージディスプレイ用ファージミドを保持した大腸菌からファージ産生が行われた。ファージ産生が行われた大腸菌の培養液に2.5 M NaCl/10%PEGを添加することによって沈殿させたファージの集団をTBSにて希釈することによってファージライブラリ液が得られた。次に、ファージライブラリ液に終濃度4%BSAおよび1.2mMカルシウムイオン濃度となるようにBSAおよびCaCl2が添加された。パンニング方法として、一般的な方法である磁気ビーズに固定化した抗原を用いたパンニング方法が参照された(J. Immunol. Methods. (2008) 332 (1-2), 2-9、J. Immunol. Methods. (2001) 247 (1-2), 191-203、Biotechnol. Prog. (2002) 18 (2) 212-20、Mol. Cell Proteomics (2003) 2 (2), 61-9)。磁気ビーズとして、NeutrAvidin coated beads(Sera-Mag SpeedBeads NeutrAvidin-coated)もしくはStreptavidin coated beads(Dynabeads M-280 Streptavidin)が用いられた。
上記の方法によって得られた大腸菌のシングルコロニーから、常法(Methods Mol. Biol. (2002) 178, 133-145)に習い、ファージ含有培養上清が回収された。
終濃度4%BSAおよび1.2 mMカルシウムイオン濃度となるようにBSAおよびCaCl2が加えられたファージを含有する培養上清が以下の手順でELISAに供された。StreptaWell 96マイクロタイタープレート(Roche)がビオチン標識抗原を含む100μLのPBSにて一晩コートされた。当該プレートの各ウェルをPBSTにて洗浄することによって抗原が除かれた後、当該ウェルが1時間以上250μLの4%BSA-TBSにてブロッキングされた。4%BSA-TBSが除かれた各ウェルに調製された培養上清が加えられた当該プレートを37℃で1時間静置することによって、ファージを提示する抗体を各ウェルに存在する抗原に結合させた。1.2 mM CaCl2/TBSTにて洗浄された各ウェルに、1.2 mM CaCl2/TBSもしくは1 mM EDTA/TBSが加えられ、当該プレートは37℃で30分間静置しインキュベートされた。1.2 mM CaCl2/TBSTにて洗浄された後に、終濃度4%のBSAおよび1.2 mMのイオン化カルシウム濃度としたTBSによって希釈されたHRP結合抗M13抗体(Amersham Pharmacia Biotech)が各ウェルに添加されたプレートを1時間インキュベートさせた。1.2 mM CaCl2/TBSTにて洗浄後、TMB single溶液(ZYMED)が添加された各ウェル中の溶液の発色反応が、硫酸の添加により停止された後、450 nmの吸光度によって当該発色が測定された。
ファージELISAの結果、Ca依存的な抗原に対する結合能があると判断されたクローン6KC4-1#85が、動物細胞発現用プラスミドへ導入された。抗体の発現は以下の方法を用いて行われた。ヒト胎児腎細胞由来FreeStyle 293-F株(Invitrogen)がFreeStyle 293 Expression Medium培地(Invitrogen)に懸濁され、1.33 x 106細胞/mLの細胞密度で6ウェルプレートの各ウェルへ3 mLずつ蒔きこまれた。調製されたプラスミドは、リポフェクション法によって細胞へ導入された。CO2インキュベーター(37度、8%CO2、90 rpm)中で4日間培養が行われた。rProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いて当業者公知の方法を用いて、上記で得られた培養上清から抗体が精製された。分光光度計を用いて精製された抗体溶液の280 nmでの吸光度が測定された。PACE法により算出された吸光係数を用いることによって、得られた測定値から抗体濃度が算出された(Protein Science (1995) 4, 2411-2423)。
(11-1)6KC4-1#85抗体のカルシウムイオン結合評価
ヒト抗体ライブラリから取得されたカルシウム依存的抗原結合抗体6KC4-1#85抗体がカルシウムと結合するか評価された。イオン化カルシウム濃度が異なる条件で、測定されるTm値が変動するか否かが参考実施例6に記載された方法で評価された。
参考実施例11の(11-1)で6KC4-1#85抗体にカルシウムイオンと結合することが示されたが、6KC4-1#85はhVk5-2配列の検討から明らかになったカルシウム結合モチーフを持たない。そこで、カルシウムイオンが6KC4-1#85抗体のどの残基とカルシウムイオンが結合しているか同定するために、6KC4-1#85抗体のCDRに存在するAsp(D)残基をカルシウムイオンの結合もしくはキレートに関与できないAla(A)残基に置換した改変重鎖(6_H1-11(配列番号:102)、6_H1-12(配列番号:103)、6_H1-13(配列番号:104)、6_H1-14(配列番号:105)、6_H1-15(配列番号:106))、または改変軽鎖(6_L1-5(配列番号:107)および6_L1-6(配列番号:108))が作製された。改変抗体遺伝子を含む発現ベクターが導入された動物細胞の培養液から、改変抗体が参考実施例6に記載された方法にしたがって精製された。精製された改変抗体のカルシウム結合が、参考実施例6に記載された方法にしたがって測定された。測定された結果を表33に示した。表33に示されているように、6KC4-1#85抗体の重鎖CDR3の95位または101位(Kabatナンバリング)をAla残基に置換することによって6KC4-1#85抗体のカルシウム結合能が失われることから、この残基がカルシウムとの結合に重要であると考えられる。6KC4-1#85抗体の改変抗体のカルシウム結合性から明らかになった6KC4-1#85抗体の重鎖CDR3のループ付け根付近に存在するカルシウム結合モチーフも、参考実施例9で記載されるようなCaライブラリのデザインの新たな要素となりうる。すなわち、参考実施例20等で具体例が挙げられた軽鎖可変領域にカルシウム結合モチーフが導入されたライブラリのほかに、たとえば6KC4-1#85抗体の重鎖CDR3に存在するカルシウム結合モチーフを含み、それ以外のアミノ酸残基にフレキシブル残基を含むライブラリが考えられる。
(12-1)ノーマルマウスを用いたin vivo試験
ノーマルマウス(C57BL/6J mouse、Charles River Japan)にhsIL-6R(可溶型ヒトIL-6レセプター:参考実施例3にて作製)を単独投与もしくは可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体を同時投与した後の可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体の体内動態が評価された。可溶型ヒトIL-6レセプター溶液(5μg/mL)、もしくは、可溶型ヒトIL-6レセプターと抗ヒトIL-6レセプター抗体の混合溶液が尾静脈に10 mL/kgで単回投与された。抗ヒトIL-6レセプター抗体としては、前記のH54/L28-IgG1、6RL#9-IgG1、FH4-IgG1が使用された。
マウス血漿中の抗ヒトIL-6レセプター抗体濃度はELISA法にて測定された。まずAnti-Human IgG(γ-chain specific) F(ab')2 Fragment of Antibody(SIGMA)をNunc-Immuno Plate, MaxiSoup(Nalge nunc International)に分注し、4℃で1晩静置することによってAnti-Human IgG固相化プレートが作成された。血漿中濃度として0.64、0.32、0.16、0.08、0.04、0.02、0.01μg/mLの検量線試料および100倍以上希釈されたマウス血漿測定試料のそれぞれが分注されたAnti-Human IgG固相化プレートが25℃で1時間インキュベーションされた。その後Biotinylated Anti-human IL-6 R Antibody(R&D)を25℃で1時間反応させた後にStreptavidin-PolyHRP80(Stereospecific Detection Technologies)を25℃で0.5時間反応させた。TMB One Component HRP Microwell Substrate(BioFX Laboratories)を基質として用いて発色反応が行われた。1N-Sulfuric acid(Showa Chemical)によって発色反応が停止された後、マイクロプレートリーダーを用いて発色液の450 nmにおける吸光度が測定された。解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて、マウス血漿中濃度が検量線の吸光度を基準として算出された。この方法で測定された静脈内投与後のノーマルマウスにおけるH54/L28-IgG1、6RL#9-IgG1、FH4-IgG1の血漿中の抗体濃度の推移を図42に示した。
マウスの血漿中可溶型ヒトIL-6レセプター濃度は電気化学発光法にて測定された。2000、1000、500、250、125、62.5、31.25 pg/mLに調整された可溶型ヒトIL-6レセプター検量線試料および50倍以上希釈されたマウス血漿測定試料と、SULFO-TAG NHS Ester(Meso Scale Discovery)でルテニウム化したMonoclonal Anti-human IL-6R Antibody(R&D)およびBiotinylated Anti-human IL-6 R Antibody (R&D)およびトシリズマブ(重鎖配列番号:109、軽鎖配列番号:83)溶液との混合液を4℃で1晩反応させた。サンプル中のFree Ca濃度を低下させ、サンプル中のほぼ全ての可溶型ヒトIL-6レセプターが6RL#9-IgG1もしくはFH4-IgG1から解離し、添加したトシリズマブと結合した状態とするために、その際のAssay bufferには10 mM EDTAが含まれていた。その後、当該反応液がMA400 PR Streptavidin Plate(Meso Scale Discovery)に分注された。さらに25℃で1時間反応させたプレートの各ウェルが洗浄された後、各ウェルにRead Buffer T(×4)(Meso Scale Discovery)が分注された。ただちに反応液はSECTOR PR 400 reader(Meso Scale Discovery)を用いて測定された。可溶型ヒトIL-6レセプター濃度は検量線のレスポンスから解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。前記の方法で測定された静脈内投与後のノーマルマウスにおける血漿中の可溶型ヒトIL-6レセプターの濃度推移を図43に示した。
(13-1)IgG抗体のFcRnへの結合に関して
IgG抗体はFcRnに結合することで長い血漿中滞留性を有する。IgGとFcRnの結合は酸性条件下(pH6.0)においてのみ認められ、中性条件下(pH7.4)においてその結合はほとんど認められない。IgG抗体は非特異的に細胞に取り込まれるが、エンドソーム内の酸性条件下においてエンドソーム内のFcRnに結合することによって細胞表面上に戻り、血漿中の中性条件下においてFcRnから解離する。IgGのFc領域に変異を導入し、酸性条件下におけるFcRnへの結合を失わせると、エンドソーム内から血漿中にリサイクルされなくなるため、抗体の血漿中滞留性は著しく損なわれる。
IgG抗体の血漿中滞留性を改善させる方法として、酸性条件下におけるFcRnへの結合を向上させる方法が報告されている。IgG抗体のFc領域にアミノ酸置換を導入し、酸性条件下のFcRnへの結合を向上させることによって、エンドソーム内から血漿中へのIgG抗体のリサイクル効率が上昇する。その結果、IgG抗体の血漿中の滞留性が改善する。アミノ酸の置換を導入する際に重要と考えられているのは、中性条件下におけるFcRnへの結合を高めないことであった。中性条件下においてFcRnに結合するIgG抗体は、エンドソーム内の酸性条件下においてFcRnに結合することによって細胞表面上に戻ることはできても、中性条件下の血漿中においてIgG抗体がFcRnから解離せず血漿中にリサイクルされないために、逆にIgG抗体の血漿中滞留性は損なわれると考えられていた。
Ca依存的に抗原に結合する抗体は、可溶型の抗原の消失を加速させ、ひとつの抗体分子が複数回繰り返し可溶型の抗原に結合する効果を有することから、極めて有用である。この抗原消失加速効果をさらに向上させる方法として、中性条件下(pH7.4)におけるFcRnに対する結合を増強する方法が検証された。
カルシウム依存的抗原結合能を有するFH4-IgG1、6RL#9-IgG1、および対照として用いられたカルシウム依存的抗原結合能を有しないH54/L28-IgG1のFc領域にアミノ酸変異を導入することによって、中性条件下(pH7.4)におけるFcRnに対する結合を有する改変体が作製された。アミノ酸の変異の導入はPCRを用いた当業者公知の方法を用いて行われた。具体的には、IgG1の重鎖定常領域に対して、EUナンバリングで表される434位のアミノ酸であるAsnがTrpに置換されたFH4-N434W(重鎖配列番号:110、軽鎖配列番号:95)と6RL#9-N434W(重鎖配列番号:111、軽鎖配列番号:93)とH54/L28-N434W(重鎖配列番号:112、軽鎖配列番号:97)が作製された。QuikChange Site-Directed Mutagenesis Kit(Stratagene)を用いて、添付説明書記載の方法を用いてそのアミノ酸が置換された変異体をコードするポリヌクレオチドが挿入された動物細胞発現ベクターが作製された。抗体の発現、精製、濃度測定は参考実施例6に記載された方法に準じて実施された。
(14-1)ノーマルマウスを用いたin vivo試験
ノーマルマウス(C57BL/6J mouse、Charles River Japan)にhsIL-6R(可溶型ヒトIL-6レセプター:参考実施例3にて作製)が単独で投与された、もしくは可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体が同時投与された後の可溶型ヒトIL-6レセプターおよび抗ヒトIL-6レセプター抗体の体内動態が評価された。可溶型ヒトIL-6レセプター溶液(5μg/mL)、もしくは、可溶型ヒトIL-6レセプターと抗ヒトIL-6レセプター抗体の混合溶液が尾静脈に10 mL/kgで単回投与された。抗ヒトIL-6レセプター抗体として、上述のH54/L28-N434W、6RL#9-N434W、FH4-N434Wが使用された。
マウス血漿中の抗ヒトIL-6レセプター抗体濃度は参考実施例12と同様のELISA法によって測定された。この方法で測定された静脈内投与後のノーマルマウスにおけるH54/L28-N434W、6RL#9-N434W、FH4-N434W抗体の血漿中の抗体濃度の推移を図44に示した。
マウスの血漿中可溶型ヒトIL-6レセプター濃度が電気化学発光法にて測定された。2000、1000、500、250、125、62.5、31.25 pg/mLに調製された可溶型ヒトIL-6レセプター検量線試料および50倍以上希釈されたマウス血漿測定試料と、SULFO-TAG NHS Ester(Meso Scale Discovery)でルテニウム化したMonoclonal Anti-human IL-6R Antibody(R&D)およびBiotinylated Anti-human IL-6 R Antibody (R&D)との混合液を4℃で1晩反応させた。サンプル中のFree Ca濃度を低下させ、サンプル中のほぼ全ての可溶型ヒトIL-6レセプターが6RL#9-N434WもしくはFH4-N434Wから解離し、free体として存在する状態とするために、その際のAssay bufferには10 mM EDTAが含まれていた。その後、当該反応液がMA400 PR Streptavidin Plate(Meso Scale Discovery)に分注された。さらに25℃で1時間反応させたプレートの各ウェルが洗浄された後、各ウェルにRead Buffer T(×4)(Meso Scale Discovery)が分注された。ただちに反応液はSECTOR PR 400 reader(Meso Scale Discovery)を用いて測定された。可溶型ヒトIL-6レセプター濃度は検量線のレスポンスから解析ソフトウェアSOFTmax PRO(Molecular Devices)を用いて算出された。前記の方法で測定された静脈内投与後のノーマルマウスにおける血漿中の可溶型ヒトIL-6レセプターの濃度推移を図45に示した。
(15-1)カルシウム依存的に抗原に結合する抗体
カルシウム依存的に抗原に結合する抗体(カルシウム依存的抗原結合抗体)はカルシウムの濃度によって抗原との相互作用が変化する抗体である。カルシウム依存的抗原結合抗体は、カルシウムイオンを介して抗原に結合すると考えられるため、抗原側のエピトープを形成するアミノ酸は、カルシウムイオンをキレートすることが可能な負電荷のアミノ酸あるいは水素結合アクセプターとなりうるアミノ酸である。こうしたエピトープを形成するアミノ酸の性質から、ヒスチジンを導入することにより作製されるpH依存的に抗原に結合する結合分子以外のエピトープをターゲットすることが可能となる。カルシウム依存的およびpH依存的に抗原に結合する性質を併せ持つ抗原結合分子を用いることで、幅広い性質を有する多様なエピトープを個々にターゲットすることが可能な抗原結合分子を作製することが可能となると考えられる。そこで、カルシウムが結合するモチーフを含む分子の集合(Caライブラリ)を構築し、この分子の集団から抗原結合分子を取得すれば、カルシウム依存的抗原結合抗体が効率的に得られると考えられる。
カルシウムが結合するモチーフを含む分子の集合の例として、当該分子が抗体である例が考えられる。言い換えればカルシウムが結合するモチーフを含む抗体ライブラリがCaライブラリである場合が考えられる。
ヒト生殖細胞系列配列を含む抗体でカルシウムイオンが結合するものはこれまで報告されていない。そこで、ヒト生殖細胞系列配列を含む抗体がカルシウムイオンと結合するか否かを判定するため、Human Fetal Spreen Poly RNA(Clontech)から調製されたcDNAを鋳型としてヒト生殖細胞系列配列を含む抗体の生殖細胞系列の配列がクローニングされた。クローニングされたDNA断片は動物細胞発現ベクターに挿入された。得られた発現ベクターの塩基配列を当業者公知の方法で決定し、その配列番号を表34に示した。配列番号:5(Vk1)、配列番号:6(Vk2)、配列番号:7(Vk3)、配列番号:8(Vk4)ならびに配列番号:4(Vk5)をコードするポリヌクレオチドが、PCR法によって天然型Kappa鎖の定常領域(配列番号:100)をコードするポリヌクレオチドと連結されたDNA断片が、動物細胞発現用ベクターに組み込まれた。また、配列番号:113(Vk1)、配列番号:114(Vk2)、配列番号:115(Vk3)、配列番号:116(Vk4)ならびに配列番号:117(Vk5)をコードするポリヌクレオチドが、PCR法によって配列番号:11で表されるIgG1のC末端2アミノ酸が欠失したポリペプチドをコードするポリヌクレオチドと連結されたDNA断片が、動物細胞発現用ベクターに組み込まれた。作製された改変体の配列は当業者公知の方法で確認された。
取得された5種類のヒト生殖細胞系列配列を含むDNA断片が挿入された動物細胞発現ベクターが動物細胞へ導入された。抗体の発現は以下の方法を用いて行われた。ヒト胎児腎細胞由来FreeStyle 293-F株(Invitrogen)がFreeStyle 293 Expression Medium培地(Invitrogen)に懸濁され、1.33 x 106細胞/mLの細胞密度で6ウェルプレートの各ウェルへ3 mLずつ蒔きこまれた。調製されたプラスミドは、リポフェクション法によって細胞へ導入された。CO2インキュベーター(37度、8%CO2、90 rpm)中で4日間培養が行われた。rProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いて当業者公知の方法を用いて、上記で得られた培養上清から抗体が精製された。分光光度計を用いて精製された抗体溶液の280 nmでの吸光度が測定された。PACE法により算出された吸光係数を用いることによって、得られた測定値から抗体濃度が算出された(Protein Science (1995) 4, 2411-2423)。
精製された抗体のカルシウムイオン結合活性が評価された。抗体へのカルシウムイオンの結合の評価の指標として、示差走査型熱量測定(DSC)による熱変性中間温度(Tm値)が測定された(MicroCal VP-Capillary DSC、MicroCal)。熱変性中間温度(Tm値)は安定性の指標であり、カルシウムイオンの結合によってタンパク質が安定化すると、熱変性中間温度(Tm値)はカルシウムイオンが結合していない場合に比べて高くなる(J. Biol. Chem. (2008) 283, 37, 25140-25149)。抗体溶液中のカルシウムイオン濃度の変化に応じた抗体のTm値の変化を評価することによって、抗体へのカルシウムイオンの結合活性が評価された。精製された抗体が20 mM Tris-HCl、150 mM NaCl、2 mM CaCl2(pH7.4)または20 mM Tris-HCl、150 mM NaCl, 3μM CaCl2(pH7.4)の溶液を外液とする透析(EasySEP、TOMY)処理に供された。透析に用いられた溶液を用いておよそ0.1 mg/mLに調製された抗体溶液を被験物質として、20℃から115℃まで240℃/hrの昇温速度でDSC測定が行われた。得られたDSCの変性曲線にもとづいて算出された各抗体のFabドメインの熱変性中間温度(Tm値)を表35に示した。
(16-1)hVk5配列
Kabatデータベース中には、hVk5配列としてhVk5-2配列のみが登録されている。以下では、hVk5とhVk5-2は同義で扱われる。WO2010/136598では、hVk5-2配列の生殖細胞系列配列中の存在比は0.4%と記載されている。他の報告でもhVk5-2配列の生殖細胞系列配列中の存在比は0~0.06%と述べられている(J. Mol. Biol. (2000) 296, 57-86、Proc. Natl. Acad. Sci. (2009) 106, 48, 20216-20221)。上記のように、hVk5-2配列は、生殖細胞系列配列中で出現頻度が低い配列であるため、ヒト生殖細胞系列配列で構成される抗体ライブラリやヒト抗体を発現するマウスへの免疫によって取得されたB細胞から、カルシウムと結合する抗体を取得することは非効率であると考えられた。そこで、ヒトhVk5-2配列を含むCaライブラリを設計する可能性が考えられるが、hVk5-2配列の物性は報告されておらず、その可能性の実現は未知であった。
hVk5-2配列は20位(Kabatナンバリング)のアミノ酸にN型糖鎖が付加する配列を有する。タンパク質に付加する糖鎖にはヘテロジェニティーが存在するため、物質の均一性の観点から糖鎖は付加されないほうが望ましい。そこで、20位(Kabatナンバリング)のAsn(N)残基がThr(T)残基に置換された改変体hVk5-2_L65(配列番号:118)が作製された。アミノ酸の置換はQuikChange Site-Directed Mutagenesis Kit(Stratagene)を用いる当業者公知の方法で行われた。改変体hVk5-2_L65をコードするDNAが動物発現用ベクターに組み込まれた。作製された改変体hVk5-2_L65のDNAが組み込まれた動物発現用ベクターは、重鎖としてCIM_H(配列番号:117)が発現するように組み込まれた動物発現用のベクターと、参考実施例6で記載した方法で共に動物細胞中に導入された。導入された動物細胞中で発現したhVk5-2_L65 およびCIM_Hを含む抗体が、参考実施例6で記載した方法で精製された。
取得された改変配列hVk5-2_L65を含む抗体が、改変に供されたもとのhVk5-2配列を含む抗体よりも、そのヘテロジェニティーが減少しているか否かが、イオン交換クロマトグラフィーを用いて分析された。イオン交換クロマトグラフィーの方法を表36に示した。分析の結果、図46に示したように糖鎖付加部位が改変されたhVk5-2_L65は、元のhVk5-2配列よりもヘテロジェニティーが減少していることが示された。
(17-1)hVk5-2配列のCDR配列を含む改変抗体の作製、発現および精製
hVk5-2_L65配列はヒトVk5-2配列のフレームワークに存在する糖鎖付加部位のアミノ酸が改変された配列である。参考実施例16で糖鎖付加部位を改変してもカルシウムイオンが結合することが示されたが、フレームワーク配列は生殖細胞系列の配列であることが免疫原性の観点から一般的には望ましい。そこで、抗体のフレームワーク配列を、当該抗体に対するカルシウムイオンの結合活性を維持しながら、糖鎖が付加されない生殖細胞系列配列のフレームワーク配列に置換することが可能であるか否かが検討された。
hVk5-2配列以外の生殖細胞系列配列(hVk1、hVk2、hVk3、hVk4)のフレームワーク配列およびhVk5-2配列のCDR配列を含む改変抗体に、カルシウムイオンが結合するか否かが実施例6に記載された方法によって評価された。評価された結果を表38に示した。各改変抗体のFabドメインのTm値は、抗体溶液中のカルシウムイオン濃度の変化によって変動することが示された。よって、hVk5-2配列のフレームワーク配列以外のフレームワーク配列を含む抗体もカルシウムイオンと結合することが示された。
(18-1)hVk5-2配列のCDR配列中の変異部位の設計
参考実施例17に記載されているように、hVk5-2配列のCDR部分が他の生殖細胞系列のフレームワーク配列に導入された軽鎖を含む抗体もカルシウムイオンと結合することが示された。この結果からhVk5-2に存在するカルシウムイオン結合部位はCDRの中に存在することが示唆された。カルシウムイオンと結合する、すなわち、カルシウムイオンをキレートするアミノ酸として、負電荷のアミノ酸もしくは水素結合のアクセプターとなりうるアミノ酸が挙げられる。そこで、hVk5-2配列のCDR配列中に存在するAsp(D)残基またはGlu(E)残基がAla(A)残基に置換された変異hVk5-2配列を含む抗体がカルシウムイオンと結合するか否かが評価された。
hVk5-2配列のCDR配列中に存在するAspおよび/ またはGlu残基がAla残基に改変された軽鎖を含む抗体分子が作製された。参考実施例16で記載されるように、糖鎖が付加されない改変体hVk5-2_L65はカルシウムイオン結合を維持していたことから、カルシウムイオン結合性という観点ではhVk5-2配列と同等と考えられる。本実施例ではhVk5-2_L65をテンプレート配列としてアミノ酸置換が行われた。作製された改変体を表39に示した。アミノ酸の置換はQuikChange Site-Directed Mutagenesis Kit(Stratagene)、PCRまたはIn fusion Advantage PCR cloning kit(TAKARA)等の当業者公知の方法によって行われ、アミノ酸が置換された改変軽鎖の発現ベクターが構築された。
得られた精製抗体がカルシウムイオンと結合するか否かが参考実施例15に記載された方法によって判定された。その結果を表40に示した。hVk5-2配列のCDR配列中に存在するAspまたはGlu残基をカルシウムイオンの結合もしくはキレートに関与できないAla残基に置換することによって、抗体溶液のカルシウムイオン濃度の変化によってそのFabドメインのTm値が変動しない抗体が存在した。Ala置換によってTm値が変動しない置換部位(32位および92位(Kabatナンバリング))はカルシウムイオンと抗体の結合に特に重要であることが示された。
(19-1)カルシウムイオン結合モチーフを有するhVk1配列の作製ならびに抗体の発現および精製
参考実施例18で記載されたAla置換体のカルシウムの結合活性の結果から、hVk5-2配列のCDR配列の中でAspやGlu残基がカルシウム結合に重要であることが示された。そこで、30位、31位、32位、50位および92位(Kabatナンバリング)の残基のみを他の生殖細胞系列の可変領域配列に導入してもカルシウムイオンと結合できるか否かが評価された。具体的には、ヒト生殖細胞系配列であるhVk1配列の30位、31位、32位、50位および92位(Kabatナンバリング)の残基がhVk5-2配列の30位、31位、32位、50位および92位(Kabatナンバリング)の残基に置換された改変体LfVk1_Ca(配列番号:131)が作製された。すなわち、hVk5-2配列中のこれらの5残基のみが導入されたhVk1配列を含む抗体がカルシウムと結合できるか否かが判定された。改変体の作製は参考実施例17と同様に行われた。得られた軽鎖改変体LfVk1_Caおよび軽鎖hVk1配列を含むLfVk1(配列番号:132)を、重鎖CIM_H(配列番号:117)と共に発現させた。抗体の発現および精製は参考実施例18と同様の方法で実施された。
上記のように得られた精製抗体がカルシウムイオンと結合するか否かが参考実施例15に記載された方法で判定された。その結果を表41に示した。hVk1配列を有するLfVk1を含む抗体のFabドメインのTm値は抗体溶液中のカルシウムの濃度の変化によっては変動しない一方で、LfVk1_Caを含む抗体配列の、Tm値は、抗体溶液中のカルシウムの濃度の変化によって1℃以上変化したことから、LfVk1_Caを含む抗体がカルシウムと結合することが示された。上記の結果から、カルシウムイオンの結合には、hVk5-2のCDR配列がすべて必要ではなく、LfVk1_Ca配列を構築する際に導入された残基のみでも十分であることが示された。
カルシウム結合モチーフとして、例えばhVk5-2配列やそのCDR配列、さらに残基が絞られた30位、31位、32位、50位、92位(Kabatナンバリング)が好適に挙げられる。他にも、カルシウムと結合するタンパク質が有するEFハンドモチーフ(カルモジュリンなど)やCタイプレクチン(ASGPRなど)もカルシウム結合モチーフに該当する。
ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型としてPCR法により抗体重鎖可変領域の遺伝子ライブラリが増幅された。抗体軽鎖可変領域部分については、参考実施例20に記載されるように、カルシウム結合モチーフを維持しカルシウム濃度依存的に抗原に対して結合可能な抗体の出現頻度を高めた抗体可変領域軽鎖部分が設計された。また、フレキシブル残基のうちカルシウム結合モチーフが導入された残基以外のアミノ酸残基として、天然ヒト抗体でのアミノ酸出現頻度の情報((KABAT, E.A. ET AL.: 'Sequences of proteins of immunological interest', vol. 91, 1991, NIH PUBLICATION)が参考にされ、天然ヒト抗体の配列中で出現頻度の高いアミノ酸を均等に分布させた抗体軽鎖可変領域のライブラリが設計された。このように作製された抗体重鎖可変領域の遺伝子ライブラリと抗体軽鎖可変領域の遺伝子ライブラリとの組合せがファージミドベクターへ挿入され、ヒト抗体配列からなるFabドメインを提示するヒト抗体ファージディスプレイライブラリ(Methods Mol Biol. (2002) 178, 87-100)が構築された。上記ライブラリの構築に際しては、ファージミドのFabとファージpIIIタンパク質をつなぐリンカー部分、および、ヘルパーファージpIIIタンパク遺伝子のN2ドメインとCTドメインの間にトリプシン切断配列が挿入されたファージディスプレイライブラリの配列が使用された。抗体遺伝子ライブラリが導入された大腸菌から単離された抗体遺伝子部分の配列が確認され、290種類のクローンの配列情報が得られた。設計されたアミノ酸分布と、確認された配列中のアミノ酸の分布が、図52に示されている。設計されたアミノ酸分布に対応する多様な配列を含むライブラリが構築された。
(22-1)Caライブラリに含まれる分子のカルシウムイオン結合活性
参考実施例14に示されているように、カルシウムイオンと結合することが示されたhVk5-2配列は生殖細胞系列配列中で出現頻度が低い配列であるため、ヒト生殖細胞系列配列で構成される抗体ライブラリやヒト抗体を発現するマウスへの免疫によって取得されたB細胞から、カルシウムと結合する抗体を取得することは非効率であると考えられた。そこで、参考実施例21でCaライブラリが構築された。構築されたCaライブラリにカルシウム結合を示すクローンが存在するか評価された。
Caライブラリに含まれるクローンが、動物細胞発現用プラスミドへ導入された。抗体の発現は以下の方法を用いて行われた。ヒト胎児腎細胞由来FreeStyle 293-F株(Invitrogen)がFreeStyle 293 Expression Medium培地(Invitrogen)に懸濁され、1.33 x 106細胞/mLの細胞密度で6ウェルプレートの各ウェルへ3 mLずつ蒔きこまれた。調製されたプラスミドは、リポフェクション法によって細胞へ導入された。CO2インキュベーター(37度、8%CO2、90 rpm)中で4日間培養が行われた。rProtein A SepharoseTM Fast Flow(Amersham Biosciences)を用いて当業者公知の方法を用いて、上記で得られた培養上清から抗体が精製された。分光光度計を用いて精製された抗体溶液の280 nmでの吸光度が測定された。PACE法により算出された吸光係数を用いることによって、得られた測定値から抗体濃度が算出された(Protein Science (1995) 4, 2411-2423)。
上記のように得られた精製抗体がカルシウムイオンと結合するか否かが参考実施例6に記載された方法で判定された。その結果を表42に示した。Caライブラリに含まれる複数の抗体のFabドメインのTmはカルシウムイオン濃度によって変動し、カルシウムイオンと結合する分子が含まれることが示された。
(23-1)pH依存的結合抗体の取得方法
WO2009/125825は抗原結合分子にヒスチジンを導入することにより、pH中性領域とpH酸性領域で性質が変化するpH依存的抗原結合抗体を開示している。開示されたpH依存的結合抗体は、所望の抗原結合分子のアミノ酸配列の一部をヒスチジンに置換する改変によって取得されている。改変する対象の抗原結合分子を予め得ることなく、pH依存的結合抗体をより効率的に取得するために、ヒスチジンを可変領域(より好ましくは抗原結合に関与する可能性がある位置)に導入した抗原結合分子の集団(Hisライブラリと呼ぶ)から所望の抗原に結合する抗原結合分子を取得する方法が考えられる。Hisライブラリから得られる抗原結合分子は通常の抗体ライブラリよりもヒスチジンが高頻度に出現するため、所望の性質を有する抗原結合分子が効率的に取得できると考えられる。
まず、Hisライブラリでヒスチジンを導入する位置が選択された。WO2009/125825ではIL-6レセプター抗体、IL-6抗体およびIL-31レセプター抗体の配列中のアミノ酸残基をヒスチジンに置換することでpH依存的抗原結合抗体を作製したことが開示されている。さらに、抗原結合分子のアミノ酸配列をヒスチジンに置換することによって、pH依存的抗原結合能を有する、抗卵白リゾチウム抗体(FEBS Letter 11483, 309, 1, 85-88)および抗ヘプシジン抗体(WO2009/139822)が作製されている。IL-6レセプター抗体、IL-6抗体、IL-31レセプター抗体、卵白リゾチウム抗体およびヘプシジン抗体でヒスチジンを導入した位置を表43に示した。表43に示した位置は、抗原と抗体との結合を制御できる位置の候補として挙げられ得る。さらに表43で示された位置以外でも、抗原と接触する可能性が高い位置も、ヒスチジンを導入する位置として適切であると考えられた。
ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型としてPCR法により抗体重鎖可変領域の遺伝子ライブラリが増幅された。実施例1に記載のHisライブラリ1として設計された抗体軽鎖可変領域の遺伝子ライブラリが、PCR法を用いて増幅された。このように作製された抗体重鎖可変領域の遺伝子ライブラリと抗体軽鎖可変領域の遺伝子ライブラリとの組合せがファージミドベクターへ挿入され、ヒト抗体配列からなるFabドメインを提示するヒト抗体ファージディスプレイライブラリが構築された。構築方法として、(Methods Mol Biol. (2002) 178, 87-100)が参考とされた。上記ライブラリの構築に際しては、ファージミドのFabとファージpIIIタンパク質をつなぐリンカー部分、および、ヘルパーファージpIIIタンパク遺伝子のN2ドメインとCTドメインの間にトリプシン切断配列が挿入されたファージディスプレイライブラリの配列が使用された。抗体遺伝子ライブラリが導入された大腸菌から単離された抗体遺伝子部分の配列が確認され、132クローンの配列情報が得られた。設計されたアミノ酸分布と、確認された配列中のアミノ酸の分布を、図53に示した。設計されたアミノ酸分布に対応する多様な配列を含むライブラリが構築された。
ヒトPBMCから作成したポリA RNAや、市販されているヒトポリA RNAなどを鋳型としてPCR法により抗体重鎖可変領域の遺伝子ライブラリが増幅された。参考実施例23に記載されるように、pH依存的抗原結合能をもつ抗体の出現頻度を向上させるため、抗体可変領域の軽鎖部分のうち、抗原接触部位となる可能性の高い部位のヒスチジン残基の出現頻度を高めた抗体可変領域軽鎖部分が設計される。また、フレキシブル残基のうちヒスチジンが導入された残基以外のアミノ酸残基として、天然ヒト抗体でのアミノ酸出現頻度の情報から特定される出現頻度の高いアミノ酸が均等に分布させた抗体軽鎖可変領域のライブラリが設計される。上記のように設計された抗体軽鎖可変領域の遺伝子ライブラリが、合成される。ライブラリの合成は商業的な受託会社等に委託して作製することも可能である。このように作製された抗体重鎖可変領域の遺伝子ライブラリと抗体軽鎖可変領域の遺伝子ライブラリとの組合せがファージミドベクターへ挿入され、公知の方法(Methods Mol Biol. (2002) 178, 87-100)に準じて、ヒト抗体配列からなるFabドメインを提示するヒト抗体ファージディスプレイライブラリが構築される。参考実施例24に記載された方法に準じて、抗体遺伝子ライブラリが導入された大腸菌から単離された抗体遺伝子部分の配列が確認される。
実施例14で見出されたFcγRIIbに対する選択性が向上されたEUナンバリングで表される238位のProがAspに置換された改変体を改変することによってFcγRIIbに対する選択性を更に増強することが試みられた。
まず、IL6R-G1dのEUナンバリングで表される238位のProをAspに置換した改変が導入されたIL6R-G1d-v1(配列番号:80)に対して、実施例14に記載されたFcγRIIbに対する選択性を増強するEUナンバリングで表される328位のLeuのGluへの置換が導入された改変体IL6R-G1d-v4(配列番号:172)が作製された。L鎖として用いられたIL6R-L (配列番号:83)と組み合わせて発現されたIL6R-G1d-v4、参考実施例2と同様の方法にしたがって調製された。ここで得られた抗体H鎖としてIL6R-G1d-v4に由来するアミノ酸配列を有する抗体はIgG1-v4と記載される。実施例14と同様の方法にしたがって評価されたIgG1、IgG1-v1、IgG1-v2、IgG1-v4のFcγRIIbに対する結合活性を表44に示した。表中の改変とはIL6R-G1dに対して導入された改変を表す。
P238D改変体に対して、実施例14でFcγRIIbに対する増強効果のあった改変(S267E/L328F)が導入された。当該改変の導入前後でのFcγRIIbに対する結合活性の変化を表45に示した。
参考実施例26で示されたように、ヒト天然型IgG1に対してEUナンバリングで表される238位のProがAspに置換されたFcに対し、さらにFcγRIIbへの結合を上げると天然型抗体の解析から予測される他の改変を組み合わせても、期待される組合せ効果は得られなかった。そこで、EUナンバリングで表される238位のProがAspに置換された改変Fcに対して網羅的改変を導入することによって、さらにFcγRIIbへの結合を増強する改変体を見出すことが試みられた。抗体H鎖として用いられたIL6R-G1d(配列番号:79)のEUナンバリングで表される252位のMetをTyrに置換する改変、EUナンバリングで表される434位のAsnをTyrに置換する改変が導入されたIL6R-F11(配列番号:174)が作製された。さらに、IL6R-F11に対してEUナンバリングで表される238位のProをAspに置換する改変が導入されたIL6R-F652(配列番号:175)が作製された。IL6R-F652に対し、EUナンバリングで表される238位の残基の近傍の領域(EUナンバリングで表される234位から237位、239位)が元のアミノ酸とシステインを除く18種類のアミノ酸にそれぞれ置換された抗体H鎖配列を含む発現プラスミドがそれぞれ調製された。抗体L鎖としてはIL6R-L(配列番号:83)が共通して用いられた。これらの改変体が参考実施例2と同様の方法により発現、精製された。これらのFc変異体はPD variantと呼ばれる。実施例14と同様の方法により各PD variantのFcγRIIa R型およびFcγRIIbに対する相互作用が網羅的に評価された。
表46に示した改変体のFcγRIa、FcγRIIaR、FcγRIIaH、FcγRIIb、FcγRIIIaVに対するKD値を実施例14と同様の方法で測定した結果を表47に示した。なお、表中の配列番号は評価された改変体のH鎖の配列番号を、また、改変とはIL6R-F11(配列番号:174)に対して導入された改変を表す。ただし、IL6R-F11を作製する際の鋳型としたIL6R-G1d/IL6R-Lについては、*として示した。また、表中のKD(IIaR)/KD(IIb)およびKD(IIaH)/KD(IIb)はそれぞれ、各改変体のFcγRIIaRに対するKD値を各改変体のFcγRIIbに対するKD値で割った値、各改変体のFcγRIIaHに対するKD値を各改変体のFcγRIIbに対するKD値で割った値を示す。親ポリペプチドのKD(IIb)/改変ポリペプチドのKD(IIb)は、親ポリペプチドのFcγRIIbに対するKD値を各改変体のFcγRIIbに対するKD値で割った値を指す。これらに加えて、各改変体のFcγRIIaRおよびFcγRIIaHに対する結合活性のうち強い方のKD値/親ポリペプチドのFcγRIIaRおよびFcγRIIaHに対する結合活性のうち強い方のKD値を表47に示した。ここで親ポリペプチドとは、IL6R-F11(配列番号:27)をH鎖に持つ改変体のことを指す。なお、表47のうち灰色で塗りつぶされたセルは、FcγRのIgGに対する結合が微弱であり、速度論的な解析では正しく解析できないと判断されたため、実施例14に記載の
〔式5〕
KD =C x Rmax / (Req - RI) - C
の式を利用して算出した値である。
先の参考実施例27に示した通り、P238Dを含むFcに対して、FcγRIIbとの結合活性を向上する、あるいはFcγRIIbへの選択性を向上させると天然型IgG1抗体の解析から予測された改変を導入しても、FcγRIIbに対する結合活性が減弱してしまうことが明らかとなり、この原因としてFcとFcγRIIbとの相互作用界面の構造がP238Dを導入することで変化していることが考えられた。そこで、この現象の原因を追及するためP238Dの変異をもつIgG1のFc(以下、Fc(P238D))とFcγRIIb細胞外領域との複合体の立体構造をX線結晶構造解析により明らかにし、天然型 IgG1のFc (以下、Fc(WT)) とFcγRIIb細胞外領域との複合体との立体構造を対比することによって、これらの結合様式が比較された。なお、FcとFcγR細胞外領域との複合体の立体構造に関する複数の報告がすでにあり、Fc(WT) / FcγRIIIb細胞外領域複合体(Nature, 2000, 400, 267-273; J.Biol.Chem. 2011, 276, 16469-16477)、Fc(WT) / FcγRIIIa細胞外領域複合体(Proc.Natl.Acad.Sci.USA, 2011, 108, 12669-126674)、およびFc(WT) / FcγRIIa細胞外領域複合体(J. Imunol. 2011, 187, 3208-3217)の立体構造が解析されている。これまでにFc(WT) / FcγRIIb細胞外領域複合体の立体構造は解析されていないが、Fc(WT)との複合体の立体構造が既知であるFcγRIIaとFcγRIIbでは細胞外領域においてアミノ酸配列の93%が一致し、非常に高い相同性を有していることから、Fc (WT) / FcγRIIb細胞外領域複合体の立体構造はFc(WT) / FcγRIIa細胞外領域複合体の結晶構造からモデリングにより推定された。
次に詳細な比較のため、Fc(P238D) / FcγRIIb細胞外領域複合体の結晶構造とFc(WT) / FcγRIIb細胞外領域複合体のモデル構造とを、FcγRIIb細胞外領域ならびにFc CH2ドメインAに対しCα原子間距離をもとにした最小二乗法により重ね合わせた(図58)。その際、Fc CH2ドメインB同士の重なりの程度は良好でなく、この部分に立体構造的な違いがあることが明らかとなった。さらにFc(P238D) / FcγRIIb細胞外領域複合体の結晶構造ならびにFc(WT) / FcγRIIb細胞外領域複合体のモデル構造を使い、抽出されたFcγRIIb細胞外領域とFc CH2ドメインBとの間でその距離が3.7Å以下の原子ペアを比較することによって、FcγRIIbとFc(WT) CH2ドメインBとの間の原子間相互作用とFcγRIIbとFc(P238D) CH2ドメインBとの間の原子間相互作用が比較された。表48に示すとおり、Fc(P238D)とFc(WT)では、Fc CH2ドメインBとFcγRIIbとの間の原子間相互作用は一致していなかった。
P238D改変を含むFcの調製は以下のように行われた。まず、hIL6R-IgG1-v1(配列番号:80)のEUナンバリングで表される220位のCysをSerに置換し、EUナンバリングで表される236位のGluからそのC末端をPCRによってクローニングした遺伝子配列Fc(P238D)を参考実施例1および2に記載された方法と同様な方法で発現ベクターの作製、発現、精製が行われた。なお、EUナンバリングで表される220位のCysは通常のIgG1においては、L鎖のCysとdisulfide bondを形成しているが、Fcのみを調製する場合にはL鎖を共発現させないことから、不要なdisulfide bond形成を回避するために当該Cys残基はSerに置換された。
FcγRIIb細胞外領域は、実施例14の方法にしたがって調製された。
結晶化に使用するため得られたFcγRIIb細胞外領域サンプル 2mgに対し、glutathione S-transferaseとの融合蛋白として大腸菌により発現精製したEndo F1(Protein Science 1996, 5, 2617-2622) 0.29mgを加え、0.1M Bis-Tris pH6.5のBuffer条件で、室温にて3日間静置することにより、FcγRIIb細胞外領域のAsnに直接結合したN-acetylglucosamine以外のN型糖鎖が切断された。次に5000MWCOの限外ろ過膜により濃縮された糖鎖切断処理が施されたFcγRIIb細胞外領域サンプルが、20mM HEPS pH7.5, 0.05M NaClで平衡化したゲルろ過カラムクロマトグラフィー(Superdex200 10/300)により精製された。さらに得られた糖鎖切断FcγRIIb細胞外領域画分にFc(P238D)をモル比でFcγRIIb細胞外領域のほうが若干過剰となるよう混合された。10000MWCOの限外ろ過膜により濃縮された前記混合液を20mM HEPS pH7.5、0.05M NaClで平衡化したゲルろ過カラムクロマトグラフィー(Superdex200 10/300)を用いて精製することによって、Fc(P238D) / FcγRIIb細胞外領域複合体のサンプルが得られた。
10000MWCOの限外ろ過膜 により約10mg/mlまで濃縮された前記のFc(P238D) / FcγRIIb細胞外領域複合体の試料を用いて、シッティングドロップ蒸気拡散法により当該複合体が結晶化された。結晶化にはHydra II Plus One (MATRIX)を用い、100mM Bis-Tris pH6.5、17% PEG3350、0.2M Ammonium acetate、および2.7%(w/v) D-Galactoseのリザーバー溶液に対し、リザーバー溶液:結晶化サンプルを0.2μl:0.2μlで混合して結晶化ドロップを作成した。シールされた当該結晶化ドロップを20℃に静置することによって、薄い板状の結晶が得られた。
得られたFc(P238D) / FcγRIIb細胞外領域複合体の単結晶一つが100mM Bis-Tris pH6.5、20% PEG3350、Ammonium acetate、2.7%(w/v) D-Galactose、Ethylene glycol 22.5%(v/v) の溶液に浸漬された。微小なナイロンループ付きのピンを用いて溶液ごとすくいとられた単結晶を液体窒素中で凍結させた。高エネルギー加速器研究機構の放射光施設フォトンファクトリーBL-1Aにて当該結晶のX線回折データが測定された。なお、測定中は常に-178℃の窒素気流中に置くことによって凍結状態を維持し、ビームラインに備え付けられたCCDディテクタQuantum 270(ADSC)によって、結晶を0.8°ずつ回転させながらトータル225枚のX線回折画像が収集された。得られた回折画像からの格子定数の決定、回折斑点の指数付け、ならびに回折データの処理には、プログラムXia2(CCP4 Software Suite)、XDS Package(Walfgang Kabsch)ならびにScala(CCP4 Software Suite)を用い、最終的に分解能2.46Åまでの当該結晶の回折強度データが得られた。本結晶は、空間群P21に属し、格子定数a=48.85Å、b=76.01Å、c=115.09Å、α=90°、β=100.70°、γ=90°であった。
Fc(P238D) / FcγRIIb細胞外領域複合体の結晶構造決定は、プログラムPhaser(CCP4 Software Suite)を用いた分子置換法によりおこなわれた。得られた結晶格子の大きさとFc(P238D) / FcγRIIb細胞外領域複合体の分子量から非対称単位中の複合体の数は一個と予想された。Fc(WT) / FcγRIIIa細胞外領域複合体の結晶構造であるPDB code:3SGJの構造座標から、A鎖239-340番ならびにB鎖239-340番のアミノ酸残基部分を別座標として取り出し、それぞれFc CH2ドメインの探索用モデルと設定した。同じくPDB code:3SGJの構造座標から、A鎖341-444番とB鎖341-443番のアミノ酸残基部分を一つの座標として取り出し、Fc CH3ドメインの探索用モデルと設定した。最後にFcγRIIb細胞外領域の結晶構造であるPDB code:2FCBの構造座標からA鎖6-178番のアミノ酸残基部分を取り出しFcγRIIb細胞外領域の探索用モデルと設定した。Fc CH3ドメイン、FcγRIIb細胞外領域、Fc CH2ドメインの順番に各探索用モデルの結晶格子内での向きと位置を、回転関数および並進関数から決定し、Fc(P238D) / FcγRIIb細胞外領域複合体結晶構造の初期モデルが得られた。得られた初期モデルに対し2つのFc CH2ドメイン、2つのFc CH3ドメインならびにFcγRIIb細胞外領域を動かす剛体精密化をおこなったところ、この時点で25-3.0Åの回折強度データに対し、結晶学的信頼度因子R値は40.4%、Free R値は41.9%となった。さらにプログラムRefmac5(CCP4 Software Suite)を用いた構造精密化と、実験的に決定された構造因子Foとモデルから計算された構造因子Fcならびにモデルから計算された位相をもとに算出された2Fo-Fc、Fo-Fcを係数とする電子密度マップを見ながらのモデル修正をプログラムCoot(Paul Emsley)でおこなった。これらの作業を繰り返すことによってモデルの精密化がおこなわれた。最後に2Fo-Fc、Fo-Fcを係数とする電子密度マップをもとに水分子をモデルに組み込み、精密化をおこなうことによって、最終的に分解能25-2.6Åの24291個の回折強度データを用い、4846個の非水素原子を含むモデルに対し、結晶学的信頼度因子R値は23.7%、Free R値は27.6%となった。
Fc(WT) / FcγRIIa細胞外領域複合体の結晶構造であるPDB code:3RY6の構造座標をベースに、プログラムDisovery Studio 3.1(Accelrys)のBuild Mutants機能を使い、FcγRIIbのアミノ酸配列と一致するように構造座標中のFcγRIIaに変異が導入された。その際、Optimization LevelをHigh、Cut Radiusを4.5とし、5つのモデルを発生させ、その中から最もエネルギースコアが良いものを採用し、Fc(WT) / FcγRIIb細胞外領域複合体のモデル構造と設定した。
参考実施例28で得られたFc (P238D)とFcγRIIb細胞外領域との複合体のX線結晶構造解析の結果に基づき、EUナンバリングで表される238位のProがAspに置換された改変FcにおいてFcγRIIbとの相互作用に影響を与えることが予測される部位(EUナンバリングで表される233位、240位、241位、263位、265位、266位、267位、268位、271位、273位、295位、296位、298位、300位、323位、325位、326位、327位、328位、330位、332位、334位の残基)に対して網羅的な改変が導入された改変体を構築することによって、P238D 改変に加えてさらにFcγRIIbとの結合を増強する改変の組合せを得ることが可能であるか検討された。
〔式5〕
KD =C x Rmax / (Req - RI) - C
の式を利用して算出した値である。
参考実施例27および29において得られた改変の中で、FcγRIIbへの結合を増強する効果もしくはFcγRIIbへの結合を維持し、他のFcγRへの結合を抑制する効果がみられた改変同士を組み合わせることによる効果が検証された。
なお、表中の改変とはIL6R-B3(配列番号:187)に対して導入した改変を表す。ただし、IL6R-B3を作製する際の鋳型としたIL6R-G1d/IL6R-Lについては、*として示した。
〔式5〕
KD =C x Rmax / (Req - RI) - C
の式を利用して算出した値である。
Claims (44)
- イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、およびpH中性域の条件下でFcRnに対する結合活性を有するFc領域を含む抗原結合分子のFc領域が、pH中性域の条件下で二分子のFcRnおよび一分子の活性型Fcγレセプターを含むヘテロ複合体を形成しないFc領域に改変することを含む、以下のいずれかの方法;
(a) 抗原結合分子の薬物動態を改善する方法、または
(b) 抗原結合分子の免疫原性を低減させる方法。 - 前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域の活性型Fcγレセプターに対する結合活性が、天然型ヒトIgGのFc領域の当該活性型Fcγレセプターに対する結合活性よりも低いFc領域に改変することを含む、請求項1に記載の方法。
- 前記活性型FcγレセプターがヒトFcγRIa、ヒトFcγRIIa(R)、ヒトFcγRIIa(H)、ヒトFcγRIIIa(V)またはヒトFcγRIIIa(F)である、請求項1または2に記載の方法。
- 前記Fc領域のアミノ酸のうちEUナンバリングで表される235位、237位、238位、239位、270位、298位、325位および329位のいずれかひとつ以上のアミノ酸を置換することを含む、請求項1から3のいずれか一項に記載の方法。
- 前記Fc領域のEUナンバリングで表されるアミノ酸であって;
234位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Met、Phe、Pro、Ser、ThrまたはTrpのいずれか、
235位のアミノ酸をAla、Asn、Asp、Gln、Glu、Gly、His、Ile、Lys、Met、Pro、Ser、Thr、ValまたはArgのいずれか、
236位のアミノ酸をArg、Asn、Gln、His、Leu、Lys、Met、Phe、ProまたはTyrのいずれか、
237位のアミノ酸をAla、Asn、Asp、Gln、Glu、His、Ile、Leu、Lys、Met、Pro、Ser、Thr、Val、TyrまたはArgのいずれか、
238位のアミノ酸をAla、Asn、Gln、Glu、Gly、His、Ile、Lys、Thr、TrpまたはArgのいずれか、
239位のアミノ酸をGln、His、Lys、Phe、Pro、Trp、TyrまたはArgのいずれか、
265位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、TyrまたはValのいずれか、
266位のアミノ酸をAla、Arg、Asn、Asp、Gln、Glu、Gly、His、Lys、Phe、Pro、Ser、Thr、TrpまたはTyrのいずれか、
267位のアミノ酸をArg、His、Lys、Phe、Pro、TrpまたはTyrのいずれか、
269位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
270位のアミノ酸をAla、Arg、Asn、Gln、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
271位のアミノ酸をArg、His、Phe、Ser、Thr、TrpまたはTyrのいずれか、
295位のアミノ酸をArg、Asn、Asp、Gly、His、Phe、Ser、TrpまたはTyrのいずれか、
296位のアミノ酸をArg、Gly、LysまたはProのいずれか、
297位のアミノ酸をAla、
298位のアミノ酸をArg、Gly、Lys、Pro、TrpまたはTyrのいずれか、
300位のアミノ酸をArg、LysまたはProのいずれか、
324位のアミノ酸をLysまたはProのいずれか、
325位のアミノ酸をAla、Arg、Gly、His、Ile、Lys、Phe、Pro、Thr、TrpTyr、もしくはValのいずれか、
327位のアミノ酸をArg、Gln、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、TyrまたはValのいずれか、
328位のアミノ酸をArg、Asn、Gly、His、LysまたはProのいずれか、
329位のアミノ酸をAsn、Asp、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Ser、Thr、Trp、Tyr、ValまたはArgのいずれか、
330位のアミノ酸をProまたはSerのいずれか、
331位のアミノ酸をArg、GlyまたはLysのいずれか、もしくは
332位のアミノ酸をArg、LysまたはProのいずれか、
のいずれかひとつ以上に置換することを含む、請求項4に記載の方法。 - 前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域の抑制型Fcγレセプターに対する結合活性が活性型Fcγレセプターに対する結合活性よりも高いFc領域に改変することを含む、請求項1に記載の方法。
- 前記抑制型FcγレセプターがヒトFcγRIIbである、請求項6に記載の方法。
- 前記活性型FcγレセプターがヒトFcγRIa、ヒトFcγRIIa(R)、ヒトFcγRIIa(H)、ヒトFcγRIIIa(V)またはヒトFcγRIIIa(F)である、請求項6または7に記載の方法。
- EUナンバリングで表される238または328のアミノ酸を置換することを含む、請求項6から8のいずれか一項に記載の方法。
- EUナンバリングで表される238のアミノ酸をAsp、または328のアミノ酸をGluに置換することを含む、請求項9に記載の方法。
- EUナンバリングで表されるアミノ酸であって;
233位のアミノ酸をAsp、
234位のアミノ酸をTrp、またはTyrのいずれか、
237位のアミノ酸をAla、Asp、Glu、Leu、Met、Phe、TrpまたはTyrのいずれか、
239位のアミノ酸をAsp、
267位のアミノ酸をAla、GlnまたはValのいずれか、
268位のアミノ酸をAsn、Asp、またはGluのいずれか、
271位のアミノ酸をGly、
326位のアミノ酸をAla、Asn、Asp、Gln、Glu、Leu、Met、SerまたはThrのいずれか、
330位のアミノ酸をArg、Lys、またはMetのいずれか、
323位のアミノ酸をIle、Leu、またはMetのいずれか、
296位のアミノ酸をAsp、
のいずれかひとつ以上に置換することを含む請求項9または10に記載の方法。 - 前記Fc領域のアミノ酸のうちEUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、298、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸が天然型Fc領域のアミノ酸と異なるアミノ酸を含むFc領域である、請求項1から11のいずれか一項に記載の方法。
- 前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyrのいずれか、
252位のアミノ酸がPhe、Trp、またはTyrのいずれか、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsn、Asp、Glu、またはGlnのいずれか、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはValのいずれか、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGluのいずれか、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
298位のアミノ酸がGly、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyrのいずれか、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThrのいずれか、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArgのいずれか、
311位のアミノ酸がAla、His、またはIleのいずれか、
312位のアミノ酸がAlaまたはHisのいずれか、
314位のアミノ酸がLysまたはArgのいずれか、
315位のアミノ酸がAla、AspまたはHisのいずれか、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHisのいずれか、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSerのいずれか、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyrのいずれか、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyrのいずれか、もしくは
436位のアミノ酸がHis、Ile、Leu、Phe、Thr、またはVal 、
のいずれかひとつ以上の組合せである、請求項12に記載の方法。 - 前記抗原結合ドメインが、カルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項1から13のいずれか一項に記載の方法。
- 前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項14に記載の方法。
- 前記抗原結合ドメインが、pHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項1から13のいずれか一項に記載の方法。
- 前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項16に記載の方法。
- 前記抗原結合ドメインが抗体の可変領域である、請求項1から17のいずれか一項に記載の方法。
- 前記抗原結合分子が抗体である、請求項1から18のいずれか一項に記載の方法。
- 前記ヘテロ複合体を形成しないFc領域に改変することが、Fc領域を構成する二つのポリペプチドの一方がpH中性域の条件下でのFcRn結合活性を有し、他方がpH中性域の条件下でのFcRn結合活性を有しないFc領域に改変することを含む、請求項1に記載の方法。
- 前記Fc領域を構成する二つのポリペプチドの一方のアミノ酸配列のうち、EUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、298、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸を置換することを含む、請求項20に記載の方法。
- 前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸をMet、
248位のアミノ酸をIle、
250位のアミノ酸をAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyr、
252位のアミノ酸をPhe、Trp、またはTyr、
254位のアミノ酸をThr、
255位のアミノ酸をGlu、
256位のアミノ酸をAsn、Asp、Glu、またはGln、
257位のアミノ酸をAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはVal、
258位のアミノ酸をHis、
265位のアミノ酸をAla、
286位のアミノ酸をAlaまたはGlu、
289位のアミノ酸をHis、
297位のアミノ酸をAla、
298位のアミノ酸をGly、
303位のアミノ酸をAla、
305位のアミノ酸をAla、
307位のアミノ酸をAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyr、
308位のアミノ酸をAla、Phe、Ile、Leu、Met、Pro、Gln、またはThr、
309位のアミノ酸をAla、Asp、Glu、Pro、またはArg、
311位のアミノ酸をAla、His、またはIle、
312位のアミノ酸をAlaまたはHis、
314位のアミノ酸をLysまたはArg、
315位のアミノ酸をAla、AspまたはHis、
317位のアミノ酸をAla、
332位のアミノ酸をVal、
334位のアミノ酸をLeu、
360位のアミノ酸をHis、
376位のアミノ酸をAla、
380位のアミノ酸をAla、
382位のアミノ酸をAla、
384位のアミノ酸をAla、
385位のアミノ酸をAspまたはHis、
386位のアミノ酸をPro、
387位のアミノ酸をGlu、
389位のアミノ酸をAlaまたはSer、
424位のアミノ酸をAla、
428位のアミノ酸をAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyr、
433位のアミノ酸をLys、
434位のアミノ酸をAla、Phe、His、Ser、Trp、またはTyr、もしくは
436位のアミノ酸をHis 、Ile、Leu、Phe、Thr、またはVal
のいずれかひとつ以上に置換することを含む、請求項21に記載の方法。 - 前記抗原結合ドメインが、カルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項20から22のいずれか一項に記載の方法。
- 前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項23に記載の方法。
- 前記抗原結合ドメインが、pHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項20から22のいずれか一項に記載の方法。
- 前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項25に記載の方法。
- 前記抗原結合ドメインが抗体の可変領域である、請求項20から26のいずれか一項に記載の方法。
- 前記抗原結合分子が抗体である、請求項20から27のいずれか一項に記載の方法。
- イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、ならびにpH中性域の条件下でFcRnに対する結合活性を有するFc領域のEUナンバリングで表されるアミノ酸であって;
234位のアミノ酸がAla、
235位のアミノ酸がAla、LysまたはArgのいずれか、
236位のアミノ酸がArg、
238位のアミノ酸がArg、
239位のアミノ酸がLys、
270位のアミノ酸がPhe、
297位のアミノ酸がAla、
298位のアミノ酸がGly、
325位のアミノ酸がGly、
328位のアミノ酸がArg、もしくは
329位のアミノ酸がLys、またはArg
の中から選択されるいずれかひとつ以上のアミノ酸を含むFc領域を含む抗原結合分子。 - 前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がLysまたはArgのいずれか、
238位のアミノ酸がLys
239位のアミノ酸がArg、または
329位のアミノ酸がLysまたはArgのいずれか、
の中から選択されるいずれかひとつ以上のアミノ酸を含む、請求項29に記載の抗原結合分子。 - イオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメイン、およびFc領域を構成する二つのポリペプチドの一方がpH中性域の条件下でのFcRnに対する結合活性を有し、他方がpH中性域の条件下でのFcRnに対する結合活性を有しないFc領域を含む抗原結合分子。
- 前記Fc領域を構成する二つのポリペプチドの一方のアミノ酸配列のうち、EUナンバリングで表される237、248、250、252、254、255、256、257、258、265、286、289、297、303、305、307、308、309、311、312、314、315、317、332、334、360、376、380、382、384、385、386、387、389、424、428、433、434、および436のいずれかひとつ以上のアミノ酸が天然型Fc領域のアミノ酸と異なるFc領域である、請求項29から31のいずれか一項に記載の抗原結合分子。
- 前記Fc領域のEUナンバリングで表されるアミノ酸であって;
237位のアミノ酸がMet、
248位のアミノ酸がIle、
250位のアミノ酸がAla、Phe、Ile、Met、Gln、Ser、Val、Trp、またはTyr、
252位のアミノ酸がPhe、Trp、またはTyr、
254位のアミノ酸がThr、
255位のアミノ酸がGlu、
256位のアミノ酸がAsn、Asp、Glu、またはGln、
257位のアミノ酸がAla、Gly、Ile、Leu、Met、Asn、Ser、Thr、またはVal、
258位のアミノ酸がHis、
265位のアミノ酸がAla、
286位のアミノ酸がAlaまたはGlu、
289位のアミノ酸がHis、
297位のアミノ酸がAla、
303位のアミノ酸がAla、
305位のアミノ酸がAla、
307位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Met、Asn、Pro、Gln、Arg、Ser、Val、Trp、またはTyr、
308位のアミノ酸がAla、Phe、Ile、Leu、Met、Pro、Gln、またはThr、
309位のアミノ酸がAla、Asp、Glu、Pro、またはArg、
311位のアミノ酸がAla、His、またはIle、
312位のアミノ酸がAlaまたはHis、
314位のアミノ酸がLysまたはArg、
315位のアミノ酸がAla、AspまたはHis、
317位のアミノ酸がAla、
332位のアミノ酸がVal、
334位のアミノ酸がLeu、
360位のアミノ酸がHis、
376位のアミノ酸がAla、
380位のアミノ酸がAla、
382位のアミノ酸がAla、
384位のアミノ酸がAla、
385位のアミノ酸がAspまたはHis、
386位のアミノ酸がPro、
387位のアミノ酸がGlu、
389位のアミノ酸がAlaまたはSer、
424位のアミノ酸がAla、
428位のアミノ酸がAla、Asp、Phe、Gly、His、Ile、Lys、Leu、Asn、Pro、Gln、Ser、Thr、Val、Trp、またはTyr、
433位のアミノ酸がLys、
434位のアミノ酸がAla、Phe、His、Ser、Trp、またはTyr、もしくは
436位のアミノ酸がHis 、Ile、Leu、Phe、Thr、またはVal、
のいずれかひとつ以上の組合せである、請求項32に記載の抗原結合分子。 - 前記抗原結合ドメインがカルシウムイオン濃度の条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項29から33のいずれか一項に記載の抗原結合分子。
- 前記抗原結合ドメインが、低カルシウムイオン濃度の条件下での抗原に対する結合活性が高カルシウムイオン濃度の条件下での抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項34に記載の抗原結合分子。
- 前記抗原結合ドメインがpHの条件によって抗原に対する結合活性が変化する抗原結合ドメインである、請求項29から33のいずれか一項に記載の抗原結合分子。
- 前記抗原結合ドメインが、pH酸性域における抗原に対する結合活性がpH中性域の条件における抗原に対する結合活性よりも低いように結合活性が変化する抗原結合ドメインである、請求項36に記載の抗原結合分子。
- 前記抗原結合ドメインが抗体の可変領域である、請求項29から37のいずれか一項に記載の抗原結合分子。
- 前記抗原結合分子が抗体である、請求項29から38のいずれか一項に記載の抗原結合分子。
- 請求項29から39のいずれか一項に記載の抗原結合分子をコードするポリヌクレオチド。
- 請求項40に記載のポリヌクレオチドが作用可能に連結されたベクター。
- 請求項41に記載のベクターが導入された細胞。
- 請求項42に記載の細胞の培養液から抗原結合分子を回収する工程を含む、請求項29から39のいずれか一項に記載の抗原結合分子の製造方法。
- 請求項29から39のいずれか一項に記載の抗原結合分子、または請求項43に記載の製造方法によって得られる抗原結合分子を有効成分として含む医薬組成物。
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ES12764620T ES2831048T3 (es) | 2011-03-30 | 2012-03-30 | Permanencia de moléculas de unión a antígeno en plasma sanguíneo y método para modificar la inmunogenicidad |
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KR1020137028357A KR102168731B1 (ko) | 2011-03-30 | 2012-03-30 | 항원 결합 분자의 혈장 체류성과 면역원성을 개변하는 방법 |
SG2013074158A SG194076A1 (en) | 2011-03-30 | 2012-03-30 | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
US14/007,947 US10618965B2 (en) | 2011-02-25 | 2012-03-30 | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
BR112013025221A BR112013025221A8 (pt) | 2011-02-25 | 2012-03-30 | retenção de moléculas de ligação a antígeno em plasma sanguíneo e método para modificação de imunogenicidade |
DK12764620.6T DK2698431T3 (da) | 2011-03-30 | 2012-03-30 | Opretholdelse af antigen-bindende molekyler i blodplasma og fremgangsmåde til modifikation af immunogenicitet |
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EP12764620.6A EP2698431B1 (en) | 2011-03-30 | 2012-03-30 | Retention of antigen-binding molecules in blood plasma and method for modifying immunogenicity |
AU2012233313A AU2012233313C1 (en) | 2011-03-30 | 2012-03-30 | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
MX2018012383A MX2018012383A (es) | 2011-03-30 | 2012-03-30 | Método para alterar la retención en plasma e inmunogenicidad de la molécula de union al antígeno. |
CA2831770A CA2831770A1 (en) | 2011-03-30 | 2012-03-30 | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
KR1020227017385A KR102639563B1 (ko) | 2011-03-30 | 2012-03-30 | 항원 결합 분자의 혈장 체류성과 면역원성을 개변하는 방법 |
CN201280026850.8A CN103703129A (zh) | 2011-03-30 | 2012-03-30 | 改变抗原结合分子的血浆中滞留性和免疫原性的方法 |
RU2013148116/10A RU2013148116A (ru) | 2011-03-30 | 2012-03-30 | Способ измерения удержания в плазме и иммуногенности антиген связывающей молекулы |
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KR1020247005528A KR20240027154A (ko) | 2011-03-30 | 2012-03-30 | 항원 결합 분자의 혈장 체류성과 면역원성을 개변하는 방법 |
EP20194883.3A EP3825325A3 (en) | 2011-03-30 | 2012-03-30 | Retention of antigen-binding molecules in blood plasma and method for modifying immunogenicity |
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TW107101834A TW201817744A (zh) | 2011-09-30 | 2012-09-27 | 具有促進抗原清除之FcRn結合域的治療性抗原結合分子 |
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EP12775324.2A EP2760890B1 (en) | 2011-09-30 | 2012-09-28 | Therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance |
EP23210057.8A EP4324850A2 (en) | 2011-09-30 | 2012-09-28 | Therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance |
BR112014007290A BR112014007290A2 (pt) | 2011-09-30 | 2012-09-28 | molécula de ligação a antígeno terapêutica com domínio de ligação a fcrn que promove eliminação de antígenos |
US14/347,187 US10253100B2 (en) | 2011-09-30 | 2012-09-28 | Therapeutic antigen-binding molecule with a FcRn-binding domain that promotes antigen clearance |
MX2014003891A MX2014003891A (es) | 2011-09-30 | 2012-09-28 | Molecula terapeutica de union al antigeno con un dominio de union a fcrn que promueve la eliminacion del antigeno. |
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PCT/JP2012/006218 WO2013046704A2 (en) | 2011-09-30 | 2012-09-28 | THERAPEUTIC ANTIGEN-BINDING MOLECULE WITH A FcRn-BINDING DOMAIN THAT PROMOTES ANTIGEN CLEARANCE |
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EP19150272.3A EP3549956A3 (en) | 2011-09-30 | 2012-09-28 | Therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance |
CA2850035A CA2850035A1 (en) | 2011-09-30 | 2012-09-28 | Therapeutic antigen-binding molecule with a fcrn-binding domain that promotes antigen clearance |
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JP2021211155A JP2022034037A (ja) | 2011-09-30 | 2021-12-24 | 抗原クリアランスを促進するFcRn結合ドメインを有する治療用抗原結合分子 |
US18/298,743 US20230257470A1 (en) | 2011-03-30 | 2023-04-11 | Method for altering plasma retention and immunogenicity of antigen-binding molecule |
US18/533,360 US20240117059A1 (en) | 2011-09-30 | 2023-12-08 | THERAPEUTIC ANTIGEN-BINDING MOLECULE WITH A FcRn-BINDING DOMAIN THAT PROMOTES ANTIGEN CLEARANCE |
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AU2012233313B2 (en) | 2017-02-02 |
EP2698431A4 (en) | 2014-10-01 |
EP3825325A2 (en) | 2021-05-26 |
AU2012233313C1 (en) | 2017-08-03 |
AU2012233313A1 (en) | 2013-10-31 |
EP2698431A1 (en) | 2014-02-19 |
EP2698431B1 (en) | 2020-09-09 |
DK2698431T3 (da) | 2020-11-30 |
EP3825325A3 (en) | 2021-10-13 |
AU2016277645B2 (en) | 2019-04-04 |
AU2016277645A1 (en) | 2017-01-19 |
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