Method of preparing ph-dependent antibodies

Abstract

To provide methods of preparing engineered antibodies exhibiting improved pH-dependent antigen binding.SOLUTION: The methods are based on introduction of histidine residues at a subset of defined amino acid positions within the antibody CDRs. The set of amino acid positions selected for histidine substitution is derived from a heat-map of histidine occurrence within the CDRs of functional antibodies from a natural antibody repertoire. The methods provide a simpler and less time-consuming approach to the identification of pH-dependent antibody variants.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure relates to the field of antibody engineering, in particular to the engineering of antibody variants that exhibit pH-dependent binding to target antigens. The present invention relates to a method for producing an antibody, as well as to an antibody prepared by said method. [Background technology]

[0002] (background) There is growing interest in engineering antibodies that exhibit pH-dependent binding to their target antigens. In particular, antibodies that exhibit reduced antigen binding at acidic pH compared to neutral pH are identified. There is growing interest in this.

[0003] Improving in vivo function of engineered antibodies by incorporating pH-sensitive antigen binding Antibodies can be engineered to exhibit high affinity antigen binding at neutral pH (e.g., pH 7.4). It may retain its binding and show reduced binding at acidic pH (e.g., pH 4.5-6.0). Once in the endothelial pathway, pH-dependent antigen binding leads to the endothelialization of the endothelial cells in acidified endosomes (approximately pH 6.0). Dissociation of antibody-antigen complexes and recycling of free antibodies mediated by FcRn are possible. This may result in enhanced antigen clearance, which may allow for lower frequency of antibody production or reduced antibody dose. This will promote the development of

[0004] Typically, pH-dependent antibody-antigen binding is due to the binding or folding of the interacting antibodies. The mechanism is dependent on the presence of ionic histidine residues, which mediate the conformational changes of histidine at low pH values. The change in electrostatic interactions induced upon protonation of stigmine may decrease binding affinity. do.

[0005] Various strategies for substituting histidines can be used to impart pH sensitivity to proteins. Various protein engineering approaches have been described that aim to insert Structural modeling-guided synthesis of colony-stimulating factor (GCSF) receptor interaction sites Rationally designed mutation sites that result in pH-sensitive variants when substituted with histidine (Sarkar et al., Nat Biotechnol, Vol. 20, pp. 908-13, However, there are distinct histidine substitutions in proteins that may mediate pH sensitivity. Evaluating a location is time consuming and unpredictable.

[0006] As an alternative approach, suitable high-throughput techniques, such as yeast surface display, can be used. Screening of combinatorial libraries using YSD or phage display has been applied to engineer pH-sensitive bonds in a variety of different protein scaffolds. Schroter et al., mAbs, vol. 7(1), 138-151, 2015, reported a reversible pH-sensitive antigen binding A general strategy for engineering antibody heavy and light chain variable domains for this purpose has been described. The approach involves the generation and sequencing of combinatorial histidine substitution libraries of heavy and light chains. This approach relies on screening. Histidine mutations are introduced into the VH and VL regions. Histidine mutations are introduced randomly into the complementarity determining regions (CDRs) of the VH domain and Histidine mutations can be introduced into any amino acid position in the CDRs of the VL domain. The rate of mutations achieved is on average 3 random mutations per variant in the library. Therefore, a complete library would theoretically contain three random histidine sequences in the CDRs. The invention encompasses variants containing all possible combinations of amino acid substitutions.

[0007] This combinatorial library approach indeed identified an adapter that exhibits pH-dependent binding to TNF. This strategy allowed for the successful identification of engineered variants of limumab. However, For possible histidine substitutions, all amino acids in the CDRs of the VH and VL domains are The large library size required to sample acid positions makes it difficult to Furthermore, this strategy introduces histidine residues at unfavorable positions. The overall antibody structure and stability due to the Summary of the Invention

[0008] (Summary of the Invention) An alternative method for preparing engineered antibody variants that exhibit pH-dependent binding to target antigens There is a need for such a method which is easier and less time consuming to perform than prior art methods. Furthermore, it exhibits pH-dependent antigen binding and does not excessively affect antigen binding ability at neutral pH. What is needed is a method that allows for the identification of engineered antibodies that do not

[0009] To address these needs, applicants have developed functional (i.e., antigen-binding) antibodies. The natural occurrence of histidine residues in the complementarity-determining regions (CDRs) of a large repertoire of variant domains. As a result of this analysis, the applicant has found that the natural occurrence of We were able to derive a "heat map" of histidine occurrence rates. This "heat map" is , "hot spot" amino acids in the CDRs where histidines may naturally occur in the antibody repertoire. A list of amino acid residues, and also "codons" in CDRs where histidine typically does not occur naturally. These "hot spots" and "hot spots" provide a list of amino acid residues. The "cold spot" list may be derived from, for example, rational design or combinatorial approaches. This method can be applied to engineer pH-dependent antibody variants in vivo.

[0010] In a first aspect, the present invention provides an engineered antibody that exhibits pH-dependent binding to its antigen. A method for preparing a parent antibody comprising: substituting at least one amino acid residue of the parent antibody with a histidine residue. preparing the engineered antibody by selecting the following hotspot list: [Table 1] at least one amino acid residue selected from the group consisting of: Dospot List: [Table 2] at least one amino acid residue from the The method further provides a method wherein the body exhibits pH-dependent binding to the antigen.

[0011] The engineered antibody has a pH greater than that of the parent antibody as a result of the histidine substitution. The ATP-dependent binding of the ATP-dependent ...

[0012] The present invention provides a method for preparing an engineered antibody that exhibits pH-dependent binding to its antigen, comprising: (a) providing a parent antibody that binds to an antigen; (b) preparing a panel of engineered variants of the parent antibody, Each of the engineered variants has at least one amino acid residue substituted with a histidine residue. It differs from the parent antibody by the following hotspot list: [Table 3] at least one amino acid residue selected from the group consisting of: Cold Spot List: [Table 4] wherein at least one amino acid residue from is not substituted with histidine; (c) screening the panel of engineered variants for pH-dependent binding to the antigen; and performing pH-dependent binding analysis on the antigen to identify engineered antibodies that exhibit pH-dependent binding to the antigen. The method further provides:

[0013] The engineered antibody identified in step (c) comprises, as a result of the substitution with the histidine, It exhibits improved pH-dependent binding (ie, higher pH dependence) compared to the parent antibody.

[0014] The present invention provides a method for preparing an engineered antibody that exhibits pH-dependent binding to its antigen, comprising: (a) identifying a parent antibody that binds to the antigen; (b) preparing a panel of engineered variants of the parent antibody, Each of the engineered variants has at least one amino acid residue substituted with a histidine residue. It differs from the parent antibody by the following hotspot list: [Table 5] at least one amino acid residue selected from the group consisting of: Cold Spot List: [Table 6] wherein at least one amino acid residue from is not substituted with histidine; (c) screening the panel of engineered variants for pH-dependent binding to the target antigen; The selection is performed by screening, whereby the presence of histidine confers pH-dependent binding to the antigen. identifying the selected amino acid position; (d) preparing one or more further engineered variants of said parent antibody, each of the ants contains a histidine at two or more of the selected amino acid positions identified in step (c). , the process; and (e) screening the further engineered variants for pH-dependent binding to the antigen thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen. The method is further provided.

[0015] The engineered antibody identified in step (e) contains, as a result of the substitution with the histidine, It exhibits improved pH-dependent binding (ie, higher pH dependence) compared to the parent antibody.

[0016] In a second aspect, the present invention is an engineered antibody that exhibits pH-dependent binding to its antigen. Thus, at least one amino acid in the CDR of the engineered antibody is a histidine residue, Hotspots list below: [Table 7] At least one amino acid residue selected from the group consisting of: List of popular spots: [Table 8] wherein at least one amino acid residue selected from the group consisting of: is not a histidine residue , the engineered antibody is provided. DETAILED DESCRIPTION OF THE INVENTION

[0017] (Detailed explanation) The present disclosure provides a method for preparing an engineered antibody that exhibits pH-dependent binding to a target antigen, comprising: Selected amino acids in the variable domains (and preferentially in the CDRs) of the parent antibody are replaced with histidine. The method is based on substituting an ionic histidine residue with an ionic histidine residue. It has been shown that introduction of a nucleotide into the VH and / or VL domain of a molecule can alter the pH dependence of antigen binding. However, there are no pH-dependent methods available to date. A method for engineering differential antigen binding involves substituting all possible histidine residues in the CDRs. Screening of random combinatorial libraries sampling for replacement by have relied heavily on training.

[0018] The method involves selecting specific amino acid residues to be substituted with histidines in the VH and VH regions of a functional antibody molecule. and histidine residues at specific amino acid positions in the VL domain, and more particularly in the CDRs thereof. The natural occurrence and natural non-occurrence of histidine residues at other amino acid positions in the CDRs The method disclosed herein differs from the prior art methods in that it is guided by: selected from a preselected subset of amino acid positions in the variable domains (and particularly the CDRs) The amino acid sequence is based on histidine substitutions at certain amino acid positions, while other residues in the CDRs are preferably histidine-free. The nucleotides are preselected to be non-substituted with dimethylaminomethyl.

[0019] The amino acid residues preselected as candidates for substitution with histidine are referred to herein as "hot" amino acid residues. These are referred to as "spot" residues (Table A). These are amino acid positions within the CDRs or in the framework adjacent to the CDRs. These are key positions where histidines naturally occur in the functional antibody repertoire. It is observed that

[0020] Table A (hot spot residue positions) [Table 9]

[0021] The amino acid residues that are preferably preselected not to be substituted with histidine are those described herein. These are amino acid positions within or near the CDRs. flanking framework positions, and histidines at these positions contribute to the functional antibody repertoire Not observed in

[0022] Table B (Cold spot residue positions) [Table 10]

[0023] The present disclosure provides a method for preparing an engineered antibody that exhibits pH-dependent binding to its antigen, comprising: At least one amino acid residue of the parent antibody is replaced with a histidine residue (which may be abbreviated herein as HIS) and preparing the engineered antibody by replacing The engineered antibody exhibits a pH-dependent cleavage compared to the parent antibody as a result of the histidine substitution. A "parent antibody" is an antibody that exhibits improved selective binding (i.e., a higher pH dependency). The parent antibody can be any antibody capable of binding to the parent. The parent antibody is typically a conventional four-chain immunoglobulin. globulins in which antigen-binding specificity resides in paired VH and VL domains. However, this method can also be used to detect other parent antibodies, as well as antigen-binding fragments, For example, Fab, F(ab'), F(ab')2, Fv, scFv, diabody, triabody, minibody, etc. and any other polypeptides containing an antigen-binding site provided by paired VH and VL domains. It is applicable to engineering pH-dependent binding in engineered immunoglobulin constructs. In the present invention, the parent antibody or its variable domains or CDRs may be, but are not limited to, a llama Camelidae species, including camels, dromedaries, alpacas, vicunas, and guanacos The preparation of camelid monoclonal antibodies is described in detail in the literature, the contents of which are incorporated herein by reference. The parent antibody and its derivatives are described in detail in WO2010 / 001251, which is incorporated herein by reference. Other preferred features of engineered antibodies are described elsewhere herein. The nature of the source is not particularly limited.

[0024] The engineered antibody replaces one or more selected amino acid residues of the parent antibody with histidine. The engineered antibody may have a total of 1, 2, 3, 4, 5, or more amino acid residues compared to the parent antibody. The amino acid sequence may contain more than one histidine substitution. The amino acid residues are typically located in framework positions in or near the CDRs of antibody variable domains. In the case of antibody molecules containing paired VH and VL domains, substitutions with histidines The residues selected for the CDR of the VH domain or the CDR of the VL domain, or both the VH domain and the VL domain, It may be located in both main CDRs.

[0025] As described herein, at least one amino acid position to be substituted with histidine is A selection must be made from the "Hot Spot" list (Table A). , at least two, at least three, at least four, or at least At least five amino acid positions are selected from a "hot spot" list. All histidine substitutions in the engineered antibody were selected from the hotspot list (Table A). That is, only the residues in the hotspot list are used for histidine and ATP. However, in other embodiments, at least one The conditions were to make two histidine substitutions at positions selected from the hot spot list (Table A). Alternatively, histidine substitutions may be included at one or more positions not in the hotspot list. By way of example, as discussed below, in certain embodiments, one or more histidine substitutions can be made to V The amino acid sequence may be included at a "cold spot" position in the H CDR3 and / or VL CDR3.

[0026] As described herein, the parent antibody preferably contains a nucleotide selected for substitution with histidine. These amino acid positions are called "cold spot" positions. The amino acid positions are shown in Table B. The engineered antibody contains a small number of unsubstituted histidines in the cold spot list (Table B). At least one amino acid position must be included. At least two, at least three, at least four or fewer spots on the Spot List (Table B) In a further embodiment, at least five amino acid positions are not substituted with histidine. None of the amino acid positions in the hot spot list (Table B) are substituted with histidine.

[0027] For VH CDR3 and VL CDR3, one or more cold spot amino acid residue positions listed in Table B Histidine residues are also introduced at these positions, and the resulting antibody variant(s) are pH-dependent. It is permissible (and in some embodiments may be beneficial) to test for specific binding. Considering the highly variable nature of the VH CDR3 and VL CDR3, In addition to or instead of these, histidine residues are inserted at cold spot positions in these CDRs. The introduction of the nucleotides into the cold spots in CDR1 and CDR2 improves the antibody structure. VH is more readily tolerated in the presence of ATP and may make an important contribution to pH-dependent antigen binding. Substitution of histidine at the listed cold spot positions in CDR3 and VL CDR3 is All aspects of the invention are acceptable.

[0028] In one specific embodiment, all amino acid residues substituted with histidine are Select from the spot list (Table A) where one or more of the following residues: H100g, H100k, H100 m, H100n, L95, L95d, L95e, L95f, L97 may be further substituted with histidine. do.

[0029] (pH dependent binding) Engineered antibodies having one or more histidine substitutions introduced according to the methods described herein may be , may exhibit pH-dependent binding to target antigens.

[0030] As used herein, the term "pH-dependent binding" in the context of antibody-antigen binding interactions refers to the ability of an antibody to bind to an antigen. This means that the antigen-binding activity of an antibody at a neutral pH is different from that at a neutral pH. .

[0031] Various measures of antigen binding activity were compared between antigen binding activity at acidic pH and antigen binding activity at neutral pH. It can be used as an indicator of the difference between

[0032] In one embodiment, the "affinity" of an antibody for its antigen is used as a measure of antigen binding activity. In a preferred embodiment, the engineered antibody can be used in its acidic pH range. The affinity for the antigen is lower than the affinity of the engineered antibody for that antigen at neutral pH In this case, the engineered antibody exhibits "pH-dependent binding."

[0033] In one embodiment, the dissociation rate constant (k d )(antibody off rate k off (also referred to as "antigen binding activity") can be used as an index of antigen binding activity. The dissociation rate constant (k d ) but operation at neutral pH The dissociation rate constant (k d ), the engineered antibody is H-dependent binding.

[0034] Dissociation rate constant (k d In embodiments where β-glucan is used as an indicator of pH-dependent binding, The dissociation rate constant (k d ) engineered antibody-antibody at neutral pH The dissociation rate constant of the original interaction (k d ) is at least 1.5, or at least 2, or It could be at least 5, or at least 10.

[0035] In other embodiments, the dissociation rate constant of the engineered antibody at acidic pH (e.g., pH 5.5) (k d ) is the dissociation rate constant (k d ) higher than that of such an embodiment. At an acidic pH (e.g., pH 5.5), d is higher than the parent antibody as a result of histidine substitutions In some embodiments, the dissociation rate of an engineered antibody-antigen interaction at acidic pH is higher than that of an antibody-antigen interaction at acidic pH. Degree constant (k d ) of the dissociation rate constant (k d ) ratio can be at least 1.5, or at least 2, or at least 5, or at least 10 Considering the in vivo antibody recycling pathway, dissociation at acidic pH (e.g., pH 5.5) Speed ​​(k d ) is a particularly valid measure of pH-dependent binding. Faster dissociation of antibody complexes allows antigen release in acidified endosomes .

[0036] In one embodiment, the equilibrium dissociation constant (K D ) should be used as an index of antigen binding activity. In a preferred embodiment, the equilibrium of the engineered antibody-antigen interaction at acidic pH can be Dissociation constant (K D ) is the equilibrium dissociation constant (K D )twist If the pH is also high, the engineered antibody exhibits "pH-dependent binding."

[0037] Equilibrium dissociation constant (K D In embodiments where β-glucan is used as an indicator of pH-dependent binding, The equilibrium dissociation constant (K D) engineered antibody-antibody at neutral pH The equilibrium dissociation constant (K D ) is greater than 1.5, or greater than 2, or greater than 5, or greater than 10. In a particularly preferred embodiment, the engineered antibody has a specificity for antigen binding at acidic pH. It can exhibit 20 to 40 times stronger antigen binding at neutral pH.

[0038] Equilibrium dissociation constant (K D In a non-limiting embodiment, The engineered antibody has a specific binding affinity to its antigen in the range of 10-20 nM at an acidic pH (e.g., pH 5.5). Equilibrium dissociation constant (K D ), whereas engineered antibodies may exhibit a neutral pH (e.g., pH 7.4 ) for that antigen D can be approximately 0.5 nM or less.

[0039] Other measures of antigen binding activity that can be used to indicate pH-dependent binding include affinity constants or affinity constants. Combined rate constant (k a ) or on-speed (k on ) is possible.

[0040] For antagonist antibodies, the measure of antigen binding activity is blocking potency, a preferred assay The IC50 at

[0041] In a preferred embodiment, the engineered antibody containing one or more histidine substitutions is a nucleotide sequence similar to that of the parent antibody. The "higher pH dependency of binding" means that The difference in antigen binding activity at acidic pH and neutral pH is This means that it is significantly greater than that of the parent antibody from which it is derived.

[0042] In some embodiments, the parent antibody exhibits a significant pH dependence of binding to its target antigen. The pH dependence was not introduced into the engineered variant as a result of one or more histidine substitutions. In other embodiments, the parent antibody may exhibit a measurable pH dependency, and this pH dependency The dependency was significantly higher in the engineered variants as a result of one or more histidine substitutions. do.

[0043] In one embodiment, the antigen-antibody binding activity at neutral pH is not significantly impaired. In some cases, it may be possible to introduce pH dependency into the resulting antibody. In such an embodiment, The engineered antibodies have significantly lower antigen-binding activity at acidic pH compared to the parent antibodies from which they are derived. whereas the antigen binding activity of the engineered antibody at neutral pH may be comparable to that of its origin at neutral pH. The antigen-binding activity of the antibody may be comparable to (i.e., not significantly different from) the antigen-binding activity of the parent antibody.

[0044] The amino acids that are candidates for histidine substitution as described herein are used to identify the functional antibody repertoire. One advantage of selecting according to the "heat map" of natural occurrence of histidine in Histidine residues are typically "hot spots" where they may naturally occur in the antibody repertoire. The goal is to introduce the nucleotide sequence only into "hot" amino acid positions and thus tolerate it in the antibody structure. Histidine substitution at the spot position significantly affected the strength of antigen binding activity at neutral pH. It has been shown that the pH dependence of antigen binding can be imparted without adversely affecting the binding affinity of the antibody.

[0045] In one embodiment, the engineered antibody exhibits an increased pH dependence of binding to its antigen compared to the parent antibody. The antibody exhibits pH-dependent activity while retaining at least 80% of the antigen-binding activity of the parent antibody at neutral pH. In some embodiments, the engineered antibody has at least the antigen-binding activity of the parent antibody at neutral pH. While still retaining 80%, at least 85%, at least 90%, at least 95%, or 100% indicates pH-dependent binding.

[0046] In a preferred embodiment, the engineered antibody has higher binding to its antigen compared to the parent antibody. while exhibiting a pH dependence of at least 80% of the affinity of the parent antibody for the antigen at neutral pH. At least 85%, at least 90%, at least 95%, or 100% are retained.

[0047] Equilibrium dissociation constant (K D In an embodiment in which the engineered The antibody reacts with the parent antibody against the target antigen at neutral pH. D 20% or less, or 15% or less, or 10% Equilibrium dissociation constant (K) for the target antigen at neutral pH less than or equal to 5% greater than D ) can be shown. In some embodiments, the engineered antibody has a K D and in reality The equilibrium dissociation constant (K) for the target antigen at approximately equal neutral pH D ) can be shown.

[0048] In other embodiments, the introduction of pH dependency allows for the engineered targeting of the antigen at neutral pH. However, the desired pH dependence of binding is achieved. The nature of the target antigen, the intended use of the antibody, and also the properties of the parent antibody, are taken into consideration, In some cases, a loss of affinity at neutral pH may be acceptable. For example, if the parent antibody has If the antibody shows particularly high affinity for the antigen, this is accompanied by a significant improvement in pH dependence. (i.e., the desired "fold difference" between binding at neutral pH and binding at acidic pH is achieved. A decrease in the affinity of the engineered antibody for its antigen at neutral pH is acceptable (provided that In some embodiments, the desired pH dependence of binding can be achieved, provided that the desired pH dependence of binding is also achieved. Thus, the affinity of an engineered antibody for its target antigen at neutral pH is the affinity of that antigen at neutral pH. The affinity of the parent antibody for the target molecule may be up to 10-fold lower, or even 20-fold lower.

[0049] In each of the above embodiments, "acidic pH" refers to a pH between 4.0 and 6.5, preferably between 4.5 and 6.5. pH 6.0, more preferably pH 5.5 to pH 6.0, and more preferably pH in the range of pH 5.5 to pH 5.8 Refers to...

[0050] In each of the above embodiments, "neutral pH" refers to a pH between 6.7 and 10.0, preferably 7.0. It refers to a pH in the range of ∼pH 8.0, more preferably 7.4.

[0051] In certain embodiments, pH-dependent binding refers to the ability of an antibody to bind antigen at an acidic pH of 5.5 to bind to a neutral This can be evaluated by comparing the antigen-binding activity of the antibody with that of the antibody at pH 7.4.

[0052] In other embodiments, pH-dependent binding refers to the ability of an antibody to bind antigen at acidic pH 6.0 to bind antigen at neutral pH. The antigen-binding activity of the antibody can be evaluated by comparing it with that of the antibody in 7.4.

[0053] Suitable methods and conditions for determining the antigen-binding activity of an antibody at a defined pH are generally As is known in the art, the antigen-binding activity of an antibody at a defined pH can be calculated by: ELISA was performed using Biacore® (GE Healthcare) as described in the accompanying experimental examples. or using the MSD platform.

[0054] (Histidine occurrence rate heat map) The amino acid residues of the parent antibodies described herein are those that correspond to the CD of functional antibodies in the natural antibody repertoire. Based on a “heat map” of the occurrence of histidine residues at specific amino acid positions in R. It may be selected for substitution with histidine or may not be substituted with histidine. This "heat map" shows the locations in the CDRs where histidines can naturally occur in the antibody repertoire. a list of "hot spot" amino acid residue positions and framework positions near the CDRs, and Also, there are "cold spot" amino acids in the CDRs where histidines typically do not occur naturally. Both the acid residue positions and a list of framework positions near the CDRs are provided.

[0055] "Antibody repertoire" refers to a population of antibodies that exhibit antigen-binding activity. A "cluster" is a population of antibodies typically produced by immunization of a host with a target antigen. These repertoires may contain antibodies against more than one target antigen. The histidine frequency "heat map" itself represents a host response using multiple different target antigens. It is derived from the diverse repertoire of llama antibodies induced by animal immunization. Antibodies isolated from these immunizations were screened by specific phage display selection. This allowed for enrichment of antibodies that exhibit pH-dependent antigen binding.

[0056] (Selection of residues to be replaced) For any given parent antibody, the selection of candidate amino acid residues for substitution with histidine is determined by the histidine Guided by a heat map of stigma occurrence rates.

[0057] The term "substitution" or "replacement" of an amino acid residue and are used interchangeably herein. An engineered antibody that is an engineered variant of a parent antibody In this context, a "substitution" refers to an amino acid residue at a defined amino acid position in a parent antibody that is different from the amino acid residue at the parent antibody. an amino acid residue, i.e., a histidine residue, that does not naturally occur at that position in the parent antibody; It is required that

[0058] The histidine occurrence "heat map" described herein shows the natural occurrence of histidine. The present invention provides a list of hotspot amino acid positions where the antibody is observed to be highly responsive to the antibody's specificity. The parent antibody from which it is derived must contain a hotspot at one or more amino acid positions on the hotspot list. In these situations, the natural histidine residues are pH-dependent. These may contribute to specific antigen binding and will generally be retained in engineered antibodies. However, in the methods described herein, this naturally occurring histidine is a histidine substitute. Therefore, the methods described herein do not include the use of engineered antibodies. At least one additional amino acid at one or more hotspot amino acid positions compared to the parent antibody It is required that the engineered antibody must contain one or more histidine residues. The introduction of an additional histidine residue in Even with respect to the parent antibody, the pH-dependent binding of the engineered antibody may be improved (i.e., higher (resulting in a pH-dependent binding to the target antigen).

[0059] Candidate amino acid residues to be substituted with histidine are amino acid positions in the CDRs of the VH and VL domains. and framework positions near the CDRs are numbered according to the KABAT numbering system (Kabat et al., "Immunological Objects"). "Sequences of Proteins of Immunological Interest," 5th ed. , Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Therefore, the engineered antibodies are selected from a list of identified "hot spots" (Table A). It must contain at least one histidine substitution at an amino acid position selected from Table A. stomach.

[0060] Various approaches can be used to confer pH-dependent antigen binding to the parent antibody of interest. The most appropriate histidine substitution can be selected based on the binding interaction of the parent antibody with its target antigen. Hot spots for histidine substitutions are identified when structural information about their effects is available. Using a "rational design" approach to select candidate amino acids from a trial list It may be appropriate to use a protein that is directly involved in the antigen-antibody binding interaction or that is located at the antigen-binding interface of the parent antibody. Hotspot residues that are likely to affect the overall structure of the protein are identified based on such structural information. Subsequently, one or more selected hot spot residues or their equivalents can be selected based on the These different combinations were used to synthesize engineered variants of the parent antibody with histidine substitutions. For example, screening for pH-dependent antigen binding using the screening methods described herein can be performed. It can be leaned.

[0061] Candidate residues for substitution with histidine are also selected from the family or subfamily of variable domains of the parent antibody. In this specification, the antibody repertoire can be selected based on its subtype. Various subtype-specific hot spots derived from histidine frequency heatmaps In one embodiment, the VH and / or VL domains of the parent antibody are listed as The variable domain family or subtype is known and candidate residues for substitution with histidine are identified. Groups are selected from the following subtype-specific or family-specific hotspot lists: These lists are not intended to be limiting, but merely to illustrate the specific variables. Domain families or subtypes most suitable for substitution with histidine For any given variable domain, the amino acid positions considered are provided as examples. The amino acid positions for possible histidine substitutions depend on the family or subtype of the variable domain. Regardless, you can choose from the complete hotspot list (Table A).

[0062] For heavy chain variable domains of the VH3 family: Table VH3A (Hotspot residues of the VH3 family) [Table 11] Table VH3B (Cold spot residues in the VH3 family) [Table 12]

[0063] For heavy chain variable domains of the VH1 family: Table VH1A (Hotspot residues of the VH1 family) [Table 13] Table VH1B (Cold spot residues in the VH1 family) [Table 14]

[0064] For heavy chain variable domains of the VH4 family: Table VH4A (Hotspot residues of the VH4 family) [Table 15] Table VH4B (Cold spot residues in the VH4 family) [Table 16]

[0065] For λ-type light chain variable domains: Table VλA (Hotspot residues of Vλ) [Table 17] Table VλB (cold spot residues of Vλ) [Table 18]

[0066] For the light chain variable domain of the Vλ1 family: Table Vλ1A (Hotspot residues of the Vλ1 family) [Table 19] Table Vλ1B (cold spot residues in the Vλ1 family) [Table 20]

[0067] For the light chain variable domain of the Vλ2 family: Table Vλ2A (Hotspot residues of the Vλ2 family) [Table 21] Table Vλ2B (Cold spot residues in the Vλ2 family) [Table 22]

[0068] For the light chain variable domain of the Vλ3 family: Table Vλ3A (Hotspot residues of the Vλ3 family) [Table 23] Table Vλ3B (Cold spot residues in the Vλ3 family) [Table 24]

[0069] For light chain variable domains of the Vλ5 family: Table Vλ5A (Hotspot residues of the Vλ5 family) [Table 25] Table Vλ5B (Cold spot residues in the Vλ5 family) [Table 26]

[0070] For the light chain variable domain of the Vλ8 family: Table Vλ8A (Hotspot residues of the Vλ8 family) [Table 27] Table Vλ8B (Cold spot residues in the Vλ8 family) [Table 28]

[0071] For kappa-type light chain variable domains: Table VKA (Hot Spot Residues of VKA) [Table 29] Table VKB (VK cold spot residues) [Table 30]

[0072] Regarding the VK1 family light chain variable domain: Table VK1A (Hotspot residues of the VK1 family) [Table 31] Table VK1B (Cold spot residues in the VK1 family) [Table 32]

[0073] Regarding the VK2 family light chain variable domain: Table VK2A (Hotspot residues of the VK2 family) [Table 33] Table VK2B (Cold spot residues in the VK2 family) [Table 34]

[0074] For the VK4 family light chain variable domain: Table VK4A (Hotspot residues of the VK4 family) [Table 35] Table VK4B (Cold spot residues in the VK4 family) [Table 36]

[0075] (Screening of a panel of variants) A particular embodiment of the method of the invention comprises constructing a panel of engineered variants of a parent antibody; The assay is then performed to identify one or more engineered variants that exhibit the desired pH-dependent binding. The method can be based on screening a panel of variants. For example, a number of possible histidine substitutions at amino acid positions defined in the heat map can be systematically This method allows for the construction of a panel of variants in a systematic manner. The heat map of histidine occurrence and thus candidates for histidine substitution were used as a guide. By sampling all possible amino acid positions and their combinations in the CDRs, The prior art does not require or make it unreasonable to do so. It is different from the binary method.

[0076] The systematic mutation analysis method described herein generally involves: (a) providing a parent antibody that binds to an antigen; (b) preparing a panel of engineered variants of the parent antibody, Each of the engineered variants has at least one amino acid residue substituted with a histidine residue. The antibody differs from the parent antibody by having a hotspot selected from a list of hotspots (Table A). At least one amino acid residue in the At least one amino acid residue selected from the list (Table B) is substituted with histidine. The step of not (c) screening the panel of engineered variants for pH-dependent binding to the antigen; and performing pH-dependent binding analysis on the antigen to identify engineered antibodies that exhibit pH-dependent binding to the antigen. nothing.

[0077] This method involves preparing a panel of engineered variants of a parent antibody of interest. Each engineered variant in the panel is selected such that at least one amino acid is a histidine. It differs from the parent antibody by substitution of histidine. The amino acid position selection was based on a heatmap of histidine occurrence. Each variant contains a small number of variants at amino acid positions selected from the hotspot list (Table A). It must contain at least one histidine substitution, while the cold spot restriction At least one amino acid residue selected from the amino acid sequence (Table B) is substituted with histidine. Preferably, each of the engineered variants in the panel has a different pattern of histidine. Include histidine substitutions in the nucleotide sequence, so that the effect of different histidine substitutions on pH-dependent antigen binding can be assessed. This allows the results to be analyzed in parallel.

[0078] In one embodiment of the method, each of the engineered variants in the panel is It may contain a single histidine substitution at one of the hotspot amino acid positions (Table A). , each of the engineered variants in the panel is located at the listed hotspot amino acid position ( Such a panel allows for the identification of individual hot spots. It is possible to analyze the effect of histidine substitution at the 5 amino acid position on pH-dependent binding. The panel of engineered variants can represent the entire hotspot list. That is, variants having a single histidine substitution at each of the amino acid positions listed in Table A. or a selected subset of the hotspot list, e.g., one or more Subsets of substitutions associated with particular variable domain families or subtypes Furthermore, the panel may represent one or more "cold spots" in the VH CDR3 or VL CDR3. " position.

[0079] In other embodiments, the panels prepared in step (b) include 2 or more, 3 or more, 4 or more, or 5 or more. These may include engineered variants containing various combinations of the above histidine substitutions. At least one of the substitutions must be at a hotspot amino acid position selected from Table A. while at least one amino acid from Table B is not substituted with histidine. Preferably, all of the histidine substitutions are hot spot substitutions selected from Table A. This panel identifies combinations of histidine residues that confer pH-dependent binding. Screening combinations (and especially combinations of hotspot histidine substitutions) Furthermore, the panel may contain one or more "cold spots" in the VH CDR3 or VL CDR3. Histidine substitutions at the "hot spot" positions, optionally with histidine substitutions at one or more hot spot amino acid positions. The present invention may include variants containing nucleotide substitutions in combination with nucleotide substitutions.

[0080] In one embodiment, substituting with histidine to confer pH-dependent binding. To identify a single amino acid position where the initial screening can be performed, a single A panel of engineered variants containing single histidine substitutions can be performed. Based on the results of this initial screening, it has already been identified as conferring pH-dependent binding. and constructing one or more further engineered variants combining the engineered histidine substitutions. Such methods are: (a) identifying a parent antibody that binds to the antigen; (b) preparing a panel of engineered variants of the parent antibody, Each of the engineered variants has at least one amino acid residue substituted with a histidine residue. The antibody differs from the parent antibody by having a hotspot selected from a list of hotspots (Table A). At least one amino acid residue in the At least one amino acid residue selected from the list (Table B) is substituted with histidine. wherein preferably each of the engineered variants has a different pattern containing histidine substitutions); (c) screening the panel of engineered variants for pH-dependent binding to the target antigen; The selection is performed by screening, whereby the presence of histidine confers pH-dependent binding to the antigen. identifying the selected amino acid position; (d) preparing one or more further engineered variants of said parent antibody, each of the ants contains a histidine at two or more of the selected amino acid positions identified in step (c). , the process; and (e) screening the further engineered variants for pH-dependent binding to the antigen thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen. obtain.

[0081] In certain embodiments of the method, the histidine substitutions in the VH and VL domains are The effects on these pH-dependent bindings can be analyzed separately. wherein step (b) comprises preparing a panel of engineered variants of the parent antibody; wherein each of the engineered variants in the panel comprises at least one amino acid sequence in the VH domain. The heavy chain hot spots differ from the parent antibody by the substitution of histidine residues for the acid residues. At least one amino acid residue selected from the pot list (Table HA) is substituted with histidine. and at least one heavy chain cold spot selected from the heavy chain cold spot list (Table HB). Furthermore, the panel may also include the steps of: Variants containing histidine substitutions at "cold spot" positions in VH CDR3 may be included.

[0082] Typically, the engineered variants contain one or more histidine residues paired with the VL domain of the parent antibody. Using such a library, the VH domain can be identified by a mutant VH domain containing a nucleotide substitution. This allows screening for histidine substitutions that confer pH-dependent binding in proteins. .

[0083] Table HA (VH hotspot residue positions) [Table 37] Table HB (VH cold spot residue positions) [Table 38]

[0084] In a further embodiment, step (b) comprises selecting a panel of engineered variants of said parent antibody. preparing a panel of engineered variants, each of which has a small number of variants in the VL domain; and a histidine residue in at least one amino acid residue of the parent antibody. The light chain hotspots are different from the light chain hotspots in that at least one amino acid residue selected from the light chain hotspot list (Table LA) is present. A few amino acids selected from the light chain hotspot list (Table LB) are substituted with stidine. and wherein at least one amino acid residue is not substituted with histidine. Additionally, the panel contains variants containing histidine substitutions at "cold spot" positions in VL CDR3. may include:

[0085] Typically, the engineered variants contain one or more histidine residues paired with the VH domain of the parent antibody. Using such a library, mutant VL domains containing amino acid substitutions can be obtained. This allows screening for histidine substitutions that confer pH-dependent binding in proteins. .

[0086] Table LA (VL hotspot residue positions) [Table 39] Table LB (VL cold spot residue positions) [Table 40]

[0087] For light chain variants of the λ class, the “hot spot” and “cold spot” residues The base position can be selected from the following list: Table VλA (Vλ hot spot residues) [Table 41] Table VλB (Vλ cold spot residues) [Table 42]

[0088] For κ class light chain variants, "hot spot" and "cold spot" residues The base position can be selected from the following list: Table VKA (VK hotspot residue positions) [Table 43] Table VKB (VK cold spot residue positions) [Table 44]

[0089] In one embodiment, the VH and VL domains identified by separate initial screening By combining the histidine substitutions of the VH and VL domains in a single engineered variant, Further screening can then be carried out to identify VH and VL that exhibit pH-dependent binding. Such a method can identify combinations of: (a) identifying a parent antibody that binds to the antigen; (b) preparing a first panel of engineered variants of the parent antibody, Each of the engineered variants in the set has at least one amino acid residue in the VH domain. The heavy chain hot spot linker differs from the parent antibody by being substituted with a histidine residue. At least one amino acid residue selected from the group consisting of α- and β-amino acids (Table HA) is substituted with histidine. and at least one amino acid selected from the heavy chain cold spot list (Table HB). the residue is not substituted with histidine; (c) screening the first panel of engineered variants for pH-dependent binding to the antigen; VH domains whereby the presence of histidine confers pH-dependent binding identifying one or more selected amino acid positions in the (d) preparing a second panel of engineered variants of the parent antibody, Each of the engineered variants in the set has at least one amino acid residue in the VL domain. The light chain hot spot linker differs from the parent antibody by being substituted with a histidine residue. At least one amino acid residue selected from the group consisting of 1 to 3 amino acids (Table LA) is substituted with histidine. and at least one amino acid residue selected from the light chain hotspot list (Table LB). the group is not substituted with histidine; (e) screening the second panel of engineered variants for pH-dependent binding to the antigen; VL domains whereby the presence of histidine confers pH-dependent binding identifying one or more selected amino acid positions in the (f) preparing one or more further engineered variants of said parent antibody, each of the ants having a sequence identical to that of one or more selected amino acid positions in the VH domain identified in step (c) which differs from the parent antibody by having an amino acid substitution with histidine, and in step (e) The amino acid at one or more selected amino acid positions in the identified VL domain is replaced with histidine. the step of: (g) screening the further engineered variants for pH-dependent binding to the antigen thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen. obtain.

[0090] This approach allows for simultaneous screening of initial (individual) VH and VL domains. It is possible to identify synergistic effects between the identified histidine mutations. To identify synergistic effects between different combinations of VL domain mutations, we used the most powerful The most highly selective VH mutations (i.e., those that confer the greatest pH dependence) were analyzed using a number of different Combine VL mutations or combine the best performing VL mutations with different VH mutations In this embodiment, the VH CDR3 or VL CDR3 and the " It is permissible to include histidine substitutions at "cold spot" positions as well.

[0091] (Screening method) Using various screening methods, engineered antibodies that exhibit pH-dependent binding to target antigens were identified. Such screening methods can: (a) determining the antigen-binding activity of the engineered antibody at acidic pH; (b) determining the antigen-binding activity of the engineered antibody at neutral pH; (c) An engineered antibody whose antigen-binding activity at acidic pH differs from its antigen-binding activity at neutral pH. selecting.

[0092] Suitable methods and conditions for determining antigen binding activity at a defined pH are generally known in the art. For example, the antigen binding activity of an antibody at a defined pH can be determined by the following methods: The results were determined by Biacore® (GE Healthcare) as described in the experimental examples. Other suitable techniques include ELISA or MSD, which can be used to measure the activity of different pH conditions. (e.g., pH 7.4 and pH 5.5) and determine antigen binding at a particular antibody concentration (e.g., EC50) It is possible.

[0093] The methods described herein measure the antigen binding activity of engineered antibodies at neutral and acidic pH. and performing a pH-dependent binding assay at a neutral pH to determine whether the engineered antibody exhibits pH-dependent binding. and / or comparing one or more parameters of binding at acidic pH.

[0094] In some embodiments, the antigen binding activity of the engineered antibodies is determined as described in the accompanying Examples. In this setup, the pH of the antibody can be measured by a dual pH ELISA. Initial binding to the body antigen is assessed at neutral pH (e.g., pH 7.4), followed by acidic pH (e.g., Washing steps are performed at pH 5.5 (e.g., pH 5.5). This allows for a rapid response to antibody association, dissociation, or both. This allows us to distinguish between the effects of pH on the binding of antibodies to their target antigens, which occurs at neutral pH and This may better represent the in vivo situation where antigen release occurs at an acidic intraendosomal pH. do.

[0095] Typically, the engineered antibody selected will bind to its antigen less at acidic pH than at neutral pH. In the initial screening, the affinity at acidic pH was Any significant difference in affinity may indicate pH-dependent binding.

[0096] The dissociation rate constant (k d ), i.e., off-rate is the dissociation rate constant (k d ) In the initial screening, (k d ) at neutral pH (k d ) Any significant difference may indicate pH-dependent binding. The dissociation rate constant (k d ) operation at neutral pH The dissociation rate constant (k d ) should be at least 1.5 or less. The number of histidine mutations can be at least 2, or at least 5, or at least 10. In combination, the overall pH dependence may increase.

[0097] The equilibrium dissociation constant (K D ) but the operation at neutral pH The equilibrium dissociation constant (K D ) may be higher than the initial screen. In the cleaning, K at acidic pH D K at neutral pH D Even if there is a significant difference Equilibrium of engineered antibody-antigen interactions at acidic pH can be shown. Dissociation constant (K D ) of the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at neutral pH D ) The ratio can be greater than 1.5, or greater than 2, or greater than 5, or greater than 10.

[0098] Combining multiple histidine mutations can increase the overall degree of pH dependence. In a particularly preferred embodiment, the histidine mutations are combined to improve the activity at acidic pH. Approximately 20 to 40 times stronger antigen binding can be achieved at neutral pH.

[0099] As described elsewhere herein, improved pH-dependent binding is achieved by increasing the affinity of the antibody to the target antigen at neutral pH. This can be achieved with or without a significant effect on compatibility.

[0100] Equilibrium dissociation constant (K D In a non-limiting embodiment, The engineered antibody has a specific binding affinity to its antigen in the range of 10-20 nM at an acidic pH (e.g., pH 5.5). Equilibrium dissociation constant (K D ), whereas engineered antibodies may exhibit a neutral pH (e.g., 7.4) K for that antigen at D can be approximately 0.5 nM or less.

[0101] In each of the above embodiments, "acidic pH" refers to a pH between 4.0 and 6.5, preferably between 4.5 and 6.5. pH 6.0, more preferably pH 5.5 to pH 6.0, and more preferably pH in the range of pH 5.5 to pH 5.8 Refers to...

[0102] In each of the above embodiments, "neutral pH" refers to a pH between 6.7 and 10.0, preferably 7.0. It refers to a pH in the range of ∼pH 8.0, more preferably 7.4.

[0103] In certain embodiments, pH-dependent binding refers to the ability of an antibody to bind antigen at an acidic pH of 5.5 to bind to a neutral This can be evaluated by comparing the antigen-binding activity of the antibody with that of the antibody at pH 7.4.

[0104] In other embodiments, pH-dependent binding refers to the ability of an antibody to bind antigen at acidic pH 6.0 to bind antigen at neutral pH. The antigen-binding activity of the antibody can be evaluated by comparing it with that of the antibody in 7.4.

[0105] The methods described herein may also or alternatively involve the preparation of parent antibodies at acidic and / or neutral pH. and measuring the antigen binding activity of the antibody, and the parameters of the binding interaction are manipulated. - comparing the results with the corresponding binding parameters of the antigen interaction.

[0106] The dissociation rate constant (k) of the engineered antibody at acidic pH (e.g., pH 5.5)d ) at the same acidic pH The dissociation rate constant (k d In such an embodiment, the acid k at pH (e.g., pH 5.5) d is higher compared to the parent antibody as a result of the histidine substitutions. The dissociation rate constant (k) of the engineered antibody-antigen interaction at acidic pH d ) at the same acidic pH The dissociation rate constant of the antibody-antigen interaction (k d ) is at least 1.5, or at least 2 , or at least 5, or at least 10.

[0107] (engineered antibodies) Also provided herein are engineered antibodies that exhibit pH-dependent binding to their antigens, wherein the engineered antibodies At least one amino acid in the CDR of the antibody is a histidine residue, and the hot spot at least one amino acid residue selected from the list (Table A) is a histidine residue; and at least one amino acid residue from the cold spot list (Table B) is a histidine residue. The engineered antibody is characterized in that it is not a group.

[0108] An "engineered antibody" is an antibody whose amino acid sequence has been intentionally altered in vitro or In the present disclosure, "engineered antibody" refers to an antibody that has been mutated. The amino acid sequence of the CDR of or mutated antibody variants.

[0109] The CDRs of antibody VL and VH domains are typically defined as comprising the following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2), and 89- 97 (CDRL3), and residues 31-35 or 31-35c in the heavy chain variable domain (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3) (Kabat et al., "Proteins of Interest in Immunology"). "Sequences of Proteins of Immunological Interest," 5th ed., Public Health h Service, National Institutes of Health, Bethesda, MD. (1991)).

[0110] The engineered antibody may be composed of a VH domain, a VL domain, or both a VH and a VL domain. A total of 1, 2, 3, 4, 5, or more histidine residues may be present in either CDR.

[0111] At least one histidine residue in the CDR of the engineered antibody is a "hot spot" residue. In one embodiment, the amino acid sequence of the present invention is selected from the amino acid sequence of the present invention (Table A). and at least two, at least three, at least four, or at least five histidine residues. The group must appear at an amino acid position selected from the hot spot list. In some embodiments, all histidine residues in the engineered antibody are included in the hot spot list. (Table A). In the CDRs of the antibody, specific amino acid positions preferably not occupied by histidine residues are These amino acid positions are shown in the "cold spot" list (Table B). The engineered antibodies are listed in the cold spot list (Table B). It must contain at least one amino acid position not occupied by a histidine residue. In some embodiments, at least two cold spots on the cold spot list (Table B) are present. At least three, at least four, or at least five amino acid positions are not histidines In a further embodiment, the amino acid positions in the cold spot list (Table B) As an exception to this, in certain embodiments, the VH CDR3 or VL CDR4 is Also include histidine substitutions in CDR3 and in "cold spot" positions in these combinations. Yes, that's fine.

[0112] The engineered antibodies exhibit pH-dependent binding to their antigens, which is due to the fact that the antibodies bind to their antigens at acidic pH. This means that the binding activity is different from the antigen-binding activity of an antibody at neutral pH.

[0113] In one embodiment, the engineered antibody binds to its antigen at acidic pH better than at neutral pH. has low affinity.

[0114] In one embodiment, the dissociation rate constant (k d ) is the dissociation rate constant (k d ) higher than

[0115] In one embodiment, the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at acidic pH is D ) is the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at neutral pH. D ) higher than

[0116] In a particularly preferred embodiment, the engineered antibodies exhibit a neutral pH response to antigen binding at acidic pH. It can exhibit 20 to 40 times stronger antigen binding.

[0117] Equilibrium dissociation constant (K D In a non-limiting embodiment, The engineered antibody has a specific binding affinity to its antigen in the range of 10-20 nM at an acidic pH (e.g., pH 5.5). Equilibrium dissociation constant (K D ), whereas engineered antibodies may exhibit a neutral pH (e.g., 7.4) K for that antigen at D can be approximately 0.5 nM or less.

[0118] (Engineered antibody structure) The engineered antibodies described herein typically have paired antigen binding specificities and six CDRs. Conventional four-chain immunoglobulins, with VH and VL domains contributing to the antigen-binding site However, the term "engineered antibody" also includes, but is not limited to, Includes Fab, F(ab'), F(ab')2, Fv, scFv, diabodies, triabodies, minibodies, etc. Antigen-binding fragments of conventional immunoglobulins and engineered antigen-binding constructs, including Any other modified immunoglobulin containing an antigen-binding site provided by the modified VH and VL domains. It encompasses the globulin structure.

[0119] The engineered antibody may comprise one or more of a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In particular, engineered antibodies may contain one or more antibody effector constant regions. The antibody may include an Fc region (composed of CH2 and CH3 domains) that may confer antibody function. The Fc region of an engineered antibody is itself engineered or modified to impart useful functional properties. It can be done.

[0120] The engineered antibodies may be of any antibody class, including: IgA, IgD, IgE, IgG, or IgM. In a preferred embodiment, the engineered antibody is an IgG. The antibodies used are: IgG of any subclass (isotype), including IgG1, IgG2, IgG3, or IgG4. It is possible.

[0121] In a preferred embodiment, the engineered antibody binds to the neonatal Fc receptor (FcRn). In particular, the Fc region may contain an Fc region that exhibits strong binding affinity to FcRn at neutral pH. It may be desirable to engineer the Fc region of an antibody to bind to the neonatal Fc receptor, FcRn. Fc modifications that confer enhanced binding to FcRn Examples include amino acid substitutions at one or more amino acid positions in the CH2 and / or CH3 domains of the Fc region. There is.

[0122] Suitable Fc mutations include those described in Vaccaro et al., Nature Biotechnology, Vol. 23, 1283-1288. 8 (2005). The Abdeg™ mutation is a human I Engineered variants of gG, including but not limited to Met252, Ser254, and Thr256 , His 433, and Asn434 to Tyr252, Thr254, Glu256, Lys433, and Phe434 (EU numbering) Abdeg™ engineered antibodies with mutations that For example, engineered human IgG1) exhibits enhanced binding to FcRn compared to its wild-type counterpart. It exhibits affinity and binds more stably to FcRn during exocytosis events at the cell surface. Preferred engineered antibodies as defined herein include those containing one or more histidine residues in the CDRs of the variable domains. The ATP substitution is combined with an Abdeg mutation in the Fc region.

[0123] Further suitable Fc mutants include the NHance™ mutants described in US 8,163,881. ance™ mutants are engineered variants of human IgG, including but not limited to: Human IgG containing mutations that change His 433 and Asn434 to Lys433 and Phe434 (EU numbering) 1. Engineered antibodies (e.g., engineered human IgG) with NHance™ mutations 1) exhibits enhanced binding affinity to FcRn compared to its wild-type counterpart. The defined preferred engineered antibodies comprise one or more histidine substitutions in the CDRs of the variable domains of the Fc Combined with the NHance™ mutation in the region.

[0124] In a preferred embodiment, the engineered antibody contains one or more histidine substitutions in the CDRs. Engineered variants of the variable domains of camelid-derived antibodies (e.g., llama antibodies). The variable domains (VH and VL) contain the Abdeg™ mutations Tyr252, Thr254, Glu256, L The Fc region may comprise the Fc region of human IgG (e.g., human IgG1) including ys433, and Phe434 (EU numbering).

[0125] In a further preferred embodiment, the engineered antibody contains one or more histidine substitutions in the CDRs. engineered variants of the variable domains of camelid-derived antibodies (e.g., llama antibodies), including The variable domains (VH and VL) are nucleotides containing the NHance™ mutations Lys433 and Phe434 ( The Fc region may comprise the Fc region of human IgG (e.g., human IgG1) including EU numbering.

[0126] Engineered to exhibit dual properties: pH-dependent antigen binding and enhanced FcRn binding affinity The antibodies can exert an active antigen-cleaving effect, removing soluble antigens from plasma in vivo. The concept of "active antigen removal" is based on the use of engineered antibodies. , which mimics the activity of cell surface endocytic receptors, thereby allowing the release of endocytic proteins from plasma. This allows for selective elimination of the target antigen. It binds to the corresponding antigen present in plasma (neutral pH) to form an antigen-antibody complex. The complexes are randomly taken up (usually by pinocytosis) into endosomes, where the pH becomes acidic. Once activated, the antibody Fc portion binds to FcRn. Therefore, the bound antigen is released into the acidic endosome and directed for degradation via the lysosomal pathway. Free antibody (still bound to FcRn but no longer bound to its antigen) continues. are recycled back to the cell surface where they undergo further rounds of antigen binding and internalization It can participate in FcRn binding at acidic pH without substantially affecting binding at pH 7.4. Fc mutations that promote binding to IgG4 can enhance this "active antigen removal" process. can.

[0127] In addition to the specific Fc mutations listed above, engineered antibodies also exhibit the ability to enhance the endosomal Improved uptake into the Fc system, e.g., pI change, charge change, Fc at pH 7.4 and / or pH 6.0 any other mutations or modifications that involve changes in Rn affinity; any other "highly efficient recyclers" Increased binding to high recycling Fc receptors, such as Fcγ receptors, especially FcγRIIb any mutation or modification that enhances immune complex formation (e.g., For example, a complement binding site may be included.

[0128] The engineered antibodies may be derived from human, mouse, rat, rabbit, camelid, or other mammalian species. The antibody may be an engineered variant of a parent antibody from any species, or a chimeric antibody. Therefore, the engineered antibodies can be used in camelid species, e.g., llamas, camels, dromedaries, and guinea pigs. Engineered variants or variable domains of camelid antibodies isolated from camelids, such as vicunas, The constant domain (or its CDRs) is derived from a camelid species (e.g., llama), and the constant domain is engineered camelid-human chimeric antibodies (e.g., llama-human chimeras) derived from humans; Camelid antibodies and camel-human chimeric antibodies can be used in combination. The production techniques are described in WO 2010 / 001251, the contents of which are incorporated herein by reference. It is listed.

[0129] The antigen-binding specificity of an engineered antibody, i.e., the nature of the target antigen to which it binds, is particularly important. Applicants believe that the methodology provided herein is not limited to the use of a large number of different antigen-binding proteins. We demonstrated that this method can be applied to engineer pH-dependent variants of various parent antibodies. The described techniques for engineering differential antigen binding serve as principles of general applicability and are not intended to be used in any particular The present invention is not limited to antibodies that bind to specific target antigens.

[0130] (Example) The present invention is further illustrated by the following examples, which should not be construed as further limiting. All references, patents and patent applications cited throughout this application are hereby incorporated by reference. The contents of the published patent application are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0131] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 illustrates pH-dependent antigen binding of engineered variants of parent antibody 18E2 as measured using a functional assay. The IC50 for inhibition of the ligand:receptor interaction was measured for parent antibody 18E2 and engineered variants of 18E2 containing one or two defined histidine mutations at both pH 7.4 and pH 5.5. [Figure 2] 1 illustrates the pH-dependent binding of histidine mutants of the parent antibody 5D1, measured using MSD technology. [Figure 3] 1 illustrates the pH-dependent binding of histidine mutants of the parent antibody 5D1, measured using MSD technology. [Figure 4] 1 illustrates the pH-dependent binding of histidine mutants of the parent antibody 5D1, measured using MSD technology. [Example]

[0132] (Example 1: Construction of heat map) Histidine residues are naturally tolerated in the V domain amino acid sequence in the large antibody repertoire. To identify the acid residue positions, a large number of functional (antibody) sequences from several target antigen projects were used. The sequences of the VH, Vλ, and Vκ domains of the original (original binding) antibodies or the corresponding Fab fragments are pooled and analyzed. analyzed.

[0133] Each target antigen project involves the use of purified antigen or DNA expressing the target (DNA vaccination). Immunization of active llamas with Fab or scFv phage display library construction and This involved the selection and screening of specific binding factors by phage display. Phage display typically involves up to three rounds of selection using trypsin as the eluent. followed by expression of Fab (or scFv) in periplasmic extracts and subsequent analysis by ELISA, MSD, or SPR. Identification of Fab (or scFv) binders by (Biacore) was performed according to standard procedures. In addition to trypsin elution, subsequent elution was performed to further enrich for antibodies with pH-dependent binding. A parallel phage display process was carried out using TBS (50 mM Tris pH 7.0). Up to three rounds of selection were performed using eluates at pH 5.5 (in 100 mM NaCl, 5.5, and 150 mM NaCl). All sequences are then pooled and sorted by type (VH, Vλ, or Vκ) and subfamily (e.g., Any strictly duplicated sequences were removed, leaving only unique sequences. The number of histidine residues at each position in the V domain was counted using the The amino acid positions in the V domain of the llama-derived antibody that accept histidine were examined. Furthermore, this data set was obtained from immunized llamas. Sequences identified by high-throughput sequencing using RNA will be used to further complete the study. It can be made complete.

[0134] Example 2: Use of heat maps to enhance pH dependence High affinity, functionality (e.g., blocking of ligand:receptor interactions), and preferably multi- A parent antibody or Fab fragment with the desired characteristics in terms of minimal pH dependence was identified using a heat map. It was selected for the histidine engineering used.

[0135] First, a small number of histidine mutations were tested on a single parent antibody. Mutations were made at position H35 in the VL domain and position L34 in the VL domain. The presence of conformational epitopes makes Biacore® incompatible. Therefore, the pH-dependent effect measured the ability of the antibody to block the ligand:receptor interaction. IC50 was measured when all proteins were incubated at pH 7.4 or pH 5.5. Results The results are shown in Table 1 and Figure 1. The data show that the introduction of a single mutation alters the pH dependence of this antibody. Interestingly, the mutated VH was significantly increased by 4-fold. By combining with a naturally mutated Vλ, the pH-dependent β-glucanase activity was enhanced without excessive effect at pH 7.4. was further improved.

[0136] Table:1 [Table 45]

[0137] From these data, amino acids were identified by histidine at the positions defined by the heat map. It was confirmed that the acid substitution was effective, i.e., it could enhance the pH dependency. Histidine mutations at other positions may have better (or worse) effects. Since it can be expected that mutations may occur, a systematic mutation analysis was performed.

[0138] Example 3: Complete mutation analysis using heatmaps to enhance pH dependence Using heat maps to introduce the pH dependence of antibody 5D1 directed against a second antigen target The heat map showed that 18 positions in the VH of 5D1 were amenable to histidine substitution. In summary, 12 positions in the Vλ of 5D1 were amenable to histidine substitution (see Table 2). Histidine residues naturally occur at positions 55 and 91 of the VL, and therefore, Note that the number of variants was further reduced. The closest germline for VL was Since it is VL8, position L49 in FR2 was mutated to histidine in the initial screening. However, based on the structural conservation of the CDR2 loop, this position is also considered to be heterodimerized. It should have been possible to mutate according to the map.

[0139] Table 2 [Table 46]

[0140] For each mutation, a synthetic gene was ordered (Geneart, Thermo Scientific). The lyophilized DNA was diluted with H2O and purified using standard DNA recloning methods (restriction enzymes and T4 ligation). Human constant domains (CH1-CH2-CH3 for VH or C for Vλ) were used to The resulting vectors were recloned into mammalian expression vectors containing the Vκ gene (Cκ for Vκ, or Cκ for Vκ). The constructs were sequence-confirmed and transiently transfected into 10 ml of suspension HEK293 cells. used to transfect engineered variant antibodies, each containing a single HIS substitution. A nozzle was created.

[0141] Each single VH 5D1 mutant was transfected individually with the wild-type light chain, while each single The VL 5D1 mutants were transfected individually with the wild-type heavy chain. Grown in style 293 expression medium (Gibco) at 37°C and 5% CO2 at an optimal density of 0.5–0.8 × 10 6 cells / mL Transfection was performed under the following conditions: The transfection mixture contained 1000kJ of HEK293 culture medium. Per mL, 40 μL of OPTI-MEM (Gibco), 1.5 μg of polyethyleneimine (PEI) (Polysci The ratios applied to heavy and light chains were 1 to 3.

[0142] Four hours after transfection, cells were cultured in 9% Primatone HS, which made up 10% of the total culture volume. / UF (Kerry BioScience / Sheffield) was added. The culture was incubated for 6 days with shaking at 100 rpm. Incubated in 1x PBS and then collected by centrifugation at 1,000 x g for 10 minutes. A 50% slurry of protein A-Sepharose beads (GE Healthcare) was added to the cell culture supernatant. The supernatant containing the beads was incubated on a rotor at 4°C for at least 2 hours at 25 rpm. The beads were centrifuged at 610 x g for 2 minutes at slow deceleration. The beads bound to functional mAB were then plated onto a 96-well 0.45 μm filter plate (PAL The cells were transferred to a 100 mL tube and subsequently diluted with 1× PBS, 1× PBS containing 0.5 M NaCl (ChemLab), and 0.15 M NaCl. The mixture was washed with 0.5x PBS containing 1 (ChemLab). A vacuum was applied between each washing step. The resulting mAB was dissolved in 50 mM sodium citrate (Sigma-Aldrich) at pH 3 containing 0.3 M NaCl (ChemLab). The eluted fraction was neutralized with 1M KHPO4 / KH2PO4 (VWR) at pH 8. Protein concentrations were measured by NanoDrop 2000 (Thermo Scientific) and by Biacore. performed the screening using the MSD platform.

[0143] Several configurations can be used for Biacore screening: In the “target” setting, antigens and purified antigens were coated on a CM5 chip (GE Healthcare). Conjugation of the antibody was performed according to the manufacturer's instructions in HBSEP buffer (0.01 M 4-(2-hydroxybenzoates) at pH 7.4). Ethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCl (ChemLab), 3 m M EDTA, 0.005% (v / v) Surfactant P20 at 25°C, pH 7.4 or pH 5.5, or Measurements were performed using the same buffer but adjusted to pH 5.5 by adding 1 M HCl. In the "capture" setting, the mutant antibody was a polyclonal anti-human antibody in all channels. The mAb is captured on an IgG-coated chip. The mAb is injected into the HBSEP to achieve an RU of 150-300. followed by various concentrations of ligand in HBSEP buffer at pH 7.4 or adjusted to pH 5.5. After each cycle, 10 mM glycine, pH 1.5, was injected for 10 seconds. The chip surface was regenerated by . All kinetic analyses were provided by GE Healthcare. The analysis was carried out using appropriate software.

[0144] For antibody testing using the MSD platform, plates (MSD) were incubated in 1x PBS at 4°C. The plates were then coated overnight with 200 μL of target solution while shaking at 600 rpm. Wash three times with 1x PBS and then add 150 μL of 1% (m / v) casein (Sigma-Aldrich) in 1x PBS. The plate was then blocked with 200 μL of 0.05% (m / v) Tween 100 for 1-2 hours at room temperature. 1x PBS containing n (Merck) (pH 7.4) or 0.05% (m / v) Tween (Merck) (pH 5.5) Citrate buffer (0.05 M citric acid (Sigma-Aldrich), 0.14 M sodium citrate (Sigma-Aldrich) The cells were washed three times with 0.15 M NaCl (ChemLab) and 0.15 M NaCl (Aldrich). 1x PBST (PBS containing 0.05% (m / v) Tween) (pH 7.4) or 0.05% ( 25% of mAB was added at various concentrations to citrate buffer (pH 5.5) containing (m / v) Tween (Merck). After incubation at room temperature for 1 hour with shaking at 600 rpm, The wells were washed four times with 1x PBST, pH 7.4 or pH 5.5. For the final washing step, PBST pH 7. 4 or citrate buffer pH 5.5 for an additional 5-10 minutes at room temperature with shaking at 600 rpm. All wells were finally washed with 1x PBS (pH 7.4) and 0.1% PBS. 25 ml of 1:2000 goat anti-human Fc Sulfo (MSD) in 1x PBS (pH 7.4) After washing, 200 μL of 1×PBST was added and the mixture was left at room temperature for 1 hour while shaking at 600 rpm. (pH 7.4), 150 μL of MSD read buffer (MSD) was added to each well, and the plate was then loaded into QuickPlex S Readings were taken on a Q 120 instrument (MSD).

[0145] In addition to a single pH setting (antibody binding and washing are performed at the same pH of 7.4 or 5.5), we A two-point pH ELISA was also tested, in which antibody binding was performed at pH 7.4 but washing was performed in citrate buffer at pH 5.5. This setup allows us to distinguish the effect of pH on association, dissociation, or both. This allows binding to occur at pH 7.4 and release of the antigen in endosomes at pH 5.5-6.0. It is more representative of the in vivo situation that must occur.

[0146] After screening all the single mutants shown in Table 2, some single mutations The 5D1VLm3, 5D1VHm appeared to have a pH-dependent effect on binding (Table 3). 6. 5D1VHm14 exhibited significant pH-dependent binding compared to 5D1wt. Furthermore, 5D1VHm13 exhibited significant pH-dependent binding. It had some improvement in pH dependence with favorable kinetics.

[0147] Table:3 [Table 47] [Table 48]

[0148] The pH dependence was also observed using MSD techniques, as shown in Figures 2 and 3 and Table 4. The effect of these mutations is visualized by the curves shown in Figure 2. In Figure 2, pH pH 5.5 was applied (maximum binding was reduced), and pH 5.5 was applied during both binding and washing. (maximal binding is lost for mutants 5D1VLm3 and 5D1VHm6). The EC50 measured at pH 7.4 remained similar to that of the wild-type parent antibody.

[0149] Table: 4 [Table 49]

[0150] These data suggest that even though pH dependence is introduced, the effect is more on the association step than on the dissociation step. Therefore, to strengthen the pH dependence, we further added histidine. Introducing mutations can be beneficial.

[0151] (Example: 4 Combination of histidine mutations to further increase pH dependency) VLm3, VHm6, and VHm13 all have effects on pH dependence, so these mutations The double HIS mutants VLm3 / VHm6 and VLm3 / VHm13 were combined in the same antibody. During transfection, simply combine the plasmids encoding each chain together. The pH dependence of the resulting antibody was tested by MSD (see Figure 4). ).

[0152] The combination of mutations had a very positive effect on pH dependence, as shown in Table 5. The double mutant showed very poor binding at pH 5.5 (pH 5.5-pH 5.5 setting). In contrast, most of the antibody bound at pH 7.4 was released during the wash at pH 5.5 (pH 7.4-pH 5.5 settings).

[0153] Table:5 [Table 50] nd: EC50 not measurable due to low binding

[0154] Example 5: Complete mutation analysis using heat maps to enhance the pH dependence of antibody 3D6 ) Using heat maps to introduce the pH dependence of antibody 3D6 directed against a second antigen target According to the heat map and CDR length, 25 positions in the VH of 3D6 were affected by histidine substitutions. and 16 positions in the Vκ of 3D6 were amenable to histidine substitution (Table 6 21 of these 25 positions were tested in the initial screen. .

[0155] Table:6 [Table 51]

[0156] For each mutation, a synthetic gene was ordered (Geneart, Thermo Scientific) and the appropriate gene was generated. The antibody was cloned into the vector and produced as shown in Example 3. In a capture setup, a Biacore T200 chip was coated with a high amount of anti-human Fc antibody (8000 RU). Subsequently, wild-type or mutant 3D6 antibodies (2 μg / ml) in HBS-EP at pH 7.4 were added. After the signal stabilized, the target was injected at 2.5 or 10 nM and washed using HBS-EP buffer at pH 7.4 or 5.5. Dissociation constant K D (M) was measured using T200 software. Since the association was performed at pH 7.4, Only the dissociation constant (kd 1 / s) was extracted from the fit at pH 5.5. The results are shown in Tables 7 and 8. Present.

[0157] Table:7 [Table 52]

[0158] Table:8 [Table 53]

[0159] These data demonstrate that pH dependence is introduced into 3D6 with minimal loss of binding at pH 7.4. To enhance the pH dependence, single histidine mutations were combined. can be beneficial.

[0160] Example 6: Combination of histidine mutations further increasing pH dependence in antibody 3D6 height) Based on the results in Tables 7 or 8, a significant pH dependency was introduced and / or the results at pH 7.4 Mutations that improve binding were combined in the same domain to create new mutants VHm23, VHm24, VHm25, VHm26, VKm16, VKm17, VKm18, and VKm19 (Table 9) were generated. For example, 3D6VHm23 3D6VHm24 has mutations VHm0 and VHm16, etc. do.

[0161] Table:9 [Table 54]

[0162] The new VH and VK mutants were synthesized by simply combining each chain during transfection of HEK293 cells. The plasmids encoding the For example, antibody 3D6VHm23 / VKm11 cotransfected VHm23 mutant DNA with mutant VKm11 DNA. After purification, the pH dependence of the resulting antibody was determined as shown in Example 1. The results were tested using Biacore® as described in 5. Excellent pH dependence and Antibodies with good affinity were detected at high target concentrations (0.04 nM; 0.1 nM; 0.37 nM; 1. The only difference was that the K at pH 7.4 D (M) was measured using T200 software. Only the dissociation constant (kd s-1) was measured at pH 5.5. The top five antibodies are shown in Table 10.

[0163] Table: 10 [Table 55]

[0164] Results from Biacore show that using the methods presented herein, loss of binding at pH 7.4 was This clearly enables rapid identification of antibodies with high pH dependency. is shown in.

Claims

1. 1. A method for preparing an engineered antibody that exhibits pH-dependent binding to an antigen, comprising: (a) identifying a parent antibody that binds to the antigen; (b) preparing a first panel of engineered variants of the parent antibody, each of the engineered variants in the panel differing from the parent antibody by substitution of at least one amino acid residue in the VH domain with a histidine residue, and comprising the following hotspot list: 【Table 1】 wherein at least one amino acid residue selected from the following cold spot list: 【Table 2】 wherein at least one amino acid residue from is not substituted with histidine; (c) screening the first panel of engineered variants for pH-dependent binding to the antigen, thereby identifying one or more selected amino acid positions in the VH domain where the presence of a histidine confers pH-dependent binding; (d) preparing a second panel of engineered variants of the parent antibody, each of the engineered variants in the panel differing from the parent antibody by substitution of at least one amino acid residue in the VL domain with a histidine residue, and comprising the following hotspot list: 【Table 3】 wherein at least one amino acid residue selected from the following cold spot list: 【Table 4】 wherein at least one amino acid residue selected from the group consisting of: (e) screening the second panel of engineered variants for pH-dependent binding to the antigen; thereby identifying one or more selected amino acid positions in the VL domain where the presence of a histidine confers pH-dependent binding; (f) preparing one or more further engineered variants of the parent antibody, each of which differs from the parent antibody by a substitution of an amino acid at one or more selected amino acid positions in the VH domain identified in step (c) with histidine, and by a substitution of an amino acid at one or more selected amino acid positions in the VL domain identified in step (e) with histidine; and (g) screening the further engineered variants for pH-dependent binding to the antigen, thereby identifying engineered antibodies that exhibit pH-dependent binding to the antigen.

2. wherein the VL domain of the parent antibody belongs to the λ class and has the following hotspot list: 【Table 5】 wherein at least one amino acid residue selected from the following cold spot list: 【Table 6】 2. The method of claim 1, wherein at least one amino acid residue from is not substituted with histidine.

3. wherein the VL domain of the parent antibody belongs to the kappa class and has the following hotspot list: 【Table 7】 wherein at least one amino acid residue selected from the following cold spot list: 【Table 8】 2. The method of claim 1, wherein at least one amino acid residue from is not substituted with histidine.

4. 4. The method of any one of claims 1 to 3, wherein the engineered antibody identified in part (g) has a lower affinity for the antigen at acidic pH than at neutral pH.

5. The engineered antibody identified in part (g) satisfies the dissociation rate constant (k d ) is the dissociation rate constant (k) of the engineered antibody-antigen interaction at neutral pH d ) or the dissociation rate constant (k) of the parent antibody-antigen interaction at acidic pH d 4. The method according to claim 1, wherein the solubility of the solubility of the

6. The engineered antibody identified in part (g) has an equilibrium dissociation constant (K D ) is the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at neutral pH. D 6. The method according to claim 1, wherein the solubility of the solubility of the

7. the further engineered variants of step (f) contain histidines at all three or more of the selected amino acid positions identified in steps (c) and (e); or 7. The method of any one of claims 1 to 6, wherein the further engineered variant of step (f) further comprises a histidine residue at one or more of the following amino acid positions: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

8. 1. An engineered antibody that exhibits pH-dependent binding to an antigen, At least one amino acid in the CDR of the engineered antibody is a histidine residue and is selected from the following hot spot list: 【Table 9】 wherein at least one amino acid residue selected from the following cold spot list: 【Table 10】 wherein at least one amino acid residue selected from the group consisting of: is not a histidine residue.

9. the engineered antibody has a lower affinity for the antigen at acidic pH than at neutral pH, and / or The equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at acidic pH D ) is the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at neutral pH. D 9. The engineered antibody of claim 8, wherein the antibody has a IgG antibody titer greater than 1000kJ / mL.

10. The dissociation rate constant (k) of the engineered antibody-antigen interaction at acidic pH d ) is the dissociation rate constant (k) of the engineered antibody-antigen interaction at neutral pH d 9. The engineered antibody of claim 8, wherein the antibody has a IgG antibody titer greater than 1000kJ / mL.

11. at least two, or at least three, or at least four amino acid residues selected from said hotspot list are histidines; and / or 11. The engineered antibody of any one of claims 8 to 10, wherein the engineered antibody further comprises a histidine residue at one or more of the following amino acid positions: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

12. At least two, or at least three, of the amino acid residues at the positions listed in Table B are not histidine; or 12. The engineered antibody of any one of claims 8-11, wherein none of the amino acid residues at the positions on the cold spot list is histidine, with the proviso that one or more of the following amino acid positions may contain histidine: H100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

13. 13. The engineered antibody of any one of claims 8 to 12, which is an engineered variant of a camelid antibody, optionally wherein the engineered antibody comprises an Fc region and wherein the engineered antibody binds to the neonatal Fc receptor (FcRn).

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