Method for producing pH-dependent antibodies

文档序号：1651928 发布日期：2019-12-24 浏览：52次中文

阅读说明：本技术 制备pH依赖性抗体的方法 (Method for producing pH-dependent antibodies ) 是由克里斯托弗·布朗什托埃里克·霍夫曼乔纳斯·德哈德雅各布斯·科内利斯·拉塞尔于 2018-05-10 设计创作，主要内容包括：公开了用于制备表现出改善的pH依赖性抗原结合的工程化抗体的方法。所述方法基于在抗体CDR内的限定的氨基酸位置的子集处引入组氨酸残基。用于组氨酸替换的所选氨基酸位置的组来源于来自天然抗体库之功能性抗体的CDR内的组氨酸存在的热图。所述方法提供了鉴定pH依赖性抗体变体的更简单且耗时更少的方法。(Methods for making engineered antibodies that exhibit improved pH-dependent antigen binding are disclosed. The method is based on the introduction of histidine residues at a subset of defined amino acid positions within the CDRs of the antibody. The set of selected amino acid positions for histidine substitutions was derived from a heatmap of histidine presence within the CDRs of functional antibodies from the natural antibody repertoire. The method provides a simpler and less time-consuming method of identifying pH-dependent antibody variants.)

1. A method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising making the engineered antibody by replacing at least one amino acid residue of a parent antibody with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1 H34，H35a，H35b，H35c VH CDR2 H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65 VH CDR3 H100g，H100k，H100m，H100n VL CDR1 L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33 VL CDR2 L50，L51a，L51b，L51c，L51d，L56 VL CDR3 L95，L95d，L95e，L95f，L97

wherein the engineered antibody exhibits pH-dependent binding to its antigen.

2. The method of claim 1, wherein the engineered antibody has a lower affinity for its antigen at acidic pH than at neutral pH.

3. The method of claim 1, wherein the off-rate constant (k) of the engineered antibody-antigen interaction at acidic pH_d) Off rate constant (k) for engineered antibody-antigen interactions above neutral pH_d)。

4. The method of claim 1, wherein the off-rate constant (k) of the engineered antibody-antigen interaction at acidic pH_d) Dissociation rate constant (k) of parent antibody-antigen interaction at higher than acidic pH_d)。

5. The method of claim 1 or claim 2, wherein the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at acidic pH_D) Equilibrium dissociation constant (K) for engineered antibody-antigen interactions above neutral pH_D)。

6. The method of any one of claims 1 to 5, wherein at least two amino acid residues selected from the hotspot list are replaced with histidine.

7. The method of any one of claims 1 to 5, wherein at least three amino acid residues selected from the hotspot list are replaced with histidine.

8. The method of any one of claims 1 to 5, wherein at least four amino acid residues selected from the hotspot list are replaced with histidine.

9. The method of any one of claims 1 to 8, wherein one or more of the following residues are also replaced with histidine: h100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

10. The method of any one of claims 1 to 9, wherein at least two amino acid residues selected from the cold spot list are not substituted with histidine.

11. The method of any one of claims 1 to 9, wherein at least three amino acid residues selected from the cold spot list are not substituted with histidine.

12. The method of any one of claims 1 to 9, wherein none of the amino acid residues selected from VHCDR1, VHCDR2, VLCDR1, and VLCDR2 of the cold spot list are substituted with histidine.

13. The method of any one of claims 1 to 12 wherein the parent antibody or CDRs thereof are derived from a species in the family camelidae.

14. A method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising the steps of:

(a) providing a parent antibody that binds to an antigen;

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

；

(c) screening the panel of engineered variants for pH-dependent binding to the antigen, and thereby identifying engineered antibodies that exhibit pH-dependent binding to the antigen.

15. The method of claim 14, wherein the engineered antibody identified in part (c) has a lower affinity for its antigen at acidic pH than at neutral pH.

16. The method of claim 14, wherein the engineered antibody identified in part (c) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Off rate constant (k) for engineered antibody-antigen interactions above neutral pH_d)。

17. The method of claim 14, wherein the engineered antibody identified in part (c) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Dissociation rate constant (k) of parent antibody-antigen interaction at higher than acidic pH_d)。

18. The method of claim 14 or claim 15, wherein the engineered antibody identified in part (c) is characterized by: equilibrium dissociation constant (K) for engineered antibody-antigen interactions at acidic pH_D) Equilibrium dissociation constant (K) for engineered antibody-antigen interactions above neutral pH_D)。

19. The method of any one of claims 14 to 18, wherein step (b) comprises preparing a set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VH domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1 H34，H35a，H35b，H35c VH CDR2 H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65 VH CDR3 H100g，H100k，H100m，H100n

。

20. the method of any one of claims 14 to 18, wherein step (b) comprises preparing a set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VL domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue selected from the following cold spot list is not replaced with histidine:

VL CDR1 L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33 VL CDR2 L50，L51a，L51b，L51c，L51d，L56 VL CDR3 L95，L95d，L95e，L95f，L97

。

21. the method of claim 20, wherein the VL domain of the parent antibody belongs to the lambda class, and wherein at least one amino acid residue selected from the following hotspot list is replaced with histidine:

CDR1 L30，L31，L32，L34， CDR2 L51，L52，L53，L55， CDR3 L89，L91，L92，L93，L95a，L95b，L95c，L96

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

CDR1 L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L33 CDR2 L50，L51a，L51b，L51c，L51d，L54，L56 CDR3 L90，L94，L95，L95d，L95e，L95f，L97

。

22. the method of claim 20, wherein the VL domain of the parent antibody belongs to the kappa class, and wherein at least one amino acid residue selected from the following hotspot list is replaced with histidine:

CDR1 L27，L27d，L29，L31，L32 CDR2 L53，L54，L55 CDR3 L89，L90，L91，L92，L93，L94，L96

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

CDR1 L24，L25，L26，L27A，L27b，L27c，L27e，127f，L28，L30，L33，L34 CDR2 L50，L51，L51a，L51b，L51c，L51d，L52，L56 CDR3 L95，L95a，L95b，L95c，L95d，L95e，L95f，L97

。

23. a method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising the steps of:

(a) identifying a parent antibody that binds to the antigen;

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

；

(c) screening the set of engineered variants for pH-dependent binding to the target antigen and thereby identifying selected amino acid positions at which the presence of histidine confers pH-dependent binding to the antigen;

(d) preparing one or more further engineered variants of said parent antibody, wherein each of said variants comprises histidine at two or more selected amino acid positions identified in step (c); and

(e) screening the further engineered variants for pH-dependent binding to the antigen; and thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen.

24. The method of claim 23 wherein the engineered antibody identified in part (e) has a lower affinity for its antigen at acidic pH than at neutral pH.

25. The method of claim 23, wherein the engineered antibody identified in part (e) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Off rate constant (k) for engineered antibody-antigen interactions above neutral pH_d)。

26. The method of claim 23, wherein the engineered antibody identified in part (e) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Dissociation rate constant (k) of parent antibody-antigen interaction at higher than acidic pH_d)。

27. The method of claim 23 or claim 24, wherein the engineered antibody identified in part (e) is characterized by: equilibrium dissociation constant (K) for engineered antibody-antigen interactions at acidic pH_D) Equilibrium dissociation constant (K) for engineered antibody-antigen interactions above neutral pH_D)。

28. The method of any one of claims 23-27, wherein each of said engineered variants in said set of step (b) comprises a single histidine substitution at an amino acid position selected from said hot spot list.

29. The method of any one of claims 23 to 28, wherein the additional engineered variant of step (d) comprises histidine residues at the three or more selected amino acid positions identified in step (c).

30. The method of any one of claims 23 to 28, wherein the additional engineered variant of step (d) comprises histidine residues at the four or more selected amino acid positions identified in step (c).

31. The method of any one of claims 23 to 28, wherein the additional engineered variant of step (d) further comprises a histidine residue at one or more of the following amino acid positions: h100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

32. A method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising the steps of:

(a) identifying a parent antibody that binds to the antigen;

(b) preparing a first set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in a VH domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1 H34，H35a，H35b，H35c VHCDR2 H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65 VH CDR3 H100g，H100k，H100m，H100n

；

(c) screening said first engineered set of variants for pH-dependent binding to said antigen; and thereby identifying one or more selected amino acid positions in the VH domain at which the presence of histidine confers pH-dependent binding;

(d) preparing a second set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VL domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue selected from the following cold spot list is not replaced with histidine:

VL CDR1 L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33 VL CDR2 L50，L51a，L51b，L51c，L51d，L56 VL CDR3 L95，L95d，L95e，L95f，L97

；

(e) screening said second engineered set of variants for pH-dependent binding to said antigen; and thereby identifying one or more selected amino acid positions in the VL domain at which the presence of histidine confers pH-dependent binding;

(f) preparing one or more further engineered variants of the parent antibody, wherein each of the variants differs from the parent antibody in that the amino acid at one or more selected amino acid positions in the VH domain identified in step (c) is replaced with histidine, and the amino acid at one or more selected amino acid positions in the VL domain identified in step (e) is replaced with histidine; and

(g) screening the additional engineered variants for pH-dependent binding to the antigen, thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen.

33. The method of claim 32, wherein the VL domain of the parent antibody belongs to the lambda class, and wherein at least one amino acid residue selected from the following hotspot list is replaced with histidine:

CDR1 L30，L31，L32，L34， CDR2 L51，L52，L53，L55， CDR3 L89，L91，L92，L93，L95a，L95b，L95c，L96

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

CDR1 L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L33 CDR2 L50，L51a，L51b，L51c，L51d，L54，L56 CDR3 L90，L94，L95，L95d，L95e，L95f，L97

。

34. the method of claim 32, wherein the VL domain of the parent antibody belongs to the kappa class, and wherein at least one amino acid residue selected from the following hotspot list is replaced with histidine:

CDR1 L27，L27d，L29，L31，L32 CDR2 L53，L54，L55 CDR3 L89，L90，L91，L92，L93，L94，L96

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

CDR1 L24，L25，L26，L27A，L27b，L27c，L27e，I27f，L28，L30，L33，L34 CDR2 L50，L51，L51a，L51b，L51c，L51d，L52，L56 CDR3 L95，L95a，L95b，L95c，L95d，L95e，L95f，L97

。

35. the method of any one of claims 32 to 34, wherein the engineered antibody identified in part (g) has a lower affinity for its antigen at acidic pH than at neutral pH.

36. The method of any one of claims 32 to 34, wherein the engineered antibody identified in part (g) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Off rate constant (k) for engineered antibody-antigen interactions above neutral pH_d)。

37. The method of any one of claims 32 to 34, wherein the engineered antibody identified in part (g) is characterized by: dissociation rate constant (k) for engineered antibody-antigen interactions at acidic pH_d) Dissociation rate constant (k) of parent antibody-antigen interaction at higher than acidic pH_d)。

38. The method of any one of claims 32 to 37, wherein the engineered antibody identified in part (g) is characterized by: equilibrium dissociation constant (K) for engineered antibody-antigen interactions at acidic pH_D) Equilibrium dissociation constant (K) for engineered antibody-antigen interactions above neutral pH_D)。

39. The method of any one of claims 32 to 38, wherein the additional engineered variant of step (f) comprises histidines at a total of three or more selected amino acid positions identified in steps (c) and (e).

40. The method of any one of claims 32 to 38, wherein the additional engineered variant of step (f) comprises histidine at a total of four or more selected amino acid positions identified in steps (c) and (e).

41. The method of any one of claims 32 to 38, wherein the additional 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.

42. The method according to any one of claims 14 to 41 wherein the parent antibody or CDRs thereof are derived from a species in the family Camelidae.

43. An engineered antibody that exhibits pH-dependent binding to its antigen, wherein at least one amino acid in a CDR of the engineered antibody is a histidine residue, characterized in that at least one amino acid residue selected from the following hot spot list is a histidine residue:

and at least one amino acid residue selected from the following cold spot list is not a histidine residue:

44. the engineered antibody of claim 43, wherein the engineered antibody has a lower affinity for its antigen at acidic pH than at neutral pH.

45. The engineered antibody of claim 43, wherein the off-rate constant (k) of the engineered antibody-antigen interaction at acidic pH_d) Off rate constant (k) for engineered antibody-antigen interactions above neutral pH_d)。

46. The engineered antibody of claim 43 or claim 44, wherein the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at acidic pH_D) Equilibrium dissociation constant for engineered antibody-antigen interactions above neutral pH(K_D)。

47. The engineered antibody of any one of claims 43 to 46, wherein at least two amino acid residues selected from the hotspot list are histidines.

48. The engineered antibody of any one of claims 43 to 46, wherein at least three amino acid residues selected from the hotspot list are histidines.

49. The engineered antibody of any one of claims 43 to 46, wherein at least four amino acid residues selected from the hotspot list are histidines.

50. The engineered antibody of any one of claims 43 to 49, further comprising a histidine residue at one or more of the following amino acid positions: h100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

51. The engineered antibody of any one of claims 43 to 50, wherein at least two amino acid residues at the positions listed in Table B are not histidines.

52. The engineered antibody of any one of claims 43 to 50, wherein at least three amino acid residues at positions selected from the cold spot list are not histidines.

53. The engineered antibody of any one of claims 43 to 50, wherein none of the amino acid residues at positions on the cold spot list are histidines, provided that histidines may be included at one or more of the following amino acid positions: h100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

54. The engineered antibody of any one of claims 43 to 53 which is an engineered variant of a Camelidae antibody.

55. The engineered antibody of any one of claims 43-54, comprising an Fc region, wherein the antibody binds to a neonatal Fc receptor (FcRn).

Technical Field

The present disclosure relates to the field of antibody engineering, and in particular to methods for engineering antibody variants that exhibit pH-dependent binding to a target antigen, and antibodies made by the methods.

Background

There is increasing interest in the engineering of antibodies that exhibit pH-dependent binding to a target antigen. In particular, it is of interest to identify antibodies that exhibit reduced antigen binding at acidic pH compared to neutral pH.

Incorporation of pH sensitive antigen binding can lead to improved function of the engineered antibody in vivo. The antibodies can be engineered to maintain high affinity antigen binding at neutral pH (e.g., pH7.4) and exhibit reduced binding at acidic pH (e.g., pH 4.5 to 6.0). Upon entry into the endosomal pathway, pH-dependent antigen binding allows dissociation of the antibody-antigen complex in acidified endosomes (about pH 6.0) and FcRn-mediated recycling of free antibody, thereby facilitating enhanced antigen clearance, which may enable less frequent or lower antibody dosing.

Generally, pH-dependent antibody-antigen binding relies on the presence of ionizable histidine residues that mediate structural shifts in the binding or folding of the interacting antibody. Changes in electrostatic interactions induced after protonation of histidine at lower pH values can lead to a decrease in binding affinity.

Various protein engineering approaches have been described with the aim of incorporating pH sensitivity into proteins by using different histidine replacement strategies. For example, rational design guided by structural modeling of the granulocyte colony-stimulating factor (GCSF) receptor interaction site enables identification of a mutation site that results in a pH sensitive variant following histidine substitution (Sarkar et al, Nat Biotechnol, Vol.20, pp908-13, 2002). However, assessing individual positions of histidine substitutions that can mediate pH sensitivity in proteins is time consuming and unpredictable.

As an alternative, screening combinatorial libraries using appropriate high throughput techniques (e.g., Yeast Surface Display (YSD) or phage display) has been applied to the engineering of pH-sensitive binding in a variety of different protein scaffolds.Etc., mAbs, vol.7(1), 138-151, 2015 describe a general strategy for engineering antibody heavy and light chain variable domains for reversible pH-sensitive antigen binding. This method relies on the generation and screening of histidine replacement libraries for both heavy and light chain combinations. In this approach, histidine mutations are randomly introduced into the complementarity-determining regions (CDRs) of the VH and VL regions. Histidine mutations may be introduced at any amino acid position in the CDRs of the VH and VL domains. The rate of histidine mutations was adjusted to achieve an average of three random mutations per library variant. Thus, a complete library theoretically contains variants that contain all possible combinations of three random histidine substitutions in the CDRs.

This combinatorial library approach did enable successful identification of engineered variants of adalimumab (adalimumab) that exhibit pH-dependent binding to TNF. However, this strategy is relatively time consuming due to the large library size required to sample all amino acid positions within the CDRs of the VH and VL domains for possible histidine substitutions. Furthermore, the overall antibody structure and stability by introducing histidine residues at unfavorable positions is not considered.

Summary of The Invention

There is a need for alternative methods for preparing engineered antibody variants that exhibit pH-dependent binding to a target antigen that are simpler and less time-consuming to perform than prior art methods. Furthermore, there is a need for methods that can identify engineered antibodies that exhibit pH-dependent antigen binding without unduly affecting antigen binding ability at neutral pH.

To meet these needs, applicants have systematically analyzed the natural presence of histidine residues within the Complementarity Determining Regions (CDRs) of a large repertoire of functional (i.e., antigen binding) antibody variable domains. The results of this analysis enabled applicants to obtain a "heat-map" of the presence of native histidine in the native antibody. The "heatmap" provides a list of not only "hot-spot" amino acid residues (where histidine may naturally occur within the antibody repertoire) within the CDRs, but also "cold-spot" amino acid residues (where histidine is not normally naturally occurring) in the CDRs. The "hot spot" and "cold spot" lists can be applied, for example, in a rational design or combined approach to the engineering of pH-dependent antibody variants.

In a first aspect, the present invention provides a method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising making the engineered antibody by replacing at least one amino acid residue of a parent antibody with a histidine residue, wherein at least one amino acid residue selected from the following hotspot list is replaced with a histidine:

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n
		VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
VL CDR2	L50，L51a，L51b，L51c，L51d，L56
		VL CDR3	L95，L95d，L95e，L95f，L97

wherein the engineered antibody exhibits pH-dependent binding to its antigen.

Due to the histidine substitution, the engineered antibody exhibits improved pH-dependent binding (i.e., greater pH-dependence) compared to the parent antibody.

The invention also provides a method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising the steps of:

(a) providing a parent antibody that binds to an antigen;

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n
		VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
VL CDR2	L50，L51a，L51b，L51c，L51d，L56
		VL CDR3	L95，L95d，L95e，L95f，L97

(c) screening the panel of engineered variants for pH-dependent binding to the antigen, and thereby identifying engineered antibodies that exhibit pH-dependent binding to the antigen.

Due to the histidine substitution, the engineered antibody identified in step (c) exhibits improved pH-dependent binding (i.e. greater pH-dependence) compared to the parent antibody.

The invention also provides a method of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising the steps of:

(a) identifying a parent antibody that binds to the antigen;

and at least one amino acid residue from the following cold spot list is not replaced with histidine:

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n

VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
		VL CDR2	L50，L51a，L51b，L51c，L51d，L56
VL CDR3	L95，L95d，L95e，L95f，L97

(e) screening the further engineered variants for pH-dependent binding to the antigen; and thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen.

Due to the histidine substitution, the engineered antibody identified in step (e) exhibits improved pH-dependent binding (i.e., greater pH-dependence) compared to the parent antibody.

In a second aspect, the present invention provides an engineered antibody that exhibits pH-dependent binding to its antigen, wherein at least one amino acid in a CDR of said engineered antibody is a histidine residue, characterized in that at least one amino acid residue selected from the following hotspot list is a histidine residue:

and at least one amino acid residue selected from the following cold spot list is not a histidine residue:

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n
		VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
VL CDR2	L50，L51a，L51b，L51c，L51d，L56
		VL CDR3	L95，L95d，L95e，L95f，L97

。

Detailed Description

The present disclosure provides methods of making engineered antibodies that exhibit pH-dependent binding to a target antigen based on the replacement of selected amino acids in the variable domain (and preferably within the CDRs) of a parent antibody with histidine residues. It is generally known in the art that the introduction of ionizable histidine residues into the VH and/or VL domains of antibody molecules can alter the pH dependence of antigen binding. However, to date, available methods for engineering of pH-dependent antigen binding have relied largely on screening random combinatorial libraries in which all amino acid residues in the CDRs are sampled for possible substitutions of histidine.

The method of the invention differs from the methods of the prior art in that the selection of a particular amino acid residue for substitution with histidine is guided by: the natural occurrence of histidine residues at specific amino acid positions within the VH and VL domains (and more particularly within the CDRs) of a functional antibody molecule; and additionally the natural absence of histidine residues at other amino acid positions in the CDRs. Thus, the methods disclosed herein are based on histidine substitutions at amino acid positions selected from a preselected subset of amino acid positions within the variable domain (and in particular the CDRs), while other residues in the CDRs are preselected to be preferably not substituted by histidine.

Amino acid residues that are preselected as candidates for substitution with histidine are referred to herein as "hot spot" residues (table a). These are amino acid positions in the CDRs, or framework positions near the CDRs, where histidine has been observed to naturally exist within a functional repertoire.

TABLE A (hotspot residue position)

Is pre-selected as preferredIs not limited toThe amino acid residue substituted by histidine is referred to herein as the "cold spot" residue (table B). These are amino acid positions in the CDRs, or framework positions near the CDRs, where no histidine was observed within the functional antibody repertoire.

TABLE B (Cold spot residue position)

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n
		VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
VL CDR2	L50，L51a，L51b，L51c，L51d，L56
		VL CDR3	L95，L95d，L95e，L95f，L97

The present disclosure provides methods of making an engineered antibody that exhibits pH-dependent binding to its antigen, comprising making the engineered antibody by replacing at least one amino acid residue of a parent antibody with a histidine residue (which may be abbreviated herein as HIS). Due to the histidine substitution, the engineered antibody exhibits improved pH-dependent binding (i.e., greater pH-dependence) compared to the parent antibody. The "parent antibody" may be any antibody capable of binding to the antigen of interest. The parent antibody is typically a conventional four-chain immunoglobulin in which paired VH and VL domains confer antigen binding specificity. However, the method is also applicable to the engineering of pH-dependent binding: other parent antibodies, and antigen-binding fragments, e.g., Fab, F (ab')₂Fv, scFv, diabodies (diabodies), triabodies (triabodies), minibodies (minibodies), etc., as well as any other modified immunoglobulin configuration that comprises an antigen binding site provided by paired VH and VL domains. In certain embodiments, the parent antibody or variable domains or CDRs thereof may be derived from a species in the family camelidae, including but not limited to: alpaca (llama), camel (camel), dromedary (dromedary), alpaca (alpaca), alpacaOr a raw camel (guanaco). The preparation of camelid monoclonal antibodies is described in detail in WO2010/001251, the content of which is incorporated herein by reference. Other preferred features of the parent antibody and the engineered antibody derived therefrom are described elsewhere herein. The nature of the antigen bound by the parent antibody is not particularly limited.

Engineered antibodies are prepared by replacing one or more selected amino acid residues of a parent antibody with histidine. The engineered antibody may comprise a total of 1, 2, 3, 4, 5, or more histidine substitutions as compared to the parent antibody. The selected amino acid residues that are replaced with histidine are typically located within the CDRs, or at framework positions near the CDRs, of the antibody variable domain. Where the antibody molecule comprises paired VH and VL domains, the selected residues for substitution with histidine may be located in the CDRs of the VH domain, or in the CDRs of the VL domain, or in the CDRs of both the VH and VL domains.

As described herein, at least one of the amino acid positions that is replaced with histidine must be selected from the "hot spot" list (table a). In certain embodiments, at least two, at least three, at least four, or at least five of the amino acid positions substituted with histidine are selected from the "hot spot" list. In certain embodiments, all histidine substitutions in the engineered antibody are made at amino acid positions selected from the hot list (table a), i.e., only residues in the hot list may be selected for substitution with histidine. However, in other embodiments, histidine substitutions may be included at one or more positions not on the hotspot list, provided that at least one histidine substitution is made at a position selected from the hotspot list (table a). For example, as discussed below, in certain embodiments, one or more histidine substitutions may be included at "cold spot" positions within the VH CDR3 and/or VL CDR 3.

As described herein, there are certain amino acid positions in the parent antibody that are preferably not selected for substitution with histidine. These amino acid positions are those shown on the "cold spot" list (table B). An engineered antibody prepared according to the methods described herein must contain at least one amino acid position on the cold spot list (table B) that is not replaced with histidine. In certain embodiments, at least two, at least three, at least four, or at least five of the amino acid positions on the cold spot list (table B) are not replaced with histidine. In other embodiments, none of the amino acid positions on the cold spot list (table B) are replaced with histidine.

In the case of VH CDR3 and VL CDR3, it is also permissible (and may be beneficial in certain embodiments) to introduce histidine substitutions at one or more of the cold-spot amino acid residue positions listed in table B, and the resulting antibody variants are tested for pH-dependent binding. Given the highly variable nature of VH CDR3 and VL CDR3, the introduction of histidine residues at cold-spot positions in these CDRs (in addition to or as an alternative to hot-spot positions) can be tolerated within antibody structures more readily than the introduction of histidine residues at cold-spot positions in CDR1 and CDR2, and can make an important contribution to pH-dependent antigen binding. Thus, histidine substitutions at the cold spot positions listed in VH CDR3 and VL CDR3 are permissible in all aspects of the invention.

In a particular embodiment, all amino acid residues that are replaced with histidine are selected from the hotspot list (table a), provided that one or more of the following residues may also be replaced with histidine: h100g, H100k, H100m, H100n, L95, L95d, L95e, L95f, L97.

pH dependent binding

Engineered antibodies into which one or more histidine substitutions have been introduced according to the methods described herein can exhibit pH-dependent binding to a target antigen.

The term "pH-dependent binding" as used herein with respect to an antibody-antigen binding interaction means that the antigen binding activity of the antibody at acidic pH differs from the antigen binding activity of the antibody at neutral pH.

Various measures of antigen binding activity can be used as an indicator of the difference in antigen binding activity at acidic versus neutral pH.

In one embodiment, the "affinity" of an antibody for its antigen can be used as an indicator of antigen binding activity. In some preferred embodiments, an engineered antibody exhibits "pH-dependent binding" if its affinity for its antigen at acidic pH is lower than its affinity for its antigen at neutral pH.

In one embodiment, the dissociation rate constant (k) of the antibody-antigen interaction_d) (also known as antibody off-rate (k)_off) Can be used as an index of antigen binding activity. In a preferred embodiment, the dissociation rate constant (k) of an antibody-antigen interaction is such that it is engineered at acidic pH_d) Higher than the dissociation rate constant (k) of the engineered antibody-antigen interaction at neutral pH_d) The engineered antibody then exhibits "pH-dependent binding".

In which the dissociation rate constant (k)_d) In some embodiments used as an indicator of pH-dependent binding, the dissociation rate constant (k) of antibody-antigen interactions is engineered at acidic pH_d) Dissociation rate constant (k) for the engineered antibody-antigen interaction at neutral pH_d) The ratio of (a) may be at least 1.5, or at least 2, or at least 5, or at least 10.

In other embodiments, the off-rate constant (k) of the engineered antibody is at acidic pH (e.g., pH 5.5)_d) Higher than the dissociation rate constant (k) of the parent antibody at the same acidic pH_d). In such embodiments, k is at an acidic pH (e.g., pH 5.5) as compared to the parent antibody due to the histidine substitution_dAnd (4) improving. In certain embodiments, the off-rate constant (k) of an antibody-antigen interaction is engineered at acidic pH_d) Dissociation rate constant (k) for parent antibody-antigen interaction at the same acidic pH_d) The ratio of (a) may be at least 1.5, or at least 2, or at least 5, or at least 10. Considering the in vivo antibody recycling pathway, the dissociation rate (k) at acidic pH (e.g., pH 5.5)_d) Is a particularly relevant measure of pH-dependent binding. Faster dissociation of the antibody-antigen complex at acidic pH (pH 5.5 to 6.0) enables the release of antigen within acidified endosomes.

In one embodiment, the equilibrium dissociation constant (K)_D) Can be used as an index of antigen binding activity. In some preferred embodiments, the equilibrium dissociation constant (K) of an antibody-antigen interaction is engineered if the interaction is performed at acidic pH_D) Higher than the equilibrium dissociation constant (K) of the engineered antibody-antigen interaction at neutral pH_D) The engineered antibody then exhibits "pH-dependent binding".

In which the dissociation constant (K) is balanced_D) In some embodiments used as an indicator of pH-dependent binding, the equilibrium dissociation constant (K) of antibody-antigen interactions is engineered at acidic pH_D) Equilibrium dissociation constant (K) for the engineered antibody-antigen interaction at neutral pH_D) The ratio of (d) may be greater than 1.5, or greater than 2, or greater than 5, or greater than 10. In some particularly preferred embodiments, the engineered antibody may exhibit 20 to 40 fold stronger antigen binding at neutral pH relative to antigen binding at acidic pH.

In which the dissociation constant (K) is balanced_D) In some non-limiting embodiments used as an indicator of antigen binding activity, engineering an antibody at acidic pH (e.g., pH 5.5) may exhibit an equilibrium dissociation constant (K) for its antigen of 10 to 20nM_D) (ii) a While K of an engineered antibody to its antigen at neutral pH (e.g., pH7.4)_DAnd may be about 0.5nM or less.

Other measures of antigen binding activity that can be used to indicate pH-dependent binding can include affinity constants or binding rate constants (k)_a) Or the rate of incorporation (k)_on)。

For antagonistic antibodies, the measure of antigen binding activity may be blocking potency, i.e., IC50 in a suitable assay.

In a preferred embodiment, an engineered antibody comprising one or more histidine substitutions exhibits greater pH dependence of binding to its antigen than the parent antibody. By "greater pH dependence of binding" is meant that the difference between the antigen binding activity at acidic pH and the antigen binding activity at neutral pH is significantly greater for the engineered antibody than for the parent antibody from which the engineered antibody is derived.

In some embodiments, the parent antibody may not exhibit significant pH dependence of binding to its target antigen, and pH dependence may be 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 dependence that is significantly increased in the engineered variant due to one or more histidine substitutions.

In certain embodiments, pH dependence can be introduced into engineered antibodies without significantly compromising the strength of antigen-antibody binding at neutral pH. In such embodiments, the engineered antibody may exhibit significantly lower antigen binding activity at acidic pH than the parent antibody from which the engineered antibody is derived, while the antigen binding activity of the engineered antibody at neutral pH may be comparable to (i.e., not significantly different from) the antigen binding activity of the parent antibody from which the engineered antibody is derived.

One advantage of the methods described herein, wherein the candidate amino acids for histidine substitution are selected according to a "heatmap" of the natural histidine presence in a functional antibody library, is that histidine residues are typically introduced only at "hot spot" amino acid positions where they may naturally occur within the antibody library, and are thus tolerated within the antibody structure. It has been observed that histidine substitutions at the hot spot positions can confer pH-dependence on antigen binding without significantly affecting the strength of antigen-binding activity at neutral pH.

In one embodiment, the engineered antibody exhibits greater pH dependence of binding to its antigen than the parent antibody, while retaining at least 80% of the antigen binding activity of the parent antibody at neutral pH. In other embodiments, the engineered antibody may retain at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the antigen binding activity of the parent antibody at neutral pH, while still exhibiting pH-dependent binding.

In a preferred embodiment, the engineered antibody exhibits greater pH dependence of binding to its antigen than the parent antibody, while retaining at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the parent antibody's affinity for the antigen at neutral pH.

In which the dissociation constant (K) is balanced_D) In some embodiments used as an indicator of antigen binding activity, the equilibrium dissociation constant (K) of an antibody to a target antigen is engineered at neutral pH_D) K of the parent antibody to the target antigen at neutral pH_DNot more than 20%, or not more than 15%, or not more than 10%, or not more than 5% larger. In other embodiments, the equilibrium dissociation constant (K) of an antibody to a target antigen is engineered at neutral pH_D) Can be substantially equal to the K of the parent antibody to the target antigen at neutral pH_D。

In other embodiments, pH-dependent introduction may result in a decrease in the affinity of the engineered antibody for its antigen at neutral pH. However, depending on the nature of the target antigen, the intended use of the antibody, and the nature of the parent antibody, the decrease in affinity at neutral pH may be acceptable provided that the desired pH dependence of binding is achieved. For example, if a parent antibody exhibits a particularly high affinity for its antigen at neutral pH, a decrease in the affinity of the engineered antibody for its antigen at neutral pH can be tolerated provided that this is accompanied by a significant increase in pH dependence (i.e., provided that the desired "fold difference" between binding at neutral pH and binding at acidic pH is achieved). In certain embodiments, the affinity of an engineered antibody for its target antigen at neutral pH may be up to 10-fold, or even 20-fold, higher than the affinity of the parent antibody for that antigen at neutral pH, provided that the desired pH dependence of binding is also achieved.

In each of the foregoing embodiments, "acidic pH" means a pH of from pH 4.0 to pH 6.5, preferably from pH 4.5 to pH 6.0, more preferably from pH5.5 to pH 6.0, and more preferably from pH5.5 to pH 5.8.

In each of the foregoing embodiments, "neutral pH" means a pH of from pH 6.7 to pH 10.0, preferably from pH 7.0 to pH 8.0, more preferably pH 7.4.

In some specific embodiments, pH-dependent binding can be assessed by comparing the antigen binding activity of an antibody at acidic pH5.5 with the antigen binding activity of the same antibody at neutral pH 7.4.

In other embodiments, pH-dependent binding can be assessed by comparing the antigen binding activity of an antibody at acidic pH 6.0 with the antigen binding activity of the same antibody at neutral pH 7.4.

Suitable methods and conditions for determining the antigen binding activity of an antibody at a defined pH are generally known in the art. For example, by(GE Healthcare) (as described in the appended experimental examples), the antigen binding activity of the antibody at a defined pH was measured by ELISA or using the MSD platform.

Heatmap of histidine presence

As described herein, amino acid residues for a parent antibody that are substituted with histidine or not substituted with histidine can be selected based on the "heat map" of the presence of histidine residues at certain amino acid positions within the CDRs of a functional antibody within the native antibody repertoire. The "heatmap" provides not only a list of "hot spot" amino acid residue positions within the CDRs and framework positions near the CDRs where histidines may naturally occur within the antibody repertoire, but also a list of "cold spot" amino acid residue positions in the CDRs and framework positions near the CDRs where histidines do not normally occur naturally.

By "antibody repertoire" is meant a population of antibodies that exhibit antigen binding activity. A "natural antibody repertoire" is typically a population of antibodies produced by immunizing a host with a target antigen. The repertoire can comprise antibodies to more than one target antigen. In the present disclosure, the "heatmap" of histidine presence is derived from a diverse repertoire of llama antibodies, which are themselves obtained by immunizing a host animal with a variety of different target antigens. Antibodies isolated from these immunizations were also screened by specific phage display selection, allowing enrichment for antibodies exhibiting pH-dependent antigen binding.

Selection of residues for substitutionSelecting

For any given parent antibody, the heatmap of histidine presence guides the selection of candidate amino acid residues for substitution with histidine.

The terms "substitution" or "substitution" of amino acid residues are used interchangeably herein. In the case of an engineered antibody that is an engineered variant of a parent antibody, a "substitution" requires the substitution of an amino acid residue at a defined amino acid position in the parent antibody with a different amino acid residue, i.e., a histidine residue, which does not naturally occur at that position in the parent antibody.

The "heatmap" of histidine presence described herein provides a list of hotspot amino acid positions where natural presence of histidine has been observed. The parent antibody of interest (from which the engineered antibody will be derived) may naturally comprise histidine residues at one or more amino acid positions on the hotspot list. In these cases, the native histidine residue will generally remain in the engineered antibody as it may promote pH-dependent antigen binding. However, for the purposes of the methods described herein, the naturally occurring histidine would not be considered a histidine substitution. Thus, the methods described herein require that the engineered antibody must comprise at least one hotspot amino acid position at which the engineered antibody is derived compared to the parent antibody (from which the engineered antibody is derived)In additionA histidine residue. The introduction of one or more additional histidine residues into the engineered antibody may improve the pH-dependent binding of the engineered antibody (i.e., result in greater pH-dependence of binding to the target antigen), even for parent antibodies that already contain hotspot histidine residues.

Candidate amino acid residues for replacement by histidine were selected from a "hot spot" list (Table A) in which the amino acid positions in the CDRs of the VH and VL domains and the framework positions in the vicinity of the CDRs were identified according to the KABAT numbering system (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The engineered antibody must comprise at least one histidine substitution at an amino position selected from table a.

Various methods can be used to select the most appropriate histidine substitution to confer pH-dependent antigen binding in the parent antibody of interest. If structural information is available about the binding interaction of a parent antibody with its target antigen, it may be appropriate to select candidate amino acids from the hot-spot list for histidine substitution using a "rational design" approach. Hotspot residues that are directly involved in the antigen-antibody binding interaction or that may affect the overall structure of the antigen binding interface of the parent antibody may be selected based on such structural information. Engineered variants of the parent antibody in which one or more selected hotspot residues, or different combinations thereof, are replaced with histidine can then be synthesized, e.g., using the screening methods described herein, and screened for pH-dependent antigen binding.

Candidate residues for histidine substitution can also be selected based on the variable domain family or subtype of the parent antibody. Described herein are various subtype-specific lists of hotspot residues, which are also derived from heat maps of histidine presence within antibody pools. In certain embodiments in which the variable domain family or subtype of the VH and/or VL domain of the parent antibody is known, the candidate residues for histidine substitution may be selected from the following subtype-specific or family-specific hotspot list. These lists are not intended to be limiting, but merely provide guidance as to the amino acid positions at which histidine substitutions for a particular variable domain family or subtype may be most suitable. For any given variable domain, amino acid positions that are likely to be substituted with histidine can be selected from the complete hot spot list (table a), regardless of the variable domain family or subtype.

For the heavy chain variable domain of the VH3 family:

TABLE VH3A (VH3 family hotspot residues)

TABLE VH3B (VH3 family Cold Point residues)

CDR1	H34，H35a，H35b，H35c
		CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H63，H64，H65
CDR3	H100g，H100h，H100i，H100k，H100I，H100m，H100n

For the heavy chain variable domain of the VH1 family:

TABLE VH1A (VH1 family hotspot residues)

CDR1	H35
		CDR2	H52
CDR3	H100h，H102

TABLE VH1B (VH1 family Cold Point residues)

For the heavy chain variable domain of the VH4 family:

TABLE VH4A (VH4 family hotspot residues)

CDR1
		CDR2	H50，H52，H63
CDR3	H95，H100f，H100i，H100l

TABLE VH4B (VH4 family Cold Point residues)

For light chain variable domains of the lambda type:

TABLE V λ A (V λ hotspot residue)

CDR1	L30，L31，L32，L34，
		CDR2	L51，L52，L53，L55，
CDR3	L89，L91，L92，L93，L95a，L95b，L95c，L96

TABLE V.lamda.B (V.lamda.Cold-spot residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L33
		CDR2	L50，L51a，L51b，L51c，L51d，L54，L56
CDR3	L90，L94，L95，L95d，L95e，L95f，L97

For light chain variable domains of the V λ 1 family:

TABLE V λ 1A (V λ 1 family hotspot residue)

CDR1	L31，L32
		CDR2
CDR3	L91，L93，L95a，L95b，L96

TABLE V λ 1B (V λ 1 family cold spot residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L30，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L53，L54，L55，L56
CDR3	L89，L90，L92，L94，L95，L95c，L95d，L95e，L95f，L97

For light chain variable domains of the V λ 2 family:

TABLE V λ 2A (V λ 2 family hotspot residues)

CDR1
		CDR2
CDR3	L95a，L96
		FR	L87

TABLE V λ 2B (V λ 2 family Cold Point residues)

For light chain variable domains of the V λ 3 family:

TABLE V λ 3A (V λ 3 family hotspot residue)

CDR1	L31，L32，L34
		CDR2	L51，L52
CDR3	L89，L91
		FR	L49，L87

TABLE V λ 3B (V λ 3 family cold spot residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L30，L33
		CDR2	L50，L51a，L51b，L51c，L51d，L53，L54，L55，L56
CDR3	L90，L92，L93，L94，L95，L95a，L95b，L95c，L95d，L95e，L95f，L96，L97

For light chain variable domains of the V λ 5 family:

TABLE V.lamda.5A (V.lamda.5 family hotspot residue)

CDR1	L30，L31，L32
		CDR2	L53
CDR3	L92，L95c，L96
		FR	L49，L87

TABLE V.lamda.5B (V.lamda.5 family Cold Point residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L54，L55，L56
CDR3	L89，L90，L91，L93，L94，L95，L95a，L95b，L95d，L95e，L95f，L97

For light chain variable domains of the V λ 8 family:

TABLE V.lamda.8A (V.lamda.8 family hotspot residue)

CDR1	L31
		CDR2	L55
CDR3	L89，L91，L95b

TABLE V.lamda.8B (V.lamda.8 family Cold Point residues)

For light chain variable domains of the kappa type:

TABLE VKA (VK hotspot residue)

CDR1	L27，L27d，L29，L31，L32
		CDR2	L53，L54，L55
CDR3	L89，L90，L91，L92，L93，L94，L96

TABLE VKB (VK Cold-Point residue)

CDR1	L24，L25，L26，L27A，L27b，L27c，L27e，127f，L28，L30，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L56
CDR3	L95，L95a，L95b，L95c，L95d，L95e，L95f，L97

For the light chain variable domain of the VK1 family:

TABLE VK1A (Hot residue of VK1 family)

CDR1	L31，L32
		CDR2
CDR3	L89，L90，L91，L93

TABLE VK1B (VK1 family cold-spot residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L30，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L53，L54，L55，L56
CDR3	L92，L94，L95，L95a，L95b，L95c，L95d，L95e，L95f，L96，L97

For the light chain variable domain of the VK2 family:

TABLE VK2A (Hot residue of VK2 family)

CDR1	L27，L27d，L32
		CDR2	L53，L54，L55
CDR3	L91，L93，L94，L96

TABLE VK2B (VK2 family cold-spot residues)

For the light chain variable domain of the VK4 family:

TABLE VK4A (Hot residue of VK4 family)

CDR1	L27，L29
		CDR2	L54
CDR3	L90，L91，L92

TABLE VK4B (VK4 family cold-spot residues)

CDR1	L24，L25，L26，L27a，L27b，L27c，L27d，L27e，L28，L30，L31，L32，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L53，L55，L56
CDR3	L89，L93，L94，L95，L95a，L95b，L95c，L95d，L95e，L95f，L96，L97

Screening panel of variants

Some embodiments of the methods of the invention may be based on constructing a panel of engineered variants of a parent antibody, which are then screened to identify one or more engineered variants that exhibit the desired pH-dependent binding. Construction of a set of variants allows systematic mutation analysis of a large number of possible histidine substitutions at amino acid positions defined by the heatmap. This method differs from prior art combinatorial methods in that the construction of the set of variants is guided by a heatmap of the presence of histidine, and therefore it is not necessary or desirable to sample all possible amino acid positions and combinations thereof in CDRs that are candidates for histidine substitution.

The mutation analysis method of the system described herein typically comprises the steps of:

(a) providing a parent antibody that binds to an antigen;

(b) preparing a set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue is replaced with a histidine residue, wherein at least one amino acid residue selected from the hotspot list (table a) is replaced with a histidine and at least one amino acid residue selected from the cold spot list (table B) is not replaced with a histidine;

(c) screening the panel of engineered variants for pH-dependent binding to the antigen, and thereby identifying engineered antibodies that exhibit pH-dependent binding to the antigen.

The method entails preparing a set of engineered variants of a parent antibody of interest, wherein each engineered variant in the set differs from the parent antibody in that at least one amino acid is replaced with histidine. The choice of amino acid positions for substitution with histidine was also based on the heatmap of the presence of histidine. Each engineered variant in the set must comprise at least one histidine substitution at an amino acid position selected from the hotspot list (table a) and not be substituted with a histidine at least one amino acid residue selected from the cold spot list (table B). Preferably, each engineered variant in the set will comprise a different pattern of histidine substitutions to allow parallel analysis of the effect of different histidine substitutions on pH-dependent antigen binding.

In one embodiment of the method, each engineered variant in the set may comprise a single histidine substitution at one of the listed hotspot amino acid positions (table a). Preferably, each engineered variant in the set comprises a different histidine substitution at one of the listed hotspot amino acid positions (table a). Such a set allows analysis of the effect of histidine substitutions at individual hotspot amino acid positions on pH-dependent binding. The set of engineered variants may represent the entire hot spot list, i.e., may comprise variants having a single histidine substitution at each amino acid position listed in table a; or may represent a selected subset of the hot spot list, such as a subset of substitutions associated with one or more specific variable domain families or subtypes. In addition, the set may also comprise variants comprising histidine substitutions at one or more "cold spot" positions within VH CDR3 or VL CDR 3.

In other embodiments, the set prepared in step (B) may comprise engineered variants comprising different combinations of two or more, three or more, four or more or five or more histidine substitutions, provided that at least one of said 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 a histidine. Preferably, all said histidine substitutions are made at hotspot amino acid positions selected from table a. This panel allowed screening for combinations of histidine substitutions (and in particular for hotspot histidine substitutions) that confer pH-dependent binding. In addition, the panel may further comprise a variant comprising a histidine substitution at one or more "cold spot" positions within VH CDR3 or VL CDR3, optionally in combination with a histidine substitution at one or more hot spot amino acid positions.

In certain embodiments, as described above, a panel of engineered variants comprising a single histidine substitution may be initially screened to identify a single amino acid position at which a histidine substitution may confer pH-dependent binding. Based on the results of this initial screen, one or more additional engineered variants can be constructed in which histidine substitutions previously identified as conferring pH-dependent binding are combined. Such a method may comprise the steps of:

(a) identifying a parent antibody that binds to the antigen;

(e) screening the further engineered variants for pH-dependent binding to the antigen; and thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen.

In some embodiments of the methods, the effect of histidine substitutions in the VH and VL domains on pH-dependent binding can be analyzed separately. Thus, in one embodiment, step (b) comprises preparing a set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VH domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the heavy chain hot spot list (table HA) is replaced with a histidine and at least one amino acid residue selected from the heavy chain cold spot list (table HB) is not replaced with a histidine. Furthermore, the panel may also comprise variants comprising histidine substitutions at "cold spot" positions within VHCDR 3.

Typically, the engineered variant will comprise a mutant VH domain paired with the VL domain of the parent antibody, which comprises one or more histidine substitutions. Such libraries allow screening of VH domains for histidine substitutions conferring pH-dependent binding.

TABLE HA (VH hotspot residue position)

TABLE HB (VH cold spot residue position)

VH CDR1	H34，H35a，H35b，H35c
		VH CDR2	H51，H52b，H52c，H54，H55，H57，H60，H61，H64，H65
VH CDR3	H100g，H100k，H100m，H100n

In another embodiment, step (b) comprises preparing a set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VL domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the light chain hot spot list (table LA) is replaced with histidine and at least one amino acid residue selected from the light chain hot spot list (table LB) is not replaced with histidine. In addition, the set may also comprise variants comprising histidine substitutions at "cold spot" positions within VL CDR 3.

Typically, the engineered variant will comprise a mutant VL domain paired with a VH domain of a parent antibody, which comprises one or more histidine substitutions. Such libraries allow screening of VL domains for histidine substitutions conferring pH-dependent binding.

TABLE LA (VL hotspot residue positions)

TABLE LB (VL cold spot residue positions)

VL CDR1	L24，L25，L26，L27a，L27b，L27c，L27e，L28，L33
		VL CDR2	L50，L51a，L51b，L51c，L51d，L56
VL CDR3	L95，L95d，L95e，L95f，L97

For lambda-like light chain variants, the "hot spot" and "cold spot" residue positions may be selected from the following list:

TABLE V λ A (V λ hotspot residue)

CDR1	L30，L31，L32，L34，
		CDR2	L51，L52，L53，L55，
CDR3	L89，L91，L92，L93，L95a，L95b，L95c，L96

TABLE V.lamda.B (V.lamda.Cold-spot residues)

CDR1	L24，L25，L26，L27，L27a，L27b，L27c，L27d，L27e，L28，L29，L33
		CDR2	L50，L51a，L51b，L51c，L51d，L54，L56
CDR3	L90，L94，L95，L95d，L95e，L95f，L97

For kappa-class light chain variants, the "hot spot" and "cold spot" residue positions may be selected from the following list:

TABLE VKA (VK hotspot residue)

CDR1	L27，L27d，L29，L31，L32
		CDR2	L53，L54，L55
CDR3	L89，L90，L91，L92，L93，L94，L96

TABLE VKB (VK Cold-Point residue)

CDR1	L24，L25，L26，L27A，L27b，L27c，L27e，127f，L28，L30，L33，L34
		CDR2	L50，L51，L51a，L51b，L51c，L51d，L52，L56
CDR3	L95，L95a，L95b，L95c，L95d，L95e，L95f，L97

In certain embodiments, the histidine substitutions of the VH and VL domains identified by the separate initial VH and VL domain screens may be combined in a single engineered variant. Additional screens can then be performed to identify VH and VL combinations that exhibit pH-dependent binding. Such a method may comprise the steps of:

(a) identifying a parent antibody that binds to the antigen;

(b) preparing a first set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in a VH domain is substituted with a histidine residue, wherein at least one amino acid residue selected from the heavy chain hot spot list (table HA) is substituted with a histidine and at least one amino acid residue selected from the heavy chain cold spot list (table HB) is not substituted with a histidine;

(d) preparing a second set of engineered variants of said parent antibody, wherein each of said engineered variants in said set differs from said parent antibody in that at least one amino acid residue in the VL domain is replaced with a histidine residue, wherein at least one amino acid residue selected from the light chain hot spot list (table LA) is replaced with a histidine and at least one amino acid residue selected from the light chain hot spot list (table LB) is not replaced with a histidine;

(g) screening the further engineered variants for pH-dependent binding to the antigen; and thereby identifying an engineered antibody that exhibits pH-dependent binding to the antigen.

This approach enables the identification of synergy between histidine mutations identified in the initial (individual) VH and VL domain screens. For example, VH mutations that exhibit the best (i.e., those that confer the greatest degree of pH dependence) can be combined with a number of different VL mutations, or VL mutations that exhibit the best can be combined with different VH mutations, to identify synergy between different combinations of VH and VL domain mutations. In this embodiment, histidine substitutions, and combinations thereof, may also be included at "cold spot" positions within the VH CDR3 or VL CDR 3.

Screening method

A variety of screening methods can be used to identify engineered antibodies that exhibit pH-dependent binding to a target antigen. Such screening methods may comprise the steps of:

(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) selecting an engineered antibody having an antigen binding activity at acidic pH that differs from the antigen binding activity at neutral pH.

Suitable methods and conditions for determining antigen binding activity at a defined pH are generally known in the art. For example, as described in the accompanying experimental examples, by(GE Healthcare) to determine the antigen binding activity of the antibody at a defined pH. Other suitable techniques (including ELISA or MSD)Can be used to determine antigen binding at specific antibody concentrations (e.g., EC50) under different pH conditions (e.g., pH7.4 and pH 5.5).

The method described herein may comprise the steps of: the antigen binding activity of the engineered antibody is measured at neutral pH and at acidic pH, and one or more binding parameters at neutral pH and/or acidic pH are compared to determine an engineered antibody that exhibits pH-dependent binding.

In certain embodiments, the antigen binding activity of the engineered antibody can be measured by a dual pH ELISA, as described in the accompanying examples. In this setup, initial binding of the antibody to the antigen is assessed at neutral pH (e.g., pH7.4), followed by a washing step at acidic pH (e.g., pH 5.5). This allows for differentiation of the effect of pH on antibody binding, dissociation or both, and may be more representative for the following in vivo situations: in this case antibody binding to the target antigen occurs at neutral pH, whereas antigen release occurs at acidic endosomal pH.

Typically, the engineered antibody selected has a lower affinity for its antigen at acidic pH than at neutral pH. In the initial screen, any significant difference in affinity at acidic pH relative to that at neutral pH can indicate pH-dependent binding.

Engineering the dissociation rate constant (k) of antibody-antigen interactions at acidic pH_d) (i.e., dissociation rate) can be higher than the dissociation rate constant (k) for engineering antibody-antigen interactions at neutral pH_d). In the initial screening, (k) at acidic pH_d) Relative to (k) at neutral pH_d) Any significant difference in (a) may be indicative of pH-dependent binding. In other embodiments, the off-rate constant (k) of the antibody-antigen interaction is engineered at acidic pH_d) Dissociation rate constant (k) for engineered antibody-antigen interaction at neutral pH_d) The ratio of (a) may be at least 1.5, or at least 2, or at least 5, or at least 10. When multiple histidine mutations are combined, the overall degree of pH dependence can be increased.

Engineering the equilibrium dissociation constant (K) of antibody-antigen interactions at acidic pH_D) Equilibrium dissociation constant (K) that can be higher than for engineering antibody-antigen interactions at neutral pH_D). In the initial screening, K at acidic pH_DRelative to K at neutral pH_DAny significant difference in (a) may be indicative of pH-dependent binding. Engineering the equilibrium dissociation constant (K) of antibody-antigen interactions at acidic pH_D) Equilibrium dissociation constant (K) with engineered antibody-antigen interaction at neutral pH_D) The ratio of (d) may be greater than 1.5, or greater than 2, or greater than 5, or greater than 10.

When multiple histidine mutations are combined, the overall degree of pH dependence can be increased. In some particularly preferred embodiments, the combination histidine mutations may achieve approximately 20 to 40 fold stronger antigen binding at neutral pH relative to antigen binding at acidic pH.

As explained elsewhere herein, improvements in pH-dependent binding can be achieved with or without significant effect on affinity for the target antigen at neutral pH.

In which the dissociation constant (K) is balanced_D) In some non-limiting embodiments used as an indicator of antigen binding activity, engineering an antibody at acidic pH (e.g., pH 5.5) may exhibit an equilibrium dissociation constant (K) for its antigen of 10 to 20nM_D) (ii) a While K of antibody to its antigen is engineered at neutral pH (e.g., 7.4)_DAnd may be about 0.5nM or less.

In each of the foregoing embodiments, "neutral pH" means a pH of from pH 6.7 to pH 10.0, preferably from pH 7.0 to pH 8.0, more preferably pH 7.4.

Additionally or alternatively, the methods described herein may comprise the steps of: the antigen binding activity of the parent antibody at acidic pH and/or at neutral pH is measured and the parameters of the binding interaction are compared to the corresponding binding parameters of the engineered antibody-antigen interaction.

Engineering the dissociation rate constant (k) of an antibody at acidic pH (e.g., pH 5.5)_d) Can be higher than the dissociation rate constant (k) of the parent antibody at the same acidic pH_d). In such embodiments, k is at an acidic pH (e.g., pH 5.5) as compared to the parent antibody due to the histidine substitution_dAnd (4) improving. Engineering the dissociation rate constant (k) of antibody-antigen interactions at acidic pH_d) Dissociation rate constant (k) for parent antibody-antigen interaction at the same acidic pH_d) The ratio of (a) may be at least 1.5, or at least 2, or at least 5, or at least 10.

Engineered antibodies

Also provided herein are engineered antibodies that exhibit pH-dependent binding to their antigen, wherein at least one amino acid in a CDR of the engineered antibody is a histidine residue, characterized in that at least one amino acid residue selected from the hot spot list (table a) is a histidine residue, and at least one amino acid residue from the cold spot list (table B) is not a histidine residue.

By "engineered antibody" is meant an antibody whose amino acid sequence has been deliberately altered or mutated in vitro. In the present disclosure, an "engineered antibody" is an antibody variant in which the amino acid sequences of the CDRs of the antibody have been deliberately altered or mutated to introduce one or more additional histidine residues.

The CDRs of antibody VL and VH domains can generally be defined as comprising the following amino acids: residues 24 to 34 (CDRL1), 50 to 56 (CDRL2) and 89 to 97 (CDRL3) in the light chain variable domain, and residues 31 to 35 or 31 to 35c (CDRH1), 50 to 65 (CDRH2) and 95 to 102 (CDRH3) in the heavy chain variable domain (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991)).

The engineered antibody may comprise a total of 1, 2, 3, 4, 5 or more histidine residues in the CDRs of the VH domain, or the VL domain, or both the VH domain and the VL domain.

At least one histidine residue in the CDRs of the engineered antibody must be present at an amino acid position selected from the "hot spot" list (table a). In certain embodiments, at least two, at least three, at least four, or at least five histidine residues must be present at amino acid positions selected from the "hot spot" list. In certain embodiments, all histidine residues in the engineered antibody are present at amino acid positions selected from the hotspot list (table a). As described herein, certain amino acid positions, which are preferred, are present in the CDRs of the engineered antibodyIs not limited toOccupied by histidine residues. These amino acid positions are those shown on the "cold spot" list (table B). The engineered antibody must contain at least one amino acid position on the cold spot list (table B) that is not occupied by a histidine residue. In certain embodiments, at least two, at least three, at least four, or at least five of the amino acid positions on the cold spot list (table B) are not histidines. In other embodiments, none of the amino acid positions on the cold spot list (table B) are histidines. In addition, in certain embodiments, it is also permissible to include histidine substitutions, and combinations thereof, at "cold spot" positions within VH CDR3 or VL CDR 3.

The engineered antibody exhibits pH-dependent binding to its antigen, meaning that the antigen-binding activity of the antibody at acidic pH differs from the antigen-binding activity of the antibody at neutral pH.

In one embodiment, the engineered antibody has a lower affinity for its antigen at acidic pH than at neutral pH.

In one embodiment, the off-rate constant (k) of the antibody-antigen interaction is engineered at acidic pH_d) Higher than the dissociation rate constant (k) for engineering antibody-antigen interactions at neutral pH_d)。

In one embodiment, inEquilibrium dissociation constant (K) for engineered antibody-antigen interactions at acidic pH_D) Higher than equilibrium dissociation constant (K) for engineering antibody-antigen interactions at neutral pH_D)。

In some particularly preferred embodiments, the engineered antibody may exhibit 20 to 40 fold stronger antigen binding at neutral pH relative to antigen binding at acidic pH.

In which the dissociation constant (K) is balanced_D) In some non-limiting embodiments used as an indicator of antigen binding activity, engineering an antibody at acidic pH (e.g., pH 5.5) may exhibit an equilibrium dissociation constant (K) for its antigen of 10 to 20nM_D) (ii) a While K of antibody to its antigen is engineered at neutral pH (e.g., 7.4)_DAnd may be about 0.5nM or less.

Structure of engineered antibodies

The engineered antibodies described herein are typically four-chain immunoglobulins of the conventional type in which the antigen binding specificity is provided by paired VH and VL domains, thereby contributing six CDRs to the antigen binding site. However, the term "engineered antibodies" also encompasses antigen-binding fragments of conventional immunoglobulins and engineered antigen-binding constructs, including but not limited to Fab, F (ab')₂Fv, scFv, diabodies, triabodies, minibodies, and the like, as well as any other modified immunoglobulin configuration that comprises an antigen binding site provided by paired VH and VL domains.

The engineered antibody may comprise a constant region comprising one or more or all of a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. In particular, the engineered antibody may comprise an Fc region (comprised of CH2 and CH3 domains) that may confer one or more antibody effector functions. The Fc region of the engineered antibody may itself be engineered or modified to impart useful functional properties.

The engineered antibody may be of any antibody class, including: IgA, IgD, IgE, IgG or IgM. In some preferred embodiments, the engineered antibody is an IgG. The engineered antibody may be an IgG of any subclass (isotype), including: IgG1, IgG2, IgG3, or IgG 4.

In a preferred embodiment, the engineered antibody may comprise an Fc region capable of binding to a neonatal Fc receptor (FcRn). In particular, the Fc region may be expected to exhibit strong binding affinity for FcRn at neutral pH. The Fc region of the engineered antibody may be modified to enhance binding to the neonatal Fc receptor FcRn. Some examples of Fc modifications that confer increased binding to FcRn include amino acid substitutions at one or more amino acid positions in the CH2 and/or CH3 domains of the Fc region.

Suitable Fc mutations include Abdeg as described by Vaccaro et al, Nature Biotechnology, Vol.23, 1283-1288(2005)^TMAnd (4) mutation. Abdeg^TMThe mutants are engineered variants of human IgG (including but not limited to human IgG1) comprising mutations of Met252, Ser254, Thr256, His 433 and Asn434 to Tyr252, Thr254, Glu256, Lys433 and Phe434(EU numbering). Carrying Abdeg^TMMutated engineered antibodies (e.g., engineered human IgG1) exhibit enhanced affinity for binding to FcRn relative to their wild-type counterpart (counterpart) and bind to FcRn more stably at the cell surface during extracellular events. Preferred engineered antibodies bind one or more histidine substitutions in the CDRs of the variable domain as defined herein to the Abdeg mutation in the Fc region.

Other suitable Fc mutations include NHance described in US 8,163,881^TMAnd (4) mutation. NHance^TMThe mutations are engineered variants of human IgG (including but not limited to human IgG1) comprising mutations of His 433 and Asn434 through Lys433 and Phe434(EU numbering). Carrying NHance^TMMutated engineered antibodies (e.g., engineered human IgG1) exhibit enhanced affinity for binding to FcRn relative to their wild-type counterpart. Preferred engineered antibodies will substitute one or more histidines in the CDRs of the variable domain as defined herein with NHance in the Fc region^TMMutation binding.

In a preferred embodiment, the engineered antibody may comprise variable domains (VH and VL) of a camelid-derived antibody (e.g. a llama antibody)Engineered variants comprising one or more histidine substitutions in a CDR, and an Fc region of a human IgG (e.g., human IgG1) comprising an Abdeg^TMMutations Tyr252, Thr254, Glu256, Lys433 and Phe434(EU numbering).

In another preferred embodiment, the engineered antibody may comprise variable domains (VH and VL) that are engineered variants of the variable domains of camelid derived antibodies (e.g. llama antibodies) comprising one or more histidine substitutions in the CDRs and an Fc region of a human IgG (e.g. human IgG1) comprising NHance^TMMutations Lys433 and Phe434(EU numbering).

Engineered antibodies that exhibit the dual properties of pH-dependent antigen binding plus enhanced binding affinity for FcRn can exert an active antigen removal (active antigen removal) effect and can be used to eliminate soluble antigens from plasma in vivo. The concept of "active antigen removal" utilizes engineered antibodies to mimic the action of cell surface endocytotic receptors, thereby enabling selective elimination of target antigens from plasma. Briefly, the engineered antibody binds to the corresponding antigen present in plasma (at neutral pH) to form an antigen-antibody complex. The complex is taken up randomly into the endosome (usually by pinocytosis) where the antibody Fc part will bind to FcRn when the pH becomes acidic. Due to the pH-dependent antigen binding properties of the engineered antibody, the bound antigen will be released in acidic endosomes and targeted for degradation by the lysosomal pathway. The free antibody (still bound to FcRn, but no longer to its antigen) will then be recycled back to the cell surface where it may participate in other rounds of antigen binding and internalization. Fc mutations that favor binding to FcRn at acidic pH without substantially affecting binding at pH7.4 can enhance this "active antigen removal" process.

In addition to the specific Fc mutations listed above, the engineered antibody may further comprise: any other mutation or modification that improves antibody uptake in endosomes, including, for example, a change in pI, a change in charge, a change in FcRn affinity at pH7.4 and/or pH 6.0; any mutant or modification that promotes increased binding to any other "highly recycled" Fc receptor (e.g., such as an Fc γ receptor, particularly Fc γ RIIb); or any mutation that enhances immune complex formation (e.g., complement fixation site).

The engineered antibody may be a parent antibody or an engineered variant of a chimeric antibody from human, murine, rat, rabbit, camelid, or other mammalian species. In some particular embodiments, the engineered antibody may be an engineered variant of a camelidae antibody isolated from a camelidae species (e.g. llama, camel, dromedary, guanaco, llama minor, etc.), or an engineered variant of a camelidae-human chimeric antibody (e.g. a llama-human chimera) in which the variable domains (or CDRs thereof) are derived from a camelidae species (e.g. llama) and the constant domains are human. Techniques for the production of camelid antibodies and camelid-human chimeric antibodies are described in WO2010/001251, the contents of which are incorporated herein by reference.

The antigen binding specificity of the engineered antibody, i.e., the nature of the target antigen to which it binds, is not particularly limited. Applicants have demonstrated that the methods provided herein are applicable to the engineering of pH-dependent variants of a number of different parent antibodies with different antigen binding properties. Thus, the described engineered techniques for pH-dependent antigen binding represent a generally applicable principle, which is not limited to antibodies binding to one specific target antigen.

Illustration of

The invention is further illustrated by the following examples, which are not to be construed as further limiting. The contents of all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Brief Description of Drawings

FIG. 1: illustrating pH-dependent antigen binding in engineered variants of the parent antibody 18E2 as measured using a functional assay. Inhibitory ligands were measured at both pH7.4 and pH5.5 for the parent antibody 18E2 and engineered variants of 18E2 containing one or two identified histidine mutations: IC50 for receptor interaction.

Fig. 2, 3 and 4: illustrating the pH-dependent binding of histidine mutants of the parent antibody 5D1 as measured using MSD technique.

Example 1: construction of heatmaps

To identify the amino acid residue positions in the V domains where histidine residues are allowed to be naturally located in large antibody repertoires, the sequences of the VH, V λ and V κ domains of a large number of functional (antigen-binding) antibodies or corresponding Fab fragments from several target antigen projects were pooled and analyzed.

Each target antigen item includes active llama immunization (DNA vaccination) with purified antigen or DNA expressing the target, Fab or scFv phage display library construction, and phage display selection and screening for specific binders. Phage display was accomplished according to standard procedures, usually using trypsin as eluent for up to three rounds of selection, followed by expression of Fab (or scFv) in periplasmic extracts and identification of Fab (or scFv) binders by ELISA, MSD or spr (biacore). In addition to trypsin elution, and in order to further enrich for antibodies with pH-dependent binding, a parallel phage display procedure was followed, wherein eluents in TBS (50mM Tris pH5.5 and 150mM NaCl) (pH 5.5) were used for up to 3 rounds of selection. All sequences were then pooled and subdivided into classes (VH, V λ or V κ) and subfamilies (e.g., VH1, VH2 and VH 3). Any exact duplicate sequences were removed and only the unique sequences were used to count the number of histidine residues at each position of the V domain. This led to the creation of a "heatmap" that shows at which amino acid positions histidine is acceptable in the V domain of immunized llama-derived antibodies.

In addition, this data set can be further supplemented with sequences identified by high-throughput sequencing using RNA from immunized llamas.

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