CN112592404B - Antibody Activity Modification and Screening Method - Google Patents

Abstract

The invention discloses a method for modifying the agonistic activity of an antibody, which comprises the step of modifying a heavy chain constant region of the antibody to improve or reduce the flexibility of the antibody, so as to respectively obtain the antibody with reduced agonistic activity and improved agonistic activity. The method is simple and convenient, and the antibody with enhanced or reduced agonistic activity can be obtained through transformation so as to adapt to the requirements of different classes of antibodies, thereby having wide application prospects.

Description

Antibody Activity Modification and Screening Method

technical field

The invention relates to the field of biopharmaceuticals, in particular to a method for modifying and screening agonistic antibodies or molecular activities.

Background technique

The development of biomacromolecular drugs has provided new methods and possibilities for the treatment of various diseases, especially molecular targeted therapy based on antibodies and heavy chain constant regions (including Fc fragments), including antibody and heavy chain constant region fusion proteins, has achieved great success in the field of biopharmaceuticals for more than 30 years and continues to be the focus of this field. The basis of all biomacromolecular drugs is their structural features, which allow them to specifically bind bioactive molecules and affect biological processes (such as immune responses). Therefore, it is of great significance to analyze the structural characteristics of biomacromolecules and establish the relationship between them and their functions for drug development.

From the mode of action, biomacromolecules can be mainly divided into three categories: effector molecules that clear targets (molecules and cells), blocking molecules that block signaling pathways involved in targets, and agonist molecules that activate downstream signaling pathways of targets. Tumor immunotherapy has made important breakthroughs in recent years. This is aided by the use of antibodies that increase the activity of immune cells to kill tumors by blocking the immunosuppressive node. However, there are still a large number of cancer patients who do not respond to existing treatments. Therefore, on the one hand, it is necessary to optimize the existing tumor immunotherapy methods; on the other hand, it is urgent to develop new tumor immunotherapy drugs. It should be pointed out that there is a class of tumor immunotherapy methods called "agonistic antibodies", which can bind to target molecules that transmit immune activation signals on the surface of immune cells and activate important immune activation signaling pathways controlled by them, thereby enhancing anti-tumor immune responses and indirectly killing tumor cells. However, although agonistic tumor immunotherapy antibodies have demonstrated their great potential in animal models and have become a widely accepted and optimistic concept of tumor immunotherapy, the development of such antibodies has not been successful so far, which is currently a major challenge in the field of tumor immunotherapy. In addition, the activation of agonistic antibodies is also a favorable means to intervene and regulate key signaling pathways in other biological processes, and has broad application prospects in the field of disease prevention and treatment. For example, activation of immunosuppressive signaling pathways may be beneficial in reducing inflammation and autoimmune symptoms.

Contents of the invention

The present invention mainly provides a method for modifying the agonistic activity of an antibody, mainly by modifying the flexibility of the antibody so that it has different agonistic activities. At the same time, the present invention also provides a method capable of detecting the flexibility of the protein. Combining the above method, antibodies with enhanced or weakened agonistic activity can be obtained simply and easily, providing a new idea for the functional modification of therapeutic antibodies.

The invention provides a method for improving the agonistic activity of an agonistic antibody, wherein the heavy chain constant region of the agonistic antibody is modified to reduce the flexibility of the agonistic antibody.

In some aspects, the agonistic antibody is no more flexible than human IgGl after modification.

In some preferred schemes, the CH1-hinge region in the heavy chain constant region is modified, preferably the hinge region in the heavy chain constant region is replaced by a less flexible sequence, more preferably the sequence is the hinge region sequence of human IgA2.

In some preferred embodiments, the modification of the antibody does not significantly reduce the affinity of the antibody for the antigen to which it is specifically targeted.

In some other preferred schemes, the Fc segment in the constant region of the heavy chain is further modified to increase the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

In some other preferred schemes, the radius of gyration (Rg) of the modified agonistic antibody is preferably /> Or less than the Rg of IgGl (eg, human IgGl).

In some other preferred schemes, the antibody specifically recognizes receptors in the TNF receptor superfamily; or the antibody is an anti-CD40 antibody or an anti-DR5 antibody.

The present invention also provides a method for reducing the agonistic activity of the antibody, modifying the heavy chain constant region of the agonistic antibody to improve the flexibility of the antibody. Preferably, the flexibility of the agonistic antibody after modification is greater than or equivalent to that of IgG3.

In some schemes, the CH1-hinge region in the heavy chain constant region is modified, preferably, a flexible linking sequence is inserted into the CH1-hinge region, preferably the flexible linking sequence is a flexible linking sequence comprising G and S, more preferably the flexible linking sequence is GSGSGS or other flexible linking sequences comprising G and S, or the CH1-hinge region is replaced with a CH1-hinge region of human IgG3 to improve the flexibility of the agonist antibody.

In some aspects, the modification to the antibody does not substantially reduce the affinity of the antibody for the antigen to which it is specifically targeted.

In some preferred embodiments, the radius of gyration (Rg) of the modified agonistic antibody is greater than

In some other preferred schemes, the Fc segment in the constant region of the heavy chain is further modified to reduce the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

In some other preferred embodiments, the antibody is a human antibody, a chimeric antibody or a humanized antibody.

The present invention also provides a method for screening the agonistic activity of antibodies, comprising the following steps:

1) providing an agonistic antibody as a parent, and providing a variant of the parent whose CH1-hinge region has been modified on the basis of the parent, wherein the antibody in which the CH1-hinge region of the parent is replaced by the CH1-hinge region of human IgG1 is called a human IgG1 variant;

2) Detect the flexibility of the female parent and the variant;

3) According to the results of flexibility detection, antibodies with flexibility not higher than human IgG1 variants were selected from all agonistic antibodies, and these antibodies had better agonistic activity.

In some schemes, the screening method further includes detecting the affinity of the parent and variants to FcγRIIB and/or the I/A ratio of the agonistic antibody, comparing the detection results, and screening out antibodies whose affinity to FcγRIIB and I/A ratio are not lower than those of the human IgG1 variant, and these antibodies have better agonistic activity.

Preferably, said step 2) includes:

1) providing an antigen capable of specifically binding to the antigen-binding portion of the agonistic antibody;

2) The antigens are respectively labeled with a pair of donor fluorescent molecules and acceptor fluorescent molecules capable of realizing fluorescence resonance energy transfer to obtain donor fluorescently labeled antigens and acceptor fluorescently labeled antigens;

3) adding the donor fluorescently-labeled antigen and acceptor fluorescently-labeled antigen to the agonistic antibody;

4) Exciting the donor fluorescent molecule and detecting the fluorescent signal emitted by the acceptor fluorescent molecule, the intensity of the fluorescent signal is positively correlated with the flexibility of the agonistic antibody.

More preferably, said step 2) includes:

1) Using the method of small-angle X-ray scattering to detect any one or more of the following parameters of the antibody parent and variant: radius of gyration (Rg), Dimensionless Kratky plots (or Kratky plots) feature, P(R)/I(0) (or P(R)) distribution, P(R)/I(0) (or P(R)) large size distribution, radius of gyration (Rg) calculated by EOM method, and maximum interatomic distance calculated by EOM method (Dmax) distribution, Rflex or Rσ value calculated by EOM method;

2) The larger the radius of gyration (Rg), the higher the upward degree of the Dimensionless Kratky plots (or Kratky plots) curve, the wider the distribution of the radius of gyration (Rg) and the maximum interatomic distance (Dmax) calculated by the EOM method, and the larger the values of Rflex and Rσ, the stronger the antibody flexibility; conversely, the weaker the antibody flexibility.

The present invention also provides a flexible detection method of biomacromolecules, comprising the following steps:

1) providing the biomacromolecules, preferably these biomacromolecules are proteins, nucleic acids, lipid molecules, sugar molecules or complexes formed by their mutual association;

2) providing two epitope recognition molecules capable of specifically binding to two separate epitopes of the biomacromolecule;

3) The two epitope recognition molecules are respectively labeled with a donor fluorescent molecule and an acceptor fluorescent molecule, and the donor fluorescent molecule and the acceptor fluorescent molecule match each other and can realize fluorescence resonance energy transfer;

4) mixing the two labeled epitope recognition molecules with the biomacromolecule;

5) Exciting the donor fluorescent molecule and detecting the fluorescent signal emitted by the acceptor fluorescent molecule, the intensity of the fluorescent signal is positively correlated with the flexibility of the antibody.

Preferably, the biomacromolecule exists in a conformation in which the distance between the two individual epitopes is between R0 and 2R0 of the donor fluorescent molecule and the ligand fluorescent molecule.

Preferably, the distance between two individual epitopes of the biomacromolecule may be between R0 and 2R0 of the donor fluorescent molecule and the ligand fluorescent molecule.

In some embodiments, the biomacromolecule is an antibody, the epitope is an antigen-binding site of the antibody, and the epitope recognition molecule is an antigen.

In some preferred schemes, the donor fluorescent molecule is Tb, and the acceptor fluorescent molecule paired with it is selected from D2, XL665, Fluorescein, GFP, Lucifer yellow, Acridine yellow, Proflavine, Atto465, Nitrobenzoxadiazole, Courmarin 6, Alexa750, Cy7, Nile red, Alexa488, Dy495, Dy490, D y648, Dy647, Oregon green, Atto488, Atto495, Alexa514, Atto520, Cy2, Rhodamine6G, Alexa700, Alexa680, Atto532, Alexa532, APC, EGFP, YFP, mPlum, Atto425, Alexa430, Coumarin 343, Acridine Orange, Tetramethyl Any of rhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Cy5, Att0590, Atto550, Cy3.5, Cy5.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, Alexa647, mCherry, mStrawberry one; or, the donor fluorescent molecule is Eu, and the acceptor fluorescent molecule paired with it is selected from APC, D2, XL665, Fluorescein, GFP, Rhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Atto550Cy3.5, Dy547, Dy548, Dy549, Any one of Dy554, Dy555, Dy556, Dy560, mCherry, mStrawberry, Alexa680, Alexa700, Alexa750, Alexa647, Cy5, Cy5.5, Cy7, Dy647, Dy648, Atto590.

The invention also provides a method of modulating antibody flexibility or modulating agonistic activity of an antibody comprising mutating the upper hinge domain of the hinge region. The mutation changes the number and/or ratio of amino acids with less hindrance in the upper hinge domain of the hinge region, or changes the length of the upper hinge domain.

In one or more embodiments, the antibody is a human antibody, a chimeric antibody, or a humanized antibody.

In one or more embodiments, the antibody is an anti-CD40 antibody or an anti-DR5 antibody.

In some embodiments, the method is a method of up-regulating antibody flexibility or down-regulating antibody agonistic activity, and the mutation increases the number and/or proportion of amino acids with less steric hindrance in the upper and/or middle hinge domain of the hinge region, or increases the length of the upper and/or middle hinge domain.

In one or more embodiments, the mutation is selected from one or more of the following, (1) inserting 1, 2, 5 or at least 6 amino acids with less hindrance in the upper hinge domain, (2) deleting amino acids with greater hindrance from the upper and/or middle hinge domain, (3) mutating amino acids with more hindrance in the upper and/or middle hinge domains to amino acids with less hindrance.

In one or more embodiments, the amino acid with less hindrance is selected from: glycine (G), alanine (A), serine (S), valine (V), threonine (T), isoleucine (I), leucine (L). Preferably, the amino acid with less hindrance is selected from G or S.

In one or more embodiments, more sterically hindered amino acids include aromatic amino acids and heterocyclyl amino acids.

In one or more embodiments, the amino acid with greater steric hindrance is selected from proline (P), hydroxyproline (O), asparagine (N), aspartic acid (D), pyroglutamic acid (U), glutamine (Q), lysine (K), glutamic acid (E), methionine (M), histidine (H), phenylalanine (F), arginine (R), tyrosine (Y), tryptophan (W).

In one or more embodiments, (1) is to insert 1, 2, 5 or at least 6 less sterically hindered amino acids after at least the 1st, at least the 2nd, at least the 3rd, at least the 4th or at least the 5th amino acid at the N-terminal of the upper hinge domain, or between the upper hinge domain and the middle hinge domain.

In one or more embodiments, the insertion in (1) is the insertion of 1, 2, 5 or at least 6 amino acids selected from G and S.

Preferably, (1) is the insertion of G, GS, SG, GSGSG, SGSGS, GGGGS, GGGSG, GGSGG, GSGGG, SGGGG, GSGSG, GSSGG, GGSSG, GSGGS, GGSGS, GGGSS, SGGSG, SGSGG, SSGGG, SGGGS, SGSGS, SGGSS, SSG between the upper hinge domain and the middle hinge domain GS, SGSSG, SSGSG, SSSGG, GSSGS, GSGSS, GGSSS, GSSSG, GSSSS, SGSSS, SSGSS, SSSGS, SSSSG or (GSGSGS) _n , where n is 1, 2 or 3.

In one or more embodiments, the at least 6 in (1) is 6, 12 or 18.

In one or more embodiments, (2) is to delete amino acids with greater steric hindrance from at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the C-terminus of the upper and/or middle hinge domain.

In one or more embodiments, (3) includes mutating the upper and/or middle hinge domains to the corresponding regions of IgG3 or mIgG2a, or the hinge region to the hinge region of IgG3 or mIgG2a, or the CH1-hinge region to the CH1 hinge region of IgG3 or mIgG2a. See Figure 15 for the amino acid sequences of the above regions.

In one or more embodiments, the flexibility of the mutated antibody is greater than or comparable to that of IgG3.

In some aspects, mutations to the antibody do not significantly reduce the affinity of the antibody for the antigen to which it is specifically targeted.

In some preferred embodiments, the radius of gyration (Rg) of the agonistic antibody after mutation is greater than

In some other preferred schemes, the Fc segment in the constant region of the heavy chain is further mutated to reduce the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

In other embodiments, the method is a method of down-regulating antibody flexibility or up-regulating antibody agonistic activity, and the mutation reduces the number and/or proportion of amino acids with less steric hindrance in the upper and/or middle hinge domain of the hinge region, or reduces the length of the upper and/or middle hinge domain.

In one or more embodiments, the mutation is selected from one or more of the following: (1) inserting 3 or 4 amino acids with less hindrance in the upper hinge domain, (2) deleting amino acids with less hindrance from the upper and/or middle hinge domain, (3) mutating amino acids with less hindrance in the upper and/or middle hinge domains to amino acids with more hindrance, (4) inserting amino acids with more hindrance in the upper and/or middle hinge domains.

In one or more embodiments, (1) is to insert 3 or 4 less sterically hindered amino acids after at least the 1st, at least the 2nd, at least the 3rd, at least the 4th or at least the 5th amino acid at the N-terminal of the upper hinge domain, or between the upper hinge domain and the middle hinge domain.

In one or more embodiments, the insertion in (1) is an insertion of 3 or 4 amino acids selected from G and S.

Preferably, (1) is the insertion of GSG, (GGS, SGG, GSS, SGS, SGG, GGGS, GGSG, GSGG, SGGG, GGSS, GSGS, SGGS, GSSG, SSGG, SGSG, SSSG, SSGS, SGSS or GSSS between the upper hinge domain and the middle hinge domain.

In one or more embodiments, (2) is to delete amino acids with less steric hindrance from at least the 1st, at least the 2nd, at least the 3rd, at least the 4th or at least the 5th amino acid from the C-terminus of the upper and/or middle hinge domain.

In one or more embodiments, (3) includes mutating the upper and/or middle hinge domain to the corresponding region of IgG1, IgG2 or IgA2, or the hinge region to the hinge region of IgG1, IgG2 or IgA2, or the CH1-hinge region to the CH1-hinge region of IgG1, IgG2 or IgA2. See Figure 15 for the amino acid sequences of the above regions.

In one or more embodiments, the antibody specifically recognizes a receptor in the TNF receptor superfamily; or the antibody is an anti-CD40 antibody or an anti-DR5 antibody.

In one or more embodiments, (4) is to insert at least 1, at least 2, at least 3, such as 1-20, 1-15 or 1-10 amino acids with relatively large steric hindrance after at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the N-terminal of the upper and/or middle hinge domain, or between the upper hinge domain and the middle hinge domain.

In one or more embodiments, the flexibility of the mutated antibody does not exceed the flexibility of human IgG1.

In some preferred embodiments, mutations to the antibody do not significantly reduce the affinity of the antibody for the antigen to which it is specifically targeted.

In some other preferred schemes, the Fc segment in the constant region of the heavy chain is further mutated to increase the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

In some other preferred schemes, the radius of gyration (Rg) of the agonistic antibody after mutation is preferably /> Or less than the Rg of IgGl (eg, human IgGl).

The present invention also provides a flexible antibody detection method, comprising the steps of: using small-angle X-ray scattering to detect any one or more of the following parameters of the antibody: radius of gyration (Rg), Dimensionless Kratky plots (or Kratky plots) features, P(R)/I(0) (or P(R)) distribution, P(R)/I(0) (or P(R)) large size distribution, radius of gyration (Rg) calculated by EOM method, and EOM method. Maximum interatomic distance (Dmax) distribution, Rflex or Rσ value calculated by EOM method; wherein, the antibody with one or more characteristics selected from the following is a flexible antibody, (1) the radius of gyration (Rg) is greater than the radius of gyration (Rg) of IgG1, (2) the radius of gyration (Rg) calculated by the EOM method is greater than the radius of gyration (Rg) calculated by the EOM method of IgG1, (3) Rflex is greater than this parameter of IgG1, (4) Rσ is greater than that of IgG1 Rσ.

In one or more embodiments, each feature of IgG1 described above is shown in the second column of FIG. 6 .

The beneficial effects of the present invention are: using the method provided by the present invention, the flexible characteristics of antibodies and other protein molecules can be analyzed more quickly; using the method provided by the present invention, an antibody modified form with significantly weakened or disappeared agonistic activity can be obtained on the basis of the parental antibody; using the method provided by the present invention, an antibody modified form with significantly enhanced agonistic activity can be obtained on the basis of the parental antibody.

Description of drawings

Figure 1. Antibody structure diagram.

Figure 2. Schematic diagram of TR-FRET analysis flexibility

Human IgG2 CH1-hinge has excellent rigidity. a. Diagram showing detection of anti-mouse CD40 antibody by TR-FRET. Anti-mouse CD40 antibody molecules mixed with CD40-Tb and CD40-D2 (mouse-derived CD40 molecules are labeled with fluorescent lanthanides Tb and D2 respectively) simultaneously bind to CD40-Tb and CD40-D2, and emit TR-FRET signals. Quantification is based on the relative ratio (Em665/Em620) of fluorescence detected at 665nm to 620nm after stimulation. b. Model diagram showing that the hinge flexibility of hIgG anti-mCD40 antibody is directly proportional to the level of TR-FRET signal. Left, anti-mouse CD40 antibody with little hinge flexibility does not trigger TR-FRET signal due to the large distance between CD40-Tb and CD40-D2; middle, hinge flexibility can bring CD40-Tb and CD40-D2 close enough to trigger TR-FRET signal; right, anti-mouse CD40 antibody with large hinge flexibility produces stronger TR-FRET signal because more molecules have closer CD40 binding sites.

Figure 3 Crystal structure of full-length antibody

Crystal structures of full-length human IgG1 (PDB: 1HZH, left) and IgG4 (PDB: 5DK3, right). The distance between the two antigen-binding sites of the two antibodies was calculated by PyMOL software as the distance between the vertices of the respective heavy chain CDR3 (W100 site of 1HZH, F103 site of 5D3K). The distance between two antigen-binding sites showing the crystalline conformation of an IgG antibody exceeds 12 nanometers.

Figure 4 TR-FRET analysis flexibility results

a-c TR-FRET signal levels of anti-mouse CD40 antibodies with corresponding constant domains. Relative TR-FRET signal levels (Em665/Em620) are shown for each concentration of control IgG or the corresponding constant domain anti-mouse CD40 antibody. Bars represent mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, analyzed by two-way ANOVA with Holm-Sidak’s post hoc analysis method.

Figure 5 SAXS analysis flexibility results

The IgG3 CH1-hinge is exceptionally flexible. a-f Dimensionless Kratky plots (a, c, e) and normalized interatomic distance distributions (P(R)/I(0)) (b,d,f) of anti-mouse CD40 antibodies (values at the highest concentration detected) for the corresponding constant domains. In the dimensionless (Dimensionless) Kratky diagram, the position of the Guinier-Kratky point (√3, 1.103) is marked with a black dashed line. g-l Distribution of Rg(g,i,k) and Dmax(h,j,l) (values for the lowest concentration of detection) in the optimized set generated in the EOM analysis of the indicated antibody.

Figure 6 SAXS analysis flexibility results (data)

IgG2 and 3 are the most rigid and most flexible subtypes of human IgG1-3 antibodies, respectively. IgG3 antibodies also had larger Rg and Dmax values (relating to the average and maximum interatomic distance, respectively) compared to IgG1 and 2 antibodies. The V11 variant with the CH1-hinge of the human IgG1-3 antibody had similar Rg and Dmax distribution characteristics and _Rflex and _Rσ ranks, respectively, to the human IgG1-3 antibody. Different SAXS distribution curves and parameters of IgG2 and 3 antibodies can be switched to a large extent together with their CH1-hinge.

Figure 7 The relationship between mouse IgG antibody agonism and hinge flexibility

(a) The flexibility of the murine IgG antibody was detected by SAXS method. Dimensionless Kratky plots analysis showed that mIgG2a has stronger flexibility than mIgG1, and the flexibility of the hinge region is also transferred through the replacement of the hinge region. (b-c) In the OT1 system, the flexible IgG2a has the weakest agonism, and the rigid IgG1 has the strongest agonism, and the IgG2a agonism is transferred to the chimeric antibody IgG1H2a through the replacement of the hinge region. In 8-week-old C57B6/L WT mice, using the OVA-specific CD8+ T cell response model, treated with 5ugDEC-OVA205 together with 30ug control or 1C10 anti-mCD40 chimeric antibody, the number of specific OT1 cells in spleen cells (b), the percentage of CD8+OT1 specific cells (c).

Figure 8 Inserting amino acid sequences in the hinge region can destroy IgG2 flexibility (TR-FRET results of IgG2 vs IgG2(GS) ₃ vs IgG2(GS) ₆ vs IgG2(GS) ₉ )

The TR-FRET experimental data showed that the linker sequence GS was inserted into the hinge region of IgG2, and the flexibility of the hinge region variants G2GS3, G2GS6, and G2GS9 gradually increased with the length of the linker sequence.

Figure 9 Inserting amino acid sequences in the hinge region can destroy the flexibility of IgG2 (SAXS results of IgG2 vs IgG2(GS) ₃ vs IgG2(GS) ₆ vs IgG2(GS) ₉ )

The SAXS experimental data showed that the linker sequence GS was inserted into the hinge region of IgG2, and the flexibility of the hinge region variants G2GS3, G2GS6, and G2GS9 gradually increased with the length of the linker sequence. (a) dimensionless Kratky analysis of hIgG2 hinge region variant SAXS. Increasing linker length gradually increases antibody flexibility. (b) Molecules with greater flexibility generally adopt a more extended conformation, which can be assessed by analyzing the normalized distribution of the interatomic distance (R) within the macromolecule, that is, P(R)/I(0).

Figure 10 Insertion of amino acid sequence in the hinge region to destroy IgG2 flexibility will affect agonistic activity (In vivo activity data of IgG2 vs IgG2(GS) ₃ vs IgG2(GS) ₆ vs IgG2(GS) ₉ )

The hinge region variants G2GS3, G2GS6, and G2GS9 were inactivated in the OT1 system. (a-b) In the hFCGRTg mouse model, using the OVA-specific CD8+ T cell response model, mice were induced with 2ugDEC-OVA205 together with 10ug control or 1C10 anti-mCD40 antibody, and the number (a) and percentage (b) of CD8+OT1-specific cells in spleen cells were analyzed.

Figure 11 Changing the amino acid sequence of the hinge region can reduce IgG antibody flexibility and affect antibody agonistic activity (IgG1 vs IgG1-A2, and other examples of reduced flexibility)

(a) TR-FRET experimental data showing that the IgG1-A2 variant (G1A2) produced by replacing the hinge region of human IgG1 with the hinge region of IgA2 is less flexible. (b) Dimensionless Kratky plots analysis of SAXS experimental data shows that the IgG1A2 variant is less flexible (more rigid).

Figure 12 Changing the amino acid sequence of the hinge region to enhance the flexibility of the IgG antibody can affect the agonistic activity of the antibody (IgG1 vs IgG1-A2, an example of enhanced activity)

Using the OVA-specific CD8 ⁺ T cell response model, FcγR human-derived mice were adoptively transferred OT-1 cells on day 1, co-immunized with DEC-OVA and control or anti-CD40 antibody intraperitoneally on day 2, and splenocytes were collected on day 7 to quantify OVA-specific CD8 ⁺ T cells. Quantification of OT-1 cells in FcyR-humanized mice were processed and analyzed as described, as well as control or anti-mouse CD40 antibodies of the indicated constant domains (30 μg per mouse). Each symbol represents an individual mouse. Bars represent mean ± SEM. **p≤0.01, ***p≤0.001, ****p≤0.0001, analyzed by two-way ANOVA with Holm-Sidak's post hoc analysis method. As shown, G1A2 antibody-treated animals had more antigen-specific OT-1 cell expansion (a, b) and CD8 cell expansion (c).

Figure 13 Different antibodies have different optimal antibody flexibility requirements ((a): 1C10:V11H2>V11H1>V11H3 activity data; (b): MD5-1:V11H1>V11H2>V11H3 activity data)

Figure 14 Insertion of amino acids that are not multiples of 3-4 in the hinge region will destroy IgG2 agonistic activity (In vivo activity data of IgG2 vs IgG2V1 vs IgG2V2 vs IgG2V3 vs IgG2V4 vs IgG2V5G vs IgG2V6)

(a) The 1C10 cloned anti-CD40 antibody stimulated the spleen cells of FcγR humanized mice in vitro, and the results showed that the hinge region variants inserted into the IgG2 hinge region that were not multiples of 3-4 amino acids were inactivated. (b) MD5-1 cloned anti-DR5 antibody stimulated MC38 tumor cells co-cultured with spleen cells of FcγR humanized mice in vitro. The results showed that the hinge region variants inserted in the hinge region of V11H2 that were not multiples of 3-4 amino acids were inactivated.

Figure 15 List of different hinge sequences

CH1 is CH1 of the heavy chain constant region, UH is the upper hinge domain of the hinge region, MH is the middle hinge domain of the hinge region, and LH is the lower hinge domain of the hinge region.

Detailed ways

Unless otherwise specified, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required herein, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures and techniques involved in cell and tissue culture, molecular biology, immunology, and protein and nucleic acid chemistry described herein are known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to conventional methods known in the art and are described in various general and more technical references that are cited and discussed in this specification unless otherwise indicated. See, e.g., Molecular Cloning: A Laboratory Manual by Sambrook et al., 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Current Protocols in Molecular Biology by Ausubel et al. (Greene Publishing Associates (1992)), and Antibiotics by Harlow and Lane. bodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990)), the contents of which are incorporated herein by reference. Enzyme reactions and purification techniques are performed according to manufacturer's instructions, generally by methods known in the art or as described herein. The nomenclatures associated with, and the experimental methods and techniques of, analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and drug delivery, and patient treatment.

Unless otherwise stated, the following terms have the following definitions:

"Antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.

The "heavy chain constant region" of an antibody, also known as CDs, consists of three domains CH1, CH2 and CH3 and a hinge region between the CH1 domain and the CH2 domain. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen.

The term "hinge region" means the part of the heavy chain constant region that connects the CH1 domain to the CH2 domain. The hinge region consists of approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into 3 distinct domains: upper, middle and lower hinge domains (Roux et al. (1998) J. Immunol. 161:4083). Some altered antibody molecules have been prepared, in which the number of cysteine residues in the hinge region has been increased to promote the formation of a fixed conformation of the antibody molecule and improve the immunomodulatory activity of agonistic antibodies (Patent No. WO2015145360A1).

"Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (CIq) of the classical complement system. Thus, an Fc region is a polypeptide comprising the portion of the constant region of an antibody heavy chain other than the first constant region immunoglobulin domain. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second (CH2) and third (CH2) constant domains of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence stretch from the amino acid residue at positions C226 or P230 of the heavy chain to the carboxy-terminus, where this numbering is according to the EU index, as in Kabat. The CH2 domain of the human IgG Fc region extends from about amino acid 231 to about amino acid 340, while the CH3 domain is located on the C-terminal side of the CH2 domain of the Fc region, ie, it extends from about amino acid 341 to about amino acid 447 of IgG. As used herein, the Fc region can be a native sequence Fc or a variant Fc. Fc may also refer to this region in isolation, or within an Fc-containing protein polypeptide, such as a "binding protein comprising an Fc region", also known as an "Fc fusion protein" (e.g., an antibody or an immunoadhesin).

"Fc receptor" or "FcR" is a receptor that binds the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants and alternatively spliced forms of these receptors. The human Fcγ receptor family includes several members: FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), FcγRIIIB (CD16b). Among them, FcγRIIB is the only inhibitory Fcγ receptor, and the others are activating Fcγ receptors. Most natural effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, whereas natural killer (NK) cells selectively express one activating Fcγ receptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitory FcγRIIB in mice and humans. These Fcγ receptors differ in their molecular structure and thus have different affinities for the various IgG antibody subclasses. Among these Fcy receptors, FcyRI is a high-affinity receptor, while FcyRIIA, FcyRIIB and FcyRIIIA are low-affinity receptors. Genetic polymorphisms also exist in these different Fcγ receptors and affect their binding affinities. The most common gene polymorphisms are polymorphic forms such as R131/H131 of FcγRIIA and V158/F158 of FcγRIIIA. Some of these polymorphic forms have been found to be associated with various diseases, and the effect of some specific therapeutic antibodies also depends on whether the patient has a specific polymorphic form of the Fcγ receptor gene.

The amino acid sequence numbers of the antibodies and their fragments or structural domains of the present invention are based on IgG Eu numbering.

The term "antigen binding site" refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen binding site of an antibody comprises amino acid residues from "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable region regions that are not hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-terminus to C-terminus, the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding and defines antibody properties. CDRs and FRs are determined according to the standard definition of Kabat et al., SEQ ID NO: uences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or residues from "hypervariable loops".

Antibodies usually specifically bind their cognate antigens (similarly, proteins of the invention can also specifically bind their epitope recognition molecules) with high affinity, which is manifested by a dissociation constant (KD) of 10 ⁻⁵ -10 ⁻¹¹ M or less. Any KD greater than about 10 ⁻⁴ M ⁻¹ is generally considered to indicate non-specific binding. As used herein, an antibody that "specifically binds" an antigen refers to an antibody that binds the antigen and substantially the same antigen with high affinity, meaning a KD of ^10-7 M or less, preferably ^10-8 M or less, even more preferably 5 x ^10-9 M or less, most preferably ^{10-8-10-10 M} or less, but does not bind with high affinity to an unrelated antigen. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity with the given antigen, for example, if it exhibits at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99% sequence identity to the given antigen's sequence.

Immunoglobulins may be from any of the generally known isotypes. IgG isotypes can be subdivided in some species into subclasses: IgG1, IgG2, IgG3 and IgG4 in humans, IgG1, IgG2a, IgG2b and IgG3 in mice. "Isotype" refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes. "Antibody" includes, for example, naturally-occurring and non-naturally-occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies;

An "agonistic antibody" is an antibody that binds to and activates a receptor, an example of the function of an agonistic antibody is binding to a receptor in the tumor necrosis factor receptor (TNFR) superfamily and inducing apoptosis in cells expressing the TNF receptor. Assays for determining induction of apoptosis are described in WO98/51793 and WO99/37684, both of which are expressly incorporated herein by reference. In a specific embodiment of the present invention, the anti-CD40 agonistic antibody can bind to the target molecule that transmits the immune activation signal on the surface of the immune cell and activate the important immune activation signaling pathway controlled by it, thereby enhancing the anti-tumor immune response to indirectly kill tumor cells. Some examples of agonistic antibodies that have entered the clinical research stage can refer to patent PCT/CN2017/087620.

"Agonist activity" refers to the activity of inducing certain specific physiological activity changes by binding the antibody to the antigen molecule, stimulating the antigen molecule to generate a signal. Antigen molecules include receptor molecules and other molecules with signaling or physiological functions. The above-mentioned specific physiological activities include, for example, proliferation activity, survival activity, differentiation activity, transcription activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation/dephosphorylation activity, redox activity, transfer activity, nucleic acid decomposition activity, dehydration activity, cell death-inducing activity and apoptosis-inducing activity, etc., but are not limited to these. The agonistic activity described herein is not limited to agonistic antibodies. In some embodiments, a reduction in agonistic activity enhances other activities of the antibody, such as inhibitory activity.

"Mother parent" refers to the object to be modified during the process of protein modification. In some embodiments, the parent is an unengineered antibody.

"Variant" refers to the protein obtained after the parent parent is modified in the process of protein engineering. Specifically, it may be a protein derived by mutation, deletion and/or addition of parent amino acids and retains some or all of the functions inherent in the parent.

"Modification" refers to the phenomenon of changing the protein structure and/or function through the introduction and/or removal of chemical groups (including amino acids or amino acid fragments, unnatural amino acids, sugar groups, acetyl groups, etc.). The "modification" of the antibody or its fragments referred to in the present invention includes mutation, deletion, side chain modification (such as glycosylation, acetylation, etc.) and/or insertion of additional amino acids in the antibody or its fragments.

"Flexibility" refers to having molecular (intra)mobility, and in the present invention mainly refers to the structural variability of proteins. As mentioned herein, when a hinge region is "flexible," it means that the two N-terminal antigen-binding regions it connects can move independently. At the same time, "flexible" and "stiff" are also used in the article to describe the degree of flexibility of the antibody. "Flexible" means that the antibody is flexible, while "stiff" means that the antibody is weak. The flexibility of biological macromolecules can be detected by the flexible detection method provided by the present invention, specifically, the small angle X-ray scattering method or the TR-FRET method provided by the present invention can be used for detection.

In this patent, when the variant is more flexible than the parent, it generally has poorer agonistic activity; when the variant is less flexible than the parent, it generally has better agonistic activity. However, for different antibodies, the optimal value or optimal value range of the flexibility required for the variant with the optimal agonistic activity is different. When the flexibility reaches the optimal value or falls within the optimal value range, the agonistic activity is the strongest. On this basis, if the flexibility is increased or weakened, the agonistic activity may be weakened. In human IgG, the flexibility of IgG3 is generally strong, and the agonistic activity of antibodies containing the CH1-hinge region of IgG3 is usually poor, while the flexibility of IgG2 and IgG1 is relatively low, so the agonistic activity of antibodies containing the CH1-hinge region of IgG2 and IgG1 is usually stronger than that of antibodies containing the CH1-hinge region of IgG3. Thus the CH1-hinge region of IgG1 or IgG2 is the natural IgG CH1-hinge region of choice. In fact, although IgG2 is less flexible than IgG1, and in some antibody embodiments, the use of IgG2 CH1-hinge region can indeed provide better agonistic activity than IgG1 CH1-hinge region and IgG3 CH1-hinge region, but in other antibody embodiments, the IgG1 CH1-hinge region with central flexibility can provide better activity than IgG2 CH1-hinge region and IgG3 CH1-hinge region. Agonist activity. Therefore, for the purpose of enhancing the agonistic activity of the antibody, it is necessary to adjust the flexibility of the antibody to a relatively low range; preferably, the degree of flexibility of the antibody should not exceed the degree of flexibility of human IgG1, but the degree of flexibility should not be too low; the variant with the best agonistic activity can be obtained from antibodies with lower flexibility through further activity screening. On the contrary, for the purpose of reducing the agonistic activity of the antibody, the flexibility of the antibody needs to be adjusted to a relatively high range; preferably, the degree of flexibility of the antibody should be greater than or equivalent to that of natural IgG3.

"Radius of gyration (Rg)", also known as the radius of inertia, refers to the distance between the assumed concentration point of the differential mass of the object and the rotation axis, which can roughly describe how tight the molecular structure is. Rg reflects the volume and shape of the protein and can be used to measure the breadth of the protein structure. The larger the Rg, the more expanded the system, and the stronger the flexibility of the antibody of the present invention. In this patent, Rg is estimated by the parameters obtained from small-angle X-ray scattering.

"Tumor necrosis factor receptor superfamily" or "TNF receptor superfamily" herein refers to receptor polypeptides that can bind to cytokines of the TNF family. In general, these receptors are type I transmembrane receptors with one or more cysteine-rich repeats in their extracellular region. Examples of cytokines in the TNF gene family include: tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor-beta (TNF-beta or lymphotoxin), CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also known as Fas ligand or CD95 ligand), Apo-2 ligand (also known as TRAIL), Apo-3 ligand (also known as TWEAK) , osteoprotegerin (OPG), APRIL, RANK ligand (also known as TRANCE), and TALL-1 (also known as BlyS, BAFF or THANK). Examples of receptors in the TNF receptor superfamily include: tumor necrosis factor receptor type 1 (TNFR1), tumor necrosis factor receptor type 2 (TNFR2), p75 nerve growth factor receptor (NGFR), B cell surface antigen CD40, T cell antigen OX-40, Apo-1 receptor (also known as Fas or CD95), Apo-3 receptor (also known as DR3, sw1-1, TRAMP and LARD), known as "transmembrane activator and CAML-interactor (inter actor)" or "TACI" receptor, BCMA protein, DR4, DR5 (or, also known as Apo-2; TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2 or KILLER), DR6, DcR1 (also known as TRID, LIT or TRAIL-R3), DcR2 (also known as TRAIL-R4 or TRUNDD), OPG, DcR3 (also known as TR6 or M68 ), CAR1, HVEM (also known as ATAR or TR2), GITR, ZTNFR-5, NTR-1, TNFL1, CD30, lymphotoxin beta receptor (LTBr), 4-1BB receptor and TR9 (EP988,371A1).

The "affinity ratio to inhibitory Fcγ receptors and activating Fcγ receptors" or "I/A ratio" described in the present invention is equal to the affinity value of the antibody heavy chain constant region and inhibitory Fcγ receptors (for example: human FcγRIIB) divided by the highest affinity value between the antibody heavy chain constant region and activating Fcγ receptors of the same species (for example: including human FcγRI, FcγRIIA, FcγRIIIA, FcγRIIIB).

The "affinity" refers to the magnitude of the binding ability between two molecules, which can usually be measured by KD. "KD"refers to the equilibrium dissociation constant for the interaction of two molecules (eg, a particular antibody and antigen or a ligand and receptor).

A "human" antibody is one whose variable regions have framework and CDR regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

A "humanized" antibody is one in which some, most or all of the amino acids outside the CDR domains of a non-human antibody have been replaced by the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of the antibody, some, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible so long as they do not eliminate the ability of the antibody to bind the particular antigen. A "humanized" antibody retains similar antigen specificity to the original antibody.

A "chimeric antibody" refers to an antibody in which the variable regions are from one species and the constant regions are from another species, eg, an antibody in which the variable regions are from a mouse antibody and the constant regions are from a human antibody.

"Flexible linking sequence" refers to an amino acid sequence having a flexible structure. In the specific embodiment of the present invention, the "flexible linking sequence" is used to add, insert, replace or modify the hinge region of the antibody to make the hinge region flexible, or to increase the flexibility of the original hinge region. Joshua S. Klein et al. provided a series of flexible linker sequences (Design and characterization of structured protein linkers with differing flexibilities, Protein Engineering, Design&Selection vol.27no.10pp.325-330, 2014), and these sequences were cited together as examples of specific flexible linker sequences in the present invention.

In addition, the inventors found that antibody flexibility can be adjusted by mutating (modifying) the upper and/or middle hinge domains of the antibody (for example, changing the number and/or ratio of amino acids with less steric hindrance, or changing the length of the upper hinge domain). The inventors also found that this regulation of antibody flexibility is closely related to the agonistic activity of the antibody; down-regulating the flexibility can increase the agonistic activity of the activating antibody, and vice versa can reduce the agonistic activity of the activating antibody. Herein, the amino acid with less steric hindrance can be selected from: glycine (G), alanine (A), serine (S), valine (V), threonine (T), isoleucine (I), leucine (L). Preferably, the amino acid with less hindrance is selected from G or S. The relatively hindered amino acids herein include aromatic amino acids and heterocyclic amino acids. In one or more embodiments, the amino acid with greater steric hindrance is selected from proline (P), hydroxyproline (O), asparagine (N), aspartic acid (D), pyroglutamic acid (U), glutamine (Q), lysine (K), glutamic acid (E), methionine (M), histidine (H), phenylalanine (F), arginine (R), tyrosine (Y), tryptophan (W).

When the adjustment is to upregulate antibody flexibility or reduce antibody agonistic activity, the mutation increases the number and/or proportion of amino acids with less steric hindrance in the upper and/or middle hinge domain of the hinge region, or increases the length of the upper and/or middle hinge domain. The mutation is selected from one or more of the following, (1) inserting 1, 2, 5 or at least 6 amino acids with less steric hindrance in the upper hinge domain, (2) deleting amino acids with larger steric hindrance from the upper and/or middle hinge domain, (3) mutating the amino acids with larger steric hindrance in the upper and/or middle hinge domain to amino acids with less steric hindrance. For example, the amino acid insertion described in (1) is located after at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the N-terminus of the upper hinge domain, or between the upper hinge domain and the middle hinge domain. The insertion described in (1) includes 1, 2, 5 or at least 6 amino acids selected from G and S. In specific embodiments, (1) is the insertion of G, GS, SG, GSGSG, SGSGS, GGGGS, GGGSG, GGSGG, GSGGG, SGGGG, GSGSG, GSSGG, GGSSG, GSGGS, GGSGS, GGGSS, SGGSG, SGSGG, SSGGG, SGGGS, SGSGS, SGGSS, SSGGS, SGSSG, SSGSG, SSSGG, GSSGS, GSGSS, GGSSS, GSSSG, GSSSS, SGSSS, SSGSS, SSSGS, SSSSG or (GSGSGS) _n , where _n is 1, 2 or 3. In a specific embodiment, (2) is to delete amino acids with greater steric hindrance from at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the C-terminus of the upper and/or middle hinge domain. In specific embodiments, (3) includes replacing amino acid residues with large steric hindrance such as (R, K, D) in the upper and/or middle hinge domains with amino acid residues with less steric hindrance (such as G, S); in some embodiments, (3) also includes mutating the upper and/or middle hinge domains to the corresponding regions of IgG3 or mIgG2a, or mutating the hinge region to the hinge region of IgG3 or mIgG2a, or mutating the CH 1 - Hinge region mutated to CH1 hinge region of IgG3 or mIgG2a. In one or more embodiments, the flexibility of the antibody after mutation is greater than or comparable to IgG3. In one or more embodiments, the mutated antibody has a radius of gyration (Rg) greater than

When the adjustment is down-regulating antibody flexibility or increasing antibody agonistic activity, the mutation reduces the number and/or ratio of amino acids with less hindrance in the upper and/or middle hinge domain of the hinge region, or reduces the length of the upper and/or middle hinge domain. The mutation is selected from one or more of the following, (1) inserting 3 or 4 amino acids with less hindrance in the upper hinge domain, (2) deleting amino acids with less hindrance from the upper and/or middle hinge domain, (3) mutating amino acids with less hindrance in the upper and/or middle hinge domains to amino acids with greater hindrance, (4) inserting amino acids with greater hindrance in the upper and/or middle hinge domains. In this case, the insertion in (1) is located after at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the N-terminal of the upper hinge domain, or between the upper hinge domain and the middle hinge domain. The insertion in (1) includes the insertion of 3 or 4 amino acids with less hindrance, wherein n is a positive integer, for example, the insertion of 3 or 4 amino acids selected from G and S. In specific embodiments, (1) is the insertion of GSG, GGS, SGG, GSS, SGS, SGG, GGGS, GGSG, GSGG, SGGG, GGSS, GSGS, SGGS, GSSG, SSGG, SGSG, SSSG, SSGS, SGSS, or GSSS between the upper hinge domain and the middle hinge domain. In a specific embodiment, (2) is to delete amino acids with less steric hindrance from at least the 1st, at least the 2nd, at least the 3rd, at least the 4th or at least the 5th amino acid from the C-terminus of the upper and/or middle hinge domain. In specific embodiments, (3) includes substituting amino acid residues with small steric hindrance such as (G, S) in the upper and/or middle hinge domains with amino acid residues with large steric hindrances (such as R, K, D); in some embodiments, (3) includes mutating the upper and/or middle hinge domains to the corresponding regions of IgG1, IgG2 or IgA2, or mutating the hinge region to the hinge of IgG1, IgG2 or IgA2 region, or mutate the CH1-hinge region to the CH1-hinge region of IgG1, IgG2 or IgA2. In a specific embodiment, (4) is to insert at least 1, at least 2, at least 3, such as 1-20, 1-15 or 1-10 relatively hindered amino acids after at least the 1st, at least 2nd, at least 3rd, at least 4th or at least 5th amino acid at the N-terminal of the upper and/or middle hinge domain, or between the upper hinge domain and the middle hinge domain. In one or more embodiments, the flexibility of the mutated antibody does not exceed the flexibility of human IgG1. In one or more embodiments, the radius of gyration (Rg) of the mutated antibody is preferably /> Or less than the Rg of IgGl (eg, human IgGl).

The term "biological macromolecules" in the present invention refers to various organic molecules that are the main active ingredients in living organisms. They usually have relatively large molecular weights (up to tens of thousands or more). Common biological macromolecules include proteins, nucleic acids, lipids, sugars, and products or systems formed by the combination of these molecules or molecular fragments, such as glycoproteins, lipoproteins, and nucleoproteins.

"Epitope", also known as an antigenic determinant, is a chemical group that exists on the surface of an antigen/protein and determines the specific structure of the antigen. It is a part of the antigen that can be recognized by the immune system (especially antibodies, B cells or T cells). An antigen molecule can have one or more different epitopes, the size of which is equivalent to the antigen-binding site of the corresponding antibody, and each epitope has only one antigen specificity.

"Epitope recognition molecule" refers to a molecule capable of specifically binding to an epitope.

"Fluorescence resonance energy transfer" (FRET) refers to the phenomenon that among different fluorophores, if the fluorescence emission spectrum of one fluorophore (donor) overlaps with the absorption spectrum of another group (acceptor), when the distance between the two groups is appropriate (generally less than 50nm, preferably less than 10nm), the phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed, that is, the fluorescence of the donor is quenched and the fluorescence of the acceptor is enhanced.

" "Radius" (R0), also known as Freund's radius, indicates the distance between the donor and the acceptor when 50% of the donor fluorescent molecules (Donor) are inactivated and energy is transferred to the acceptor fluorescent molecule (Acceptor) in FRET.

"TR-FRET" in the present invention is the abbreviation of time-resolved fluorescence resonance energy transfer, time-resolved fluorescence resonance energy transfer analysis (Time-Resolved Fluorescence Resonance Energy Transfer assay, TR-FRET analysis), the main principle of action is that time-resolved fluorescent molecules are used as donor fluorescent molecules, which can transfer energy to adjacent acceptor fluorescent molecules at a certain distance, and the acceptor fluorescent molecules will be excited to generate fluorescence signals of specific wavelengths (that is, the "TR-FRET signal" described in the present invention) for the experimenter to test. Specifically, the lanthanide element with a longer half-life of the emitted light is used as the donor fluorescent molecule, and the light energy (A) emitted by the donor fluorescent molecule can be transferred to the adjacent acceptor fluorescent molecule at a certain working distance, and the acceptor fluorescent molecule will be excited to generate an emitted fluorescent signal (B) of a specific wavelength (the (B/A) ratio is the "TR-FRET signal" described in the present invention) for the experimenter to detect.

The "donor fluorescent molecule" and "acceptor fluorescent molecule" of the present invention can be directly labeled with epitope recognition molecules (such as antigens), and then respectively bind to two epitopes (such as: antigen binding sites) of proteins (such as antibodies); the same effect can also be achieved by indirect labeling of epitope recognition molecules, or by direct or indirect labeling of other molecules that can bind to epitopes.

The donor fluorescent molecules and ligand fluorescent molecules used in the TR-FRET analysis of the present invention can be selected and paired in the following manner:

1) When the donor fluorescent molecule is Eu, the paired acceptor fluorescent molecule can be selected from APC, D2, XL665, Fluorescein, GFP, Rhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Atto550Cy3.5, Dy547, Dy548, Dy549 , any one of Dy554, Dy555, Dy556, Dy560, mCherry, mStrawberry, Alexa680, Alexa700, Alexa750, Alexa647, Cy5, Cy5.5, Cy7, Dy647, Dy648, Atto590;

or,

2) When the donor fluorescent molecule is Tb, the paired acceptor fluorescent molecule can be selected from: D2, XL665, Fluorescein, GFP, Lucifer yellow, Acridine yellow, Proflavine, Atto465, Nitrobenzoxadiazole, Courmarin 6, Alexa750, Cy7, Nile red, Alexa488, Dy495, Dy490, Dy 648, Dy647, Oregon green, Atto488, Atto495, Alexa514, Atto520, Cy2, Rhodamine6G, Alexa700, Alexa680, Atto532, Alexa532, APC, EGFP, YFP, mPlum, Atto425, Alexa430, Coumarin 343, Acridine Orange, Tetramethylr Hodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Cy5, Att0590, Atto550, Cy3.5, Cy5.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, Alexa647, mCherry, mStrawberry .

The present invention includes following embodiments:

Item 1. A method for increasing the agonistic activity of an agonistic antibody, which is characterized in that the heavy chain constant region of the agonistic antibody is modified to reduce the flexibility of the agonistic antibody. Preferably, the modification is selected from one or more of the following, (1) inserting 1, 2, 5 or at least 6 amino acids with less hindrance in the upper hinge domain, (2) deleting amino acids with greater hindrance from the upper and/or middle hinge domain, (3) mutating amino acids with greater hindrance in the upper and/or middle hinge domains to amino acids with less hindrance. More preferably, (1) inserts 1, 2, 5 or at least 6 less hindered amino acids after at least the first, at least the second, at least the third, at least the fourth or at least the fifth amino acid at the N-terminal of the upper hinge domain, or between the upper hinge domain and the middle hinge domain, preferably, the at least 6 are 6, 12 or 18, (2) at least the first, at least the second, At least the 3rd, at least the 4th or at least the 5th amino acid begins to delete the amino acid with greater steric hindrance, (3) including mutation of the upper and/or middle hinge domain to the corresponding region of IgG3 or mIgG2a, or mutation of the hinge region to the hinge region of IgG3 or mIgG2a, or mutation of the CH1-hinge region to the CH1-hinge region of IgG3 or mIgG2a.更加优选的是，(1)中的插入是插入1、2、5或至少6个选自G和S的氨基酸，或者(1)是在上部铰链结构域和中间铰链结构域之间插入G、GS、SG、GSGSG、SGSGS、GGGGS、GGGSG、GGSGG、GSGGG、SGGGG、GSGSG、GSSGG、GGSSG、GSGGS、GGSGS、GGGSS、SGGSG、SGSGG、SSGGG、SGGGS、SGSGS、SGGSS、SSGGS、SGSSG、SSGSG、SSSGG、GSSGS、GSGSS、GGSSS、GSSSG、GSSSS、SGSSS、SSGSS、SSSGS、SSSSG或(GSGSGS) _n ，其中n为1、2或3。

Item 2. The method according to Item 1, wherein the flexibility of the agonistic antibody after modification is no more than that of human IgG1.

Item 3. The method according to Item 1, characterized in that the CH1-hinge region in the heavy chain constant region is modified.

Item 4. The method according to Item 1, characterized in that the hinge region in the heavy chain constant region is replaced by a less flexible sequence, preferably the sequence is the hinge region sequence of human IgA2.

Item 5. The method according to any one of Items 1-4, wherein the modification of the antibody does not significantly reduce the affinity of the antibody to the antigen it specifically targets.

Item 6. The method according to Item 1-4, characterized in that the Fc segment in the constant region of the heavy chain is further modified to increase the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

Item 7. The method as described in Item 1-4, characterized in that the radius of gyration (Rg) of the modified agonistic antibody is preferably /> Or less than the Rg of human IgG1.

Item 8. The method according to any one of Items 1-4, wherein the antibody specifically recognizes a receptor in the TNF receptor superfamily.

Item 9. The method according to any one of items 1-4, wherein the antibody is an anti-CD40 antibody or an anti-DR5 antibody.

Item 10. A method for reducing the agonistic activity of an antibody, which is characterized in that the heavy chain constant region of the agonistic antibody is modified to increase the flexibility of the antibody. Preferably, the modification is selected from one or more of the following, (1) inserting 3 or 4 amino acids with less hindrance in the upper hinge domain (2) deleting amino acids with less hindrance from the upper and/or middle hinge domain, (3) mutating amino acids with less hindrance in the upper and/or middle hinge domains to amino acids with greater hindrance, (4) inserting amino acids with greater hindrance in the upper and/or middle hinge domain; more preferably, (1) is in the upper hinge domain At least the 1st, at least the 2nd, at least the 3rd, at least the 4th or at least the 5th amino acid at the N-terminal of the N-terminus, or after inserting 3 or 4 amino acids with less steric hindrance, or between the upper hinge domain and the middle hinge domain, (2) from the C-terminal of the upper and/or middle hinge domain. Mutation to the corresponding region of IgG1, IgG2, or IgA2, or mutation of the hinge region to the hinge region of IgG1, IgG2, or IgA2, or mutation of the CH1-hinge region to the CH1-hinge region of IgG1, IgG2, or IgA2, (4) after at least the 1st, at least the 2nd, at least the 3rd, at least the 4th, or at least the 5th amino acid at the N-terminus of the upper hinge domain, or after the upper hinge At least 1, at least 2, at least 3, such as 1-20, 1-15 or 1-10 amino acids with relatively large steric hindrance are inserted between the chain domain and the middle hinge domain. More preferably, the insertion in (1) is the insertion of 3 or 4 amino acids selected from G and S, or (1) is the insertion of GSG, GGS, SGG, GSS, SGS, SGG, GGGS, GGSG, GSGG, SGGG, GGSS, GSGS, SGGS, GSSG, SSGG, SGSG, SSSG, SSGS, SGSS or GSSS between the upper hinge domain and the middle hinge domain.

Item 11. The method according to Item 10, wherein the flexibility of the agonistic antibody after modification is greater than or equivalent to IgG3.

Item 12. The method according to Item 10, wherein the CH1-hinge region in the heavy chain constant region is modified to increase the flexibility of the agonistic antibody.

Item 13. The method according to item 12, characterized in that a flexible linking sequence is inserted into the CH1-hinge region, preferably the flexible linking sequence is a flexible linking sequence comprising G and S, more preferably the flexible linking sequence is GSGSGS, or the CH1-hinge region is replaced with a CH1-hinge region of human IgG3.

Item 14. The method according to any one of Items 10-13, characterized in that the modification of the antibody does not significantly reduce the affinity of the antibody to the antigen it specifically targets.

Item 15. The method according to Item 10-13, characterized in that the radius of gyration (Rg) of the modified agonistic antibody is greater than

Item 16. The method according to Items 10-13, characterized in that the Fc segment in the constant region of the heavy chain is further modified to reduce the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody.

Item 17. The method according to any one of Items 1-16, wherein the antibody is a human antibody, a chimeric antibody or a humanized antibody.

Item 18. A method for screening the agonistic activity of an antibody, comprising the following steps:

1) providing an agonistic antibody as a parent, and providing a variant of the parent whose CH1-hinge region has been modified on the basis of the parent, wherein the antibody in which the CH1-hinge region of the parent is replaced by the CH1-hinge region of human IgG1 is called a human IgG1 variant;

2) Detect the flexibility of the female parent and the variant;

3) According to the results of flexibility detection, antibodies with flexibility not higher than human IgG1 variants were selected from all agonistic antibodies, and these antibodies had better agonistic activity.

Item 19. The screening method as described in Item 18, which is characterized in that it also includes detecting the affinity of the parent and variants to FcγRIIB and/or the I/A ratio of the agonistic antibody, comparing the detection results, and screening out antibodies whose affinity to FcγRIIB and I/A ratio are not lower than those of human IgG1 variants, and these antibodies have better agonistic activity.

Item 20. The screening method as described in item 18, characterized in that said step 2) includes:

1) providing an antigen capable of specifically binding to the antigen-binding portion of the agonistic antibody;

2) The antigens are respectively labeled with a pair of donor fluorescent molecules and acceptor fluorescent molecules capable of realizing fluorescence resonance energy transfer to obtain donor fluorescently labeled antigens and acceptor fluorescently labeled antigens;

3) adding the donor fluorescently-labeled antigen and acceptor fluorescently-labeled antigen to the agonistic antibody;

4) Exciting the donor fluorescent molecule and detecting the fluorescent signal emitted by the acceptor fluorescent molecule, the intensity of the fluorescent signal is positively correlated with the flexibility of the agonistic antibody.

Item 21. The screening method as described in item 18, characterized in that said step 2) includes:

1) Using the method of small-angle X-ray scattering to detect any one or more of the following parameters of the antibody parent and variant: radius of gyration (Rg), Dimensionless Kratky plots (or Kratky plots) feature, P(R)/I(0) (or P(R)) distribution, P(R)/I(0) (or P(R)) large size distribution, radius of gyration (Rg) calculated by EOM method, and maximum interatomic distance calculated by EOM method (Dmax) distribution, Rflex or Rσ value calculated by EOM method;

2) The larger the radius of gyration (Rg), the higher the upward degree of the Dimensionless Kratky plots (or Kratky plots) curve, the wider the distribution of the radius of gyration (Rg) and the maximum interatomic distance (Dmax) calculated by the EOM method, and the larger the values of Rflex and Rσ, the stronger the antibody flexibility; conversely, the weaker the antibody flexibility.

Item 22. A flexible detection method for biomacromolecules, comprising the following steps:

1) providing the biomacromolecule;

2) providing two epitope recognition molecules capable of specifically binding to two separate epitopes of the biomacromolecule;

3) The two epitope recognition molecules are respectively labeled with a donor fluorescent molecule and an acceptor fluorescent molecule, and the donor fluorescent molecule and the acceptor fluorescent molecule match each other and can realize fluorescence resonance energy transfer;

4) mixing the two labeled epitope recognition molecules with the biomacromolecule;

5) Exciting the donor fluorescent molecule and detecting the fluorescent signal emitted by the acceptor fluorescent molecule, the intensity of the fluorescent signal is positively correlated with the flexibility of the antibody.

Item 23. The detection method as described in Item 22, wherein the biomacromolecule is protein, nucleic acid, lipid molecule, sugar molecule or a complex formed by their mutual combination.

Item 24. The detection method according to Item 22, wherein the biomacromolecule has a conformation in which the distance between the two individual epitopes is between R0 and 2R0 of the donor fluorescent molecule and the ligand fluorescent molecule.

Item 25. The detection method according to item 22, wherein the biomacromolecule is an antibody, the epitope is an antigen binding site of an antibody, and the epitope recognition molecule is an antigen.

Item 26. The detection method as described in Item 22, wherein the donor fluorescent molecule is Tb, and the acceptor fluorescent molecule paired with it is selected from D2, XL665, Fluorescein, GFP, Lucifer yellow, Acridineyellow, Proflavine, Atto465, Nitrobenzoxadiazole, Courmarin 6, Alexa750, Cy7, Nilered, Alexa488, Dy4 95, Dy490, Dy648, Dy647, Oregon green, Atto488, Atto495, Alexa514, Atto520, Cy2, Rhodamine6G, Alexa700, Alexa680, Atto532, Alexa532, APC, EGFP, YFP, mPlum, Atto425, Alexa430, Coumarin 343, Acrid Ine Orange, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Cy5, Att0590, Atto550, Cy3.5, Cy5.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, Alexa647, mCh erry, any one of mStrawberry; or, the donor fluorescent molecule is Eu, and the acceptor fluorescent molecule paired with it is selected from APC, D2, XL665, Fluorescein, GFP, Rhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Cy3, Atto550Cy3.5, Dy547, Dy Any one of 548, Dy549, Dy554, Dy555, Dy556, Dy560, mCherry, mStrawberry, Alexa680, Alexa700, Alexa750, Alexa647, Cy5, Cy5.5, Cy7, Dy647, Dy648, Atto590.

The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, so as to define the protection scope of the present invention more clearly.

Example

Materials and Methods

1. Mice

FcγR-deficient (FcγRα ^-/- ) and FcγR humanized (FcγRα ^-/- /hFcγRI ⁺ /hFcγRIIAR131 ⁺ /hFcγRIIB ⁺ /hFcγRIIIAF158 ⁺ /hFcγRIIIB ⁺ , or “hFCGRTg”) mice (Mouse model recapitulating human Fcgamma receptor structural and functional diversity, Proc Natl Acad Sci USA, vol.109, no.16, pp.6181-6186, 2012) and OT1 mice (Inhibitory Fcgamma receptor is required for the maintenance of tolerance through distinct mechanisms, J Immunol, vol.192, no.7, pp.3021 -3028, 2014) was kindly provided by Dr. Jeffrey Ravetch (Rockefeller University). For FcγR humanized mice, breeding mice and chimeric mice whose bone marrow cells were adoptively transferred were used and confirmed to give the same results. To generate bone marrow chimeric mice, 8–10 week-old wild-type C57BL/6 mice (SLAC, Shanghai, China) were lethally irradiated with 8 Gy using an RS2000pro X-ray bioirradiator (Rad Source Technologies, Inc, USA), and 2 × 10 donor ^bone marrow cells were transferred by tail vein injection. Two months after transplantation, the peripheral blood of the bone marrow-reconstituted mice was collected, and the reconstitution of human FcγRIIA/B expression levels in B cells and CD11b ⁺ cells was confirmed to be more than 95% by flow cytometry analysis. All mice were housed and maintained in the Specific Pathogen Free Animal Facility of the Department of Animal Science, Shanghai Jiaotong University School of Medicine. All animal care and research were performed in accordance with institutional and NIH guidelines and have been approved by the SJTUSM Institutional Animal Care and Use Committee (Protocol Registry Number: A-2015-014).

2. Antibodies

Anti-mouse CD40 antibody (clone 1C10) with different heavy chain constant regions, anti-human CD40 antibody (patent number: clones 21.4.1 and 3.1.1 of US7,338,660) and anti-mouse DR5 antibody (clone MD5-1) were produced according to published methods (Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumoractivities of agonistic CD 40 antibodies, Science (New York, NY), vol.333, no.6045, pp.1030-1034, 2011; Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fcγreceptor engagement, Proceedings of the National Academy of Sciences of the United States of America, vol.109, no.pp.10966-10971, 2012). In brief, heavy chain expression constructs for anti-CD40 and anti-DR5 antibodies were obtained by subcloning human IgG constant region sequences into mammalian expression vectors with 1C10 and DR5 heavy chain gene variable domains, i.e., heavy chain expression constructs for anti-CD40 antibodies were obtained by subcloning human IgG or murine IgG constant region sequences into mammalian expression vectors with 1C10 heavy chain gene variable domains, or anti-DR5 antibody heavy chain expression constructs were obtained by subcloning human IgG constant region sequences Into a mammalian expression vector with the variable domain of the DR5 heavy chain gene (InhibitoryFcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies, Science (New York, NY), vol.333, no.6045, pp.1030-1034, 2011; Apoptotic and antitum or activity of death receptor antibodies require inhibitory Fcγreceptor engagement, Proceedings of the National Academy of Sciences of the United States of America, vol.109, no.pp.10966-10971, 2012), or directly through site-directed mutagenesis (Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies, Science (New York, NY), vol.333, no.6045, pp.1030-1034, 2011). Human IgG2, 3, 4 constant region sequences (CD sequences) were obtained by gene synthesis (Biosune, Shanghai, China) based on the human IgG sequences in the IMGT database http://www.imgt.org/. The sequences G3(H2) and G2(H3) of the chimeric constant region were also synthesized based on the IMGT sequence, wherein "G1-G3" respectively refers to the CH2-CH3 region of the IgG1-3 heavy chain constant region, and "H1-H3" respectively refers to the CH1-hinge region of the IgG1-3 heavy chain constant region. V11(H1) is a previously described human IgG1 heavy chain constant region variant carrying G237D/P238D/H268D/P271G/A330R mutations (Engineered antibody Fc variant with selectively enhanced FcgammaRIIb binding over both FcgammaRIIa(R131) and FcgammaRIIa(H1 31), Protein Eng Des Sel, vol.26, no.10, pp.589-598, 2013). V11(H2) and V11(H3) used the CH1-hinge region of the human IgG2 and IgG3 heavy chain constant regions and the CH2-CH3 region of the V11 variant, respectively. V11(H1)-V11(H3) were synthesized according to the sequence. Based on the mouse IgG sequence in the IMGT database http://www.imgt.org/, the mouse IgG1 and 2a constant region sequences (CD sequences) were obtained by gene synthesis (Biosune, Shanghai, China). The sequence G1 (H2a) of the chimeric constant region is obtained by fusion molecular cloning method according to IgG1 and IgG2 sequences. Anti-CD40 and anti-DR5 antibody light chain expression constructs have been described in previously published articles and patents of the present invention (Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies, Science (New York, NY), vol.333, no.6045, pp.1030-1034, 2 011; Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fcgamma receptor engagement, Proc Natl Acad SciU SA, vol.109, no.27, pp.10966-10971, 2012). For antibody production, antibody heavy and light chain expression vectors were transiently transfected into 293T cells, and antibodies secreted into the supernatant were purified using Protein G Sepharose 4 Fast Flow (GE Healthcare) and dialyzed into phosphate buffered saline (PBS). LPS (endotoxin) levels were analyzed by endotoxin limulus assay (Thermo Scientific) and confirmed to be <0.1 EU μg ⁻¹ . SEC analysis was performed on the antibody product to assess the level of multimeric aggregates, and antibodies were used in the absence of discernible multimers.

The forward primer (IgG2(GS)3f) and reverse primer (IgG2(GS)3r) used for mutagenesis to produce IgG2(GS)3 heavy chain were:

IgG2(GS)3f: 5'ggtagcggaagcggtagttgttgtgtcgagtgcccaccg3' (SEQ ID NO: 1);

IgG2(gs)3r: 5'actaccgcttccgctacctttgcgctcaactgtcttgtc3' (SEQ ID NO: 2).

The forward primer (igg2v1f) and reverse primer (igg2v1r) used for mutagenesis to produce IgG2v1 or v11h2v1 heavy chain are:

igg2v1f:

5'acagttgagcgcaaaggttgttgtgtcgagtgcccaccgtgccca3' (SEQ ID NO: 3);

igg2v1r:

5' gcactcgacacaacaacctttgcgctcaactgtcttgtccacctt3' (SEQ ID NO: 4).

The forward primer (igg2v2f) and reverse primer (igg2v2r) used for mutagenesis to produce IgG2v2 or v11h2v2 heavy chain are:

igg2v2f:

5'aagacagttgagcgcaaaggtagctgttgtgtcgagtgcccaccgtgccca3' (SEQ ID NO: 5);

igg2v2r:

5'gcactcgacacaacagctacctttgcgctcaactgtcttgtccacctt3' (SEQ ID NO: 6).

Forward primer (igg2v3f) and reverse primer (igg2v3r) used for mutagenesis to produce IgG2v3 or v11h2v3 heavy chain are:

igg2v3f:

5'aagacagttgagcgcaaaggtagcggatgttgtgtcgagtgcccaccgtgccca3' (SEQ ID NO: 7);

igg2v3r:

5'tgggcactcgacacaacatccgctacctttgcgctcaactgtcttgtccacctt3' (SEQ ID NO: 8).

Forward primer (igg2v4f) and reverse primer (igg2v4r) used for mutagenesis to produce IgG2v4 or v11h2v4 heavy chain are:

igg2v4f:

5'aagacagttgagcgcaaaggtagcggaagctgttgtgtcgagtgcccaccgtgc3' (SEQ ID NO: 9);

igg2v4r:

5'tgggcactcgacacaacagcttccgctacctttgcgctcaactgtcttgtccacctt3' (SEQ ID NO: 10).

The forward primer (igg2v5f) and reverse primer (igg2v5r) used for mutagenesis to produce IgG2v5 or v11h2v5 heavy chain are respectively:

igg2v5f:

5'gagcgcaaaggtagcggaagcggttgttgtgtcgagtgccca3' (SEQ ID NO: 11);

igg2v5r:

5'gacacaacaaccgcttccgctacctttgcgctcaactgtctt3' (SEQ ID NO: 12).

3. ova-specific CD8 ⁺ T cell responses

CD45.1 ⁺ OT-1 splenocytes (2×10 ⁶ cells per mouse, suspended in 200 μl PBS) were adoptively transferred to mice by tail vein injection on day 1, and 2 μg DEC-OVA (Differential antigen processing by dendritic cell subsets in vivo, Science (New York, NY), vol.315, no.580 by intraperitoneal injection on day 2 8, pp.107-111, 2007) and control or anti-CD40 antibody (3.16-100 μg per mouse, as described in the legend). On day 7, splenocytes were harvested, and after lysing erythrocytes, single cell suspensions were stained with anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD45.1 (A20) antibodies, anti-TCR-Vα2 (B20.1) to quantify OVA-specific OT-1CD8 ⁺ T cells. OT-1CD8 ⁺ T cells were defined as CD45.1 ⁺ CD8 ⁺ TCR-Vα2 ⁺ cells. For intracellular IFN-γ staining, splenocytes were cultured in medium (RPMI containing 10% fetal bovine serum, 1% Pen-Strep, 10 mM HEPES, 50 μM 2-mercaptoethanol) containing 1 μg ml ^-1 CD28 antibody and 1 μg ml ^-1 OVA peptide (SIINFEKL) at 37°C, 5% CO for 1 hour, then added brefeldin A (BFA) to a final concentration of 10 μg ml ^-1 , and Splenocytes were cultured for an additional 5 hours. Cultured splenocytes were surface stained for CD4 (clone RM4-5) and CD8 (clone 53-6.7) and intracellularly stained for IFN-γ (clone XMG1.2) according to the manufacturer's instructions (BD Biosciences) and analyzed by flow cytometry on a BD FACSCanto II (BD Biosciences).

4. Flow Cytometry

Harvest spleen, prepare single cell suspension and lyse red blood cells, resuspend about 1~4× ¹⁰⁶ splenocytes in 50 μl FACS buffer (1xPBS containing 0.5% FBS, 2mM EDTA and 0.1% NaN3) containing staining antibody, and incubate on ice for 15 minutes, then wash cells twice by FACS buffer, resuspend in 200 μl FACS buffer containing DAPI or 7AAD, and flow through Cytometry analysis. For intracellular IFN-γ staining, an additional staining step was performed using the Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions. For analysis of reconstitution levels in hFCGR ^Tg chimeric mice, heparinized blood was collected from mouse orbits and stained with anti-CD19 (clone 1D3), anti-CD11b (clone M1/70), anti-human CD32 (clone FLI8.26); additionally stained with anti-human CD40 (clone 5C3) to analyze reconstitution levels of human FcγR and human CD40.

5. Small angle X-ray scattering (SAXS)

SAXS data were collected at the National Center for Protein Science (NCPSS) at the Shanghai Synchrotron Radiation Facility (SSRF) on beamline BL19U2 equipped with a Pilatus 1M detector (DECTRIS Ltd) (The new NCPSS BL19U2 beamline at the SSRF for small-angle X-ray scattering from biological macromolecules in solution, J Appl Crystallogr, vol.4 9, no. Pt 5, pp. 1428-1432, 2016). Purified monomeric anti-CD40 antibodies were verified by size exclusion chromatography (SEC). A series of diluted antibody samples (0.4-2.8mg ml ^-1 ) were collected, all samples were in HBS buffer (150mM sodium chloride, 10mM HEPES pH7.4), 60ul of each concentration sample, and the samples were exposed to a capillary tube of 240×80μm X-ray beam. Data reduction, beamline intensity normalization and buffer subtraction were performed using BioXTAS RAW software (version 1.2.1) developed by Cornel High Energy Synchrotron Source (CHESS). SAXS data analysis was performed using software from the ATSAS program suite (version 2.8.4 (r10553)) (ATSAS 2.8: a comprehensive data analysis suite for small-anglescattering from macromolecular solutions, J Appl Crystallogr, vol. 50, no. Pt 4, pp. 1212-1225, 2017). The SAXS data obtained from the highest concentration sample were used for Guinier analysis, P(R) analysis and Kratky plot, while the data obtained from the highest and lowest concentration samples were used for EOM (Advanced ensemble modeling of flexible macromolecules using X-ray solution scattering, IUCrJ, vol.2, no.Pt 2, pp.207-217, 2015) analysis. The radius of gyration (Rg) and I(0) were determined by the "Autorg"("radius of gyration" of the program PRIMUSQT from the ATSAS program suite (version 2.8. ") function calculation (Analysis of X-ray and neutronscattering from biomacromolecular solutions, Curr Opin Struct Biol, vol.17, no.5, pp.562-571, 2007), using GNOM (Determination of the regularization parameter in indirect-transform methods using perceptual criteria, Journal of Applied Crystallography, vol.25, no.4, pp.495-503, 1992) ("distance distribution" function of program PRIMUSQT (PRIMUS: a Windows PC-based system for small-angle scattering data analysis, Journal of Applied Crystallography, vol.36, no.5, pp.1277-1282, 20 03)) Determine the pairing distribution function P(R) and the maximum interatomic distance (Dmax), and also estimate the Porod volume (VP) value using the "distance distribution" function of the program PRIMUSQT, using the Bayesian inference method (Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data, Sci Rep, vol.8, no.1, pp.7204, 2018) (program PRIM The "Molecular Weight" module of USQT) calculates molecular weight (Mr) values.

To apply EOM (EOM 2.1) (Advanced ensemble modeling of flexible macromolecules using X-ray solution scattering, IUCrJ, vol.2, no.Pt 2, pp.207-217, 2015) to SAXS data of human IgG antibodies, a published method (In-depth analysis of subclass-specific conformation al preferences of IgG antibodies, IUCrJ, vol.2, no.Pt 1, pp.9-18, 2015). In brief, each antibody was considered as five rigid bodies connected by flexible linkers: 2 extracted from the hinge of PDB 1HZH (Crystal structure of a neutralizing human IgG against HIV-1: template for vaccine design, Science (New York, NY), vol.293, no.5532, pp.1155-1159, 2001). An immobilized CPPC fragment mimics a disulfide bond, 2 Fabs and 1 Fc domain. The extracted CPPC fragment was also used as a model for the second "CPRC" in the IgG3 hinge region, for the PDB files of the Fab and Fc domains or from the crystal structure (PDB 1HZH for IgG1 Fc (Crystal structure of a neutralizing human IG against HIV-1: a template for vaccine design, Science (New York, NY), vol.293, no.5 532, pp.1155-1159, 2001); PDB 4HAG (IgG2 Fc structure and the dynamic features of the IgG CH2-CH3 interface, Mol Immunol, vol.56, no.1-2, pp.131-139, 2013)) extraction for IgG2 Fc, or by using SWISS-MODEL Workspace (The SWISS-MODELworkspace: a web-based environment for protein structure homology modeling, Bioinformatics, vol.22, no.2, pp.195-201, 2006) generated by homology modeling. For homology modeling, choose PDB 5W38 (Structural characterization of the Man5 glycoform of human IgG3 Fc, Mol Immunol, vol.92, no.pp.28-37, 2017) as the template for IgG3 and V11 Fc, and choose PDB 6AMM (Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFbeta1 antibody, MAbs, vol.10, no.3, pp.444-452, 2018) was used for all IgG Ab domains. Perform EOM with default settings.

6. Time-resolved FRET (TR-FRET)

The extracellular domain of mouse CD40 (His-tagged, Novoprotein, China) was stained with terbium (Tb) and D2 ( Chemistry, Cisbio Bioassays, China) labeling to obtain CD40-Tb (1.5Tb/CD40) and CD40-D2 (0.3D2/CD40). CD40-Tb, CD40-D2 and control or anti-CD40 antibodies were diluted to optimal concentrations in TPBS-BSA (5x PBS+0.2% BSA+0.05% Tween-20) and mixed in a Proxi Plate™-384F Plus 384-Well plate (PerkinElmer, Cat. No.: 6008260) to a final volume of 20 μl. The final concentration of mouse CD40-Tb was 2.6nM, and the final concentration of mouse CD40-D2 was 41.6nM (concentration ratio, CD40-Tb:CD40-D2=1:16). The control and anti-mouse CD40 monoclonal antibodies were serially diluted 2-fold from 512 nM to 4 nM, the plate was incubated at room temperature for 1 hour, and then the TR-FRET signal was read using a Synergy neo microplate reader (Biotech Instruments, Inc., USA) with the following settings: excitation at 330 nm, delay 50 μs before recording, fluorescence counting 400 μs through 620 nm (for Tb) and 665 nm (for D2) emission filters The signal of "Em 665nm/Em 620nm" was analyzed as the TR-FRET signal.

7. In vitro pro-apoptotic activity of anti-DR5 antibody

MC38 cells (density ~80%) were plated in a flat-bottomed 96-well tissue culture plate (Thermo, Cat. No. 167008), with a density of 8×10 cells per well ^, 200 μl of complete medium (DMEM+10% fetal bovine serum+1% Pen/Strep), and cultured overnight.轻轻吸出培养基后，加入重悬于100μl完全培养基的4×10 ⁵个裂解红细胞的FcγRα ^-/-或hFCGR ^Tg B6小鼠脾细胞，然后加入100μl含有1μgml ^-1对照IgG，αDR5：hIgG1，αDR5：hIgG2，αDR5：hIgG3，αDR5：hIgG4，αDR5：hIgG V11(H1)，hIgG V11(H2)，hIgG V11(H2)V1，hIgG V11(H2)V2，hIgG V11(H2)V3，hIgG V11(H2)V4，hIgG V11(H2)V5，hIgG V11(H2)V6或αDR5：hIgGV11(H3)伴随加入或不加1μgml ^-1的2B6的培养基(Jackson Immuno Research，货号009-000-003)。 After 4 hours, cells were harvested and stained with anti-mouse CD45.2 antibody (BD, Cat. No. 560691), followed by Annexin V/PI staining and intracellular activated caspase-3 staining (clone C92-605; BD Biosciences) using Annexin V FITC Apoptosis Detection Kit I (BD Biosciences, Cat. No. 556547) according to the manufacturer's instructions. Samples were analyzed with a BD FACSCalibur™ or LSRFortessa™ X-20 flow cytometer. MC38 cells were circled based on forward, side scatter and CD45.2 negative, and the percentage of AnnexinV ⁺ PI ^- or activated caspase-3 ⁺ apoptotic cells was analyzed.

8. Hepatotoxicity

To study the hepatotoxic effect of anti-DR5 antibody, mice were treated with 100 μg anti-DR5 antibody intravenously and the survival rate was monitored for 1 month. Six days after treatment, mouse serum was analyzed for aspartate aminotransferase levels using the MaxDiscovery Assay Kit (Bioo Scientific) aspartate aminotransferase enzymatic method according to the manufacturer's instructions.

9. Statistical Analysis

Statistical analyzes were performed with GraphPad Prism (version 6.01 for windows), and p values less than 0.05 were considered statistically significant. Asterisks indicate statistical comparisons with the control group unless otherwise stated in the figure (*p≤0.05, **p≤0.01, ***p≤0.001, ***p≤0.0001).

Example 1 TR-FRET can analyze the principle of antibody flexibility

Previous work has shown that the relationship between the agonistic activities of human IgG1, 2, and 3 antibodies is IgG2>IgG1>IgG3 (Chinese patent application number: 201710429281.6, PCT/CN2017/087620). In order to study the relationship between the flexibility or activity of antibodies, the TR-FRET method was used to analyze the flexibility of antibodies. Taking the anti-mouse CD40 antibody as an example, the mouse CD40 is excited (Tb) and accepted (D2) by TR-FRET respectively, and then labeled with a fluorophore and then mixed with the anti-mouse CD40 antibody (Figure 2a). The arms of some antibodies can bind to CD40-Tb and CD40-D2 respectively. According to the principle of TR-FRET, if the distance between Tb and D2 is less than twice Radius (2R0), exciting Tb can generate TR-FRET signal, and the intensity of the signal is determined by the distribution of the distance between Tb and D2. For Tb and D2 fluorophores, the distance of 2R0 is 11.6 nm (Reference (Terbium-based time-gatedForster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes, Dalton transactions (Cambridge, England: 2003), vol.44, no.11, pp.4994 -5003, 2015) (Resonance energy transfer: methods and applications, Analytical biochemistry, vol. 218, no. 1, pp. 1-13, 1994) and Cisbio.com). According to the two antibodies with complete crystal structures, the distance between their antigen-binding sites is 12-17 nanometers (Reference (Crystal structure of a neutralizing human IgG against HIV-1: a template for vaccine design, Science (New York, NY), vol.293, no.5532, pp.1155-1159, 2001; Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab, Nature structural&molecular biology, vol.22, no.12, pp.953-958, 2015) and Figure 3), exceeding the minimum distance capable of generating a TR-FRET signal (Figure 2b). Under this condition, the flexibility of the antibody hinge region allows the antibody arms and antigen to swing in solution, making it possible to reduce the distance between CD40-Tb and CD40-D2 to within 2R0 (11.6 nm) (Fig. 2b). Importantly, when the distance between Tb and D2 decreases from 2R0, the signal of TR-FRET increases exponentially (Reference (Resonance energy transfer: methods and applications, Analytical biochemistry, vol.218, no.1, pp.1-13, 1994)), which greatly enhances the signal of TR-FRET. Therefore, there are more molecules in a more flexible antibody with a smaller Tb-D2 distance, which can generate a stronger TR-FRET signal; conversely, a stiff antibody hinge region will limit the swing of the antibody's arms, making Tb-D2 more often The distance is relatively large, thereby reducing the TR-FRET signal (Figure 2b).

Example 2 TR-FRET analysis showed that the IgG3 antibody with the most flexible hinge region had the highest TR-FRET signal, while the IgG2 antibody with the least flexible hinge region had the lowest TR-FRET signal ( FIG. 4 ). To analyze the flexibility of different human native IgG antibodies, we compared IgG1, IgG2, and IgG3 antibodies in parallel using the TR-FRET method described above. After anti-mouse CD40 antibodies were expressed in IgG1, IgG2 and IgG3 formats, respectively, TR-FRET signals were measured after mixing with CD40-Tb and CD40-D2 as described above (Fig. 2a). The results showed that among the three natural IgG antibodies, the TR-FRET signal of the IgG3 antibody was the strongest, while the TR-FRET signal of the IgG2 antibody was the weakest (Fig. 4a), suggesting that the IgG3 antibody was the most flexible, while the IgG2 antibody was stiff enough to limit the swinging of its arms to trigger the TR-FRET signal. To further analyze whether the factor affecting the flexibility of IgG antibodies is the hinge region, we expressed V11(H1), V11(H2) and V11(H3) antibodies with the same variable region and Fc region and hinge regions from IgG1, IgG2 and IgG3, respectively. Parallel comparison results showed that the TR-FRET signal characteristics of these three antibodies were consistent with those of the corresponding IgG antibodies (Figure 4b), suggesting that the flexibility of these IgG antibodies was mainly affected by the antibody hinge region. Further analysis of the chimeric antibodies (G2(H3) and G3(H2) antibodies) generated by IgG2 and IgG3 swapping the hinge region showed that the grafted antibody hinge region sequence could graft the corresponding flexible features (Fig. 4c). These results suggest that TR-FRET analysis can distinguish the different flexible properties of different IgG antibodies due to different hinge regions, in which the most flexible IgG3 hinge region corresponds to the highest TR-FRET signal, while the least flexible IgG2 hinge region corresponds to the lowest TR-FRET signal.

Example 3 Small-angle X-ray scattering (small-angle X-ray scattering, SAXS) analysis confirmed that IgG3 antibodies are more flexible and IgG2 antibodies are less flexible (Figures 5-6).

Small-angle X-ray scattering technique is a widely accepted method for researching biomacromolecules but requires high hardware facilities. It can analyze the flexible characteristics of biomacromolecules in solution (How Random are Intrinsically Disordered Proteins? A Small Angle Scattering Perspective, Curr Protein PeptSc, vol.13, no.1, pp.55-75, 2012). The anti-CD40 antibody monomer molecules purified by molecular sieves were serially diluted and then analyzed by small angle scattering ( FIG. 5 ). The results of Dimensionless Kratky plots show that in IgG1, IgG2 and IgG3 antibodies, the apexes of the curves of IgG1 and IgG2 are very close to the Guinier–Kratky point ( 1.103) (Figure 5a), indicating that both IgG1 and IgG2 antibodies have the characteristics of globular proteins (NADPH oxidase activator P67(phox) behavesin solution as a multidomain protein with semi-flexible linkers, J StructBiol, vol.169, no.1, pp.45-53, 2010; How Random are In Trinsically Disordered Proteins? A Small Angle Scattering Perspective, Curr Protein Pept Sc, vol.13, no.1, pp.55-75, 2012); at the same time, the vertex of the Dimensionless Kratky plots of the IgG3 antibody moved to the upper right obviously, indicating that IgG3 is a multifunctional domain protein linked by a flexible linker sequence (How Random are Intrinsically Disordered Proteins? A Small Angle Scattering Perspective, CurrProtein Pept Sc, vol.13, no.1, pp.55-75, 2012), confirmed the analysis results of TR-FRET on IgG3 antibodies; at the same time, the results of P(R)/I(0), Rg, Dmax, etc., which can reflect the molecular size, also support the larger molecular size of IgG3 antibodies (Figure 5b, Figure 6), which has a more loose structure. Further analysis of V11(H1), V11(H2) and V11(H3) antibodies showed that their small-angle scattering characteristics were consistent with those of natural IgG1, IgG2 and IgG3 antibodies (Figure 5c, Figure 5d), suggesting that the flexibility of these IgG antibodies is mainly affected by the antibody hinge region. Further analysis of the chimeric antibodies (G2(H3) and G3(H2) antibodies) produced by IgG2 and IgG3 swapping the hinge region showed that the transplanted antibody hinge region sequence could transplant the corresponding small-angle scattering flexibility characteristics (Figure 5e, Figure 5f, Figure 6). These results further support that TR-FRET analysis is an effective means to analyze the flexibility of antibody molecules. It should be pointed out that the Dimensionless Kratky plots of IgG1 and IgG2 antibodies are relatively similar, and it is difficult to clearly judge the difference in flexibility between them based on this.

Further use of the Ensemble optimization method (EOM) method developed for small-angle scattering data analysis can quantitatively evaluate the flexibility of the sample (In-depth analysis of subclass-specific conformational preferences of IgG antibodies, IUCrJ, vol.2, no.Pt 1, pp.9-18, 2015; Advanced ensemble modeling of flexible macromolecules using X-raysolution scattering, IUCrJ, vol.2, no.Pt2, pp.207-217, 2015). The results of EOM analysis showed that among the analyzed IgG1, IgG2 and IgG3 natural antibodies, the distribution of the radius of gyration (Rg) and the maximum interatomic distance (Dmax) of the IgG2 antibody was the narrowest (Figure 5g, Figure 5h), which was consistent with previous research results (In-depth analysis of subclass-specific conformational preferences of IgG antibodies, IUCrJ, vol.2, no.Pt 1, pp.9-18, 2015), while the Rg and Dmax distributions of IgG3 antibodies are the broadest, suggesting that IgG2 and IgG3 antibodies are the least flexible and the most flexible of the three antibodies, respectively. In addition, the Rflex and Rσ values obtained by EOM analysis, which can quantify the reaction flexibility, further support the flexibility relationship of the three antibodies as IgG3>IgG1>IgG2 (Figure 6). Finally, further analysis of the V11(H1), V11(H2) and V11(H3) antibodies showed that their distributions of Rg and Dmax, as well as the relative relationship of Rflex and Rσ were consistent with those of the corresponding natural IgG1, IgG2 and IgG3 antibodies (Figure 5i, Figure 5j, Figure 6), and swapping the hinge regions of IgG2 and IgG3 antibodies also exchanged these features (Figure 5k, Figure 5l, Figure 6).

On the one hand, the above results further support that the relative flexibility of the three natural IgG antibodies is IgG3>IgG1>IgG2 and is mainly determined by their hinge regions; on the other hand, these results further verify the analysis results of TR-FRET on antibody flexibility, indicating that TR-FRET is a very sensitive analysis method that can distinguish the flexibility of IgG1 and IgG2 without complex simulation analysis.

Example 4 Mouse IgG flexibility affects mouse IgG agonism (Figure 7)

The flexibility of the murine IgG antibody was detected by the SAXS method, and the Dimensionless Kratky plots analysis showed that mIgG2a showed an upward characteristic, indicating that mIgG2a has stronger flexibility than mIgG1, and the flexibility of the hinge region is also transferred through the replacement of the hinge region (Figure 7a).

In order to analyze the relationship between the agonistic activity of murine agonist antibodies and the hinge region flexibility, the immune activation activities of anti-CD40 murine IgG antibody (clone 1C10) with different natural IgGs (IgG1, IgG2a) and hinge region swapped variants (IgG1(H2a)) were compared. The results showed that IgG1 antibodies with weak flexibility (strong rigidity) had the strongest agonistic activity, while IgG2a antibodies with strong flexibility had the weakest agonistic activity (Fig. 7b, Fig. 7c). At the same time, the hinge region is replaced, and the flexible hinge region of IgG2a can provide the variant IgG1 (H2a) with lower agonism than the parent IgG1. These results suggest that in the 1C10 anti-CD40 murine IgG antibody, the least flexible IgG1 hinge region can provide the best agonistic activity, whereas the most flexible IgG2a hinge region supports the least agonistic activity.

Example 5 TR-FRET analysis of IgG2 hinge region mutants with enhanced flexibility, and confirmation by SAXS (Fig. 8-9)

In order to further verify the TR-FRET method for analyzing antibody flexibility, 1 to 3 "GSGSGS" linking sequences were inserted into the hinge region of the IgG2 antibody to generate three IgG2 variants: IgG2(GS)3, IgG2(GS)6, and IgG2(GS)9 (Figure 15). It is expected that the longer the length of the inserted sequence, the more flexible the antibody will be. The results of TR-FRET analysis showed that with the increase of the inserted sequence, the TR-FRET signal of the antibody increased significantly (Figure 8), indicating that the flexibility increased, which was consistent with expectations. Small-angle scattering analysis of the IgG2 antibody and these variants showed that the longer the inserted "GSGSGS" link sequence, the more upward the DimensionlessKratky plots of the antibody (Figure 9), indicating higher flexibility. Therefore, the analysis of TR-FRET and small-angle X-ray scattering of IgG2 and IgG2 variants such as IgG2(GS)3, IgG2(GS)6, and IgG2(GS)9 further validated that TR-FRET is a sensitive and quantitative flexible analysis method. At the same time, these results also show that changing the length or amino acid sequence of the IgG antibody hinge region can change the flexibility of the antibody, and increasing the length of the hinge region can enhance the flexibility of the antibody.

Example 6 Enhancing the flexibility of the IgG2 hinge region can reduce the agonistic activity of IgG2 antibodies ( FIG. 10 ).

Further analysis of the immune activation activity of IgG2 and IgG2 (GS) 3, IgG2 (GS) 6, IgG2 (GS) 9 and other IgG2 variant anti-CD40 antibodies showed that the immune activation activity of the more flexible IgG2 variant anti-CD40 antibodies was significantly lower than that of IgG2 antibodies, manifested in fewer antigen-specific CD8 positive T cells and the proportion (Figure 10). These results suggest that enhancing the flexibility of IgG antibodies can affect the agonistic activity of IgG antibodies. When the flexibility of the IgG2 antibody increases to a certain extent, the antibody loses its agonistic activity.

Example 7 Changing the amino acid sequence of the hinge region can enhance the flexibility of IgG antibodies (IgG1 vs IgG1-A2, and other examples of enhancing flexibility) (Figure 11)

Hinge region sequences with different flexibility characteristics can be screened from natural hinge regions and mutants by TR-FRET analysis. Among them, the IgG1 antibody grafted with the IgA2 hinge region sequence (IgG1A2) exhibited a lower TR-FRET signal than the IgG1 antibody, reflecting its lower flexibility (Fig. 11a). The flexibility of IgG1A2 was further verified by SAXS method analysis. Dimensionless Kratky plots analysis showed that the IgG1A2 curve was lower than that of the maternal IgG1, showing that IgG1A2 was less flexible (more rigid) than IgG1 (Figure 11b).

Example 8 Changing the amino acid sequence of the hinge region to reduce the flexibility of the IgG antibody can affect the agonistic activity of the antibody (Figure 12)

Further analysis of the immune activation activity of the G1A2 anti-CD40 antibody showed that G1A2 had a stronger immune activation activity than IgG1 ( FIG. 12 ). These results suggest that weakening the flexibility of IgG antibodies can also affect the agonistic activity of IgG antibodies. When the flexibility of IgG1 antibody is reduced, the antibody can acquire stronger agonistic activity characteristics.

Example 9 The best natural hinge region of 1C10 clone human IgG antibody is IgG2 hinge region (V11H2>V11H1>V11H3)

In order to analyze the relationship between the agonistic activity of agonistic antibodies and the flexibility of the hinge region, the immune activation activities of three forms (V11(H1), V11(H2) and V11(H3)) of anti-CD40 antibody (clone 1C10) with the same variable region and Fc region and different native IgG hinge regions were compared in parallel. The results showed that the least flexible (most rigid) V11(H2) antibody had the strongest agonistic activity, while the most flexible V11(H3) antibody had the weakest agonistic activity (Fig. 13a). These results suggest that in the 1C10 antibody, the least flexible IgG2 hinge region was able to provide the best agonistic activity, whereas the most flexible IgG3 hinge region supported the least agonistic activity.

Example 10 The best natural hinge region of MD5-1 clone human IgG antibody is the IgG1 hinge region (V11H1>V11H2>V11H3)

In order to further analyze the relationship between the agonistic activity of the agonistic antibody and the flexibility of the hinge region, the agonistic activity of three forms (V11(H1), V11(H2) and V11(H3)) of the anti-DR5 antibody (clone MD5-1) with the same variable region and Fc region and different native IgG hinge regions were compared in parallel (the agonistic activity of the anti-DR5 antibody was expressed as the ability to induce apoptosis). The results showed that the V11(H1) antibody with the middle flexibility had the strongest agonistic activity, while the V11(H3) antibody with the most flexible had the weakest agonistic activity (Fig. 13b). These results suggest that in the MD5-1 antibody, the flexible centered IgG1 hinge region is able to provide the best agonistic activity, whereas the most flexible IgG3 hinge region supports the weakest agonistic activity.

Example 11 Insertion of amino acids that are not multiples of 3-4 in the hinge region will destroy human IgG2 agonistic activity

Structurally, the hinge region, as the connection sequence between Fab and Fc, is a highly flexible fragment. Since this region is rich in proline, it is prone to stretching and distortion to a certain extent, which is conducive to the complementary binding between the antigen binding site of the antibody and the antigen epitope. The number of inserted amino acids is likely to change the angle of torsion and affect subtle changes in the secondary structure.

In order to study the effect of hinge region length change on agonism, we sequentially added glycine G (Glycine, Gly) and serine S (Serine, Ser) between the upper and middle hinges of the 1C10 anti-CD40 human IgG2 antibody to generate six IgG2 variants: IgG2V1, IgG2V2, IgG2V3, IgG2V4, IgG2V5 and IgG2V6 (that is, before Body and G2(GS)3). In order to further analyze the immune activation activity of IgG2 and IgG2V1-6 IgG2 variant anti-CD40 antibodies, the humanized FcγRs spleen cell model was stimulated in vitro, the spleen cells of hFCGRTg mice were isolated, and 30ug/ml control antibody or 1C10 clone anti-CD40 hinge region variants were added for incubation. After 48 hours, the activation level of antigen-presenting cells was detected by flow cytometry. The results showed that, based on the IgG2 hinge region, the addition of non-3 or 4 amino acids would significantly destroy the agonistic effect of the antibody on the activation of antigen-transmitting cells ( FIG. 14 a ). In order to verify that the addition of non-3-4 amino acids will significantly destroy the rigidity of the hinge region and whether it is applicable to other agonistic antibodies, we inserted 1-6 amino acids in the "GSGSGS" link sequence into the hinge region of the MD5-1 anti-DR5 IgGV11(H2) antibody in turn, and generated 6 V11H2 variants: IgGV11(H2)V1, IgGV11(H2)V2, IgGV11(H2)V 3. IgGV11(H2)V4, IgGV11(H2)V5 and IgGV11(H2)V6. Using an in vitro pro-apoptotic model, we co-cultured MC38 cells with FcγR humanized C57B6/L or Fcgrα-/-C57B6/L mouse spleen cells, stimulated them with control IgG or MD5-1 anti-mouse DR5 antibody for 2 hours, and quantified the percentage of Annexin V+PI-apoptotic MC38 cells by flow cytometry. It was shown that, based on the V11H2 hinge region, adding non-3 or 4 amino acids would significantly destroy the antibody activity ( FIG. 14 b ), which was consistent with the 1C10 clone anti-mCD40 antibody species. Taken together, these results indicate that when modifying the antibody hinge region, adding 3-4 amino acids may provide higher agonistic activity than adding amino acids that are not a multiple of 3-4.

Combining the research results of 1C10 antibody and MD5-1 antibody, it shows that the antibody hinge regions that support different antibodies to obtain the best agonistic activity are not necessarily the same, but the flexibility of these optimal hinge regions is at the weaker end, and the agonistic activity of antibodies using the most flexible IgG3 hinge region among the three natural hinge regions is the worst. These results suggest that, on the one hand, in order to destroy the agonistic activity of the antibody, it can be achieved by enhancing the flexibility of the antibody to a certain extent; on the other hand, in order to enhance the agonistic activity of the antibody, it is necessary to screen the optimal hinge region sequence among hinge region sequences with different flexibility for different antibody clones.

Although specific embodiments of the invention have been described above for purposes of illustration, those skilled in the art will appreciate that many changes may be made in the details without departing from the invention as described in the claims.

sequence listing

<110> Shanghai Jiao Tong University School of Medicine

<120> Antibody Activity Modification and Screening Method

<130> 207634

<141> 2020-09-21

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39

<212>DNA

<213> Artificial Sequence

<400> 1

ggtagcggaa gcggtagttg ttgtgtcgag tgcccaccg 39

<210> 2

<211> 39

<212>DNA

<213> Artificial Sequence

<400> 2

actaccgctt ccgctacctt tgcgctcaac tgtcttgtc 39

<210> 3

<211> 45

<212>DNA

<213> Artificial Sequence

<400> 3

acagttgagc gcaaaggttg ttgtgtcgag tgcccaccgt gccca 45

<210> 4

<211> 45

<212>DNA

<213> Artificial Sequence

<400> 4

gcactcgaca caacaacctt tgcgctcaac tgtcttgtcc acctt 45

<210> 5

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 5

aagacagttg agcgcaaagg tagctgttgt gtcgagtgcc caccgtgccc a 51

<210> 6

<211> 48

<212>DNA

<213> Artificial Sequence

<400> 6

gcactcgaca caacagctac ctttgcgctc aactgtcttg tccacctt 48

<210> 7

<211> 54

<212>DNA

<213> Artificial Sequence

<400> 7

aagacagttg agcgcaaagg tagcggatgt tgtgtcgagt gcccaccgtg ccca 54

<210> 8

<211> 54

<212>DNA

<213> Artificial Sequence

<400> 8

tgggcactcg acacaacatc cgctaccttt gcgctcaact gtcttgtcca cctt 54

<210> 9

<211> 54

<212>DNA

<213> Artificial Sequence

<400> 9

aagacagttg agcgcaaagg tagcggaagc tgttgtgtcg agtgcccacc gtgc 54

<210> 10

<211> 57

<212>DNA

<213> Artificial Sequence

<400> 10

tgggcactcg acacaacagc ttccgctacc tttgcgctca actgtcttgt ccacctt 57

<210> 11

<211> 42

<212>DNA

<213> Artificial Sequence

<400> 11

gagcgcaaag gtagcggaag cggttgttgt gtcgagtgcc ca 42

<210> 12

<211> 42

<212>DNA

<213> Artificial Sequence

<400> 12

gacacaacaa ccgcttccgc tacctttgcg ctcaactgtc tt 42

<210> 13

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 13

Val Asp Lys Arg Val

1 5

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 14

Glu Pro Lys Ser Cys Asp Lys Thr His Thr

1 5 10

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 15

Cys Pro Pro Cys Pro

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 16

Ala Pro Glu Leu Leu Gly Gly Pro

1 5

<210> 17

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 17

Cys Pro Val Pro Pro Pro Pro Pro Pro Cys Cys

1 5 10

<210> 18

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 18

Val Asp Lys Thr Val

1 5

<210> 19

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 19

Glu Arg Lys

1

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 20

Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5

<210> 21

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 21

Ala Pro Pro Val Ala Gly Pro

1 5

<210> 22

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 22

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr

1 5 10

<210> 23

<211> 50

<212> PRT

<213> Artificial Sequence

<400> 23

Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

1 5 10 15

Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro

20 25 30

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

35 40 45

CysPro

50

<210> 24

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 24

Glu Ser Lys Tyr Gly Pro Pro

1 5

<210> 25

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 25

Cys Pro Ser Cys Pro

1 5

<210> 26

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 26

Asp Val Thr Val

1

<210> 27

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 27

Glu Arg Lys Gly Ser Gly Ser Gly Ser

1 5

<210> 28

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 28

Glu Arg Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10 15

<210> 29

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 29

Glu Arg Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly

1 5 10 15

Ser Gly Ser Gly Ser

20

<210> 30

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 30

Glu Arg Lys Gly

1

<210> 31

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 31

Glu Arg Lys Gly Ser

1 5

<210> 32

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 32

Glu Arg Lys Gly Ser Gly

1 5

<210> 33

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 33

Glu Arg Lys Gly Ser Gly Ser

1 5

<210> 34

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 34

Glu Arg Lys Gly Ser Gly Ser Gly

1 5

<210> 35

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 35

Val Asp Lys Lys Ile

1 5

<210> 36

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 36

Val Pro Arg Asp

1

<210> 37

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 37

Cys Gly Cys Lys Pro Cys Ile Cys Thr

1 5

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 38

Glu Pro Arg Val Pro Ile Thr Gln Asn Pro

1 5 10

<210> 39

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 39

Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala

1 5 10

Claims (8)

1. A method for up-regulating antibody flexibility or down-regulating the Fc receptor-dependent agonistic activity of an antibody, comprising mutating the upper and/or middle hinge domains of the CH1 hinge region, wherein the mutation is selected from one or more of the following: (a) Insert G, GS, GSGSG, GSGSGS, (GSGSGS) ₂ , (GSGSGS) ₃ at the C-terminus of the upper hinge domain ERK, (b) Change the upper hinge domain VPRD to EPRVPITQNP, and the middle hinge domain CGCKPCICT to CPPLKECPPCA, (c) Mutate the upper and/or middle hinge domain to the corresponding region of IgG3 or mIgG2a, or mutate the hinge region to the hinge region of IgG3 or mIgG2a, or mutate the CH1-hinge region to the CH1-hinge region of IgG3 or mIgG2a, wherein the pre-mutation sequence of the upper hinge domain is selected from one or more of the following: EPKSCDKTHT, P, ERK, ESKYGPP, V PRD, the pre-mutation sequence of the middle hinge domain is selected from one or more of the following: CPPCP, CPVPPPPCC, CCVECPPCP, CPSCP, CPVPPPPCC, CGCKPCICT, and the upper hinge domain of IgG3 is ELKTPLGDTTHT, and the middle hinge domain is CPRCP (EPKSCDTPPPCPRCP)₃, the upper hinge domain of mIgG2a is EPRVPITQNP, and the middle hinge domain is CPPLKECPPCA. 2. The method of claim 1, wherein the antibody is an agonistic antibody, and/or The flexibility of the antibody after mutation is greater than or equivalent to IgG3, and/or Mutations to the antibody do not significantly reduce the affinity of the antibody for the antigen it specifically targets, and/or The radius of gyration (Rg) of the antibody after mutation is greater than and / or The antibody is a human antibody, a chimeric antibody or a humanized antibody. 3. A method for down-regulating antibody flexibility or up-regulating the Fc receptor-dependent agonistic activity of an antibody, comprising mutating the upper and/or middle hinge domains of the CH1 hinge region, the mutation being selected from one or more of the following: (a) inserting GSG or GSGS at the C-terminus of the upper hinge domain ERK, (b) Change the upper hinge domain EPKSCDKTHT to P and the middle hinge domain CPPCP to CPVPPPPCC. (c) Mutating the upper and/or middle hinge domain to the corresponding region of IgG1, IgG2 or IgA2, or mutating the hinge region to the hinge region of IgG1, IgG2 or IgA2, or mutating the CH1-hinge region to the CH1-hinge region of IgG1, IgG2 or IgA2, wherein the pre-mutation sequence of the upper hinge domain is selected from one or more of the following: ELKTPLGDTTHT, ESKY GPP, VPRD, EPRVPITQNP, the pre-mutation sequence of the middle hinge domain is selected from one or more of the following: CPRCP (EPKSCDTPPPCPRCP) 3, CPSCP, CGCKPCICT, CPPLKECPPCA, and the upper hinge domain of IgG1 is EPKSCDKTHT, the middle hinge domain is CPPCP, the upper hinge domain of IgG2 is ERK, and the middle hinge domain is CCVECPPC P, the upper hinge domain of IgA2 is P, and the middle hinge domain is CPVPPPPCC. 4. The method of claim 3, wherein, the antibody is an agonistic antibody, and/or After mutation, the antibody is no more flexible than human IgG1, and/or Mutations to the antibody do not significantly reduce the affinity of the antibody for the antigen it specifically targets, and/or The radius of gyration (Rg) of the antibody after mutation is or less than the Rg of human IgG1, and/or The antibody is a human antibody, a chimeric antibody or a humanized antibody, and/or The antibody specifically recognizes a receptor in the TNF receptor superfamily, and/or The antibody is an anti-CD40 antibody or an anti-DR5 antibody. 5. The method of claim 4, wherein the radius of gyration (Rg) of the antibody after mutation is 6. The method according to any one of claims 1-5, wherein the Fc segment in the heavy chain constant region is further modified to increase the affinity between the agonistic antibody and FcγRIIB or the I/A ratio of the agonistic antibody. 7. The method according to any one of claims 1-5, characterized in that the method regulates the flexibility of the antibody or regulates the Fc receptor-dependent agonistic activity of the antibody under the condition that the immune system expresses human Fc receptors. 8. The method of any one of claims 1-5, wherein the Fc receptor is a human Fc receptor.