WO2023036127A1

WO2023036127A1 - Antibody or antigen binding fragment thereof targeting il-23p19, and use thereof

Info

Publication number: WO2023036127A1
Application number: PCT/CN2022/117259
Authority: WO
Inventors: 郎国竣; 胡宇豪; 刘婵娟; 曹国庆; 王文蓉; 闫鑫甜; 吴琪
Original assignee: 三优生物医药(上海)有限公司
Priority date: 2021-09-10
Filing date: 2022-09-06
Publication date: 2023-03-16
Also published as: CN115785267A; CN115785267B

Abstract

Provided are an antibody or antigen binding fragment thereof targeting IL-23p19, and the use thereof. The antibody or antigen binding fragment thereof targeting IL-23p19 specifically binds to a p19 subunit of IL-23, but does not bind to a p40 subunit, and the binding ability to IL-23 is comparable to or better than that of control antibody Guselkumab, the ability to block the binding of IL-23 to receptor IL-23R is comparable to or better than that of control antibody Guselkumab, and the ability to inhibit the activation of cell STAT3 phosphorylation by IL-23 and the ability to inhibit the activation of IL-17 secretions of mouse splenocytes by IL-23 are comparable to or better than those of control antibody Guelkumab.

Description

Antibody targeting IL-23p19 or antigen-binding fragment thereof and application thereof

technical field

The present invention belongs to the field of antibodies, and in particular relates to an IL-23 binding molecule, especially an antibody and its fragment specifically recognizing IL-23p19 subunit. Furthermore, the present invention also relates to nucleic acids or host cells comprising such antibodies or fragments thereof, as well as therapeutic and diagnostic methods or uses using these antibodies and fragments.

Background technique

Interleukin-23 (IL-23) is a cytokine produced by activated dendritic cells, macrophages, monocytes, etc. component. When the two subunits of p19 and p40 form a heterodimer, they can exert biological functions, but they do not have biological functions when they exist alone.

IL-23 can bind to receptors on the cell surface, activate downstream signaling pathways and exert biological functions. The IL-23 receptor consists of two subunits, IL-23R and IL12β1, which bind to the p19 and p40 subunits of IL-23, respectively. IL-23 mainly acts on Th17 cells, plays an important role in the stability and proliferation of Th17 cells, and can promote Th17 cells to secrete inflammation-related cytokines such as IL-17A, IL-17F and IL-22, and act on keratinocytes , leading to its hyperactivation and proliferation. Activated keratinocytes will produce a large number of cytokines, chemokines and antimicrobial peptides, recruit and activate immune cells such as T cells, and finally cause the psoriatic phenotype.

At present, clinical drug research on this target is still very limited, so the development of new and effective antibody drugs targeting this target has important theoretical and practical significance.

Contents of the invention

The invention discloses an IL-23-targeting antibody or an antigen-binding fragment thereof and applications thereof, in particular an IL-23 binding molecule that specifically binds to the p19 subunit but not to the p40 subunit.

The present invention therefore provides a novel IL-23p19-binding antibody, as well as antigen-binding fragments thereof.

In some embodiments, the anti-IL-23p19 antibodies of the present invention have one or more or all of the following properties:

(i) specifically binds to the p19 subunit of IL-23 and does not bind to the p40 subunit;

(ii) have excellent cross-binding properties with human, mouse (such as mouse) and cynomolgus IL-23;

(iii) the ability to block the binding of IL-23 to the receptor IL-23R is equivalent to or better than that of a known control antibody (such as Guselkumab);

(iv) The ability to inhibit IL-23-activated cellular STAT3 phosphorylation is better than known control antibodies (such as Guselkumab);

(v) Inhibit IL-23's ability to activate splenocytes (eg, mouse splenocytes) to secrete IL-17 better than known control antibodies (eg, Guselkumab).

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to IL-23p19, comprising 3 heavy chain CDRs (HCDRs) of the sequence shown in SEQ ID NO: 12, and/or as shown in SEQ ID NO : 3 light chain CDRs (LCDRs) of the sequence shown in 16.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to IL-23p19, comprising 3 heavy chain CDRs (HCDRs) of the sequence shown in SEQ ID NO: 20, and/or as shown in SEQ ID NO : 3 light chain CDRs (LCDRs) of the sequence shown in 16.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to IL-23p19, comprising 3 heavy chain CDRs (HCDRs) of the sequence shown in SEQ ID NO: 23, and/or as shown in SEQ ID NO : 3 light chain CDRs (LCDRs) of the sequence shown in 27.

In some embodiments, the invention provides a nucleic acid encoding an antibody of the invention or a fragment thereof, a vector comprising the nucleic acid, a host cell comprising the vector.

In some embodiments, the invention provides methods of making an antibody or fragment thereof of the invention.

In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions, and combinations comprising antibodies of the invention.

The present invention also provides methods for blocking IL-23-mediated signaling pathways in a subject using the antibodies of the present invention, and methods for preventing or treating IL-23-related diseases, such as immune system diseases (such as autoimmune diseases or inflammation) method.

The present invention also relates to methods for detecting IL-23p19 in a sample.

The invention is further illustrated in the following figures and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes easily conceived by those skilled in the art will be included in the spirit of the present invention and the protection scope of the appended claims.

Description of drawings

Figure 1 shows the binding of antibody A44 to the recombinant protein human IL-23.

Figure 2 shows that antibody A44 blocks the binding of human IL-23 to human IL-23R.

3A-3B show the binding of the antibody to the recombinant protein human IL-23 after antibody engineering.

Figures 4A-4B show that the antibody blocks the binding of the recombinant protein human IL-23 to human IL-23R after antibody engineering.

Figures 5A-5C show antibody binding to human IL12-p40 subunits after antibody engineering.

Figures 6A-6C show antibody binding to monkey IL-23 after antibody engineering.

Figures 7A-7C show antibody binding to mouse IL-23 after antibody engineering.

Figures 8A-8B show that antibodies compete with Guselkumab for IL-23 binding after antibody engineering.

Figures 9A-9B show that antibodies inhibit intracellular STAT3 phosphorylation after antibody engineering.

Figures 10A-10C show that the antibodies inhibit the secretion of IL-17 by mouse splenocytes after antibody engineering.

Detailed description of the invention

I. Definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to explain this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation of combining the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

The p19 subunit of IL-23 (also referred to herein as "IL-23p19" and "p19 subunit") is a 189 amino acid polypeptide containing a leader sequence of 21 amino acids (Oppmann et al., Immunity 13:715 (2000)), and contains four packed alpha helices called A, B, C, and D, with an up-up-down-down topology. The 4 helices are connected by 3 polypeptide loops. The A-B and C-D loops are made relatively long because they connect the parallel helices. A short B-C loop connects the antiparallel B and C helices. The pl9 subunit of IL-23 is a member of the IL-6 family of helical cytokines. This cytokine family binds to its cognate receptors through three conserved epitopes (sites I, II and III; Bravo and Heath (2000) EMBO J. 19:2399-2411). The p19 subunit interacts with three cytokine receptor subunits to form a competent signaling complex. When expressed in cells, the p19 subunit first forms a complex with the p40 subunit, which shares the p40 subunit with IL-12. The p19p40 complex is secreted from cells as a heterodimeric protein and is known as IL-23. In one embodiment, the IL-23p19 of the invention is from human (UNIPROT accession number Q9NPF7) or cynomolgus monkey (UNIPROT accession number G7PIH8) or murine (eg mouse, UNIPROT accession number: Q9EQ14).

As used herein, the terms "anti-IL-23p19 antibody", "anti-IL-23p19", "IL-23p19 antibody" or "IL-23p19-binding antibody" refer to an antibody that is capable of binding to (human or cynomolgus or murine) IL-23p19 subunit or a fragment thereof such that the antibody can be used as a diagnostic and/or therapeutic agent targeting (human or cynomolgus or murine) IL-23p19. In one embodiment, the anti-IL-23p19 antibody does not bind the p40 subunit of IL-12.

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.

II. Antibodies

In some embodiments, an anti-IL-23p19 antibody or fragment thereof of the invention binds IL-23p19 with high affinity (e.g., the human IL-23p19 subunit in human IL-23 or the cynomolgus monkey IL-23 in cynomolgus monkey IL-23p19 subunit or the murine IL-23p19 subunit in murine IL-23), for _example , binds IL-23p19 with an equilibrium dissociation constant (K _D ) that is less than about 1 nM, preferably less than Or equal to about 0.5nM, 0.4nM, 0.3nM or 0.2nM, most preferably, said _KD is less than or equal to about 0.09nM, 0.08nM, 0.07nM, 0.06nM. In some embodiments, the anti-IL-23p19 antibodies of the invention bind IL-23p19 with a _KD of 0.01-0.2 nM, preferably 0.05 nM-0.15 nM. In some embodiments, the IL-23p19 is human IL-23p19. In some embodiments, the IL-23p19 is cynomolgus IL-23p19. In some embodiments, the IL-23p19 is murine IL-23p19, eg, mouse IL-23p19. In some embodiments, antibody binding affinity is determined using bio-optical interferometry (eg, surface plasmon resonance (Biacore) measurement).

In some embodiments, an anti-IL-23p19 antibody or fragment thereof of the invention binds human IL-23p19, cynomolgus IL-23p19, and/or murine IL-23p19. In some embodiments, an anti-IL-23p19 antibody or fragment thereof of the invention binds human IL-23p19, cynomolgus IL-23p19, and murine IL-23p19. In some embodiments, an anti-IL-23pl9 antibody or fragment thereof of the invention blocks binding of human or cynomolgus or murine IL-23 to its receptor IL-23R. In some embodiments, anti-IL-23p19 antibodies or fragments thereof of the invention block human, cynomolgus and murine IL-23 binding to its receptor IL-23R.

In some embodiments, the anti-IL-23p19 antibody or fragment thereof of the present invention binds human IL-23 (IL-23p19) with high affinity (preferably the binding ability is better than that of known anti-IL-23p19 antibodies, such as Guselkumab) , and/or bind cynomolgus monkey IL-23 (IL-23p19) with high affinity (preferably, the binding ability is equivalent to or better than that of known anti-IL-23p19 antibodies, such as Guselkumab), and/or with better affinity Binds murine IL-23 (IL-23p19).

In some embodiments, an anti-IL-23p19 antibody or fragment thereof of the invention inhibits the ability of IL-23 (e.g., human IL-23, cynomolgus IL-23, or mouse IL-23) to activate cellular STAT3 phosphorylation, preferably , said inhibition was superior to known anti-IL-23p19 antibodies such as Guselkumab.

In some embodiments, the anti-IL-23p19 antibody or fragment thereof of the present invention has better tissue specificity and substantially no binding to cell surface membrane proteins.

In some embodiments, the antibodies of the present invention or fragments thereof inhibit the secretion of IL-17 by cells (such as splenocytes) by binding to IL-23p19, for example, the inhibitory rate of the antibodies or fragments thereof of the present invention on the secretion of IL-17 by cells Reach 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, 99%, 100%.

In some embodiments, antibodies or fragments thereof of the invention (optionally in combination with therapeutic modalities and/or other therapeutic agents, such as immunomodulators) are capable of preventing or treating IL-23-associated diseases, such as immune system diseases (e.g., Rohn's disease, moderately to severely active ulcerative colitis, psoriatic arthritis, palmoplantar pustulosis, and psoriasis).

In some embodiments, the anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, derived from the heavy chain variable region.

In some embodiments, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, derived from the light chain variable region.

In some embodiments, the anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region. ).

In some aspects, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL). In some aspects, an anti-IL-23pl9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region.

In some embodiments, the anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region. In some embodiments, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention further comprises an antibody light chain constant region. In some embodiments, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region and a light chain constant region.

In some embodiments, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises an antibody heavy chain (HC). In some embodiments, an anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention further comprises an antibody light chain (LC). In some embodiments, an anti-IL-23pl9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain.

In some embodiments, an antibody heavy chain of the invention comprises an antibody heavy chain variable region and an antibody heavy chain constant region. In some embodiments, an antibody light chain of the invention comprises an antibody light chain variable region and an antibody light chain constant region.

In some embodiments, the heavy chain variable region of the invention

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence selected from SEQ ID NO: 12, 20, 23 an amino acid sequence of identity or consisting of said amino acid sequence; or

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 12, 20, 23; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence selected from SEQ ID NO: 12, 20, 23 The amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) is or consists of said amino acid sequences, preferably, said amino acid changes do not occur in the CDR region.

In some embodiments, the light chain variable region of the invention

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 16, 27 The amino acid sequence of or consists of said amino acid sequence; or

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 16, 27; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 16, 27 (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of said amino acid sequence, preferably, said amino acid change does not occur in the CDR region.

In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the invention, HCDR1, HCDR2 and HCDR3 are selected from

(i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:12,

(ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:20,

(iii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:23,

or

(iv) With respect to the sequence of any one of (i)-(iii), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions) are included in the three HCDR regions , preferably conservative substitutions).

In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region of the invention, LCDR1, LCDR2 and LCDR3 are selected from

(i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:16,

(ii) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27,

or

(iii) relative to the sequence of any one of (i)-(ii), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions) are included in the three LCDR regions , preferably conservative substitutions).

In some embodiments, HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or HCDR1 comprises one, two or three changes (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.

In some embodiments, HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 2 or 18, or HCDR2 comprises one, two or three amino acid sequences compared to the amino acid sequence of SEQ ID NO: 2 or 18 Amino acid sequence of a change (preferably amino acid substitution, preferably conservative substitution).

In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11, 19 or 22, or HCDR3 comprises one, two or three amino acid sequences compared to the amino acid sequence of 11, 19 or 22 Amino acid sequence of a change (preferably amino acid substitution, preferably conservative substitution).

In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or 25, or LCDR1 comprises one, two or three amino acid sequences compared to the amino acid sequence of SEQ ID NO: 14 or 25 Amino acid sequence of a change (preferably amino acid substitution, preferably conservative substitution).

In some embodiments, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or the LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 7 (preferably Amino acid substitutions, preferably conservative substitutions) amino acid sequence.

In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 26, or the LCDR3 comprises one, two or three amino acids compared to the amino acid sequence of SEQ ID NO: 15 or 26 Amino acid sequence of a change (preferably amino acid substitution, preferably conservative substitution).

In some embodiments, the heavy chain constant region of an antibody of the invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region of the present invention is a lambda or kappa light chain constant region, preferably a lambda light chain constant region.

In some preferred embodiments, the antibody heavy chain constant region of the present invention

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 31 A specific amino acid sequence or consists of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 31; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 31 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.

In some embodiments, the amino acid changes occur in the Fc region.

In some embodiments, the antibody light chain constant region of the invention

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 32 A specific amino acid sequence or consists of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 32; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 32 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.

In some specific embodiments of the present invention, the anti-IL-23p19 antibody or antigen-binding fragment thereof of the present invention comprises:

(i) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:12, and three complementarity-determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO:16 LCDR2 and LCDR3;

(ii) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:20, and three complementarity-determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO:16 LCDR2 and LCDR3; or

(iii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 23, and three complementarity determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO: 27 LCDR2 and LCDR3.

(i) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 11, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 14, 7, and 15;

(ii) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 18, and 19, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 14, 7, and 15; or

(iii) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 22, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 25, 7, and 26.

(i) VH comprising the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO: 16 Amino acid sequences at least 90% identical or VL consisting of said amino acid sequences;

(ii) VH comprising the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO: 16 Amino acid sequences at least 90% identical or VL consisting of said amino acid sequences; or

(iii) VH comprising the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO:27 Amino acid sequences at least 90% identical or a VL consisting of said amino acid sequences.

In some embodiments, the anti-IL-23p19 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence selected from SEQ ID NO: 13, 21 or 24 or an amino acid sequence with 99% identity or consisting of said amino acid sequence;

(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 13, 21 or 24; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence selected from SEQ ID NO: 13, 21 or 24 The amino acid sequence of each) amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of said amino acid sequence, preferably, said amino acid change does not occur in the CDR region of the heavy chain, more preferably, said Amino acid changes do not occur in the heavy chain variable region;

and / or

(b) light chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence selected from SEQ ID NO: 17 or 28 % identity amino acid sequences or consist of said amino acid sequences;

(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 17 or 28; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence selected from SEQ ID NO: 17 or 28 The amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) or consists of said amino acid sequences, preferably, said amino acid changes do not occur in the CDR region of the light chain, more preferably, said amino acid changes Does not occur in the light chain variable region.

In a preferred embodiment, the present invention provides an anti-IL-23p19 antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the following An amino acid sequence or consisting of said amino acid sequence:

(i) SEQ ID NO: 13 and SEQ ID NO: 17;

(ii) SEQ ID NO: 21 and SEQ ID NO: 17; or

(iii) SEQ ID NO:24 and SEQ ID NO:28.

In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitutions are conservative substitutions. A conservative substitution is one in which an amino acid is replaced by another within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid replacement.

In certain embodiments, substitutions occur in the CDR regions of the antibody. Typically, the resulting variant is modified (eg, improved) relative to the parent antibody in certain biological properties (eg, increased affinity) and/or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substitutional variants are affinity matured antibodies.

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to enhance, for example, the affinity of the antibody or the effectiveness of treating a disease. Fc region variants may include human Fc region sequences (eg, human IgGl, IgG2, IgG3 or IgG4 Fc regions) comprising amino acid changes (eg, substitutions) at one or more amino acid positions.

In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, eg, "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the anti-IL-23p19 antibodies or antigen-binding fragments thereof of the present invention also include antibodies or antigen-binding fragments having one or more of the following properties:

(i) exhibit the same or similar binding affinity and/or specificity to IL-23p19 as the antibody of the present invention;

(ii) inhibit (eg, competitively inhibit) the binding of an antibody of the invention to IL-23p19;

(iii) bind the same or overlapping epitope with the antibody of the present invention;

(iv) compete with the antibody of the present invention for binding to IL-23p19;

(v) have one or more biological properties of the antibodies of the invention.

In some embodiments, the anti-IL-23p19 antibody of the invention is an IgG1 format antibody or an IgG2 format antibody or an IgG4 format antibody.

In some embodiments, the anti-IL-23p19 antibody is a monoclonal antibody.

In some embodiments, the anti-IL-23p19 antibody is a human antibody. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol 20:450-459 (2008).

In some embodiments, the anti-IL-23p19 antibody is a chimeric antibody.

In some embodiments, at least a portion of the framework sequence of the anti-IL-23p19 antibody is a human consensus framework sequence. In one embodiment, the anti-IL-23p19 antibody of the present invention also encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv) or ( Fab') ₂ , single domain antibody, diabody, fragmented antibody dAb or linear antibody.

In certain embodiments, the anti-IL-23p19 antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the second binding specificity also includes antigenic proteins that can specifically bind classes such as pro-inflammatory cytokines and chemokines. As used herein, the term "pro-inflammatory cytokine" refers to a class of cytokines secreted by immune cells or other types of cells that promote inflammation. Pro-inflammatory cytokines are mainly produced by helper T cells (Th) and macrophages and participate in the upregulation of inflammatory responses. Examples of proinflammatory cytokines include, but are not limited to, IL-1β, IL-6, G-CSF, GM-CSF, TNF-α. Chemokines include, but are not limited to, IL-8, GRO-α, and MCP-1. In one embodiment, the second antigen binding moiety specifically binds IL-1β, IL-6, IL-13, TNF-α or BAFF. In one embodiment, the bispecific antibody molecule has a first binding specificity for IL-23p19 and a second binding specificity for TNF (eg, TNFα). In one embodiment, the bispecific antibody molecule binds IL-23p19 and TNF. Multispecific antibody molecules can have any combination of binding specificities for the aforementioned molecules.

As defined herein, the term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, antibody fragments are antigen-binding fragments. "Antigen-binding fragment" refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; dAb (domain antibody); linear antibodies; single chain antibodies (e.g. scFv); ; a diabody or a fragment thereof; or a camelid antibody.

As defined herein, an "antibody that binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% of The binding of said reference antibody to its antigen above, conversely, the reference antibody blocks more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen in a competition assay.

As defined herein, an antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks more than 50%, 60%, 70%, 80%, 90%, or 95% of said antibody in a competition assay. Reference is made to the binding of an antibody to its antigen. Conversely, a reference antibody blocks greater than 50%, 60%, 70%, 80%, 90%, or 95% of the binding of that antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Assays, biophotometric interferometry (eg Fortebio) or surface plasmon resonance (Biacore), etc.

As defined herein, an antibody that inhibits (e.g. competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits more than 50%, 60%, 70%, 80%, 90% or 95% of all Binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits more than 50%, 60%, 70%, 80%, 90%, or 95% of the binding of that antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (eg, the equilibrium dissociation constant). Methods for determining affinity are known in the art.

An antibody exhibiting the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or more than 95% of the reference antibody affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.

As defined herein, a "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or or regions containing antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and based on affinity propagation clustering using a large number of crystal structures North CDR definition.

For example, according to different CDR determination schemes, the residues of each CDR are as follows.

A CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways.

Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it is meant according to the AbM numbering system number position.

In one embodiment, the heavy chain variable region CDRs of the antibodies of the present invention are determined according to AbM rules.

In one embodiment, the light chain variable region CDRs of the antibodies of the present invention are determined according to AbM rules.

It should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. Thus, where reference is made to defining antibodies with a particular CDR sequence as defined in the present invention, the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the methods of Kabat, Chothia, AbM, Contact and North, the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. A minimal binding unit may be a subsection of a CDR. As will be apparent to those skilled in the art, the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.

The term "Fc region" is used herein to define the CH2 and CH3 constant regions of an immunoglobulin heavy chain and the term includes native sequence Fc regions and variant Fc regions.

"An antibody in IgG form" refers to the IgG form to which the heavy chain constant region of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in IgG4 form means that its heavy chain constant region is from IgG4, or an antibody in IgGl form means that its heavy chain constant region is from IgGl.

"Human antibody" or "fully human antibody" or "fully human antibody" are used interchangeably and refer to an antibody having an amino acid sequence corresponding to that of an antibody derived from a human Or human cells are produced or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

As used herein, the term "bind" or "specifically bind" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry (Biacore). Or MSD assay or surface plasmon resonance (SPR) assay.

III. Nucleic acids of the invention and host cells comprising them

In one aspect, the invention provides a nucleic acid encoding any of the above anti-IL-23p19 antibodies or fragments thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

In one aspect, the invention provides nucleic acid encoding any of the anti-IL-23p19 antibodies or fragments thereof described herein. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain variable region and/or a heavy chain variable region of an antibody, or a nucleic acid comprising an amino acid sequence encoding a light chain and/or a heavy chain of an antibody.

For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 12, 13, 16, 17, 20, 21, 23, 24, 27 or 28, or encoding and being selected from SEQ ID NO: The amino acid sequence shown in any one of ID NO: 12, 13, 16, 17, 20, 21, 23, 24, 27 or 28 has at least 85%, 90%, 91%, 92%, 93%, 94% A nucleic acid having an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical.

In one embodiment, one or more vectors comprising said nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In one embodiment, the vector is pcDNA3.3.

In one embodiment, a host cell comprising said vector is provided. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293F or 293 cells, as for example Graham et al., J. Gen Virol. 1977) and others. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980), CHO-S cells, etc.; and bone marrow Tumor cell lines such as Y0, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. ), pp. 255-268 (2003).

IV. Production and Purification of Antibody Molecules of the Invention

In one embodiment, the present invention provides a method for preparing an anti-IL-23p19 antibody or fragment thereof (preferably an antigen-binding fragment), wherein said method comprises a method suitable for expressing said antibody or fragment thereof (preferably an antigen-binding fragment). ), and optionally isolating the antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-IL-23p19 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, there is provided a method of making an anti-IL-23p19 antibody, wherein the method comprises, under conditions suitable for expression of the antibody, culturing a host cell comprising a nucleic acid encoding said antibody, as provided above, and The antibody is optionally recovered from the host cell (or host cell culture medium). For the recombinant production of anti-IL-23p19 antibodies, nucleic acid encoding the antibodies, such as those described above, is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the antibody heavy and light chains).

V. Assay

Anti-IL-23p19 antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibodies of the present invention are tested for their antigen-binding activity, for example, by known methods such as ELISA, Western blotting, and the like. Binding to IL-23p19 can be assayed using methods known in the art, exemplary methods are disclosed herein. In some embodiments, a biophotometric interferometry (eg, Fortebio affinity measurement) or MSD assay is used.

In another aspect, competition assays can be used to identify antibodies that compete with any of the anti-IL-23p19 antibodies disclosed herein for binding to IL-23p19. In certain embodiments, such competing antibodies bind the same or overlapping epitopes (eg, linear or conformational epitopes) as any of the anti-IL-23p19 antibodies disclosed herein.

The invention also provides assays for identifying biologically active anti-IL-23p19 antibodies. Biological activities may include, for example, binding to IL-23p19 (eg, binding to human and/or cynomolgus and/or mouse IL-23p19), inhibiting the induction of IL-17 secretion by IL-23, blocking IL-23 signaling pathways (eg, Inhibition of IL-23 activates cellular STAT3 phosphorylation), etc. Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activities.

Cells for use in any of the above in vitro assays include cell lines that either naturally express IL-23p19 or have been engineered to express IL-23p19. Such cells also include cell lines transfected with DNA encoding IL-23p19 that express IL-23p19 and that do not normally express IL-23p19.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-IL-23p19 antibodies.

It will be appreciated that any of the above assays can be performed using anti-IL-23p19 antibodies and additional active agents.

VI. Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising any of the anti-IL-23p19 antibodies provided herein and other substances, such as therapeutic agents, including cytokines, other antibodies, small molecule drugs, or immunomodulators ( such as anti-inflammatory agents or immunosuppressants).

In some embodiments, the immunoconjugate is used to prevent or treat an IL-23-associated disease, such as a disease of the immune system (eg, autoimmune disease or inflammation).

VII. Pharmaceutical Compositions and Pharmaceutical Formulations

In some embodiments, the present invention provides a composition, preferably a pharmaceutical composition, comprising any of the anti-IL-23p19 antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, a composition, eg, a pharmaceutical composition, comprises an anti-IL-23p19 antibody or fragment thereof or immunoconjugate thereof of the invention in combination with one or more other therapeutic agents.

The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-IL-23p19 antibodies or immunoconjugates thereof, or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding anti-IL-23p19 antibodies pharmaceutical preparations). In certain embodiments, the composition comprises one or more antibodies or fragments thereof that bind IL-23p19, or one or more polynucleotides encoding one or more antibodies or fragments thereof against IL-23p19 . These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

The compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

Pharmaceutical formulations comprising an antibody described herein can be prepared by mixing an antibody of the invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

The pharmaceutical compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators, among others. The active ingredients are suitably present in combination in amounts effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.

VIII. Drug Combinations and Kits

In some embodiments, the present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising the anti-IL-23p19 antibody of the present invention or a fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, and one or Various other therapeutic agents (eg, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, etc.).

Another object of the present invention is to provide a kit of parts comprising the pharmaceutical combination of the present invention, preferably said kit is in the form of pharmaceutical dosage units. Dosage units may thus be presented according to a dosing regimen or interval between drug administrations.

In one embodiment, the kit of parts of the invention comprises in the same package:

- a first container containing a pharmaceutical composition comprising an anti-IL-23p19 antibody or fragment thereof;

- a second container containing a pharmaceutical composition comprising an additional therapeutic agent.

In some embodiments, the combination is used to prevent or treat an IL-23-associated disease, such as a disease of the immune system (e.g., an autoimmune disease or inflammation).

IX. USES AND METHODS

One aspect of the present invention provides a method for treating IL-23-related diseases in a subject, comprising administering to the subject an effective amount of the anti-IL-23p19 antibody of the present invention or an antigen-binding fragment thereof, an immunoconjugate, a drug Composition or combination product.

In some embodiments, the IL-23-associated diseases described herein include diseases of the immune system, such as autoimmune diseases and inflammatory diseases, such as Crohn's disease, moderately to severely active ulcerative colitis, psoriatic arthritis , palmoplantar pustulosis and psoriasis.

In one embodiment, the immune system disease of the invention is a disease with elevated nucleic acid/protein levels of IL-23 (eg IL-23p19).

In one embodiment, the disorder of the immune system is a disorder expressing elevated levels of IL-23 (eg, elevated expression levels of IL-23p19).

In other aspects, the present invention provides the use of an anti-IL-23p19 antibody or a fragment thereof in the manufacture or preparation of a medicament for the treatment of the related diseases or conditions mentioned herein.

In some embodiments, an antibody or antibody fragment or immunoconjugate or composition or product of the invention delays the onset of a disorder and/or symptoms associated with a disorder.

In some embodiments, the methods of prevention or treatment described herein further comprise administering to said subject or individual in combination an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein, and one or more other therapies, For example treatment modalities and/or other therapeutic agents.

In some embodiments, treatment modalities include surgery, radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ), or focused irradiation), etc.

In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators. Exemplary immunomodulators include immunosuppressants or anti-inflammatory agents.

As used herein, "chemotherapeutic agents" include chemical compounds useful in the treatment of diseases of the immune system.

As used herein, the term "small molecule drug" refers to a low molecular weight organic compound capable of modulating biological processes. "Small molecules" are defined as molecules having a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants.

An "immunosuppressant" as used herein is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

In some embodiments, the antibody combinations described herein can be administered separately, eg, as separate antibodies, or linked (eg, as a bispecific or trispecific antibody molecule).

Such combination therapy encompasses combined administration (e.g., two or more therapeutic agents contained in the same formulation or in separate formulations), and separate administration, in which case the additional therapeutic and/or pharmaceutical agents may be administered Administration of an antibody of the invention occurs before, simultaneously with, and/or after.

IX. Methods and Compositions for Diagnosis and Detection

In certain embodiments, any of the anti-IL-23p19 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of IL-23, particularly IL-23p19, in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, a biological sample comprises cells or tissues. In some embodiments, the biological sample is from a lesion associated with an immune system disorder (eg, autoimmune disease or inflammation).

In one embodiment, an anti-IL-23p19 antibody for use in a method of diagnosis or detection is provided. In another aspect, methods of detecting the presence of IL-23p19 in a biological sample are provided. In certain embodiments, the methods comprise detecting the presence of IL-23p19 protein in a biological sample. In certain embodiments, the IL-23p19 is human IL-23p19 or cynomolgus or murine (eg, mouse) IL-23p19. In certain embodiments, the method comprises contacting a biological sample with an anti-IL-23p19 antibody as described herein under conditions that allow the binding of the anti-IL-23p19 antibody to IL-23p19, and detecting the presence of anti-IL-23p19 antibody Whether a complex is formed between IL-23p19 and IL-23p19. Complex formation indicates the presence of IL-23p19. The method can be an in vitro or in vivo method. In one embodiment, an anti-IL-23p19 antibody is used to select a subject suitable for treatment with an anti-IL-23p19 antibody, eg, wherein IL-23p19 is the biomarker used to select said subject.

These and other aspects and embodiments of the invention are described in the figures (a brief description of the figures immediately following) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are presented by way of illustration and not limitation and that various modifications may be made by those skilled in the art.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Embodiment 1 raw material preparation

In this example, the following seven recombinant proteins were prepared or purchased: human IL-23-His (human IL-23 sequence: UniProt accession number #Q9NPF7-1: Arg20-Pro189 (IL-23A) and UniProt accession number #P29460 -1: Ile23-Ser 328 (IL-23B)), human IL-23-His-biotin, human IL-23-Fc, monkey IL-23 (purchased from AcroBio, ILB-CM52W8), mouse IL-23 ( Novoprotein, CI18), human IL-12-His (UniProt accession #P29460-1: Ile 23-Ser 328 (IL-12B) and UniProt accession #P29459-1: Arg 23-Ser 219 (IL-12A)) and human IL-23R (UniProt accession #Q5VWK5-1: Gly 24-Gly 355).

In this embodiment, the following control antibodies were prepared: Guselkumab (Janssen Biotech, US7491391, the encoded amino acid sequence is shown in SEQ ID NO: 33, 34), IL-12 antibody (Ustekinumab, purchased from self-improvement).

1.1 Plasmid construction

Each human IL-23 sequence, human IL-23R sequence, human IL-12 sequence and the light/heavy chain sequence of Guselkumab were used for gene synthesis of the target fragment, PCR amplification was performed after obtaining the target fragment, and then homologous recombination The method is constructed to the eukaryotic expression vector pcDNA3.4, wherein human IL-23-Fc is the C-terminal of the synthetic human IL-23 gene fragment with an Fc tag (the encoded amino acid sequence is shown in SEQ ID NO: 35 ), human IL-23-His and human IL-12-His are His tags on the C-terminus of the synthetic human IL-23 gene or human IL-12 gene fragment.

1.2 Plasmid preparation

The constructed expression vectors were respectively transformed into Escherichia coli SS320, cultured overnight at 37°C, and plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic express use.

1.3 Expression and purification of recombinant protein

Human IL-23-His, human IL-23R-His, human IL-23-Fc and human IL-12-His were expressed through the Expi-HEK293 transient expression system (ThermoFisher, A14635), and the specific method was as follows: On the day of transfection, confirm that the cell density is about 4.5×10 ⁶ to 5.5×10 ⁶ viable cells per ml, and the cell viability is greater than 95%. At this time, use fresh Expi293 expression medium preheated at 37°C to adjust the cells to a final concentration of For 3×10 ⁶ cells per milliliter, dilute the target plasmid with 4°C pre-cooled Opti-MEM ^TM (the ratio of the amount of plasmid to the expression volume is 1 μg/mL), and dilute ExpiFectamine ^TM 293 reagent with Opti-MEM ^TM at the same time, and then the two Mix them and gently pipette and mix to prepare ExpiFectamine ^TM 293 reagent/plasmid DNA mixture, incubate at room temperature for 10-20 minutes, slowly add to the prepared cell suspension, shake gently at the same time, and finally place in cell culture shaker bed at 37°C, 8% CO ₂ .

Add ExpiFectamine ^TM 293 Transfection Enhancer 1 and 2 within 18-22 hours after transfection, place the shaker flask in a shaker at 32°C and 5% CO ₂ to continue culturing, and 5-7 days after transfection, the cell supernatant 15000g high-speed centrifugation for 10 minutes, the Fc-labeled antigen expression supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100mM sodium acetate (pH 3.0), and then neutralized with 1M Tris-HCl, The finally eluted proteins were replaced by ultrafiltration concentrator tubes (Millipore, UFC901096) into PBS (pH 7.4) buffer; His-tagged antigen expression supernatants were collected using nickel column Ni Smart Beads 6FF (Changzhou Tiandirenhe Biotechnology Co., Ltd., SA036050) was purified and eluted with imidazole solution, and the finally eluted proteins were replaced with PBS (pH 7.4) buffer through ultrafiltration concentrator tubes (Millipore, UFC901096). After being identified by SDS-PAGE and activity identification, it was put into storage and frozen.

1.4 Human IL-23 Antigen Biotinylation

The human IL-23-His prepared in Example 1.3 was mixed with activated biotin (Thermofisher, 21335) at a molar ratio of 1:20, and the reaction tube was placed in ice and reacted for 2 hours to complete biotin coupling. At 4° C. and 5000 g, the labeled protein was replaced into PBS buffer using an ultrafiltration centrifuge tube (PALL, OD010C35) to remove unconjugated biotin. After being identified by SDS-PAGE and activity identification, it was put into storage and frozen.

1.5 Control antibody expression and purification

The control antibody Guselkumab was expressed using the Expi-CHO transient expression system, and the main materials used included: Gibco medium (Cat. No.: A29100-01), Gibco transfection kit (Cat. No.: A29129).

The plasmid containing the light chain of the Guselkumab antibody and the plasmid of the heavy chain were mixed according to the molar ratio of 2:1. In the 25mL expression system, the above plasmid mixture (5μg) was mixed with the transfection reagent according to the standard procedure and added dropwise to 25mL of Expi-CHO cell expression system. After mixing well, express in a cell culture incubator at 37°C for 18-22 hours. Subsequently, feed medium was added to the above-mentioned transfection mixture and placed in a 32°C cell culture incubator to continue culturing. On the 5th day after transfection, the second feed was added, and the cells were placed in a 32°C cell incubator to continue culturing for 10-12 days.

The expressed cell suspension was subjected to high-speed centrifugation and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 μm filter membrane, and purified by Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100mM glycine salt (pH 3.0), concentrated, replaced with buffer solution (pH 7.4), aliquoted, identified by SDS-PAGE, SEC, and activity, and stored in the warehouse after passing the identification. For the biotinylation method of the control antibody Guselkumab, refer to Example 1.4 to prepare Guselkumab-biotin.

Example 2 Antibody library sea selection

In this example, human IL-23-His was used as the positive screening antigen, and human IL-12-His was used as the negative screening antigen. Multiple antibody molecules specifically binding to IL-23p19 subunit were obtained in the library.

2.1 Construction of phage display antibody gene library

For the construction of phage display antibody gene library, refer to patent CN202010236256.8.

2.2 Screening of antibody gene phage display library

2.2.1 Magnetic bead method to screen antibody gene phage display library

Incubate with streptavidin-coupled magnetic beads with human IL-23His-biotin (human-IL-23-His-biotin prepared in Example 1) at a concentration of 100 nM, so that IL-23-His-biotin Binding to magnetic beads. The magnetic beads bound to human IL-23-His-biotin and the constructed phage library were incubated at room temperature for 2 hours. After washing with PBST for 6-8 times, non-specifically adsorbed phages were removed, and trypsin (Gibco, 25200072) was added to gently mix and react for 20 minutes to elute specifically bound antibody-displayed phages. The eluted phages infect logarithmic SS320 cells (Lucigen, MC1061 F) and let stand for 30 minutes, then culture at 220rpm for 1 hour, then add VSCM13 helper phage and let stand for 30 minutes, continue at 220rpm Cultivate under conditions for 1 hour, centrifuge and replace into C+/K+2-YT medium, and the finally obtained phages will continue to be used for the next round of sea selection. From the second round of sea selection, human IL-12-His was added as a negative screen to remove phages that specifically bind to the p40 subunit. The concentration of human IL-23-His-biotin used in the second round, the third round and the fourth round decreased successively to 30nM, 10nM and 1nM respectively. 12, 16 and 20 times.

The phage library eluted in each round was detected by ELISA to evaluate the effect of enrichment, and 10 clones were randomly selected from the library screened in each round for sequence analysis to evaluate the proportion of unique sequences.

2.2.2 Immunotube screening of phage display antibody gene phage library

The specific implementation method is as follows:

In the first round of screening, 1 mL of human IL-23-His (prepared in Example 1) with a concentration of 100 μg/mL was added to the immunotube, coated overnight at 4°C, and the coating solution was discarded the next day, and 5% degreased protein was added. Milk was blocked for 2 hours, rinsed twice with PBS, then added a total of 10 ¹⁴ phages from the fully human antibody library, incubated for 2 hours, washed 8 times and 2 times with PBS and PBST successively to remove non-specifically bound phages, and then Add 0.8 mL of 0.05% EDTA trypsin digestion solution to the immune tube to elute the phages that specifically bind to the target antigen, and then infect the SS320 cells grown to the logarithmic phase (Lucigen, 60512-1 ), stand at 37°C for 30 minutes, then culture at 220rpm for 1 hour, then add VSCM13 helper phage, let stand for 30 minutes, continue to culture at 220rpm for 1 hour, centrifuge and replace with C+/K+2-YT medium and continue culturing overnight at 30°C and 220rpm. From the second round of sea selection, human IL-12-His was added as a negative screen to remove phages that specifically bind to the p40 subunit. The coating concentration of human IL-23-His used in the second, third and fourth rounds decreased successively, respectively 30 μg/mL, 10 μg/mL and 3 μg/mL, and the washing intensity of PBS was also gradually increased. The number of elutions was 12, 16 and 20 times.

2.3 Selection of monoclonal

The phages eluted in the third and fourth rounds were infected with Escherichia coli and plated, single clones were selected, ELISA binding evaluation, sequencing and sequence analysis, and finally the full-length sequence of an optimal clone was constructed.

Example 3 Construction, expression and purification of full-length antibody

In this example, one Fab with affinity activity obtained in Example 2 was constructed as a human IgG1 type, the light chain was Lambda, and the antibody type was a fully human antibody.

Plasmid construction: From the screened Fab antibody-containing strains, PCR amplification was used to obtain antibody light and heavy chain variable region fragments (see the sequence listing for the sequence), and were constructed to contain light and heavy chain constant region fragments by homologous recombination method On the eukaryotic expression vector plasmid (pcDNA3.0) of (SEQ ID NO: 32 and SEQ ID NO: 31), the expression plasmids respectively containing the complete antibody light and heavy chain full-length genes are formed.

Plasmid preparation: Transform the constructed expression plasmids containing the full-length genes of the antibody light and heavy chains into Escherichia coli SS320, culture overnight at 37°C, and extract the plasmids using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) , to obtain endotoxin-free antibody light chain plasmids and heavy chain plasmids for eukaryotic expression.

Expression and purification of the antibody: through the Expi-CHO transient expression system (Thermo Fisher, A29133), use the obtained plasmid for expression, the specific method is as shown in Example 1.5, the prepared antibody containing the complete antibody light chain heavy chain constant region is numbered for A44.

Example 4 Affinity blocking ability of antibody

In this example, the affinity activity of antibody A44 to the recombinant protein human IL-23 and the ability to block the binding of human IL-23 and human IL-23R were tested.

4.1 Detection of antibody affinity based on ELISA

On a 96-well ELISA plate, coated with anti-human IgG (Fab specific) goat antibody (Sigma, I5260-1ML), 2 μg/mL, incubated overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 2% BSA (Sanko, A600332-0100) was added to block for 2 hours. Subsequently, after the orifice plate was washed 3 times with PBST, the antibody A44 prepared above and the control antibody Guselkumab were added in a gradient dilution (the initial concentration of the gradient dilution was 10 μg/mL, the dilution factor was 3 times, and the gradient dilution was 8 times), Incubate for 1 hour. Subsequently, the well plate was washed 3 times with PBST, human IL-23-His-biotin prepared as above in Example 1 was added at 2 μg/mL, and incubated for 1 hour. Subsequently, avidin-HRP (Thermo Fisher, 434423) diluted 1:5000 was added and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The value at OD450 was read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 1. The results showed that the binding ability of antibody A44 to human IL-23 was slightly weaker than Guselkumab.

4.2 Detection of blocking ability of antibodies based on ELISA

In this example, the blocking system parameters with both sensitivity and signal-to-noise ratio were used to detect the ability of A44 to block the binding of human IL-23 and human IL-23R, as follows:

On a 96-well ELISA plate, coat the human IL-23R protein prepared above at 8 μg/mL and incubate overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 2% BSA (Sanko, A600332-0100) was added to block for 2 hours. Subsequently, the orifice plate was washed 3 times with PBST, and the antigen IL23-biotin (prepared in Example 1.4) was premixed with antibody A44 or Guselkumab in equal volume before adding to the orifice plate, wherein the final concentration of the antigen was 0.5 μg/mL, and the antibody A44 or Guselkumab was serially diluted (initial concentration was 10 μg/mL, 3-fold serial dilution, diluted 8 times), and then the mixture of the above antigen and antibody was added to the PBST-washed well plate and incubated for 1 hour. Subsequently, the well plate was washed 3 times with PBST, and avidin-HRP (Thermo Fisher, 434423) diluted 1:5000 was added and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The value at OD450 was read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 2. The results show that antibody A44 blocks the ability of human IL-23 to bind to human IL-23R Slightly weaker than Guselkumab.

Example 5 Antibody Engineering

In order to improve the ability of the antibody to inhibit the phosphorylation of STAT3 in cells, antibody engineering was carried out on antibody A44 in this example.

In this example, the parent molecule A44 was modified for affinity maturation to improve affinity and biological activity. The affinity maturation transformation process is as follows: transform the CDR defined by AbM. The affinity maturation transformation is based on the M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon is composed of NNK) to introduce mutations in the CDR region, and co-construct 4 phage display libraries, library 1 and library 2 are single point combination mutation, library 1 is CDRL1+CDRL3+CDRH3 combination mutation, library 2 is CDRL2+CDRH1+CDRH2 combination mutation; library 3 and library 4 are double point saturation mutation, Library 3 is a double point saturation mutation of CDRL3 and library 4 is a double point saturation mutation of CDRH3. The storage capacity of the library is shown in Table 1.

Using the parental molecule as a template to obtain a single mutation fragment in the CDR region by PCR, and then by overlapping PCR to obtain the Fab full-length antibody (VL-CL-linker-VH-CH1), and then by double enzyme digestion (HindⅢ and NotⅠ) and double The point mutation antibody was connected to the phage display vector by sticky end ligation, and finally the antibody sequence with the mutation site was transferred into Escherichia coli (SS320) by electroporation.

After the four constructed libraries were packaged into phages, they were panned and screened in liquid phase: the phages displaying antibody Fab bound to the biotinylated target antigen, and the magnetic beads adsorbed the phage by capturing the biotinylated target antigen , by reducing the pressure of the amount of antigen input into biotinylation to select antibodies with higher affinity, after panning, elution, and infecting Escherichia coli (SS320) for the next round of panning; solid phase panning: coated with The antigen on the immunotube binds to the phage displaying antibody Fab, and the antibody with high affinity is panned by reducing the amount of coated antigen under pressure, after panning, elution, and infecting Escherichia coli (SS320) for the next cycle of panning; screening method Referring to the screening part in Example 2.2, after 2-3 rounds of panning, single clones were selected for affinity ELISA detection, and clones with stronger affinity were screened to construct full-length IgG for expression in mammalian cells. So far, one round of affinity maturation process has been completed.

For the sequences of the modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L obtained after repeating the above-mentioned affinity maturation process for 2-3 rounds, refer to the sequence listing.

Table 1 Library design and storage capacity table

文库名称library name	文库设计library design	突变设计mutation design	库容大小 Storage capacity

库1Library 1	CDRL1+CDRL3+CDRH3CDRL1+CDRL3+CDRH3	单点组合突变single point combinatorial mutation	1E+81E+8
库2Library 2	CDRL2+CDRH1+CDRH2CDRL2+CDRH1+CDRH2	单点组合突变single point combinatorial mutation	1E+81E+8
库3Library 3	CDRL3CDRL3	双点饱和突变double point saturation mutation	1E+31E+3
库4Library 4	CDRH3CDRH3	双点饱和突变double point saturation mutation	1E+31E+3

Example 6 Affinity blocking ability of modified antibody

In this example, the affinity activity of the modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L to the recombinant protein human IL-23 and the ability to block the binding of human IL-23 and human IL-23R were tested.

6.1 Detection of the affinity of the modified antibody based on ELISA

Coat human IL-23-His (prepared in Example 1) on a 96-well ELISA plate at 2 μg/mL and incubate overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 5% skimmed milk was added to block for 2 hours. Subsequently, after the orifice plate was washed 3 times with PBST, the modified antibody S5-5HWTL, S5-15HWTL, S5-4H6L or control antibody Guselkumab (initial concentration 1 μg/mL, from initial concentration Initially, the 2nd and 8th concentration points are 9-fold dilutions on the basis of the previous concentration, and the other concentration points are 3-fold dilutions of the previous concentration point), and incubated for 1 hour. Subsequently, the well plate was washed 3 times with PBST, goat anti-human λ chain antibody-HRP (Abcam, ab200966) diluted 1:5000 was added, and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The value at OD450 was read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Fig. 3A-Fig. 3B, and the results showed that the binding ability of engineered antibodies S5-5HWTL, S5-15HWTL, S5-4H6L and human IL-23 was better than Guselkumab.

6.2 Detection of blocking ability of antibodies based on ELISA

For the detection method, see Example 4.2, and the results are shown in Figure 4A-Figure 4B. The results show that the ability of the modified antibodies S5-5HWTL, S5-15HWTL, and S5-4H6L to block the binding of human IL-23 and human IL-23R is slightly weaker for Guselkumab.

Example 7 Combination of modified antibody and human IL12-p40 subunit

In this example, the ability of the modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L to bind to the p40 subunit was tested.

Coat human IL-12-His (prepared in Example 1) on a 96-well ELISA plate at 2 μg/mL and incubate overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 5% skimmed milk (Erie) was added to block for 2 hours. Subsequently, after the orifice plate was washed 3 times with PBST, a gradient dilution was added (initial concentration 3 μg/mL, starting from the initial concentration at the second concentration point, and the eighth concentration point was a 9-fold dilution on the basis of the previous concentration. , the other concentration points are 3-fold dilutions based on the previous concentration point), the modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L, the positive antibody of the control antibody Guselkumab and IL-12 anti-human IL-12 antibody Incubate with the same subtype control IgG1 for 1 hour. Subsequently, the well plate was washed 3 times with PBST, goat anti-human IgG-Fc-HRP (Abcam, ab97225) diluted 1:5000 was added, and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The values at OD450 were read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 5A-Figure 5C. 4H6L does not bind to the p40 subunit of human IL-12, that is, S5-5HWTL, S5-15HWTL and S5-4H6L specifically bind to the p19 subunit of human IL-23.

Example 8 Binding of modified antibody to monkey IL-23

In this example, the binding of engineered antibodies S5-5HWTL, S5-15HWTL and S5-4H6L to monkey IL-23 was tested.

On a 96-well ELISA plate, monkey IL-23-His (AcroBio, ILB-CM52W8) was coated at 2 μg/mL and incubated overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 5% skimmed milk was added to block for 2 hours. Subsequently, after the orifice plate was washed 3 times with PBST, a gradient dilution was added (initial concentration 3 μg/mL, the second concentration point, and the eighth concentration point were 9-fold dilutions based on the previous concentration point, and other concentration points were at The modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L, the control antibody Guselkumab and the same subtype control IgG1 were incubated for 1 hour. Subsequently, the well plate was washed 3 times with PBST, goat anti-human IgG-Fc-HRP (Abcam, ab97225) diluted 1:5000 was added, and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The values at OD450 were read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 6A-Figure 6C. The binding ability of 4H6L to monkey IL-23 is comparable to Guselkumab.

Example 9 Binding of modified antibody to mouse IL-23

In this example, the binding of engineered antibodies S5-5HWTL, S5-15HWTL and S5-4H6L to murine IL-23 was tested.

Coat mouse IL-23-His (Novoprotein, CI18) on a 96-well ELISA plate at 2 μg/mL and incubate overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 5% skimmed milk (Erie) was added to block for 2 hours. Subsequently, after the orifice plate was washed 3 times with PBST, a gradient dilution was added (initial concentration 3 μg/mL, the second concentration point, and the eighth concentration point were 9-fold dilutions based on the previous concentration point, and other concentration points were at The modified antibodies S5-5HWTL, S5-15HWTL and S5-4H6L, the control antibody Guselkumab and the same subtype control IgG1 were incubated for 1 hour. Subsequently, the well plate was washed 3 times with PBST, goat anti-human IgG-Fc-HRP (Abcam, ab97225) diluted 1:5000 was added, and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The value at OD450 was read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 7A-Figure 7C. 4H6L has a good binding ability to mouse IL-23, while Guselkumab basically does not bind to mouse IL-23.

Example 10 Antibody epitope analysis after modification

In this example, the difference in binding to human IL-23 epitopes between the engineered antibodies S5-5HWTL, S5-15HWTL and S5-4H6L and Guselkumab was detected.

Coat human IL-23-His (prepared in Example 1) on a 96-well ELISA plate at 2 μg/mL and incubate overnight at 4°C. The next day, the well plate was washed 3 times with PBST, and 5% skimmed milk was added to block for 2 hours. Subsequently, after the well plate was washed 3 times with PBST, the modified antibodies S5-5HWTL, S5-15HWTL, S5-4H6L, control antibody Guselkumab and the same isotype control IgG1 were incubated for 1 hour. Subsequently, Guselkumab-biotin (Guesekumab coupled with biotin (Thermofisher), see Example 1.4 for the preparation method) was added to each well and incubated for 1 hour. The well plate was washed 3 times with PBST, 1:5000 diluted NeutrAvidin-HRP (Thermo Fisher, 434423) was added and incubated for 1 hour. Subsequently, the well plate was washed 6 times with PBST, TMB (SurModics, TMBS-1000-01) was added and the color was developed in the dark for 5-10 minutes. According to the color development, 2M HCl was added to terminate the reaction. The value at OD450 was read by a microplate reader (Molecular Devices, SpecterMax 190) and fitted with four parameters. The results are shown in Figure 8A-Figure 8B. 4H6L binds to the same epitope of human IL-23 as Guselkumab, and the smaller IC50 value of the modified antibody may be related to its higher affinity.

Example 11 Modified antibody inhibits intracellular STAT3 phosphorylation

In this example, the effects of the transformed antibodies S5-5HWTL, S5-15HWTL, and S5-4H6L on inhibiting STAT3 phosphorylation in human IL-23-activated cells were tested.

Binding of IL-23 to its receptor stimulates TyK2 and JAK2 signaling pathways, activates phosphorylation of STAT3, and subsequently produces SEAP secreted alkaline phosphatase. Neutralization of IL-23 by antibodies can inhibit the activation of the STAT3 signaling pathway and thereby inhibit the production of secreted alkaline phosphatase. In this example, based on the above principles, the luciferase gene reporter system HEK-Blue ^TM IL-23 cells (InvivoGen, hkb-il23) were selected.

Collect HEK-Blue ^TM IL-23 cells in the exponential growth phase, digest and count the cells, centrifuge to remove the supernatant, resuspend in 10% FBS/DMEM medium and count, adjust the cell density to 5× with 10% FBS/DMEM 10 ⁵ cells/mL were added to a 96-well flat-bottomed cell culture plate at a cell volume of 100 μL/well. Use 10% FBS/DMEM to serially dilute the antibody or prepare the IL-23-His prepared in Example 1, and remodel the antibody after the serially diluted antibody ((initial concentration 1 μg/mL, 3-fold serial dilution, diluted 8 times)) S5-5HWTL, S5-15HWTL, S5-4H6L, the control antibody Guselkumab and the same subtype control IgG1 were premixed with human IL-23-His for 30 minutes, then added to a 96-well flat-bottomed cell culture plate, 100 μL per well, and incubated at 37°C The chamber was incubated for 20-24 hours (the final concentration of human IL-23-His was 8 ng/mL). Prepare QUANTI-Blue Solution in advance, wherein, QB reagent:QB buffer:sterile water=1:1:98, vortex and shake to mix, and incubate at room temperature for 10 minutes. Add the prepared QUANTI-Blue Solution to a 96-well flat-bottomed cell culture plate, 20 μL per well, and incubate in a 37°C incubator for 3-6 hours to detect OD630. The results are shown in Figure 9A-9B, and the results show that the engineered antibodies S5-5HWTL, S5-15HWTL, and S5-4H6L are far superior to Guselkumab in inhibiting the phosphorylation of STAT3 in cells activated by IL-23.

Example 12 Modified antibody inhibits mouse splenocytes from secreting IL-17

In this example, the ability of the transformed antibodies S5-5HWTL, S5-15HWTL, and S5-4H6L to inhibit IL-23 from activating mouse splenocytes to secrete IL-17 was tested.

IL-23 is an important cytokine that mediates the body's inflammatory response. Human IL-23 can promote the secretion of IL-17 in mouse splenocytes. In this example, IL-23 and IL-23 neutralizing antibodies (i.e. modified antibodies S5-5HWTL, S5-15HWTL, S5-4H6L) were added to mouse splenocytes, and IL-23 antibodies could neutralize IL -23 activity, inhibits IL-23-induced secretion of IL-17 in mouse splenocytes. The neutralizing activity of the IL-23 antibody can be detected by measuring the concentration of mouse IL-17A in the culture supernatant.

Extract the spleen of a mouse (C57/BL6 mouse, Shanghai Jiesijie), grind it, filter it with a 100 μm cell strainer, centrifuge to remove the supernatant, add red blood cell lysate for 5 minutes, centrifuge to remove the supernatant, and culture it with mouse spleen cells Wash the base again. Resuspend and count with mouse spleen cell culture medium, and adjust the cell density to 2.5× ¹⁰⁶ cells/mL with detection medium, wherein the components of detection medium are RPMI-1640 medium, 10% fetal bovine serum, 1% non- Essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 50 μM β-mercaptoethanol, 50 ng/mL rhIL-2. Use the detection medium to serially dilute the antibody (initial concentration 0.5 μg/mL, three-fold serial dilution, a total of 6 points) or IL-23-His, and the serially diluted modified antibodies S5-5HWTL, S5-15HWTL, S5- 4H6L, the control antibody Guselkumab and the same subtype control IgG1 were premixed with the human IL-23-His prepared in Example 1 for 30 minutes, and then added to a 96-well flat-bottomed cell culture plate, 100 μL per well (wherein, human IL-23-His The final concentration is 8ng/mL). Subsequently, the mouse splenocytes were added to a 96-well flat-bottomed plate, 100 μL per well, cultured at 37°C for 4 days, the cell supernatant was collected, and the ELISA pre-coated plate (R&D, DY421-05) was used according to the standard procedure (operated according to the kit instructions) Process) to quantify the content of mouse IL-17A. The results are shown in Fig. 11A-Fig. 11C, the results show that the modified antibody S5-5HWTL is at a dose concentration of 0.063 μg/mL, the modified antibody S5-15HWTL is at a dose concentration of 0.167 μg/mL, and the modified antibody S5-4H6L is at a dose concentration of 0.167 μg/mL. At a dose concentration of 0.056 μg/mL, the ability to inhibit IL-23 from activating mouse splenocytes to secrete IL-17 can be achieved better than that of Guselkumab.

Example 13 Antibody thermal stability detection after transformation

In this example, the thermal stability of the transformed antibodies S5-5HWTL, S5-15HWTL, and S5-4H6L was tested.

Prepare antibody solution, 0.25mg/mL, 19μL/well, set three parallel wells for each test product, and use PBS and IPI as reference. Then add 1 μL of SYPRO orange dye at a concentration of 100× to each well, ready to go on the machine. Use ABI 7500FAST RT-PCR instrument to test, the test type selects melting curve, adopts continuous mode, scans the temperature range from 25 to 95 ℃, the heating rate is 1%, 25 ℃ equilibrates for 5 minutes, collects data during the heating process, and reports the base Select ROX as the group, select None as the quenching group, and the reaction volume is 20 μL. The temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the denaturation temperature of the candidate antibody. The results are shown in Table 2. The results show that the Tm values of the modified antibodies S5-5HWTL and S5-15HWTL are close to 70°C, and the druggability is very good.

Table 2 Summary of Physicochemical Properties of Antibodies

the	DSF(℃)DSF(°C)
S5-5HWTLS5-5HWTL	69.969.9
S5-15HWTLS5-15HWTL	68.268.2

Antibody affinity detection after embodiment 14 transformation

In this example, the affinity of the modified antibodies S5-5HWTL, S5-15HWTL, S5-4H6L and the human IL-23-His prepared in Example 1 was detected by using Biacore. The results are shown in Table 3. The results show that the modified antibodies S5-5HWTL and S5-4H6L are better than Guselkumab in K _on , slightly worse than Guselkumab in K _off , and the overall K _D is comparable to Guselkumab.

Table 3 Summary table of antibody affinity kinetics

the	KD(M)KD(M)	Ka(1/MS)Ka(1/MS)	Kd(1/S)Kd(1/S)
GuselkumabGuselkumab	2.46E-112.46E-11	8.74E+048.74E+04	2.15E-062.15E-06
S5-5HWTLS5-5HWTL	6.25E-116.25E-11	1.11E+051.11E+05	6.97E-066.97E-06
S5-15HWTLS5-15HWTL	1.08E-101.08E-10	1.09E+051.09E+05	1.18E-051.18E-05
S5-4H6LS5-4H6L	5.89E-115.89E-11	1.02E+051.02E+05	6.03E-066.03E-06

Table 4: Sequence Listing

Claims

An anti-IL-23p19 antibody or antigen-binding fragment thereof comprising:

(i) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:12, and three complementarity-determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO:16 LCDR2 and LCDR3, or variants comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the 6 CDR regions;

(ii) three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:20, and three complementarity-determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO:16 LCDR2 and LCDR3, or variants comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the 6 CDR regions; or

(iii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 23, and three complementarity determining regions LCDR1, HCDR1, and HCDR1 contained in VL as shown in SEQ ID NO: 27 LCDR2 and LCDR3, or variants comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the 6 CDR regions.
An anti-IL-23p19 antibody or antigen-binding fragment thereof comprising:

(i) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 11, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 14, 7, and 15;

(ii) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 18, and 19, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 14, 7, and 15; or

(iii) HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 22, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 25, 7, and 26.
The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence selected from SEQ ID NO: 12, 20, 23 or an amino acid sequence that is 99% identical or consists of said amino acid sequence; or

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 12, 20, 23; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence selected from SEQ ID NO: 12, 20, 23 Amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) or consist of amino acid sequences, preferably, the amino acid changes do not occur in the CDR region; and/or

light chain variable region

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence selected from SEQ ID NO: 16, 27 % identity amino acid sequences or consist of said amino acid sequences; or

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 16, 27; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence selected from SEQ ID NO: 16, 27 (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of said amino acid sequence, preferably, said amino acid change does not occur in the CDR region.
The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

(i) VH comprising the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO: 16 Amino acid sequences at least 90% identical or VL consisting of said amino acid sequences;

(ii) VH comprising the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO: 16 Amino acid sequences at least 90% identical or VL consisting of said amino acid sequences; or

(iii) VH comprising the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90% identity therewith or consisting of said amino acid sequence, and comprising or having an amino acid sequence shown in SEQ ID NO:27 Amino acid sequences at least 90% identical or a VL consisting of said amino acid sequences.
The antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a heavy chain constant region and/or a light chain constant region.
The antibody or antigen-binding fragment thereof of claim 5, wherein

heavy chain constant region

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 31 A specific amino acid sequence or consists of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 31; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 31 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of said amino acid sequence; and/or

light chain constant region

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 32 A specific amino acid sequence or consists of said amino acid sequence;

(ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 32; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 32 The amino acid sequence that is altered (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.
The IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein said antibody is an antibody or antigen-binding fragment in IgG1 form or IgG2 form or IgG3 form or IgG4 form.
The IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein said antibody is in IgG1 format.
The IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.
The IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a human antibody or a chimeric antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g. scFv) , (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein said antibody or antigen-binding fragment thereof has one or more of the following properties:

(i) specifically binds to the p19 subunit of IL-23 and does not bind to the p40 subunit;

(ii) have excellent cross-binding properties with human, mouse and cynomolgus monkey IL-23;

(iii) the ability to block the binding of IL-23 to the receptor IL-23R is equivalent to or better than that of the control antibody Guselkumab;

(iv) The ability to inhibit the phosphorylation of STAT3 in cells activated by IL-23 is better than that of the control antibody Guselkumab;

(v) The ability of inhibiting IL-23 to activate mouse splenocytes to secrete IL-17 is better than that of the control antibody Guselkumab.
An isolated nucleic acid encoding the light chain variable region or the heavy chain variable region, or the light chain or the heavy chain, in the IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.
A vector comprising the nucleic acid of claim 13, preferably said vector is an expression vector.
A host cell comprising the nucleic acid of claim 13 or the vector of claim 14, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO-S cells or HEK293 cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
A method for preparing an IL-23p19-binding antibody or an antigen-binding fragment thereof, the method comprising under conditions suitable for expressing a nucleic acid encoding an IL-23p19-binding antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 Cultivating the host cell of claim 15, optionally isolating said antibody or antigen-binding fragment thereof, optionally said method further comprising recovering said IL-23p19-binding antibody or antigen-binding fragment thereof from said host cell.
An immunoconjugate comprising an IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and other substances, such as cytotoxic agents.
A pharmaceutical composition comprising the IL-23p19-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the immunoconjugate of claim 17, and optionally one or more other therapeutic agents, For example chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.
A pharmaceutical combination comprising an IL-23p19-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or an immunoconjugate according to claim 17, and one or more other therapeutic agents, such as chemotherapeutic agents, Cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.
Preventing or treating an immune system disorder (e.g., an autoimmune disease or inflammatory disease, such as Crohn's disease, moderately to severely active ulcerative colitis, psoriatic arthritis, palmoplantar pustulosis, and psoriasis) in a subject ) method comprising administering to said subject an effective amount of an IL-23p19-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 12, or an immunoconjugate of claim 17, Or the pharmaceutical composition of claim 18, or the pharmaceutical combination of claim 19.
The method of claim 20, wherein said method further comprises administering to the patient one or more therapies, such as a treatment modality and/or other therapeutic agents, preferably, the therapeutic modality includes surgery and/or radiation therapy, and the other therapeutic agent is selected from From chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
A method for detecting IL-23p19 in a sample, the method comprising

(a) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the immunoconjugate of claim 17; and

(b) detecting complex formation between the antibody or antigen-binding fragment thereof and IL-23p19; optionally, the antibody is detectably labeled.