WO2023165514A1

WO2023165514A1 - Antigen-binding molecule specifically binding to flt3 and cd3 and pharmaceutical use thereof

Info

Publication number: WO2023165514A1
Application number: PCT/CN2023/079002
Authority: WO
Inventors: 张玲; 朱亚丽; 金薪盛; 王姝; 杨莉; 应华; 陶维康
Original assignee: 江苏恒瑞医药股份有限公司; 上海恒瑞医药有限公司
Priority date: 2022-03-01
Filing date: 2023-03-01
Publication date: 2023-09-07
Also published as: TW202342549A

Abstract

Provided are an antigen-binding molecule specifically binding to FLT3 and CD3 and pharmaceutical use thereof. The antigen-binding molecule can be used for treating tumor-associated diseases.

Description

Antigen-binding molecule specifically binding to FLT3 and CD3 and its medical use

technical field

The present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to antigen-binding molecules and applications thereof.

Background technique

The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

FMS-like tyrosine kinase 3 (FLT3, also known as CD135, FLK2, STK1) is a receptor tyrosine kinase. Compared with healthy cells, it is overexpressed on most of the bulk cells of patients with acute myeloid leukemia (AML), and is highly expressed on the leukemic hematopoietic stem cells of most patients. Additionally, FLT3 is the most frequently mutated gene in AML patients, and mutations that result in constitutive activation of the receptor are associated with poor prognosis.

CD3 is an isotype or heterodimer antigen expressed on T cells. Functional CD3 is formed by dimer association of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ/ε, δ/ε, and ζ/ζ. CD3 binds to the T cell receptor complex (TCR) and is required for T cell activation. Therefore, anti-CD3 antibodies that activate T cells have been proposed for therapeutic purposes. However, administration of anti-CD3 antibodies may trigger T cell activation and associated cytokine release. Excessive cytokine release leads to severe cytokine release syndrome (CRS), which is an important challenge in the clinical application of anti-CD3 antibodies.

Therefore, there is an unmet need to provide FLT3/CD3 bispecific antibodies with high activity and low cytokine release.

Contents of the invention

The present disclosure provides an antigen-binding molecule that specifically binds FLT3 and CD3 and an antibody that specifically binds FLT3.

In one aspect, the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety that specifically binds FLT3 and at least one antigen-binding moiety that specifically binds CD3, the antigen-binding moiety that specifically binds FLT3 comprising a heavy Chain variable region (FLT3-VH) and light chain variable region (FLT3-VL), the antigen-binding module that specifically binds CD3 comprises heavy chain variable region (CD3-VH) and light chain variable region (CD3 -VL).

In some embodiments, the antigen-binding molecule comprises one or two antigen-binding moieties that specifically bind FLT3 and one antigen-binding moiety that specifically binds CD3, and the antigen-binding moiety that specifically binds FLT3 comprises a heavy chain Variable region (FLT3-VH) and light chain variable region (FLT3-VL), the antigen-binding module that specifically binds CD3 comprises heavy chain variable region (CD3-VH) and light chain variable region (CD3-VL ).

In some embodiments, the antigen-binding molecule comprises an antigen-binding moiety that specifically binds FLT3 and an antigen-binding moiety that specifically binds CD3, and the antigen-binding moiety that specifically binds FLT3 comprises a heavy chain variable region ( FLT3-VH) and a light chain variable region (FLT3-VL), the antigen-binding module specifically binding to CD3 comprises a heavy chain variable region (CD3-VH) and a light chain variable region (CD3-VL).

In some embodiments, the antigen binding molecule as described above, wherein

(i) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83 or 22, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or 23 and comprising the amino acid of SEQ ID NO: 24 sequence FLT3-LCDR3, or

(ii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or .

(iii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 76 or 77 and comprising the amino acid of SEQ ID NO: 9 sequence of FLT3-HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, 78, 79 or 80, FLT3- comprising the amino acid sequence of SEQ ID NO: 11, 81 or 82 LCDR2 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antigen-binding molecule as described above, wherein the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and FLT3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO:24.

In some embodiments, for the antigen-binding molecule according to any one of the preceding items, the FLT3-HCDR1, FLT3-HCDR2, FLT3-HCDR3, FLT3-LCDR1, FLT3-LCDR2 and FLT3-LCDR3 are defined according to the Kabat numbering convention.

In a specific embodiment, the antigen-binding molecule described herein that specifically binds FLT3 and CD3 comprises at least one antigen-binding moiety that specifically binds FLT3 and at least one antigen-binding moiety that specifically binds CD3, and that specifically binds FLT3 The antigen binding module of the heavy chain variable region comprises the heavy chain variable region FLT3-VH and the light chain variable region FLT3-VL, and the antigen binding module specifically binding to CD3 comprises the heavy chain variable region CD3-VH and the light chain variable region CD3-VH VL; where

(i) the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 19, FLT3-HCDR2 shown in SEQ ID NO: 20 and FLT3-HCDR3 shown in SEQ ID NO: 21, and the FLT3- The VL comprises FLT3-LCDR1 set forth in SEQ ID NO: 83 or 22, FLT3-LCDR2 set forth in SEQ ID NO: 84 or 23, and FLT3-LCDR3 set forth in SEQ ID NO: 24, or

(ii) the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 13, FLT3-HCDR2 shown in SEQ ID NO: 14 and FLT3-HCDR3 shown in SEQ ID NO: 15, and the FLT3- VL comprising FLT3-LCDR1 set forth in SEQ ID NO: 16, FLT3-LCDR2 set forth in SEQ ID NO: 17, and FLT3-LCDR3 set forth in SEQ ID NO: 18, or

(iii) said FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 7, FLT3-HCDR2 shown in SEQ ID NO: 8, 76 or 77, and FLT3-HCDR3 shown in SEQ ID NO: 9, and The FLT3-VL comprises FLT3-LCDR1 shown in SEQ ID NO: 10, 78, 79 or 80, FLT3-LCDR2 shown in SEQ ID NO: 11, 81 or 82, and FLT3-LCDR2 shown in SEQ ID NO: 12 LCDR3.

In some embodiments, the antigen-binding molecule according to any one of the preceding, which binds to human FLT3 with a KD of less than 1×10 ^-8 M, 5×10 ^-9 M at 25°C, the KD is obtained through the surface measured by plasmon resonance.

In some embodiments, the antigen binding molecule of any one of the preceding, wherein:

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 5, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 3, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 4, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 2, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 amino acid sequence.

In some embodiments, the antigen binding molecule of any one of the preceding, wherein

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 36, 37, 38, 39, 40 , 41, 42, 43 or 44 amino acid sequence.

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 52, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 55, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 54, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 5, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 6; or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 49, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 46, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 47, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 48, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 51, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 3, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 4; or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 36, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 37, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 30, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 31, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 32, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 33, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 38, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 34, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 39, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 40, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 41, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 42, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 43, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 35, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 44, or

The FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, and the FLT3-VL comprises the amino acid sequence of SEQ ID NO: 2.

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50.

In a specific embodiment, an antigen binding molecule described herein comprises at least one antigen binding moiety that specifically binds FLT3 and at least one antigen binding moiety that specifically binds CD3, wherein:

(i) the antigen-binding moiety specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, 5, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56, 6 or 55, or

(ii) the antigen-binding module specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 45, 3, 46, 47 or 48, and FLT3 shown in SEQ ID NO: 50, 4, 49 or 51 -VL, or

(iii) the antigen-binding module specifically binding to FLT3 comprises the FLT3-VH shown in SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 2, 36, 37 , 38, 39, 40, 41, 42, 43 or 44 FLT3-VL;

specifically,

(i) the antigen-binding moiety that specifically binds FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56 or 55, or

(ii) the antigen-binding moiety specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 45, 46, 47 or 48, and FLT3-VL shown in SEQ ID NO: 50, 49 or 51, or

(iii) the antigen-binding module that specifically binds FLT3 comprises FLT3-VH shown in SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 36, 37, 38, 39 , 40, 41, 42, 43 or 44 FLT3-VL;

More specifically,

(i) the antigen-binding module specifically binding to FLT3 comprises FLT3-VH shown in SEQ ID NO: 53, and FLT3-VL shown in SEQ ID NO: 56, or

(ii) the antigen-binding module specifically binding to FLT3 comprises the FLT3-VH shown in SEQ ID NO: 45, and the FLT3-VL shown in SEQ ID NO: 50.

The antigen-binding module specifically binding to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 57, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 58, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 59; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 60 , CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:61, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:62.

In some embodiments, the CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:64.

The antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the CD3-VH comprises CD3-HCDR1 shown in SEQ ID NO: 57, SEQ ID NO : CD3-HCDR2 shown in 58, and CD3-HCDR3 shown in SEQ ID NO: 59; and said CD3-VL comprises CD3-LCDR1 shown in SEQ ID NO: 60, CD3 shown in SEQ ID NO: 61 -LCDR2, and CD3-LCDR3 shown in SEQ ID NO: 62;

specifically,

The antigen-binding module specifically binding to CD3 comprises CD3-VH shown in SEQ ID NO: 63, and CD3-VL shown in SEQ ID NO: 64.

In some embodiments, the antigen binding molecule according to any one of the preceding, wherein the antigen binding molecule comprises an Fc region (including an IgG Fc region or an IgG ₁ Fc region). In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors, compared to wild-type Fc regions. In some embodiments, the Fc region is a human IgG ₁ Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.

In some embodiments, the antigen-binding molecule according to any one of the preceding, wherein the antigen-binding molecule comprises an Fc region comprising a first subunit (Fc1) and a second subunit (Fc1) capable of associating with each other ( Fc2), each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the Fc1 and Fc2 each independently have one or more amino acid substitutions according to the pestle-and-hole technique. In some embodiments, the Fc1 has a convex structure according to the knob-and-hole technique, and the Fc2 has a pore structure according to the knob-and-hole technique. In some embodiments, the Fc1 has a pore structure according to the knob-and-hole technique, and the Fc2 has a convex structure according to the knob-and-hole technique. In some embodiments, The amino acid residue at position 366 of the Fc1 is W; and the amino acid residue at position 366 of the Fc2 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, numbered according to for the EU index. In some embodiments, the amino acid residue at position 354 of the Fc1 is C; and the amino acid residue at position 349 of the Fc2 is C, numbered according to the EU index. In some embodiments, the Fcl has the amino acid sequence of SEQ ID NO:27, and the Fc2 has the amino acid sequence of SEQ ID NO:28.

In some embodiments, the antigen binding molecule of any one of the preceding, wherein the antigen binding moiety that specifically binds FLT3 is a Fab, and the antigen binding moiety that specifically binds CD3 is a substituted Fab; or The antigen-binding moiety specifically binding to FLT3 is a substituted Fab, and the antigen-binding moiety specifically binding to CD3 is a Fab; the substituted Fab comprises a Titin chain and an Obscurin chain capable of forming a dimer. In some embodiments, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 103, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104 to SEQ ID NO: 144 The amino acid sequence of the group. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 103, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 138.

In some embodiments, the antigen-binding molecule according to any one of the preceding, wherein the antigen-binding molecule comprises an antigen-binding moiety that specifically binds to FLT3 and an antigen-binding moiety that specifically binds to CD3, and the antigen-binding moiety that specifically binds to FLT3 The antigen-binding moiety of is a Fab; the antigen-binding moiety that specifically binds CD3 is a substituted Fab.

In some embodiments, the antigen-binding molecule according to any one of the preceding items comprises a first chain having a structure represented by formula (a), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (c) the third strand of the structure shown and a fourth strand with the structure shown in formula (d),

(a) [FLT3-VH]-[CH1]-[Fc1],

(b)[FLT3-VL]-[CL],

(c) [CD3-VH]-[Linker 1]-[Titin chain]-[Fc2],

(d) [CD3-VL]-[Linker 2]-[Obscurin Chain],

The structures represented by formulas (a), (b), (c) and (d) are arranged from N-terminal to C-terminal, and the linker 1 and the linker 2 are the same or different peptide linkers. In some embodiments, said Linker 1 and said Linker 2 have the same number of amino acid residues. In some embodiments, said Linker 1 and said Linker 2 are the same peptide linker. In some embodiments, said linker 1 and linker 2 comprise the amino acid sequence shown in SEQ ID NO: 66.

In some embodiments, the antigen-binding molecule of any one of the preceding items has: a first chain comprising the amino acid sequence of SEQ ID NO: 72, a second chain comprising the amino acid sequence of SEQ ID NO: 73, A third strand comprising the amino acid sequence of SEQ ID NO:74 and a fourth strand comprising the amino acid sequence of SEQ ID NO:75.

In some embodiments, the antigen-binding molecule of any one of the preceding, wherein the antigen-binding molecule The subcontains two antigen-binding moieties specifically binding to FLT3 and one antigen-binding moiety specifically binding to CD3, the antigen-binding moiety specifically binding to FLT3 is a Fab; the antigen-binding moiety specifically binding to CD3 is replaced Fab.

In some embodiments, the antigen-binding molecule according to any one of the preceding items comprises a first chain having a structure represented by formula (e), two second chains having a structure represented by formula (b), and a chain having a structure represented by formula (b). The 3rd chain of structure shown in formula (f) and a 4th chain with structure shown in formula (g),

(e) [FLT3-VH]-[CH1]-[Fc2],

(b)[FLT3-VL]-[CL],

(f) [FLT3-VH]-[CH1]-[Linker 3]-[CD3-VL]-[Linker 4]-[Titin chain]-[Fc1],

(g) [CD3-VH]-[Linker 5]-[Obscurin chain],

The structures shown in formulas (e), (b), (f) and (g) are arranged from the N-terminal to the C-terminal, and the linker 3, the linker 4 and the linker 5 are the same or different Peptide linker. In some embodiments, the linker 4 and the linker 5 have the same number of amino acid residues. In some embodiments, the linker 3 comprises the amino acid sequence shown in SEQ ID NO: 146, the linker 4 comprises the amino acid sequence shown in SEQ ID NO: 145, and the linker 5 Comprising the amino acid sequence shown in SEQ ID NO:66.

In some embodiments, the antigen-binding molecule according to any one of the preceding items has: a first chain comprising the amino acid sequence of SEQ ID NO: 68, two second chains comprising the amino acid sequence of SEQ ID NO: 69 , a third strand comprising the amino acid sequence of SEQ ID NO:70 and a fourth strand comprising the amino acid sequence of SEQ ID NO:71.

In another aspect, the present disclosure also provides an isolated antibody capable of specifically binding to FLT3, said antibody comprising a heavy chain variable region FLT3-VH and a light chain variable region FLT3-VL, wherein

(ii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or

(iii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 76 or 77, and the amino acid comprising SEQ ID NO: 9 sequence of FLT3-HCDR3, and said FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, 78, 79 or 80, the amino acid sequence comprising SEQ ID NO: 11, 81 or 82 The amino acid sequence of FLT3-LCDR2 and the FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO:12.

In some embodiments, an antibody provided herein comprises a heavy chain variable region FLT3-VH and a light chain variable region FLT3-VL, wherein

(ii) the FLT3-VH comprises FLT3-HCDR1 shown in SEQ ID NO: 13, FLT3-HCDR2 shown in SEQ ID NO: 14 and FLT3-HCDR3 shown in SEQ ID NO: 15, and the FLT3- The VL comprises FLT3-LCDR1 set forth in SEQ ID NO: 16, FLT3-LCDR2 set forth in SEQ ID NO: 17, and FLT3-LCDR3 set forth in SEQ ID NO: 18, or

In some embodiments, the aforementioned antibody, wherein the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and comprising The FLT3-HCDR3 of the amino acid sequence of SEQ ID NO: 21, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 and comprising FLT3-LCDR3 of the amino acid sequence of SEQ ID NO:24.

In some embodiments, the FLT3-HCDR1, FLT3-HCDR2, FLT3-HCDR3, FLT3-LCDR1, FLT3-LCDR2, and FLT3-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, the antibody according to any one of the preceding, which binds to human FLT3 with a KD of less than 1×10 ^-8 M, 5×10 ^-9 M at 25°C, said KD is obtained by surface plasmon measured by the resonance method.

In some embodiments, the antibody of any one of the preceding, wherein

In some embodiments, provided herein is an isolated antibody wherein

(i) said antibody comprises the FLT3-VH shown in SEQ ID NO: 53, 5, 52 or 54, and the FLT3-VL shown in SEQ ID NO: 56, 6 or 55, or

(ii) the antibody comprises the FLT3-VH shown in SEQ ID NO: 45, 3, 46, 47 or 48, and the FLT3-VL shown in SEQ ID NO: 50, 4, 49 or 51, or

(iii) the antibody comprises FLT3-VH shown in SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 2, 36, 37, 38, 39, 40, FLT3-VL as shown in 41, 42, 43 or 44;

specifically,

(i) the antibody comprises FLT3-VH shown in SEQ ID NO: 53, 52 or 54, and FLT3-VL shown in SEQ ID NO: 56 or 55, or

(ii) the antibody comprises the FLT3-VH shown in SEQ ID NO: 45, 46, 47 or 48, and the FLT3-VL shown in SEQ ID NO: 50, 49 or 51, or

(iii) the antibody comprises FLT3-VH shown in SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, FLT3-VL as shown in 43 or 44;

More specifically,

(i) the antibody comprises FLT3-VH shown in SEQ ID NO: 53, and FLT3-VL shown in SEQ ID NO: 56, or

(ii) the antibody comprises FLT3-VH shown in SEQ ID NO: 45, and FLT3-VL shown in SEQ ID NO: 50.

In some embodiments, the antibody of any one of the preceding items, wherein said antibody is a bispecific antibody. In some embodiments, the bispecific antibody specifically binds FLT3 and CD3.

In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of the antigen-binding molecule of any of the foregoing or the antibody of any of the foregoing, and one or more pharmaceutical acceptable carrier, diluent, buffer or excipient. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

In another aspect, the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any of the foregoing or the antibody of any of the foregoing.

In another aspect, the present disclosure also provides a host cell comprising the aforementioned isolated nucleic acid.

In another aspect, the present disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject in need a therapeutically effective amount of any of the aforementioned antigen-binding molecules or any of the aforementioned antigen-binding molecules. Said antibody or composition thereof.

In another aspect, the present disclosure also provides the use of the antigen-binding molecule described in any one of the foregoing or the antibody or composition thereof in the preparation of a drug for treating or preventing diseases.

In another aspect, the present disclosure also provides the antigen-binding molecule of any one of the foregoing or the antibody of any one of the foregoing or a composition thereof for use as a medicament. In some embodiments, the medicament is used to treat or prevent a disease.

In some embodiments, the disease of any one of the preceding is a proliferative disease, a tumor, or an immune disease.

In some embodiments, the disease of any of the preceding is a tumor or cancer.

In some embodiments, the disease of any one of the preceding is multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's myeloma, acute monocytic leukemia, plasma cell leukemia, plasmacytoma, B prolymphocytic leukemia, hairy cell leukemia, B cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, Large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymus) large B cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, lymph node marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell-rich/histiocytic large B-cell lymphoma, primary Central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma from HHV-8-associated multicentric Casterman's disease, features intermediate between diffuse large B-cell lymphoma and primary Unclassified B-cell lymphoma between Kitt lymphoma, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and classic Hodgkin lymphoma, and other hematopoietic cell-associated cancers , and breast cancer, gastric cancer, liver cancer, lung cancer (such as non-small cell lung cancer, small cell lung cancer), cervical cancer, colorectal cancer (such as colon cancer, rectal cancer), endometrial cancer, head and neck cancer, mesothelioma, ovarian cancer cancer, pancreatic cancer, prostate cancer, skin cancer, kidney cancer, bladder cancer and other related cancers.

In some embodiments, the aforementioned disease is a disease associated with FLT3; in some embodiments, the aforementioned disease is a disease expressing FLT3.

The antigen-binding molecule provided by the present disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).

Description of drawings

Figure 1A: Schematic diagram of structure-1; Figure 1B: Schematic diagram of structure-2.

Detailed ways

the term

The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. 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 disclosure belongs.

Throughout the specification and claims, the words "including "includes", "has", "includes", etc. are understood to be inclusive rather than exclusive or exhaustive; that is, "including but not limited to". Unless otherwise stated, "comprising" includes "consisting of ...composition". For example, for FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, it specifically covers the FLT3-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 7.

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

The term "and/or", eg "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for both or either meaning.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure (i.e., the alpha carbon bonded to a hydrogen, carboxyl, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

The term "amino acid mutation" includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct so long as the final construct possesses the desired properties, such as reduced or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and/or carboxyl termini of the polypeptide chain. Specific amino acid mutations may be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, that is, replacing one amino acid with another amino acid having different structural and/or chemical properties. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) . Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, are also available. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be expressed in the form of position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 is mutated from the original T to W.

The term "antigen-binding molecule" is used in the broadest sense and encompasses various molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins fused thereto, as long as they exhibit the desired antigen-binding activity. The antigen binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen binding domain. Exemplarily, the antigen-binding molecules herein are bispecific antigen-binding molecules (eg, bispecific antibodies).

The term "antibody" is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity . For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons composed of two identical light chains and two identical heavy chains joined by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also called variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).

The term "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) capable of specifically binding to two different antigens or at least two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art, which can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies according to the integrity of the IgG molecule, and can be divided into bivalent bispecific antibodies according to the number of antigen-binding regions. , trivalent, tetravalent or more valent bispecific antibodies, according to whether the structure is symmetrical, can be divided into symmetrical structure bispecific antibodies and asymmetric structure bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, which have lower immunogenicity, and Small molecular weight, high tumor tissue permeability, typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragments), This type of antibody has a relatively large molecular weight. The Fc fragment is helpful for the purification of the antibody and improves its solubility and stability. The Fc part may also bind to the receptor FcRn to increase the serum half-life of the antibody. A typical bispecific antibody structure model Such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG , DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4 -Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F.Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585–608(2019); Chen S1 et al., J Immunol Res.2019 Feb 11 ; 2019:4516041).

The term "variable region" or "variable domain" refers to the antigen-binding domain of an antigen-binding molecule. Herein, the heavy chain variable region in the antigen-binding module that specifically binds to FLT3 is designated as FLT3-VH, and the light chain variable region is designated as FLT3-VL; the heavy-chain variable region in the antigen-binding module that specifically binds to CD3 Denoted as CD3-VH and light chain variable region as CD3-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region in the variable domain that mainly contributes to binding to the antigen; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3. In this paper, the three CDR regions in FLT3-VH are marked as FLT3-HCDR1, FLT3-HCDR2 and FLT3-HCDR3; the three CDR regions in FLT3-VL are marked as FLT3-LCDR1, FLT3-LCDR2 and FLT3-LCDR3 ; The three CDR regions in CD3-VH are marked as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively; The three CDR regions in CD3-VL Labeled as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, respectively. Each VH and VL is sequenced from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

The amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia" numbering sequence, "ABM" numbering sequence, "Contact" numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P., etc., Dev.Comp.Immunol., 27, 55-77 (2003); Front Immunol.2018 Oct 16; 9:2278) etc.; familiar. The numbering rules of this disclosure are shown in Table 1.

Table 1. Relationship between CDR numbering systems

Unless otherwise stated, the "Kabat" numbering convention is applicable to the variable regions and CDR sequences in the examples of the present disclosure.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody molecule (e.g. scFv); and multispecific antibodies formed from antibody fragments.

As described herein, the terms "Fab" or "Fab fragment" are used interchangeably and refer to a fragment of a monovalent antibody consisting of VL, VH, CL and CH1 domains; herein, an antibody, such as an anti-FLT3 antibody The Fab monovalent antibody fragment can be linked to the Fc1 subunit through the C-terminal of its CH1, and the formed Fab-Fc1 can be further linked to another antibody, such as an anti-CD3 antibody, through a disulfide bond at the N-terminal of the Fc1 subunit. Fab-Fc2 forms bispecific antibodies.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions. In some embodiments, the Fc region comprises the same or different two subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.

The term "Titin chain" refers to a section of Titin protein that is 78-118 amino acids in length and contains Titin Ig- Like 152 domain peptides or functional variants thereof, the Titin chain can combine with Obscurin Ig-like 1 or Obscurin-like Ig-like 1 domain to form a dimerization complex. The term "Obscurin chain" refers to a peptide segment of 87-117 amino acids on the Obscurin protein that contains the Obscurin Ig-like 1 domain or a functional variant thereof, or a segment of the Obscurin-like 1 protein that is 78-118 amino acids in length An amino acid peptide segment comprising an Obscurin-like Ig-like 1 domain or a functional variant thereof, the Obscurin chain can combine with a Titin Ig-like 152 domain to form a dimerization complex. The Titin chain and Obscurin chain disclosed herein can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S), and the replacement does not affect the binding of the antigen-binding molecule to the antigen.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from a different source or species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).

The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the on-rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the off-rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and is expressed as molarity (M). KD values for antibodies can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, ie, the antibody molecules comprised in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically comprise multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Monoclonal" denotes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent of an antigen binding protein (eg, an antibody). An antigen may have one or more epitopes capable of interacting with different antigen binding proteins (eg antibodies).

The term "epitope" refers to an area (area or region) on an antigen capable of specifically binding to an antibody or antigen-binding fragment thereof. An epitope may be formed from contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), such that non-contiguous amino acids are spatially separated by folding of the antigen (i.e. by tertiary folding of the antigen in proteinaceous nature). near. The difference between conformational epitopes and linear epitopes is: In the presence of solvent, antibody binding to conformational epitopes is lost. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind a particular epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot.Sci.9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

The term "capable of specifically binding", "specifically binds" or "binds" means that an antibody is capable of binding to a certain antigen or epitope with a higher affinity than to other antigens or epitopes. Typically, an antibody binds an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 x 10 ^-8 M or less (eg, about 5 x 10 ^-9 M or less). In some embodiments, the antibody binds an antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g. (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.

The term "non-binding" means that the antibody cannot bind to a certain antigen or an epitope within the antigen in the above-mentioned specific binding manner. For example, when the antibody binds the antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 1 x 10 ^-6 M or greater.

The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds an antigen of interest. Particular antigen binding moieties include the antigen binding domain of an antibody, eg, comprising a heavy chain variable region and a light chain variable region. The term "antigen binding moiety that specifically binds FLT3" refers to a moiety that is capable of binding FLT3 with sufficient affinity such that molecules containing this moiety are useful as diagnostic and/or therapeutic agents targeting FLT3. For example, an antigen binding moiety that specifically binds FLT3 has an equilibrium dissociation constant (KD) of <about 10 nM, as measured by a surface plasmon resonance assay. Antigen binding moieties include antibody fragments as defined herein, eg Fab, substituted Fab or scFv.

The term "linker" refers to a linking unit that joins two polypeptide fragments. Herein, linkers appearing in the same formula may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in the structural formula, it means that the units on both sides are directly connected by covalent bonds.

"Tm" is the melting denaturation temperature (intrinsic fluorescence). When the protein is denatured (heating or denaturant action), the tertiary structure is opened, and the microenvironment of the aromatic amino acid changes, resulting in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence changes to half of the maximum value.

"Tonset" is the denaturation initiation temperature. It means the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.

"Tagg" is the aggregation onset temperature. By static light scattering, at two wavelengths of 266nm and 473nm Under Detect Aggregation, monitor the temperature at which the sample begins to aggregate. Tagg 266 refers to the aggregation initiation temperature monitored at 266nm.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to the reference nucleic acid, and defined in Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA ). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location. An isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or in separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in the host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the corresponding artificial chemical mimetic of a naturally occurring amino acid, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "identity" of sequences refers to the degree (percentage) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps may be introduced as necessary to obtain the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term "fused" or "linked" refers to the covalent linking of components, such as an antigen binding module and an Fc domain, directly or via a linker.

The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing the expression of one or more heterologous coding regions operatively linked thereto and/or controlling (along with the host cell). A vector of nucleic acid sequences. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Herein, the term includes mutant progeny that have the same function or biological activity as the cells screened or selected for among the primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia willow salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum , Physcomitrella patens and Neurospora crassa. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luke Mold (Chrysosporium lucknowense), any Fusarium species, Yarrowia lipolytica, and Neurospora crassa. The host cells of this patent do not include objects that are not authorized under the patent law.

As used in this disclosure, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed "Cell" includes primary subject cells and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny will have the exact same DNA content due to deliberate or unintentional mutations .comprising mutant progeny that have the same function or biological activity as the original transformed cell from which they were screened.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

The term "pharmaceutical composition" means a mixture comprising one or more of the antigen binding molecules or antibodies described herein and other chemical components such as physiological/pharmaceutical acceptable carriers and excipients.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless expressly stated otherwise. As used herein, the term "cyno" or "cynomolgus" refers to Macaca fascicularis. In certain embodiments, the individual or subject is a human.

"Administration" or "administration", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the interaction of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human , subjects, cells, tissues, organs or biological fluids.

The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane secretions of secretory tissues and organs, vaginal secretions, ascites , pleura, pericardium, peritoneum, peritoneal and other body cavity fluids, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ condition culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

"Treatment" and "treatment" (and grammatical variants thereof) refer to clinical intervention intended to be applied to the individual being treated, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or improved prognosis . In some embodiments, the molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or their underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate the impairment caused by or associated with the disease state (e.g. lung disease). In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or in any way related to the disease state The amount of progression of any other undesirable symptoms associated with the disease. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms . A complete therapeutic or prophylactic effect does not necessarily occur after one dose, but may occur after a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. "Therapeutically effective amount" and "prophylactically effective amount" can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.

Antigen binding molecules of the present disclosure

The present disclosure provides antigen-binding molecules that have many favorable properties, such as high affinity for FLT3 and FLT3-expressing cells, in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties, and druggability (such as yield, purity and stability, etc.).

Exemplary Antigen Binding Molecules

Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (eg, bispecific antibodies) and anti-FLT3 antibodies that specifically bind FLT3 and CD3. In particular, the antigen-binding molecules of the present disclosure have any of the following properties:

a. High affinity for FLT3. The antigen-binding molecule binds to human FLT3 with a KD of less than 1×10 ^-7 M, 1×10 ^-8 M, and 5×10 ^-9 M at 25°C, and the KD is measured by surface plasmon resonance .

b. High affinity for FLT3 on the surface of FLT3-expressing cells. The antibodies bind cell surface FLT3 with an _EC50 of less than 10 _pM , 1 pM or 0.5 pM, as measured by ELISA. The cells in question are Monomac-6.

c. It has in vitro specific killing activity on FLT3 positive cells.

d. Induction of low levels of cytokine (IL6 and IFNγ) release.

e. Stronger therapeutic activity in vivo.

The present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety specifically binding to FLT3 and at least one antigen-binding moiety specifically binding to CD3, the antigen-binding moiety specifically binding to FLT3 comprising FLT3-VH and FLT3 - VL, said antigen binding moiety specifically binding to CD3 comprising CD3-VH and CD3-VL. The present disclosure also provides an isolated antibody capable of specifically binding to FLT3, said antibody comprising FLT3-VH and FLT3-VL. Specifically, the Examples herein disclose antibody series 18, 99 and 125. The antigen-binding molecule containing antibody 125 will be described below as an example.

The FLT3-VH of the antigen-binding molecule or anti-FLT3 antibody that specifically binds to FLT3 and CD3 has: FLT3-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 19, FLT3-HCDR1 having an amino acid sequence as shown in SEQ ID NO: 20 HCDR2 and the amino acid sequence of FLT3-HCDR3 as shown in SEQ ID NO: 21, and the FLT3-VL has: the amino acid sequence of FLT3-LCDR1 as shown in SEQ ID NO: 83 or 22, the amino acid sequence as shown in SEQ ID NO: 84 Or FLT3-LCDR2 shown in 23 and amino acid sequence such as SEQ ID NO: FLT3-LCDR3 shown in 24. In some embodiments, the FLT3-VH has: the amino acid sequence of FLT3-HCDR1 shown in SEQ ID NO: 19, the amino acid sequence of FLT3-HCDR2 shown in SEQ ID NO: 20 and the amino acid sequence of SEQ ID NO: The FLT3-HCDR3 shown in 21, and the FLT3-VL have: the amino acid sequence of FLT3-LCDR1 shown in SEQ ID NO: 83, the amino acid sequence of FLT3-LCDR2 shown in SEQ ID NO: 84, and the amino acid sequence of SEQ ID NO: 84 ID NO: FLT3-LCDR3 shown in 24.

In some embodiments, the antigen-binding molecule or antibody as described above, the FLT3-VH and/or the FLT3-VL is murine or humanized. In some embodiments, the FLT3-VH and/or the FLT3-VL are humanized. In some embodiments, FR1, FR2 and FR3 of the humanized FLT3-VH have at least 60%, 70% or 80% sequence identity to FR1, FR2 and FR3 of SEQ ID NO: 5, said FR4 of humanized FLT3-VH has at least 80% or 90% sequence identity to FR4 of SEQ ID NO: 5, and FR1, FR2 and FR3 of said humanized FLT3-VL are identical to SEQ ID NO: 6 FR1, FR2 and FR3 have at least 60%, 70% or 80% sequence identity and/or FR4 of said humanized FLT3-VL has at least 80% or 90% sequence identity with FR4 of SEQ ID NO:6 sequence identity. In some embodiments, the FLT3-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ1*01, and it is unsubstituted or has a compound selected from 1E, 37L, 48I , 67A, 69W, 71V and 78A in the group consisting of one or more amino acid substitutions; and/or the FLT3-VL has FR1, FR2, FR3 derived from IGKV6-21*01 and FR4 derived from IGKJ4*01 , and it is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2N, 47W, 49Y and 71Y. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

In some embodiments, the antigen binding molecule or antibody of any one of the preceding, wherein the amino acid sequence of FLT3-VH has at least 85%, 90%, 91% of SEQ ID NO: 53, 5, 52 or 54 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of said FLT3-VL has at least SEQ ID NO: 56, 6 or 55 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, 5, 52 or 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, 6 or 55 . In some embodiments, the amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, 52 or 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56 or 55.

In some embodiments, the antigen binding molecule or antibody of any preceding one, wherein

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 52, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 53, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 55, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 54, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 52, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 56, or

The amino acid sequence of the FLT3-VH is shown in SEQ ID NO: 5, and the amino acid sequence of the FLT3-VL is shown in SEQ ID NO: 6.

In some embodiments, the antigen-binding molecule or antibody according to any one of the preceding items, wherein the amino acid sequence of the FLT3-VH is as shown in SEQ ID NO: 53, and the amino acid sequence of the FLT3-VL is as in SEQ ID NO :56.

In some embodiments, the antigen-binding molecule as described above, the CD3-VH has: the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 57, the amino acid sequence of CD3-HCDR1 shown in SEQ ID NO: 58 HCDR2, and CD3-HCDR3 with an amino acid sequence as shown in SEQ ID NO: 59; and the CD3-VL has: CD3-LCDR1 with an amino acid sequence as shown in SEQ ID NO: 60, and an amino acid sequence as shown in SEQ ID NO: 61 The CD3-LCDR2 shown, and the CD3-LCDR3 shown in SEQ ID NO:62 with amino acid sequence.

In some embodiments, the antigen binding molecule according to any one of the preceding, said CD3-VH and/or said CD3-VL is murine or humanized. In some embodiments, the CD3-VH and/or the CD3-VL are humanized. In some embodiments, the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 63, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 64. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

According to the technical solutions of antibody series 18 and 99 disclosed in the examples herein, these antibodies have a similar technical solution range to antibody 125 described above.

Structure of Antigen Binding Molecule

The bispecific antigen-binding molecule of the present disclosure is not limited to a specific molecular structure as long as it has the desired antigen-binding function. For example, the bispecific antigen binding molecules herein can be bivalent (1+1) or trivalent (2+1). The antigen-binding moiety in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, which is fused via a peptide linker. The peptide linker of the present disclosure may be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, a peptide linker can be a flexible peptide of 1-50, or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has a structure of L ₁ -(GGGGS)nL ₂ , wherein L ₁ is a bond, A, GS, GGS, GGGS, SGGGGS, GGGTKLTVLGGG, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is a bond, G, GG, GGG or GGGG, and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, each of the peptide linkers independently has the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the peptide linker is ASTKG (SEQ ID NO: 66), GGGGS (SEQ ID NO: 145), GGGGSGGGGS (SEQ ID NO: 146) or GGGGSGGGGSGGGGS (SEQ ID NO: 147).

Exemplarily, the antigen-binding molecule of the present disclosure comprises a first chain having a structure represented by formula (a), a second chain having a structure represented by formula (b), and a chain having a structure represented by formula (c-1). The third strand of the structure shown and a fourth strand with the structure shown in formula (d-1),

(a) [FLT3-VH]-[CH1]-[Fc1],

(b)[FLT3-VL]-[CL],

(c-1)[CD3-VH]-[ASTKG]-[Titin chain]-[Fc2],

(d-1)[CD3-VL]-[ASTKG]-[Obscurin chain],

The structures represented by formulas (a), (b), (c-1) and (d-1) are arranged from N-terminal to C-terminal.

Exemplary bivalent antigen binding molecules have:

The antigen-binding molecule has: a first chain with an amino acid sequence as shown in SEQ ID NO:72, a second chain with an amino acid sequence as shown in SEQ ID NO:73, and an amino acid sequence as shown in SEQ ID NO:74 The third strand and a fourth strand with an amino acid sequence as shown in SEQ ID NO:75.

Variants of Antigen Binding Molecules

In certain embodiments, amino acid sequence variants of the antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.

Substitution, insertion, and deletion variants

In certain embodiments, antigen binding molecule variants having one or more amino acid substitutions are provided. Substitutions can be made in CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 2. Amino Acid Substitutions

According to common side chain properties, amino acids can be grouped as follows:

(1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

A non-conservative substitution would refer to the substitution of a member of one class for a member of another class.

One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variant selected for further study will have an altered (e.g. improved) certain biological property (e.g. increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Some of the biological properties of the parental antibody are retained. An exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity). Alterations (eg, substitutions) can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e. residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and/or residues that contact antigen, while making changes to the resulting variant VH or VL test for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.

In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs as long as Such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (eg, conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this approach, a residue or group of residues (e.g. charged residues such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g. Ala or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions showing functional sensitivity to the initial substitution. In addition, contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include: amino- and/or carboxy-terminal fusions of one residue or polypeptides of 100 or more residues in length; and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of antibodies) at the N- or C-terminus of the antibody.

Fab Transformation

In one aspect, in the antigen-binding molecules of the present disclosure, one of the antigen-binding moiety that specifically binds FLT3 and the antigen-binding moiety that specifically binds CD3 is a substituted Fab comprising Heavy chain variable region, light chain variable region, Titin chain and Obscurin chain. In the replaced Fab, the original CH1 and CL of the Fab are replaced by Titin chain and Obscurin chain. Exemplarily, the sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2.

Table 3-1. Amino acid sequence of Titin chain

Table 3-2. Amino acid sequences of Obscurin chains

Modification of the Fc region

In one aspect, the Fc region of an antigen binding molecule of the disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fcγ receptor, and reduce or Eliminate effector functions. A native IgG Fc region, specifically an IgG ₁ Fc region or an IgG ₄ Fc region, may result in the targeting of an antigen binding molecule of the present disclosure to cells expressing Fc receptors, rather than cells expressing antigen. The engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a binding affinity for Fc receptors that is reduced by more than 50%, 80%, 90%, or 95% compared to a native Fc region. In some embodiments, the Fc receptor is an Fc gamma receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, eg, FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to a native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region. In some embodiments, the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cells primed. For the IgG ₁ Fc region, amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function. In some embodiments, the Fc region is a human IgG ₁ Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index. For the IgG ₄ Fc region, amino acid residue substitutions at positions such as 228 may reduce effector function.

Antigen binding molecules may also comprise disulfide bond engineering, eg, 354C of the first subunit and 349C of the second subunit. To increase the serum half-life of the antigen binding molecule, mutations at 252Y, 254T and 256E can be introduced.

Undesirable homodimerization may result when the antigen binding molecule comprises different binding modules fused to the two subunits of the Fc region. In order to increase yield and purity, it would therefore be advantageous to introduce modifications in the Fc region of the antigen binding molecules of the present disclosure that promote heterodimerization. In some embodiments, the Fc region of the present disclosure comprises modifications according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of the first subunit and the introduction of a knob at the interface of the second subunit. A hole structure (hole) is introduced at the interface of the base. This enables the protrusion structure to be positioned in the hole structure, promotes the formation of heterodimers and inhibits the generation of homodimers. The bulge structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains such as tyrosine or tryptophan. Instead, the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine. Protrusion structures and hole structures are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in the table below:

Table 4. KIH mutation combinations

Besides the knob-and-hole technique, other techniques for modifying the CH3 domain of a heavy chain to achieve heterodimerization are also known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO2007/147901 , WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.

The C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK; it may also be a truncated C-terminus, for example, one or two C-terminal amino acid residues are removed from the truncated C-terminus. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending in PG. Thus, in some embodiments, a composition of intact antibodies can include a population of antibodies from which all K447 residues and/or G446+K447 residues have been removed. In some embodiments, a composition of intact antibodies can include a population of antibodies in which the K447 residue and/or the G446+K447 residues have not been removed. In some embodiments, the composition of intact antibodies has a population of antibodies with and without a mixture of antibodies with the K447 residue and/or G446+K447 residues.

Recombination method

Antigen binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen binding molecule are provided.

In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

In the case of bispecific antibodies with heterodimeric heavy chains, for example four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc region polypeptide, one One for the second light chain, and one for the second heavy chain comprising a second heteromonomeric Fc region polypeptide. These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, ie one vector may contain more than one of these nucleic acids.

In one embodiment, the present disclosure provides an isolated nucleic acid encoding an antibody as previously described. Such nucleic acids may independently encode any of the aforementioned polypeptide chains. In another aspect, the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids. In another aspect, the disclosure provides host cells comprising such nucleic acids. In one embodiment, there is provided a method of making an antigen binding molecule, wherein said 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 optionally The antibody is recovered from the host cell (or host cell culture medium).

For recombinant production of antigen-binding molecules, nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be 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), or produced recombinantly or obtained by chemical synthesis.

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when the antibody does not require glycosylation and Fc effector functions. After expression, antibodies can be isolated from bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", This results in the production of antibodies with partially or fully human glycosylation patterns. Suitable host cells for expressing (glycosylated) antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified for use in combination with insect cells, especially for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g. US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, eg mammalian cell lines adapted for growth in suspension. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) liver cells ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004) , pp. 255-268.

Immunoconjugate

The present disclosure also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents such as chemotherapeutics or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof) of bacterial, fungal, plant or animal origin, or radioactive isotopes.

Diagnostic and Therapeutic Compositions

In certain embodiments, the antigen binding molecules provided by the present disclosure can be used to detect the presence of FLT3 and/or CD3 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue, such as tumor tissue.

In one embodiment, an antigen binding molecule for use in a diagnostic or detection method is provided. In yet another aspect, methods of detecting the presence of FLT3 and/or CD3 in a biological sample are provided. In certain embodiments, the method comprises contacting a biological sample with an antigen-binding molecule under suitable conditions, and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo methods. In one embodiment, antigen binding molecules are used to select subjects suitable for treatment, eg, FLT3 and/or CD3 are biomarkers used to select patients.

Exemplary disorders that can be diagnosed using the antigen binding molecules of the disclosure, such as tumors or cancers.

In certain embodiments, labeled antigen binding molecules are provided. Labels include, but are not limited to, labels or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection (e.g., indirect detection via enzymatic reactions or molecular interactions). modules, such as enzymes or ligands).

In additional aspects, pharmaceutical compositions comprising the antigen binding molecules are provided, eg, for use in any of the following methods of treatment. In one aspect, a pharmaceutical composition comprises any of the antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the antigen binding molecules provided herein and at least one additional therapeutic agent.

Pharmaceutical compositions of antigen-binding molecules described in the present disclosure are prepared by mixing such antigen-binding molecules having the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical composition In the form of a lyophilized composition or an aqueous solution. Formulations for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filters.

Methods of treatment and routes of administration

Any of the antigen binding molecules provided herein can be used in methods of treatment.

In yet another aspect, the present disclosure provides the use of an antigen binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of tumors or cancer. And the drug is in the form of an effective amount for the above diseases. In some embodiments, the effective amount is a unit daily dose or a unit weekly dose. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents agent). A "subject" according to any of the above embodiments may be a human.

In yet another aspect, there is provided a pharmaceutical composition comprising said antigen binding molecule, eg, for any of the above pharmaceutical uses or methods of treatment. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

The antigen binding molecules of the present disclosure can be used alone or in combination with other agents for therapy. For example, an antigen binding molecule of the disclosure can be co-administered with at least one additional therapeutic agent.

The antigen binding molecules of the present disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if local treatment is desired, intralesional. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The antigen binding molecules of the present disclosure will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor. Antigen binding molecules need not, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage, and any route empirically/clinically determined to be suitable.

In order to prevent or treat diseases, the antigen-binding molecules of the present disclosure (when used alone or in combination with one or more when used in combination with two other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, previous therapy, the patient's Clinical history and response to therapeutic molecules, and the judgment of the attending physician. The therapeutic molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of the antigen binding molecule may be an initial candidate dose for administration to the patient, whether for example by one or more divided administrations or by continuous infusion . A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Correspondingly, taking a body weight of 50kg as an example, the exemplary unit daily dose is 50 μg-5g.

products

In another aspect of the present disclosure, an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Containers can be formed from various materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, for the treatment, prophylaxis and/or diagnosis of a condition, and may have a sterile access opening (e.g., the container may have a stopper pierceable by a hypodermic needle). IV solution bag or vial). At least one active agent in the composition is an antigen binding molecule of the present disclosure. The label or package insert indicates that the composition is used to treat the condition of choice. Additionally, the article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen binding molecule of the present disclosure; and (b) a second container having a composition therein, wherein the combination The drug contains an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Example and test case

The present disclosure is further described below in conjunction with examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. For experimental methods not specified in the examples and test examples of this disclosure, conventional conditions are usually followed, such as Cold Spring Harbor’s Antibody Technology Experiment Manual, Molecular Cloning Manual; or the conditions suggested by raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.

Example 1. Antigen-binding molecules containing Titin chain/Obscurin chain

The Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758A1 (incorporated herein by reference) and CN202110527339.7 and patents (incorporated herein by reference) as priority documents . Construct a bispecific antibody, wherein CL is the kappa light chain constant region in WO2021139758A1, the amino acid sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2, and the linker sequence includes GGGGS (SEQ ID NO: 145), ASTKG (SEQ ID NO: 66) or RTVAS (SEQ ID NO: 67), the amino acid sequences of Fc1, Fc2, and CH1 in this example are shown below.

>Example 1-CH1 (SEQ ID NO: 65)

>Example 1 - Fc1 (knob, SEQ ID NO: 27)

>Example 1-Fc2 (hole, SEQ ID NO: 28)

1.1 DI bispecific antibody

Referring to Example 5 of WO2021139758A1, construct DI bispecific antibodies against hNGF and hRANKL: DI-2 to DI-20, which comprise the first heavy chain, the second heavy chain, the first light chain and the second Light chain:

The first heavy chain: from N-terminal to C-terminal: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2],

The first light chain: from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],

Second heavy chain: from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and

The second light chain: from N-terminal to C-terminal: [VL2-D]-[CL];

Among them, VH1-I and VL1-I are respectively the heavy chain variable region and light chain variable region of I0 in WO2021139758A1, and VH2-D and VL2-D are respectively the heavy chain variable region and light chain variable region of D0 in WO2021139758A1. district. The structures of Obscurin chain, Titin chain, linker 1 and linker 2 in the DI bispecific antibody in this example are shown in the table below.

Table 5. Obscurin chain/Titin chain and linker correspondence table in DI bispecific antibody

Note: See Table 3-1 and Table 3-2 for the numbers of Titin chains and Obscurin chains in the table.

The method in Test Example 4 of WO2021139758A1 was used to detect the binding activity of DI-2 to DI-20 bispecific antibodies and their antigens. Thermostability studies were performed on antibodies. Research method: The concentration of the antibody was diluted to 5 mg/mL with PBS, and its thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit). The experimental results showed that the antigen-binding activity of the engineered bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) have been significantly improved, and the thermal stability of the bispecific antibody is better.

Table 6. Binding Activity Detection of DI Bispecific Antibodies

Table 7. Thermal stability test results of DI bispecific antibodies

Use 10mM acetic acid, pH 5.5, 9% sucrose buffer to prepare a solution containing DI bispecific antibody, place the solution in a 40°C incubator and incubate for four weeks, then concentrate the antibody concentration to the concentration at the beginning of the incubation, observe solution precipitation. The experimental results showed that the solution of DI-2 bispecific antibody group precipitated, and DI-3 to DI-7 had better stability than DI-2.

Table 8. Precipitation of DI bispecific antibodies

1.2 PL bispecific antibody

Construction of PL bispecific antibodies against hPDL1 and hCTLA4: PL-1 to PL-19 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:

The first heavy chain: from N-terminal to C-terminal: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],

The first light chain: from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],

Second heavy chain: from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and

The second light chain: from N-terminal to C-terminal: [VL2-L]-[CL];

Wherein, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1 respectively, and the amino acid sequences of VH2-L and VL2-L are as follows.

>VH2-L (SEQ ID NO: 148)

>VL2-L (SEQ ID NO: 149)

The structure of Obscurin chain, Titin chain, linker 1 and linker 2 in the PL bispecific antibody in this example is shown in the table below.

Table 9. Obscurin chain/Titin chain and linker correspondence table in PL bispecific antibody

The binding activity of the PL bispecific antibody was detected by referring to the ELISA method in Test Example 4 in WO2021139758A1, wherein the hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermostability studies were performed on antibodies. Methods: The concentration of the antibody was diluted to 1.4-3 mg/mL with PBS, and its thermal stability was measured with a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit). The experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1(°C), Tagg 266(°C), Tonset(°C) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of the bispecific antibody is better.

Table 10. Detection of binding activity of PL bispecific antibodies

Table 11. Thermal stability test results of PL bispecific antibodies

1.3 HJ bispecific antibody

Construction of HJ bispecific antibodies against hIL5 and hTSLP: HJ-3 to HJ11 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as follows:

The first heavy chain: from N-terminal to C-terminal: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],

The first light chain: from N-terminal to C-terminal: [VL1-H]-[Linker 2]-[Obscurin chain],

Second heavy chain: from N-terminal to C-terminal: [VH2-J]-[CH1]-[Fc2], and

The second light chain: from N-terminal to C-terminal: [VL2-J]-[CL];

Among them, VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in WO2021139758A1, respectively, and VH2-J and VL2-J are the heavy chain variable region and light chain variable region of J1 in WO2021139758A1, respectively. district. The structure of Obscurin chain, Titin chain, linker 1 and linker 2 in the HJ bispecific antibody in this example is shown in the table below.

Table 12. Obscurin chain/Titin chain and linker correspondence table in HJ bispecific antibody

The antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in WO2021139758A1. To study the thermal stability of the antibody, the method: prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH5.5 and 9% sucrose, and then concentrate the bispecific antibody by ultrafiltration to obtain different concentrations The HJ bispecific antibody solution (see Table 13-2 for the concentration of the HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation. On day 0 (that is, before the incubation at 40°C, D0), Day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21) and day 28 (day 21 of incubation at 40°C, 28 days, D28) to test the SEC purity of the sample, and after incubation at 40°C for 28 days, immediately take a sample to test the CE-SDS purity of the sample. Experimental results show that this disclosure constructs The binding activity of the HJ bispecific antibodies to antigens did not change significantly; and, compared with HJ-3, the thermal stability of the HJ-5 to HJ-11 bispecific antibodies was better.

Table 13-1. Detection of binding activity of HJ bispecific antibody

Table 13-2. Accelerated stability test results of HJ bispecific antibody

Embodiment 2: Preparation of mouse anti-human FLT3 (ECD) monoclonal antibody

SJL/Balb/C mice were immunized with human FLT3-ECD (Sino Biological, 10445-H08H), FLT3-E6-mFc, FLT3-E6-Fc and CHOK1-hFLT3-E56, CHOK1-hFLT3-E6 stable transfection cells. After 3 times of immunization, blood was taken to measure the titer of the antibody in the serum, and the mice with high antibody titer in the serum and the titer tended to plateau were selected for splenocyte fusion, and the fused hybridoma cells were plated in a 96-well cell culture plate , placed in a 37°C, 5% CO ₂ incubator for cultivation. The cell culture supernatant was taken for detection by laser scanning microplate cell analyzer mirrorball and flow cytometer FACS. The screened positive clones were expanded, cryopreserved and subcloned two to three times until single-cell clones were obtained. The selected hybridoma clones were further prepared and purified by serum-free cell culture method. The obtained hybridoma antibody was detected by FACS for binding to human FLT3 protein and competition with FLT3 ligand, and hybridoma cell lines with strong binding activity and competitiveness were selected. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, Cat#15596-018), and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification and then sent to a sequencing company for sequencing to obtain three antibodies, mAb18, mAb99 and mAb125.

> Amino acid sequence of mAb18 heavy chain variable region (SEQ ID NO: 1)

>Amino acid sequence of mAb18 light chain variable region (SEQ ID NO: 2)

> Amino acid sequence of mAb99 heavy chain variable region (SEQ ID NO: 3)

>Amino acid sequence of mAb99 light chain variable region (SEQ ID NO: 4)

> Amino acid sequence of mAb125 heavy chain variable region (SEQ ID NO: 5)

>Amino acid sequence of mAb125 light chain variable region (SEQ ID NO: 6)

Table 14. List of antibody CDR sequences

The variable region sequences of the above mAb18, mAb99 and mAb125 candidate molecules were respectively amplified by PCR to amplify the VH/VK sequences, and then carried out the same with the expression vector pHr (with signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment). Source recombination, the construction of recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr, and then obtain its chimeric antibodies Ch18, Ch99 and Ch125.

>Amino acid sequence of human IgG1 heavy chain constant region (L234A/L235A) (SEQ ID NO: 25)

> Amino acid sequence of human light chain kappa constant region (SEQ ID NO: 26)

Example 3: Humanized design of anti-human FLT3 monoclonal antibody

Search for the VH/VL homologous sequence of the murine antibody from the human germline database, graft the CDR region of the murine antibody onto the human template, and mutate some residues of VL and VH, and transfer the murine antibody The constant regions were replaced with human constant regions to obtain the final humanized molecule.

3-1. Humanization of antibody mAb18

The humanized antibody of mouse antibody mAb18 selects FR1, FR2, FR3 of IGHV1-69*02, and FR4 of IGHJ6*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV2-40*01 FR4 of 01 served as the template for the framework region of the light chain. Optionally, amino acid residues at positions 1, 24, 27, 28, 30, 38, 40, 54, 55, 69 and/or 93 of the heavy chain variable region of the humanized antibody are substituted (according to Kabat numbering system numbering, the same below) to replace the amino acid residues; and/or to replace the amino acid residues at positions 2, 28, 29, 45, 55 and/or 56 on the light chain variable region of the humanized antibody .

Table 15-1. Mutations of humanized antibody 18

>h18 CDR2 of VH4, VH6 and VH7 (SEQ ID NO: 76)

>CDR2 of h18 VH5 (SEQ ID NO: 77)

> CDR1 of h18 VL3 (SEQ ID NO: 78)

CDR1 of >h18 VL4, VL6 and VL8 (SEQ ID NO: 79)

CDR1 of >h18 VL5, VL7 and VL9 (SEQ ID NO: 80)

CDR2 of >h18 VL6 and VL7 (SEQ ID NO: 81)

CDR2 of >h18 VL8 and VL9 (SEQ ID NO: 82)

The sequence of the antibody variable region is as follows:

>h18VH1 (SEQ ID NO: 29)

>h18VH2 (SEQ ID NO: 30)

>h18VH3 (SEQ ID NO: 31)

>h18VH4 (SEQ ID NO: 32)

>h18VH5 (SEQ ID NO: 33)

>h18VH6 (SEQ ID NO: 34)

>h18VH7 (SEQ ID NO: 35)

>h18VL1 (SEQ ID NO: 36)

>h18VL2 (SEQ ID NO: 37)

>h18VL3 (SEQ ID NO: 38)

>h18VL4 (SEQ ID NO: 39)

>h18VL5 (SEQ ID NO: 40)

>h18VL6 (SEQ ID NO: 41)

>h18VL7 (SEQ ID NO: 42)

>h18VL8 (SEQ ID NO: 43)

>h18VL9 (SEQ ID NO: 44)

3-2. Humanization of antibody mAb99

The humanized antibody of mouse antibody mAb99 selects FR1, FR2, FR3 of IGHV1-46*01, and FR4 of IGHJ6*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV1-33*01 FR4 of 01 served as the template for the framework region of the light chain. Optionally, the amino acid residues at positions 1, 12, 40, 69, 71, 76 and/or 78 of the heavy chain variable region of the humanized antibody are substituted; and/or the light Amino acid residues at positions 41, 42, 43, 44, 49 and/or 71 of the chain variable region are substituted.

Table 15-2. Mutations of humanized antibody 99

The sequence of the antibody variable region is as follows:

>h99VH1 (SEQ ID NO: 45)

>h99VH2 (SEQ ID NO: 46)

>h99VH3 (SEQ ID NO: 47)

>h99VH4 (SEQ ID NO: 48)

>h99VL1 (SEQ ID NO: 49)

>h99VL2 (SEQ ID NO: 50)

>h99VL3 (SEQ ID NO: 51)

3-3. Humanization of antibody mAb125

The humanized antibody of mouse antibody mAb125 selects FR1, FR2, FR3 of IGHV1-46*01, and FR4 of IGHJ1*01 as the template of the heavy chain framework region; selects FR1, FR2, FR3 and IGKJ4* of IGKV6-21*01 FR4 of 01 served as the template for the framework region of the light chain. Optionally, the amino acid residues at positions 1, 37, 48, 67, 69, 71 and/or 78 of the heavy chain variable region of the humanized antibody are substituted; and/or the light Amino acid residues at positions 2, 29, 47, 49, 56 and/or 71 of the chain variable region are substituted.

Table 15-3. Mutations of humanized antibody 125

> CDR1 of h125 VL2 (SEQ ID NO: 83)

> CDR2 of h125 VL2 (SEQ ID NO: 84)

The sequence of the antibody variable region is as follows:

>h125VH1 (SEQ ID NO: 52)

>h125VH2 (SEQ ID NO: 53)

>h125VH3 (SEQ ID NO: 54)

>h125VL1 (SEQ ID NO: 55)

>h125VL2 (SEQ ID NO: 56)

The heavy chain variable region and the light chain variable region of each of the above groups are combined with the constant regions shown in SEQ ID NO: 25 and SEQ ID NO: 26 to obtain a complete humanized antibody.

Table 15-4.18 series of humanized antibodies

Example 4: Preparation of anti-FLT3-CD3 bispecific antibody

The CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Specifically, the embodiment of the present disclosure adopts S107E.

Table 16-1. CDRs of S107E

The S107E variable region sequence is as follows:

>S107E-VH (SEQ ID NO: 63)

>S107E-VL (SEQ ID NO: 64)

>IgG ₁ (CH1) (SEQ ID NO: 65)

>CL (SEQ ID NO: 26)

>Titin (T.18, SEQ ID NO: 103)

>Obscurin (0.27, SEQ ID NO: 138)

> IgG ₁ Fc (Knob) (SEQ ID NO: 27)

>IgG ₁ Fc (Hole) (SEQ ID NO: 28)

> linker a (SEQ ID NO: 66)

> linker b (SEQ ID NO: 146)

> linker c (SEQ ID NO: 145)

The present disclosure employs two antibody structures. in,

Structure-1 is an asymmetric structure molecule, and its schematic diagram is as shown in Figure 1A (Ob represents Obscurin) which contains

Chain 1: VH(anti-FLT3) _-IgG1 (CH1) _-IgG1Fc (Knob);

Chain 2: VL(anti-FLT3)-CL;

Chain 3: VH(S107E)-Linker α-Titin-IgG ₁ Fc (Hole); and

Strand 4: VL(S107E)-linker a-Obscurin.

Structure-2 is an asymmetric structure molecule, and its schematic diagram is shown in Figure 1B, which contains

Chain 1: VH(anti-FLT3) _-IgG1 (CH1) _-IgG1Fc (Hole);

Chain 2 (two): VL (anti-FLT3)-CL;

Chain 3: VH (anti-FLT3)-IgG ₁ (CH1 )-Linker b-VL(S107E)-Linker c-Titin-IgG ₁ Fc (Knob);

Strand 4: VH(S107E)-linker a-Obscurin.

Table 16-2. Variable regions of bispecific antibodies

Exemplarily, the bispecific antibody BsAb-Hu99-5 (ie, BsAb-99) includes five chains.

> BsAb-99 chain 1 (SEQ ID NO: 68)

>BsAb-99 chain 2 (two) (SEQ ID NO: 69)

> BsAb-99 chain 3 (SEQ ID NO: 70)

> BsAb-99 chain 4 (SEQ ID NO: 71)

The bispecific antibody BsAb-Hu125-4 (ie BsAb-125) comprises four chains.
> BsAb-125 chain 1 (SEQ ID NO: 72)

> BsAb-125 chain 2 (SEQ ID NO: 73)

> BsAb-125 chain 3 (SEQ ID NO: 74)

> BsAb-125 chain 4 (SEQ ID NO: 75)

The positive control molecule used in this disclosure is AMG427, the amino acid sequence of which is shown in SEQ ID NO: 863 in WO2017021362A1.

test case

Test Example 1: Binding of an antigen-binding molecule to FLT3 on the cell surface

The centrifuged Monomac-6 cells were resuspended with 2% FBS (ThermoFisher Scientific, 10099-141), and the cell content in the cell suspension was 1-2×10 ⁶ cells/mL. Add 50 μL/well of cell suspension and 50 μL/well of serially diluted antibody solution to a 96-well cell plate, and incubate at 4°C for 1 hour. The 96-well cell plate was centrifuged at room temperature at 300 g for 5 min, and the plate was washed 3 times with PBS. Add 100 μL/well anti mouse IgG(H+L)-A488 (invitrogen, A11001, 1:1000 dilution) to the 96-well cell plate, incubate at 4°C for 1 hour hour. The 96-well cell plate was centrifuged at room temperature at 300 g for 5 min, and the plate was washed 3 times with PBS. 100 μL/well of PBS was added to the 96-well cell plate, detected by flow cytometry and analyzed by Flowjo software.

Table 17-1. Binding of 18 series antibodies to Monomac-6

The above ratio values set the _EC50 of m18 to 1.

Table 17-2. Binding of 99 series bispecific antibodies to Monomac-6

The above ratio values set the _EC50 of BsAb-m99 as 1.

Table 17-3. Binding of 125 series bispecific antibodies to Monomac-6

The above ratio values will take the _EC50 of BsAb-m125 as 1.

The experimental results showed that the humanized antibody maintained the ability to bind to the natural cell line Monomac-6 expressing the FLT3 antigen.

Test Example 2: Biacore Antibody Affinity Experiment

Biacore T200, Cytiva instrument was used to measure the affinity of AMG427 and BsAb-125 bispecific antibody to human FLT3 antigen, human and monkey CD3.

Use Protein A biosensor chip (Cat.#29127556, Cytiva) to affinity capture a certain amount of antibody to be tested, and then flow through a series of concentration gradients of human FLT3 antigen (Sino Biological, 10445-H08H), human The source CD3 antigen (Sino Biological, CT038-H2508H) and the monkey-derived FLT3 antigen (ACRO, CDD-C52W4) were used to detect the reaction signal in real time using a Biacore instrument (Biacore T200, Cytiva) to obtain the binding and dissociation curves. After each cycle of dissociation, the biosensor chip was washed and regenerated with 10mM Glycine-HCl (pH1.5). The buffer solution used in the experiment is HBS-EP+10× buffer solution (pH 7.4 (Cat.#BR-1006-69, Cytiva) diluted to 1× (pH 7.4) with D.I.Water. The data obtained in the experiment are used BIAevaluation version , Cytiva software fitted the (1:1) Langmuir model to obtain the affinity value.

Table 18. Affinity Assays

Test Example 3: In vitro PBMC-natural cell line killing experiment

Bispecific antibodies can bind to tumor cells expressing FLT3 antigen through FLT3-terminal antibody molecules, and on the other hand, can recruit and activate T cells through CD3-terminal antibody molecules, and substances such as perforin and granzyme secreted by T cells can kill cancer cells. The killing ability of the bispecific antibody molecule is evaluated by detecting the change in the fluorescence signal intensity of the tumor cell after the bispecific antibody molecule, T cells, tumor cells and PBMC are co-incubated. In this experiment, the effect of FLT3-CD3 bispecific antibody on natural cell lines expressing FLT3 antigen (Monomac-6, MoLM-13 and MV-4- 11, FLT3 expression level from high to low), and the killing effect of natural cell line K562 cells not expressing FLT3 antigen.

The plasmid of PCDH/lucG was transfected into Monomac-6 (Nanjing Kebai, CBP60521), MoLM-13 (DSMZ, ACC 554), MV-4-11 (ATCC, CRL-9591 ^TM ), K562 (ATCC, CCL- 243) cells, construct a cell line stably expressing luciferase and GFP. Culture Monomac-6/lucG cells with 1640 (Gibco, 11875119) + 10% FBS (ThermoFisher Scientific, 10099-141) + 1 × insulin (Gibco, 41400-045) medium; culture with 1640 + 10% FBS complete medium MoLM-13/lucG, K562/lucG cells; MV-4-11/lucG cells were cultured with IMDM (Gibco, 12440061)+10% FBS complete medium. The above cells were passaged 2-3 times a week at a ratio of 1:10. use 1640+10% FBS complete medium to resuspend the revived PBMCs (Miaoshun Biology), and culture them in a T75 culture flask for 4 hours (at a density of 2×10 ⁶ cells/mL). The centrifuged PBMCs were resuspended with 1640+10% FBS, and 50 μL was added to each well (the density was 1.5×10 ⁶ cells/mL). The above target cells were collected, and 25 μL was added to each well (at a density of 3×10 ⁵ cells/mL), ensuring that the E:T Ratio was 10:1. After diluting the antibody with 1640+10% FBS medium, add 25 μL to each well (initial concentration is 2 nM, 3-fold dilution, 9 dose points). The treated cells were cultured in an incubator at 37°C with 5% CO ₂ for 48 hours. The culture plate was centrifuged at room temperature at 1000 rpm for 3 minutes, 50 μL of the supernatant was pipetted into a new 3788 plate (Corning, 3903) for the detection of cytokines, and stored at -20°C. Add 20 μL ONE-GloTM Luciferase (promega, E6120) to the culture plate, incubate at room temperature for 5 minutes, use Vendor to detect Luminescence, and calculate the killing effect of the antibody at different concentrations. Graphpad Prism8.0 software was used to analyze data and calculate antibody-mediated killing EC ₅₀ . The wells with only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate of the antibody was calculated.

Table 19. Killing of PBMC by bispecific antibodies

The experimental results show that BsAb-125 has good consistent killing activity on FLT3-positive natural cell lines, and BsAb-125 has no killing activity on cell lines that do not express FLT3 antigen. It is safer than AMG427 and has a significantly wider therapeutic window.

Test Example 4: In Vitro PBMC-HSPCs Killing Experiment

It has been reported in the literature that there is relatively low FLT3 expression on the surface of normal hematopoietic stem cells HSPCs. In this experiment, BsAb-125 or AMG427 was incubated with HSPCs and PBMCs to detect the changes in the fluorescence signal intensity of the cells to evaluate the damage of bispecific antibody molecules to HSPCs.

Resuspend the revived PBMCs (Miaoshun Biology) with 1640 (Gibco, 11875119) + 10% FBS (ThermoFisher Scientific, 10099-141) complete medium, and place them in a T75 culture flask for 4 hours (at a density of 2×10 ⁶ cells/ mL). The centrifuged PBMCs were resuspended with 1640+10% FBS, and 50 μL was added to each well (the density was 1.5×10 ⁶ cells/mL). Resuspend HSPCs (Lonza, 2M-101C) with IMDM+15% FBS (solution B, FBS inactivated), add 25 μL to each well (density 3×10 ⁵ cells/mL), E:T Ratio is 10:1. After diluting the antibody with solution B, add 25 μL to each well (the initial concentration is 2 nM, 5× dilution, 6 points). The treated cells were cultured in an incubator at 37°C with 5% CO ₂ for 48 hours. Centrifuge the culture plate at 1000rpm at room temperature for 3 minutes, remove the residual medium, resuspend the cells in 300μL of FACS buffer (2% FBS+PBS), centrifuge at 1200rpm at room temperature for 3min, remove the supernatant; add APC-Anti at a ratio of 1:20 -CD38(sino, 10818-MM27-A) and FITC-Anti-CD34(sino, 68035-XM01-F) into FACS buffer, Cells were resuspended in 100 μL/well, incubated at 4°C for 30 minutes, added 300 μL FACS buffer, and centrifuged at room temperature at 1200 rpm for 3 minutes. After the supernatant was removed, 200 μL FACS buffer was added to resuspend the cells, and FACSVerse was used for flow detection. The data were analyzed using Flowjo, and the EC50 of antibody-mediated killing was calculated with Graphpad Prism8.0 software. The wells with only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate of the antibody was calculated.

Table 20. Killing of HSPCs by bispecific antibodies

In this experiment, AMG427 has obvious killing activity on hematopoietic stem cells HSPCs, while BsAb-125 has no killing activity on HSPCs. Therefore, BsAb-125 can protect the normal hematopoietic stem cells of the patient's body from being killed, and has high safety.

Test Example 5: In Vitro Cytokine Release Experiment

In order to test whether the screened antibody has good safety, we tested the cytokine release of the antibody molecule in vitro. The specific method is to dilute the supernatant collected in the cell killing experiment (test example 3) 10 times with 1640+10% FBS medium for use. Dilute the IL-6 and IFNγ standards with 1640+10% FBS medium to the required concentration. Equilibrate the kit of the cytokine to be detected (Human IL6kit: CISBIO, 62HIL06PEG; Human IFNγkit: CISBIO, 62HIFNGPEG) to room temperature, and use the detection buffer to dilute the two detection antibodies in the kit (20-fold dilution). Add 16 μL of cell supernatant (stock solution for IL-6 detection, 10-fold dilution for IFNγ detection) and IL-6 or IFNγ standard into a 96-well plate, add 4 μL of diluted corresponding detection antibody; shake and mix, room temperature 1000rpm Centrifuge for 1 minute and incubate overnight at room temperature. The samples incubated overnight were taken out, centrifuged at 1000 rpm at room temperature for 1 minute, and the absorbance values at 665nm and 620nm were read using a PHERAstar multifunctional microplate reader. Data were analyzed with Graphpad Prism 8.

Table 21. Cytokine release by bispecific antibodies

The experimental results showed that the release of IL-6 and IFNγ cytokines of BsAb-125 was much lower than that of AMG427 in natural cell lines with high or low expression of FLT3 antigen, which has better safety.

Biological evaluation of in vivo activity

Test Example 6: Effect of FLT3-CD3 Antibody on Tumor Growth of NOG Mice Bearing Human Acute Monocytic Leukemia Cell Monomac-6 Tumor

In this experiment, NOG mice were subcutaneously inoculated with human acute monocytic leukemia Monomac-6 cells, and intraperitoneally injected with hPBMCs (Miaoshun Biotechnology, ID#: P121081003C) one day before the inoculation, to detect the effect of bispecific antibody BsAb-125 on subcutaneously transplanted tumors. growth effects. 5×10 ⁶ hPBMCs were intraperitoneally injected into female NOG female mice (4-8 weeks old), and 1×10 ⁶ cells/mouse/100 μL (containing 50 μL Matrigel) Monomac-6 cells were inoculated into the right side of the mouse one day later. Under the skin of the ribs. Eight days after inoculation, the mice were randomly divided into 3 groups: Vehicle (PBS), AMG427 0.2mpk group, BsAb-125 0.3mpk group (equal molar dose with AMG427 0.2mpk) (n=8/group). The day of grouping was defined as Day 0 of the experiment, and each antibody (Biw) was administered intraperitoneally, ip, for a total of 4 times, and the tumor volume and animal weight were monitored and recorded twice a week. All data were drawn and statistically analyzed using Excel and GraphPad Prism 5 software.

Table 22. Effect of FLT3-CD3 double antibody on tumor growth of NOG mice bearing Monomac-6 tumor

The experimental results showed that both tested antibodies could significantly inhibit the growth of Monomac-6 subcutaneous xenograft tumors, and the tumor inhibitory effect of BsAb-125 was significantly better than that of AMG427.

Test Example 7: Effect of FLT3-CD3 antibody on tumor growth of NDG mice bearing human acute monocytic leukemia cell MV-4-11-LucG orthotopically transplanted tumor

In this experiment, NDG mice (Biocytogen) were used to establish the MV-4-11-LucG orthotopic xenograft tumor model, and hPBMCs were injected intraperitoneally to evaluate the effect of FLT3-CD3 bispecific antibody BsAb-125 on tumor growth, and compared with Molecule AMG427 for comparison.

MV-4-11-LucG cells were inoculated into female NDG mice (6-8 weeks old) at 1×10 ⁷ cells/mouse/200 μL tail vein, and each mouse was intraperitoneally injected with bioluminescent substrate about 10 days after inoculation. (15mg/mL, 10mL/kg), after 10 minutes, it was imaged by a small animal imaging system. When the Total Flux reached 4-5×10 ⁷ p/s, the hPBMCs (Sai Li Commercial, ID#: SC12220A) were collected at 3.5 ×10 ⁶ cells/mouse were injected into the peritoneal cavity of mice. After 5 days, the fluorescence was taken again, and the mice were randomly divided into 5 groups according to the fluorescence signal: Vehicle (PBS), AMG427 0.2/0.7mpk group, BsAb-125 0.3/1mpk group (respectively with AMG427 0.2mpk and 0.7mpk equimolar dose) (n=8/group). The day of the grouping was defined as Day 0 of the experiment, and each antibody (Biw) was administered intraperitoneally, ip, for a total of 4 times, and the fluorescence intensity and animal weight were monitored twice a week and the data were recorded. All data were drawn and statistically analyzed using Excel and GraphPad Prism 5 software. The bioluminescence signal value is Total Flux (unit, p/s), the average value is calculated in avg; the SD value is calculated in STDEV; the SEM value is calculated in STDEV/SQRT (number of animals in each group).

Table 23. MV-4-11-LucG orthotopic tumor efficacy test results

The experimental results showed that AMG427 high-dose and low-dose (0.7, 0.2mpk) groups showed strong anti-tumor effects, which were significantly different from the solvent group (p<0.01), and the tested antibody BsAb-125 had the strongest anti-tumor effect , and the anti-tumor effects were stronger than the equimolar amount of AMG427. In this experiment, the body weight of the tumor-bearing mice remained basically stable, and the antibodies showed no obvious toxicity and were well tolerated.

Test Example 8: In vivo safety evaluation of FLT3/CD3 bispecific antibody

Divide 6 cynomolgus monkeys (half male and half male, weighed the day before administration) into two groups, calculate the dosage according to the body weight, select a syringe of appropriate specification, and the error of the actual dosage should not exceed 5% of the theoretical dosage . Taking the day of administration as D0, pre-administration (0h), post-administration 0.083h, 2h, 4h, 6h, 24h, 48h, 96h, 168h, 240h, 336h, 504h, 672h were the PK blood collection points of each group. Take about 1 mL of whole blood from the front or hind limb vein (non-administered limb) of cynomolgus monkeys into an inert separation gel coagulation tube, let it stand at room temperature for about 30 minutes until the serum is precipitated, centrifuge at 2600g for 10 minutes at 4°C, and use the collected blood after centrifugation. Antibody concentration and release of cytokines IL-2, IL-4, IL-5, IL-6, TNF-α and IFNγ were detected in serum.

Table 24. Results of cytokine release assay data in cynomolgus monkeys

The results show that the PK of the BsAb-125 molecule of the present disclosure is good, and the release of cytokines is much lower than that of AMG427, so the BsAb-125 has higher safety.

Although the foregoing invention has been described in detail by means of drawings and examples for clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims

An antigen-binding molecule that specifically binds FLT3 and CD3, comprising at least one antigen-binding moiety that specifically binds FLT3 and at least one antigen-binding moiety that specifically binds CD3, the antigen-binding moiety that specifically binds FLT3 comprises a heavy chain Variable region FLT3-VH and light chain variable region FLT3-VL, the antigen-binding module specifically binding to CD3 comprises heavy chain variable region CD3-VH and light chain variable region CD3-VL; wherein

(i) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83 or 22, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or 23 and comprising the amino acid of SEQ ID NO: 24 sequence FLT3-LCDR3, or

(ii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or

(iii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 76 or 77 and comprising the amino acid of SEQ ID NO: 9 sequence of FLT3-HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, 78, 79 or 80, FLT3- comprising the amino acid sequence of SEQ ID NO: 11, 81 or 82 LCDR2 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12;

Preferably,

The antigen-binding molecule binds to human FLT3 with a KD of less than 1×10 −8 M at 25° C., and the KD is measured by surface plasmon resonance.
The antigen binding molecule according to claim 1, wherein:

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 5, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 3, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 4, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 2, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 amino acid sequence;

Preferably,

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 36, 37, 38, 39, 40 , 41, 42, 43 or 44 amino acid sequence;

More preferably,

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50.
The antigen-binding molecule according to claim 1 or 2, wherein

The antigen-binding module specifically binding to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 57, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 58, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 59; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 60 , CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 62;

Preferably,

Said CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and said CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule further comprises an Fc region, the Fc region is preferably an IgG Fc region, more preferably an IgG 1 Fc region;

Preferably, the Fc region comprises one or more amino acid substitutions capable of reducing the binding of the Fc region to Fcγ receptors;

More preferably, the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
The antigen-binding molecule according to claim 4, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, and each of the Fc1 and Fc2 independently has one or more reduced Fc regions Amino acid substitutions for homodimerization;

Preferably, said Fc1 has a convex structure according to the pestle and socket technique, and said Fc2 has a pore structure according to the pestle and socket technique, or said Fc1 has a pore structure according to the pestle and socket technique, and said Fc2 has a protrusion according to the pestle and socket technique structure;

More preferably, the amino acid residue at position 366 of the Fc1 is W; and the amino acid residue at position 366 of the Fc2 is The amino acid residue at position 368 is A, and the amino acid residue at position 407 is V, and the numbering is based on the EU index.
The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding moiety that specifically binds FLT3 is a Fab, and the antigen-binding moiety that specifically binds CD3 is a substituted Fab; or The antigen-binding module that specifically binds FLT3 is a replaced Fab, and the antigen-binding module that specifically binds CD3 is a Fab; the replaced Fab includes a Titin chain and an Obscurin chain capable of forming a dimer;

Preferably,

The Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 103, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104 to SEQ ID NO: 144;

More preferably,

The Titin chain comprises the amino acid sequence of SEQ ID NO: 103, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 138.
The antigen-binding molecule according to claim 6, which comprises an antigen-binding module that specifically binds FLT3 and an antigen-binding module that specifically binds CD3, the antigen-binding module that specifically binds FLT3 is a Fab, and the specificity The antigen binding moiety that binds CD3 is a substituted Fab;

Preferably,

The antigen-binding molecule comprises a first chain having a structure shown in formula (a), a second chain having a structure shown in formula (b), a third chain having a structure shown in formula (c) and a chain having a structure shown in formula (c) The fourth chain of the structure shown in formula (d),

(a) [FLT3-VH]-[CH1]-[Fc1],

(b)[FLT3-VL]-[CL],

(c) [CD3-VH]-[Linker 1]-[Titin chain]-[Fc2],

(d) [CD3-VL]-[Linker 2]-[Obscurin chain],

The structures shown in formulas (a), (b), (c) and (d) are arranged from the N-terminal to the C-terminal, and the linker 1 and the linker 2 are the same or different peptide linkers;

More preferably,

The antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 72, a second strand comprising the amino acid sequence of SEQ ID NO: 73, a third strand comprising the amino acid sequence of SEQ ID NO: 74 strand and a fourth strand comprising the amino acid sequence of SEQ ID NO:75.
The antigen-binding molecule according to claim 6, which comprises two antigen-binding moieties specifically binding to FLT3 and one antigen-binding moiety specifically binding to CD3, said anti-FLT3 specifically binding The original binding moiety is a Fab, and the antigen-binding moiety that specifically binds CD3 is a substituted Fab; preferably,

The antigen-binding molecule comprises a first chain having a structure shown in formula (e), two second chains having a structure shown in formula (b), a third chain having a structure shown in formula (f) and a A fourth chain having a structure shown in formula (g),

(e) [FLT3-VH]-[CH1]-[Fc2],

(b)[FLT3-VL]-[CL],

(f) [FLT3-VH]-[CH1]-[Linker 3]-[CD3-VL]-[Linker 4]-[Titin chain]-[Fc1],

(g) [CD3-VH]-[Linker 5]-[Obscurin chain],

The structures shown in formulas (e), (b), (f) and (g) are arranged from the N-terminal to the C-terminal, and the linker 3, the linker 4 and the linker 5 are the same or different peptide linker;

More preferably,

The antigen-binding molecule has: a first chain comprising the amino acid sequence of SEQ ID NO: 68, two second chains comprising the amino acid sequence of SEQ ID NO: 69, a second chain comprising the amino acid sequence of SEQ ID NO: 70 Three strands and a fourth strand comprising the amino acid sequence of SEQ ID NO:71.
An isolated antibody capable of specifically binding to FLT3, said antibody comprising a heavy chain variable region FLT3-VH and a light chain variable region FLT3-VL, wherein

(i) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 83 or 22, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or 23 and comprising the amino acid of SEQ ID NO: 24 sequence FLT3-LCDR3, or

(ii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and FLT3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 LCDR3, or

(iii) the FLT3-VH has: FLT3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, FLT3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, 76 or 77 and comprising the amino acid of SEQ ID NO: 9 sequence of FLT3-HCDR3, and the FLT3-VL has: FLT3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, 78, 79 or 80, FLT3- comprising the amino acid sequence of SEQ ID NO: 11, 81 or 82 LCDR2 and FLT3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12;

Preferably,

The antibody binds to human FLT3 with a KD of less than 1×10 −8 M at 25° C., and the KD is measured by surface plasmon resonance.
The isolated antibody of claim 9, wherein

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 5, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 3, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 4, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 1, 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 2, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 amino acid sequence;

Preferably,

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, 52 or 54, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56 or 55, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50, 49 or 51, or

(iii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33, 34 or 35, and said FLT3-VL comprises SEQ ID NO: 36, 37, 38, 39, 40 , 41, 42, 43 or 44 amino acid sequence;

More preferably,

(i) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 53, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 56, or

(ii) said FLT3-VH comprises the amino acid sequence of SEQ ID NO: 45, and said FLT3-VL comprises the amino acid sequence of SEQ ID NO: 50.
The isolated antibody of claim 9 or 10, wherein said antibody is a bispecific antibody;

Preferably, the bispecific antibody specifically binds FLT3 and CD3.
A pharmaceutical composition comprising:

A therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 8 or the antibody of any one of claims 9 to 11, and one or more pharmaceutically acceptable carriers, diluents, buffers agent or excipient;

Preferably, said pharmaceutical composition further comprises at least one second therapeutic agent.
An isolated nucleic acid encoding the antigen-binding molecule of any one of claims 1-8 or the antibody of any one of claims 9-11.
A host cell comprising the isolated nucleic acid of claim 13.
A method for treating or preventing a disease, the method comprising administering a therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 8 or any one of claims 9 to 11 to a subject in need thereof. The antibody or the step of the pharmaceutical composition described in claim 12;

Preferably, the disease is a tumor or cancer;

More preferably, the disease is selected from the group consisting of multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, Acute monocytic leukemia, plasma cell leukemia, plasmacytoma, B-prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, ex Somatic B-lymphoblastic lymphoma, myeloid leukemia, WM, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocyte Lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia, lymph node marginal zone B-cell lymphoma, splenic margin District lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell/histiocytic large B-cell lymphoma, primary central nervous system lymphoma, Primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK positive Large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma from HHV-8-associated multicentric Castleman disease, features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma unclassified B-cell lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, breast cancer, gastric cancer, liver cancer, lung cancer, cervical cancer, nodal Cancers of the rectum, endometrium, head and neck, mesothelioma, ovary, pancreas, prostate, skin, kidney and bladder.