Method for generating bispecific antibodies based on common light chain

Abstract

Relates to a novel platform method for preparing a bispecific antibody, in particular to a platform method for preparing a bispecific antibody with a common light chain, the obtained bispecific antibody and application of the bispecific antibody.

Description

Methods for generating bispecific antibodies based on a common light chain Technical Field

[0001] The present application relates to a new platform method for preparing bispecific antibodies, and specifically to a platform method for preparing bispecific antibodies with a common light chain, as well as the bispecific antibodies obtained thereby and their uses. Background Art

[0002] Camelids possess both heavy-chain-only nanobodies and traditional antibodies. Nanobodies have attracted significant attention due to their excellent druggability and ease of constructing multispecific antibodies. However, when testing serum titers in alpacas, it is often not possible to obtain only the results for nanobodies. In fact, even when the serum titer is detected, the nanobodies are not detected. Furthermore, studies have found that even within the same alpaca, the epitopes recognized by nanobodies and traditional antibodies produced by them differ. This is due to the differences in their antigen-binding modes: nanobodies are more likely to bind to cracks or cavities on the protein surface, while traditional antibodies bind to larger epitopes. If only alpaca nanobodies are screened, antibodies that bind to specific epitopes are likely to be missed. In camelids, the traditional IgG1 antibody accounts for approximately 50% of the total IgG, and its heavy and light chains share over 90% identity with human antibodies, offering significant therapeutic potential. To this end, we provide a new method for constructing bispecific antibodies containing traditional alpaca antibodies. This method avoids light chain mispairing, improves the purity of bispecific antibodies, increases yield, reduces production costs, and provides a better method for the development of bispecific antibodies.

[0003] Summary of the Invention

[0004] The present application has developed a new construction platform for preparing bispecific antibodies using a common light chain, that is, first by constructing a hybrid scFv library with a common light chain by combining the heavy chain variable region of a traditional antibody for a first antigen with the light chain variable region of an antibody for a second antigen, then screening the hybrid scFv library for immunogen (first antigen) to obtain the target hybrid scFv, the light chain carried by these target scFvs is identical to the light chain of the antibody for the second antigen, and then preparing the heavy chain variable region of the target scFv into full-length heavy chains and expressing them in combination with the antibody for the second antigen, thus obtaining a bispecific antibody with a common second antibody light chain. This platform is a completely new method for constructing bispecific antibodies. This method avoids the common light chain mispairing problem when preparing bispecific antibodies, reduces by-products, and therefore not only improves the purity of the target bispecific antibodies, increases yield, but also reduces production consumption and improves production efficiency.

[0005] This platform can be applied to a variety of immune animals, such as mice, rats, rabbits, and camelids. It is preferred to prepare bispecific antibodies using the above method in camelids. Generally, using camelids (e.g., alpacas) can obtain a richer immune library.

[0006] Because camelid (e.g., alpacas) antibody genes share up to 80% similarity with human antibody genes, and the key amino acid residues at the VH / VL interface are largely identical, it is preferred that the antibody against the second target be a humanized or human-derived antibody. Thus, the camelid VH targeting the first target can form a stable antibody structure with the human VL targeting the second target, resulting in low immunogenicity.

[0007] Therefore, the novel platform for preparing bispecific antibodies provided by the present invention has at least the following advantages:

[0008] 1. Avoiding the light chain mispairing problem during the preparation process, thereby producing a high concentration of the target bispecific antibody product and improving efficiency;

[0009] 2. The camelid antibody heavy chain variable region and the human antibody light chain variable region can form a stable hybrid scFv, avoiding further modification of the heavy chain variable region, simplifying the preparation process and improving production efficiency;

[0010] 3. High-abundance immune libraries can be obtained;

[0011] 4. The bispecific antibody of interest obtained through screening can be exempted from humanization modification;

[0012] 5. The obtained bispecific antibody has low immunogenicity.

[0013] In one aspect, the present invention provides a method for preparing a bispecific antibody, comprising: 1) obtaining all heavy chain variable regions of a library of corresponding camelid immunoglobulin antibodies (first antibodies) against a first antigen, and obtaining light chain variable regions of a second antibody against a second antigen; 2) co-expressing all heavy chain variable regions of the first antibody library with light chain variable regions of the second antibody to obtain a hybrid library; 3) screening the hybrid library against the first antigen to obtain heavy chain variable regions that specifically recognize the first antigen; 4) preparing heavy chains from the heavy chain variable regions that recognize the first antigen obtained in step 3); and 5) co-expressing the heavy chain obtained in step 4) with the heavy chain and light chain of the second antibody to form a bispecific antibody that specifically binds to the first antigen and the second antigen and has the same light chain of the second antibody. In one embodiment, the bispecific antibody prepared by the method of the present application is composed of two different heavy chains and two identical light chains, wherein the first heavy chain is derived from a traditional antibody that recognizes a first antigen (preferably a traditional antibody of the camelid family), the second heavy chain is derived from an antibody that recognizes a second antigen, and the two identical light chains are derived from an antibody that recognizes the second antigen, wherein the two heavy chains respectively interact with the two light chains to form binding arms that specifically bind to the first antigen and the second antigen.

[0014] In one embodiment, the hybrid library is a hybrid scFv library or a hybrid Fab library. In a preferred embodiment, the nucleic acids encoding all heavy chain variable regions of the first antibody library are respectively inserted into an expression vector containing a nucleic acid encoding the light chain variable region of the second antibody and co-expressed, thereby obtaining a hybrid scFv library. In another embodiment, the nucleic acids encoding all heavy chain variable regions of the first antibody library and the nucleic acids encoding the light chain variable region of the second antibody are respectively inserted into different expression vectors and co-expressed, thereby obtaining a hybrid Fab library.

[0015] In one embodiment, the immunoglobulin in step 1) is IgG. In one embodiment, the second antibody is a human antibody.

[0016] In one embodiment, the camelid immunoglobulin antibody library in step 1) can be an immune library, a natural library, or a synthetic antibody library (for example, but not limited to, a yeast library or a phage library). In one embodiment, the immunoglobulin antibody library in step 1) is obtained by conventional methods. In a specific embodiment, the immunoglobulin antibody library in step 1) is an immune library. In a specific embodiment, the immune library is obtained by immunizing a camelid with a first antigen, optionally testing the camelid serum, and collecting peripheral blood mononuclear cells (PBMCs) after obtaining a titer that meets the requirements. All heavy chain variable region sequences or their encoding nucleotide sequences of the first antibody library against the first antigen are obtained from the peripheral blood mononuclear cells.

[0017] In one embodiment, the hybrid scFv library in the above method is prepared as follows: the encoding nucleotide sequences of all heavy chain variable regions of the first antibody library are separately constructed into a vector containing a nucleotide sequence encoding the light chain variable region of the second antibody; the obtained vector is transfected into a host cell and the vector is expressed under appropriate conditions, thereby obtaining a hybrid scFv library. In one embodiment, the hybrid Fab library in the above method is prepared as follows: the nucleotide sequence encoding the heavy chain variable region of the first antibody and the nucleotide sequence encoding the light chain variable region of the second antibody are separately constructed in different vectors; then the host cell is co-transfected and co-expressed under appropriate conditions, thereby obtaining the corresponding hybrid Fab library. In a preferred embodiment, the vector is an expression vector, such as a phagemid vector. In one embodiment, the hybrid scFv library can be, for example, a yeast library or a phage library. In one embodiment, the hybrid Fab library can be, for example, a yeast library or a phage library.

[0018] In one embodiment, the camelid is selected from dromedary, Bactrian camel, alpaca, guanaco, llama, etc., preferably alpaca.

[0019] In one embodiment, in step 4) of the above method, the heavy chain is formed by combining the heavy chain variable region of the first antibody with the IgG antibody constant region. Preferably, the IgG antibody constant region is an IgG1 antibody constant region. In another embodiment, the constant region has amino acid modifications, such as modifications that improve constant region interactions, modifications that improve ADCC activity, modifications that remove ADCC effector functions, etc. For example, in one embodiment, the modifications can be selected from mutations T366W, S354C, L234A L235A, D265A P329A, Y407V, T366S, L368A, Y349C. In one embodiment, the heavy chain constant region of the second antibody is modified, such as modifications that improve constant region interactions, modifications that improve ADCC activity, modifications that remove ADCC effector functions, etc. For example, in one embodiment, the modification can be selected from the group consisting of mutations T366W, S354C, L234A L235A, D265A P329A, Y407V, T366S, L368A, Y349C. In one embodiment, the nucleic acid sequence encoding the first antibody heavy chain variable region is constructed into a vector comprising a nucleic acid sequence encoding an IgG antibody constant region.

[0020] In one embodiment, the first antigen is FGFR2IIIB and the second antigen is VEGFR2. In another embodiment, the first antigen is LILIRB4 and the second antigen is CD3.

[0021] In one embodiment, the present application provides a method for preparing a bispecific antibody against FGFR2IIIB and VEGFR2, comprising: 1) immunizing a camelid (preferably an alpaca) with FGFR2IIIB to obtain all heavy chain variable regions of an anti-FGFR2IIIB immunoglobulin antibody (first antibody) library; 2) obtaining a light chain variable region of a second antibody against VEGFR2; 3) co-expressing all heavy chain variable regions of the anti-FGFR2IIIB antibody library with the light chain variable region of the anti-VEGFR2 antibody to obtain a bispecific antibody having covalent bonds. The method further comprises the steps of: preparing a hybrid scFv library having the same light chain variable region as an anti-VEGFR2 antibody; 4) screening the hybrid scFv library against FGFR2IIIB to obtain a hybrid scFv that specifically recognizes FGFR2IIIB; 5) preparing the heavy chain variable region in the hybrid scFv obtained in step 4) into a heavy chain; 6) co-expressing the heavy chain obtained in step 5) with the heavy chain and light chain of the anti-VEGFR2 antibody, thereby forming a bispecific antibody that specifically binds to FGFR2IIIB and VEGFR2 and has the same anti-VEGFR2 antibody light chain. In an alternative embodiment, the above step 3) obtains a hybrid Fab library having a common light chain variable region of an anti-VEGFR2 antibody, screening the hybrid Fab library in step 4) to obtain a hybrid Fab that specifically recognizes FGFR2IIIB, and preparing the heavy chain variable region in the hybrid Fab obtained in step 4) into a heavy chain in step 5).

[0022] In a specific embodiment, the generated anti-FGFR2 IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2 IIIB and VEGFR2, respectively, wherein:

[0023] 1) The antibody arm that binds to FGFR2IIIB comprises the HCDR1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, the HCDR3 of SEQ ID NO: 3, the LCDR1 of SEQ ID NO: 45, the LCDR2 of SEQ ID NO: 46, and the LCDR3 of SEQ ID NO: 47; wherein the antibody arm that binds to VEGFR2 comprises the HCDR1 of SEQ ID NO: 48, the HCDR2 of SEQ ID NO: 49, the HCDR3 of SEQ ID NO: 50, the LCDR1 of SEQ ID NO: 45, the LCDR2 of SEQ ID NO: 46, and the LCDR3 of SEQ ID NO: 47; or

[0024] 2) The antibody arm that binds to FGFR2IIIB comprises the HCDR1 shown in SEQ ID NO:8, the HCDR2 shown in SEQ ID NO:9, the HCDR3 shown in SEQ ID NO:10, the LCDR1 shown in SEQ ID NO:45, the LCDR2 shown in SEQ ID NO:46, and the LCDR3 shown in SEQ ID NO:47; wherein the antibody arm that binds to VEGFR2 comprises the HCDR1 shown in SEQ ID NO:48, the HCDR2 shown in SEQ ID NO:49, the HCDR3 shown in SEQ ID NO:50, the LCDR1 shown in SEQ ID NO:45, the LCDR2 shown in SEQ ID NO:46, and the LCDR3 shown in SEQ ID NO:47.

[0025] In a specific embodiment, the generated anti-FGFR2 IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2 IIIB and VEGFR2, respectively, wherein:

[0026] 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; or

[0027] 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 11 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15.

[0028] In a specific embodiment, the anti-FGFR2IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein:

[0029] 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO: 7 and a light chain comprising the sequence shown in SEQ ID NO: 6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 16 and a light chain comprising the sequence shown in SEQ ID NO: 6; or

[0030] 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO: 13 and a light chain comprising the sequence shown in SEQ ID NO: 6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 16 and a light chain comprising the sequence shown in SEQ ID NO: 6.

[0031] In one embodiment, the present application provides a method for preparing a bispecific antibody against LILIRB4 and CD3, comprising: 1) immunizing a camelid (preferably an alpaca) with LILIRB4 to obtain all heavy chain variable regions of an anti-LILIRB4 immunoglobulin antibody (first antibody) library; 2) obtaining a light chain variable region of a second antibody against CD3; 3) co-expressing all heavy chain variable regions of the anti-LILIRB4 antibody library with the light chain variable regions of an anti-CD3 antibody to obtain a hybrid scFv library having a common anti-CD3 antibody light chain variable region; 4) screening the hybrid scFv library for LILIRB4 to obtain a hybrid scFv that specifically recognizes LILIRB4; 5) preparing the heavy chain variable region in the hybrid scFv obtained in step 4) into a heavy chain; 6) co-expressing the heavy chain obtained in step 5) with the heavy chain and light chain of an anti-CD3 antibody, thereby forming a bispecific antibody that specifically binds to LILIRB4 and CD3 and has the same anti-CD3 antibody light chain. In an alternative embodiment, the above step 3) obtains a hybrid Fab library having a common anti-CD3 antibody light chain variable region, the hybrid Fab library is screened in step 4) to obtain a hybrid Fab that specifically recognizes LILIRB4, and in step 5), the heavy chain variable region in the hybrid Fab obtained in step 4) is prepared into a heavy chain.

[0032] In a specific embodiment, the anti-LILIRB4 and CD3 bispecific antibody comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0033] 1) The antibody arm that binds to LILIRB4 comprises the HCDR1 of SEQ ID NO: 18, the HCDR2 of SEQ ID NO: 19, the HCDR3 of SEQ ID NO: 20, the LCDR1 of SEQ ID NO: 54, the LCDR2 of SEQ ID NO: 55, and the LCDR3 of SEQ ID NO: 56; wherein the antibody arm that binds to CD3 comprises the HCDR1 of SEQ ID NO: 51, the HCDR2 of SEQ ID NO: 52, the HCDR3 of SEQ ID NO: 53, the LCDR1 of SEQ ID NO: 54, the LCDR2 of SEQ ID NO: 55, and the LCDR3 of SEQ ID NO: 56;

[0034] 2) the antibody arm that binds to LILIRB4 comprises the HCDR1 of SEQ ID NO:25, the HCDR2 of SEQ ID NO:26, the HCDR3 of SEQ ID NO:27, the LCDR1 of SEQ ID NO:54, the LCDR2 of SEQ ID NO:55, and the LCDR3 of SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises the HCDR1 of SEQ ID NO:51, the HCDR2 of SEQ ID NO:52, the HCDR3 of SEQ ID NO:53, the LCDR1 of SEQ ID NO:54, the LCDR2 of SEQ ID NO:55, and the LCDR3 of SEQ ID NO:56; or

[0035] 3) The antibody arm that binds to LILIRB4 comprises the HCDR1 shown in SEQ ID NO:32, the HCDR2 shown in SEQ ID NO:33, the HCDR3 shown in SEQ ID NO:34, the LCDR1 shown in SEQ ID NO:54, the LCDR2 shown in SEQ ID NO:55, and the LCDR3 shown in SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises the HCDR1 shown in SEQ ID NO:51, the HCDR2 shown in SEQ ID NO:52, the HCDR3 shown in SEQ ID NO:53, the LCDR1 shown in SEQ ID NO:54, the LCDR2 shown in SEQ ID NO:55, and the LCDR3 shown in SEQ ID NO:56.

[0036] In a specific embodiment, the anti-LILIRB4 and CD3 bispecific antibody comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0037] 1) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 21 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 39 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40;

[0038] 2) the antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 39 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; or

[0039] 3) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 35 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 39 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40.

[0040] In a specific embodiment, the anti-LILIRB4 and CD3 bispecific antibody comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0041] 1) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 24 and a light chain comprising the sequence set forth in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 41 and a light chain comprising the sequence set forth in SEQ ID NO: 23;

[0042] 2) the antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 31 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: 23; or

[0043] 3) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 38 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: 23.

[0044] In a second aspect, the present invention provides a bispecific antibody having identical antibody light chains prepared by the method of the first aspect, which is composed of two different heavy chains and two identical light chains, wherein the first heavy chain is derived from an immunoglobulin antibody of the camelid family that recognizes a first antigen, the second heavy chain is derived from a human antibody that recognizes a second antigen, and the two identical light chains are derived from a human antibody that recognizes the second antigen, wherein the two heavy chains interact with the two light chains to form binding arms that specifically bind to the first antigen and the second antigen. In one embodiment, the first antigen is FGFR2IIIB and the second antigen is VEGFR2. In another embodiment, the first antigen is LILIRB4 and the second antigen is CD3.

[0045] In a specific embodiment, the present invention provides a bispecific antibody against FGFR2IIIB and VEGFR2, wherein the bispecific antibody against FGFR2IIIB and VEGFR2 comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein:

[0046] 1) The antibody arm that binds to FGFR2IIIB comprises the HCDR1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, the HCDR3 of SEQ ID NO: 3, the LCDR1 of SEQ ID NO: 45, the LCDR2 of SEQ ID NO: 46, and the LCDR3 of SEQ ID NO: 47; wherein the antibody arm that binds to VEGFR2 comprises the HCDR1 of SEQ ID NO: 48, the HCDR2 of SEQ ID NO: 49, the HCDR3 of SEQ ID NO: 50, the LCDR1 of SEQ ID NO: 45, the LCDR2 of SEQ ID NO: 46, and the LCDR3 of SEQ ID NO: 47; or

[0047] 2) The antibody arm that binds to FGFR2IIIB comprises the HCDR1 shown in SEQ ID NO:8, the HCDR2 shown in SEQ ID NO:9, the HCDR3 shown in SEQ ID NO:10, the LCDR1 shown in SEQ ID NO:45, the LCDR2 shown in SEQ ID NO:46, and the LCDR3 shown in SEQ ID NO:47; wherein the antibody arm that binds to VEGFR2 comprises the HCDR1 shown in SEQ ID NO:48, the HCDR2 shown in SEQ ID NO:49, the HCDR3 shown in SEQ ID NO:50, the LCDR1 shown in SEQ ID NO:45, the LCDR2 shown in SEQ ID NO:46, and the LCDR3 shown in SEQ ID NO:47.

[0048] In a specific embodiment, the anti-FGFR2IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein:

[0049] 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; or

[0050] 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 11 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15.

[0051] In a specific embodiment, the anti-FGFR2IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein:

[0052] 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO: 7 and a light chain comprising the sequence shown in SEQ ID NO: 6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 16 and a light chain comprising the sequence shown in SEQ ID NO: 6; or

[0053] 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO: 13 and a light chain comprising the sequence shown in SEQ ID NO: 6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 16 and a light chain comprising the sequence shown in SEQ ID NO: 6.

[0054] In one embodiment, the present application provides a bispecific antibody against LILIRB4 and CD3, comprising antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0055] 1) The antibody arm that binds to LILIRB4 comprises the HCDR1 of SEQ ID NO: 18, the HCDR2 of SEQ ID NO: 19, the HCDR3 of SEQ ID NO: 20, the LCDR1 of SEQ ID NO: 54, the LCDR2 of SEQ ID NO: 55, and the LCDR3 of SEQ ID NO: 56; wherein the antibody arm that binds to CD3 comprises the HCDR1 of SEQ ID NO: 51, the HCDR2 of SEQ ID NO: 52, the HCDR3 of SEQ ID NO: 53, the LCDR1 of SEQ ID NO: 54, the LCDR2 of SEQ ID NO: 55, and the LCDR3 of SEQ ID NO: 56;

[0056] 2) the antibody arm that binds to LILIRB4 comprises the HCDR1 of SEQ ID NO:25, the HCDR2 of SEQ ID NO:26, the HCDR3 of SEQ ID NO:27, the LCDR1 of SEQ ID NO:54, the LCDR2 of SEQ ID NO:55, and the LCDR3 of SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises the HCDR1 of SEQ ID NO:51, the HCDR2 of SEQ ID NO:52, the HCDR3 of SEQ ID NO:53, the LCDR1 of SEQ ID NO:54, the LCDR2 of SEQ ID NO:55, and the LCDR3 of SEQ ID NO:56; or

[0057] 3) The antibody arm that binds to LILIRB4 comprises the HCDR1 shown in SEQ ID NO:32, the HCDR2 shown in SEQ ID NO:33, the HCDR3 shown in SEQ ID NO:34, the LCDR1 shown in SEQ ID NO:54, the LCDR2 shown in SEQ ID NO:55, and the LCDR3 shown in SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises the HCDR1 shown in SEQ ID NO:51, the HCDR2 shown in SEQ ID NO:52, the HCDR3 shown in SEQ ID NO:53, the LCDR1 shown in SEQ ID NO:54, the LCDR2 shown in SEQ ID NO:55, and the LCDR3 shown in SEQ ID NO:56.

[0058] In a specific embodiment, the anti-LILIRB4 and CD3 bispecific antibody comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0059] 1) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 21 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 39 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40;

[0060] 2) the antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 39 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 40; or

[0061] 3) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 35 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 39 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40.

[0062] In a specific embodiment, the anti-LILIRB4 and CD3 bispecific antibody comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein:

[0063] 1) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 24 and a light chain comprising the sequence set forth in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 41 and a light chain comprising the sequence set forth in SEQ ID NO: 23;

[0064] 2) the antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 31 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: 23; or

[0065] 3) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 38 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: 23.

[0066] In a third aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB, wherein the antibody or antigen-binding fragment thereof comprises:

[0067] 1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 45, LCDR2 shown in SEQ ID NO: 46, and LCDR3 shown in SEQ ID NO: 47; or

[0068] 2) HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:9, HCDR3 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47.

[0069] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB comprises:

[0070] 1) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; or

[0071] 2) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 11 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15.

[0072] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB comprises:

[0073] 1) a heavy chain comprising the sequence shown in SEQ ID NO: 5 and a light chain comprising the sequence shown in SEQ ID NO: 6; or

[0074] 2) A heavy chain comprising the sequence shown in SEQ ID NO: 12 and a light chain comprising the sequence shown in SEQ ID NO: 6.

[0075] In a fourth aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to LILIRB4, wherein the antibody or antigen-binding fragment thereof comprises:

[0076] 1) HCDR1 set forth in SEQ ID NO: 18, HCDR2 set forth in SEQ ID NO: 19, HCDR3 set forth in SEQ ID NO: 20, LCDR1 set forth in SEQ ID NO: 54, LCDR2 set forth in SEQ ID NO: 55, and LCDR3 set forth in SEQ ID NO: 56;

[0077] 2) HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; or

[0078] 3) HCDR1 shown in SEQ ID NO:32, HCDR2 shown in SEQ ID NO:33, HCDR3 shown in SEQ ID NO:34, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56.

[0079] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 comprises:

[0080] 1) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40;

[0081] 2) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 28 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; or

[0082] 3) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 35 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40.

[0083] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 comprises:

[0084] 1) a heavy chain comprising the sequence shown in SEQ ID NO: 22 and a light chain comprising the sequence shown in SEQ ID NO: 23;

[0085] 2) a heavy chain comprising the sequence shown in SEQ ID NO: 29 and a light chain comprising the sequence shown in SEQ ID NO: 23; or

[0086] 3) a heavy chain comprising the sequence shown in SEQ ID NO: 36 and a light chain comprising the sequence shown in SEQ ID NO: 23.

[0087] In one embodiment, the antigen-binding fragment described in the third or fourth aspect of the present invention is, for example, a Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment.

[0088] In one embodiment, the antibodies or antigen-binding fragments thereof described in the second, third, and fourth aspects herein may have amino acid modifications, such as amino acid modifications in the antibody constant region, such as modifications that improve constant region interactions, modifications that improve ADCC activity, or modifications that eliminate ADCC effector function. For example, in one embodiment, the modifications are selected from the group consisting of mutations T366W, S354C, L234A, L235A, D265A, P329A, Y407V, T366S, L368A, and Y349C.

[0089] In one embodiment, the two heavy chain constant regions in the bispecific antibody of the invention dimerize to form a dimeric heavy chain constant region. Preferably, the two heavy chain constant regions are different and heterodimerize to form a heterodimeric heavy chain constant region.

[0090] In some embodiments, the first and second heavy chain constant regions are identical. In other embodiments, the first heavy chain constant region and the second heavy chain constant region are different and the two pair and heterodimerize.

[0091] The heavy chain constant region fragments suitable for use in the antibody molecules of the present invention can be any antibody heavy chain constant region. The heavy chain constant region can include native sequence heavy chain constant regions and variant heavy chain constant regions. The native sequence heavy chain constant region encompasses naturally occurring various immunoglobulin heavy chain constant region sequences, such as various Ig subtypes and their allotypes.

[0092] Preferably, the heavy chain constant region of the antibody of the present invention comprises, from N-terminus to C-terminus: CH1-hinge region-CH2-CH3. In some embodiments, the heavy chain constant region suitable for use in the antibodies or bispecific antibodies of the present invention is a human IgG heavy chain constant region, for example, a human IgG1, IgG, IgG3, or human IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region, for example, comprising or consisting of an amino acid sequence having at least 90% identity thereto, for example, 95%, 96%, 97%, 99% or higher identity thereto. Preferably, the heavy chain constant region comprises an Fc region.

[0093] The Fc region of the antibody or bispecific antibody of the present invention can be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0094] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0095] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0096] As will be appreciated by those skilled in the art, according to the intended use of the antibody molecule of the present invention, the antibody molecule of the present invention may also include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has one or more of the L234A / L235A mutation, the D265A mutation, or the P329A mutation that reduces binding to the Fcγ receptor. In some embodiments, the Fc region used in the present invention has a L234A / L235A mutation, the D265A mutation, and the P329A mutation that reduces binding to the Fcγ receptor. In a preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the heavy chain constant region of the Fc region comprising a mutation that reduces binding to Fcγ receptors comprises or consists of the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity. In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 57 and comprises L234A / L235A mutations, D265A mutations, and P329A mutations.

[0097] As will be appreciated by those skilled in the art, in order to promote the formation of the bispecific antibodies of the present invention as heterodimers, the heavy chain constant regions comprised by the bispecific antibodies of the present invention may comprise mutations that are conducive to heterodimerization. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0098] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the first and second CH3 regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0099] Preferably, corresponding knob mutations and hole mutations are introduced into the first monomer Fc region and the second monomer Fc region, respectively, based on the Knob-in-Hole technique. For this technique, see, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0100] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index).

[0101] In a further embodiment, in the CH3 region of one Fc region, the knob mutation comprises or consists of substitution of the threonine residue at position 366 with a tryptophan residue (T366W) and substitution of the serine residue at position 354 with a cysteine ​​residue (S354C) or substitution of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, substitution of the serine residue at position 354 with a cysteine ​​residue); and in the CH3 region of another Fc region, the hole mutation comprises or consists of substitution of the tyrosine residue at position 407 with a valine residue (Y407V), optionally substitution of the threonine residue at position 366 with a serine residue (T366S) and substitution of the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the EU index), optionally substitution of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0102] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W (knob mutation), and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (hole mutation) (numbering is according to the EU index).

[0103] Thus, in a specific embodiment, the bispecific antibody of the present invention comprises two Fc regions that are heterodimerized, wherein

[0104] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0105] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0106] c) one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C;

[0107] Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, eg, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; eg, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0108] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0109] In a specific embodiment, the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein

[0110] the first Fc region comprises a knob mutation, and the heavy chain constant region comprising the first Fc region comprises the amino acid sequence of SEQ ID NO:60, or comprises an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical, to SEQ ID NO:60, or consists of SEQ ID NO:60; in some embodiments, the heavy chain constant region comprises an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical, to SEQ ID NO:60 and comprises a knob mutation (e.g., S354C and T366W);

[0111] The second Fc region comprises a hole mutation, and the heavy chain constant region comprising the second Fc region comprises the amino acid sequence of SEQ ID NO: 61, or comprises an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 61, or consists of SEQ ID NO: 61. In some embodiments, the heavy chain constant region comprises an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 61 and comprises a hole mutation.

[0112] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the second, third, and fourth aspects of the present application; a vector comprising the nucleic acid molecule; and a host cell comprising the nucleic acid molecule or the vector.

[0113] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in aspects 2, 3, or 4 of this application; and uses of the antibody or antigen-binding fragment thereof, or pharmaceutical composition, described in aspects 2, 3, or 4 in treating a disease. In one embodiment, the present invention provides uses of the antibody or antigen-binding fragment thereof, or pharmaceutical composition, described in aspects 2, 3, or 4, or the pharmaceutical composition of aspect 5, in preparing a medicament for treating a disease.

[0114] In a specific embodiment, the disease is associated with abnormal expression of the antigen to which the antibody specifically binds, for example, the disease is associated with abnormal expression of FGFR2IIIB, abnormal expression of VEGFR2, abnormal expression of LILIRB4, or abnormal expression of CD3.

[0115] In one embodiment, the present invention provides a method for treating a disease in a subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding fragment thereof of the second, third, or fourth aspects, or the pharmaceutical composition of the fifth aspect to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG1 shows the binding activity of the positive clones in Example 4 to KATOIII and SNU-16 cells expressing human FGFR2IIIB.

[0117] FIG2 shows the binding activity of the specific antibody in Example 7 to cells specifically expressing cynomolgus monkey FGFR2IIIB.

[0118] FIG3 shows the sandwich ELISA assay for binding of the bispecific antibody to FGFR2IIIB and VEGFR2 in Example 10.

[0119] FIG4 shows the sandwich ELISA method of Example 16 for detecting the bispecific antibody and human LILRB4 protein, which can also bind to human CD3 protein.

[0120] FIG5 shows the SEC detection results of the specific hybrid antibodies B4-Ram-KH antibody and 1B4-Ram-KH antibody obtained in Example 8.

[0121] FIG6 shows the SEC detection results of the specific hybrid antibodies 1B2-SP34-KH antibody, 1D5-SP34-KH and 1H4-SP34-KH antibody obtained in Example 16.

[0122] Detailed Description of the Invention

[0123] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0124] 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. For the purposes of the present invention, the following terms are defined below.

[0125] I. Definition

[0126] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0127] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0128] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0129] The term "antibody" is used in the broadest sense herein to refer to a protein comprising an antigen binding site, and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain circumstances, for example, antibodies naturally occurring in camels may comprise only heavy chains.

[0130] The terms "immunoglobulin," "conventional antibody," "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably herein and refer to a glycoprotein having a structure substantially similar to that of a natural antibody, comprising two full-length heavy chains (H) and two full-length light chains (L) interconnected by disulfide bonds. The immunoglobulin heavy chain comprises a heavy chain variable region (VH, also called a heavy chain variable domain) and a heavy chain constant region (CH, also called a heavy chain constant domain), while the light chain comprises a light chain variable region (VL, also called a light chain variable domain) and a light chain constant region (CL, also called a light chain constant domain).

[0131] Immunoglobulins can be divided into five classes based on the amino acid sequence of the constant region of their heavy chains: IgA, IgD, IgE, IgG, and IgM. Some classes can be further divided into subclasses, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4 subclasses, and IgA, which can be divided into IgA1 and IgA2 subclasses. Based on the amino acid sequence of the constant domain of their light chains, immunoglobulins can be divided into one of two types: kappa and lambda.

[0132] IgG immunoglobulins are essentially heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. The IgG heavy chain constant region comprises three domains: CH1, CH2, and CH3, with CH1 and CH2 connected by a hinge region. The CH2 and CH3 domains typically comprise the Fc region of IgG immunoglobulins, mediating various effector functions through interaction with their receptors (FcγRs).

[0133] The qualifier "hybrid" appearing in the terms "hybrid antibody", "hybrid scFv" or "hybrid Fab" means that the antibody heavy chain variable region and the light chain variable region in the corresponding antibody, scFv or Fab are derived from the heavy chain variable region and the light chain variable region of antibodies, scFv or Fab directed against different targets, respectively.

[0134] The term "antibody fragment" refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and is capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain antibodies (e.g., scFv); single-domain antibodies; and camelid antibodies (heavy-chain antibodies). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by papain digestion of an intact antibody.

[0135] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.

[0136] "Complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs in the variable domain are typically referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and affinity propagation clustering based on a large number of crystal structures. The North CDR definition of propagation clustering (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0137] For example, different schemes are employed to define different extents of CDR regions using the Kabat and Chothia numbering systems.

[0138] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined by any of the above schemes.

[0139] CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the position determined according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0140] The term "immune library" refers to a library of immunoglobulin (Ig) variable region (V) genes obtained by immunizing an animal with a specific immunogen. These V genes are highly biased toward producing antibodies that recognize that immunogen. Animals that can be immunized include alpacas, rabbits, and rodents (e.g., mice and rats).

[0141] The term "natural library" refers to a library prepared from immunoglobulin variable region (V) genes from people or animals that have not been immunized with a specific immunogen; the antibodies encoded by the V genes have no bias in recognizing specific antigens, and the antibodies screened generally have low affinity.

[0142] The term "synthetic library" refers to a library obtained by preparing synthetic V region genes using bioengineering methods (such as the polymerase chain reaction (PCR) method).

[0143] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by conventional methods known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0144] The term "treat" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

[0145] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0146] II. Preparation of Antibodies of the Invention

[0147] In one embodiment, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or its antigen-binding fragment or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the antibody or its antigen-binding fragment, and optionally isolating the antibody or its antigen-binding fragment. In a certain embodiment, the method further comprises recovering the antibody or its antigen-binding fragment from the host cell (or host cell culture medium).

[0148] In order to recombinantly produce the antibodies or antigen-binding fragments thereof of the present invention, nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention are first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, using oligonucleotide probes that specifically bind to nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention.

[0149] The antibodies or antigen-binding fragments thereof of the present invention, prepared as described herein, can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibodies or antigen-binding fragments thereof of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. Example

[0150] The present invention is further illustrated by the following examples; however, it should be understood that the examples are described in an illustrative rather than a limiting sense and that various modifications may be made by those skilled in the art.

[0151] Unless expressly stated to the contrary, the present invention will be practiced using conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art. The experimental materials used, unless otherwise specified, are commercially available. Where specific techniques or conditions are not specified in the examples, the experiments were performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the corresponding product specifications. Where the manufacturer of the reagents or instruments used is not specified, all are conventional products that can be purchased through regular channels.

[0152] Example 1 Animal Immunization

[0153] Two alpacas, A and B (Suzhou Aikangde Biotechnology Co., Ltd.), were selected for immunization. Before immunization, 5 mL of blood was collected from each alpaca, and serum was collected as a negative control for immune titer testing. Immunization method: For the first immunization, complete Freund's adjuvant (Sigma-Aldrich, Catalog No. F5881) was mixed with 0.25 mg of human FGFR2IIIB-his protein (ACRO, Catalog No. FGB-H5223) in a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. Twenty-one days after the first immunization, a second immunization was performed with 0.25 mg of human FGFR2IIIB-his protein mixed with incomplete Freund's adjuvant (Sigma-Aldrich, Catalog No. F5506) in a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. A total of five immunizations were performed, with a 3-week interval between each immunization. The protocol for immunizations 3-5 was the same as for the second.

[0154] Starting from the second immunization, 5 mL of peripheral blood was collected every 10 days after each immunization to monitor the immune response. After the immunization was completed, peripheral blood mononuclear cells (PBMCs) were obtained from the alpacas for library construction.

[0155] Example 2. Construction of a phagemid vector containing the light chain variable region of Ramucirumab

[0156] The nucleotide sequence of the light chain variable region of ramucirumab (an anti-VEGFR2 antibody) was amplified by PCR using primers containing the restriction sites BamHI and HindIII. The nucleotide sequence was then inserted into a phagemid vector modified to contain restriction sites (BamHI and HindIII) using enzyme ligation (BamHI and HindIII). The constructed phagemid vector, named pSid-Rmab-VL, contains a (G4S)4 linker between the multiple cloning site (MCS) and the nucleotide sequence of the light chain variable region. The NheI and NotI regions of the multiple cloning site serve as insertion sites for the heavy chain variable region. The heavy chain variable region (VH) containing the restriction site can be ligated to the N-terminus of the linker-VL using enzyme ligation (NheI and NotI), ultimately forming an scFv.

[0157] Example 3. Immune library construction and screening

[0158] 1. Construction of a library containing VH of traditional camelid antibodies

[0159] Total RNA was extracted from the peripheral blood PBMC of the alpaca obtained in Example 1 using the Trizol method, and 5 μg of RNA was reverse transcribed and analyzed using PrimeScript TMcDNA was obtained using the II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A). The reverse-transcribed cDNA was amplified by nested PCR. The first round of PCR amplification used primers located on the signal peptide and CH2 to obtain 1) a 1000-bp fragment containing the conventional VH (VH+CH1+CH2) and 2) a 750-bp fragment containing the Nanobody (VHH+CH2). The 1000-bp fragment was recovered by agarose gel electrophoresis. A second round of PCR amplification of the VH fragment was performed using primers containing NheI and NotI restriction sites. The VH fragment was purified using a standard DNA product purification kit. The VH fragment was ligated into pSid-Rmab-VL using enzyme digestion and ligation (NheI and NotI digestion) (preferably a 1:3 molar ratio of pSid-Rmab-VL:VH). The obtained vector was electroporated into competent Escherichia coli TG1 (Lucigen, Cat. No. 60502). Immediately after electroporation, 1 mL of 2YT medium (preheated at 37°C) was added to the electroporation cup for recovery. The electroporation product was aspirated and the electroporation cup was washed with 2YT medium to obtain a total of 10 mL of recovery product. The product was recovered at 37°C, 180 rpm for 60 min, and 100 μL of the recovery product was diluted to 10 -3 and 10 -4 The number of transformants in the library was determined. The next day, the library capacity was determined to be 2 × 10e8 CFU, with an insertion rate of 100%. A phage library containing Camelidae VH was then obtained using conventional methods.

[0160] The primers used in the first round of PCR are as follows:

[0161] The primers used in the second round of PCR are as follows:

[0162] 2. Library Screening

[0163] The phage library was then screened against the antigen human FGFR2IIIB-his protein using phage display technology through two rounds of solid-phase screening to obtain a hybrid scFv containing a camelid VH that can interact with ramucirumab VL and bind to FGFRIIIB.

[0164] After two rounds of screening, the eluted phage was diluted and infected into TG1 cells in the logarithmic phase. The cells were then plated on 2YT (50 μg / mL carbenicillin + 2% glucose) plates and incubated overnight at 37°C. The next day, a single colony was picked from the plate and plated into a deep-well plate containing 400 μL of 2YT (50 μg / mL carbenicillin) liquid medium. The culture was continued at 37°C, 220 rpm, until the logarithmic phase was reached. 1 mM IPTG was added and the cells were induced overnight at 30°C. The next day, the plates were centrifuged at 500g for 5 minutes, and the supernatant was collected for ELISA analysis.

[0165] Human FGFR2IIIB-his was diluted to a concentration of 4 μg / mL with PBS buffer at pH 7.4 and added to a 96-well ELISA plate at a volume of 50 μL / well. The plate was placed at 4°C overnight. The next day, the liquid was discarded and 200 μL / well of 1% skim milk blocking solution diluted with PBS was added. The plates were incubated at 37°C for 1 hour for blocking. After blocking, the blocking buffer was discarded and the plate was washed twice with PBST buffer (pH 7.4 PBS containing 0.005% tween-20). 50 μL / well of supernatant was added and the plate was incubated at 37°C for 1 hour. The reaction solution was discarded and the plate was washed twice with PBST. 50 μL / well of diluted HRP-conjugated mouse anti-HA tag secondary antibody (SinoBiological, Cat. No. 100028-MM10) was added and incubated at 37°C for 1 hour. The plate was washed twice with PBST and 50 μL / well of TMB was added for 5 minutes. The reaction was terminated by adding 50 μL / well of 1 M H2SO4. The absorbance was read at 450 nm using a microplate reader to count the number of clones binding to human FGFR2IIIB. Candidate clones were induced to express antibodies, and the supernatants were collected for ELISA analysis.

[0166] Positive clones binding to human FGFR2IIIB and cynomolgus monkey FGFR2IIIB were selected for sequencing and identification. All antibodies with different sequences were selected as candidates. After screening, the antibody scFv clones numbered B4 and 1B4 were finally selected for further study. Their CDR sequences and VH sequences are shown in Table A below.

[0167] Table A:

[0168] Example 4. Cell Binding Activity

[0169] Cell binding experiments were performed to determine whether the supernatants of the corresponding prokaryotic cultures expressing the positive antibody scFv clones B4 and 1B4 of the present invention could bind to the FGFR2IIIB protein endogenously expressed on the surface of KATO-III (Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences, Cat# TCHu229) and SNU-16 (Nanjing Kebai Biotechnology Co., Ltd., Cat# CBP60502).

[0170] KATO-III and SNU-16 cells were collected to obtain single cell suspensions, and 1×10 5 Cells were seeded / well in a 96-well V-bottom plate and centrifuged at 400g for 5 minutes at room temperature. The supernatant was discarded and the cell pellet was resuspended in supernatant from prokaryotic cultures expressing B4 and 1B4 scFv, the positive control bemarituzumab (10 μg / mL), and an irrelevant IgG1 negative control (10 μg / mL). After incubation at 4°C for 30 minutes, the cells were centrifuged at 400g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended and washed once with PBS. Centrifuge at 400g for 5 minutes at room temperature, and discard the supernatant. Cell pellets incubated with prokaryotic supernatant were resuspended in a 1:150 dilution of PE anti-HA.11 Epitope Tag Antibody (Biolegend, Cat#901518). Cell pellets incubated with positive and negative antibodies were resuspended in a 1:150 dilution of R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098). Incubate at 4°C in the dark for 30 minutes, centrifuge at 400g for 5 minutes at room temperature, and discard the supernatant. Cell pellets were washed twice with PBS. Finally, cells were resuspended in 100 μL of PBS per well, and fluorescence signals in the PE channel were measured using a Beckman CytoFlex (Beckman) flow cytometer.

[0171] The flow cytometry results are shown in Figure 1 . 1B4 binds significantly to both cells, and B4 binds to KATOⅢ cells that highly express human FGFR2IIB.

[0172] Example 5. Construction of specific hybrid antibodies against FGFR2IIIB

[0173] The nucleotide sequences encoding the heavy chain variable regions of B4 and 1B4 scFv were constructed into a eukaryotic expression vector containing the IgG1 antibody constant region (amino acid sequence SEQ ID NO: 42), which was then co-transfected with the Rmab-VL eukaryotic expression vector encoding the ramucirumab light chain into ExpiCHO-S cells to obtain the corresponding specific hybrid antibodies containing the interacting ramucirumab light chain and the anti-human FGFR2IIIB heavy chain: B4-IgG antibody and 1B4-IgG antibody, whose heavy and light chain sequences are shown in Table B below.

[0174] Table B:

[0175] Example 6. Affinity detection

[0176] In this experiment, the binding affinity of specific antibodies B4-IgG and 1B4-IgG to human FGFR2IIIB protein (SEQ ID NO: 30) and cynomolgus monkey FGFR2IIIB protein (SEQ ID NO: 37) was detected using the ForteBio Octet RED96 according to the manufacturer's instructions. Briefly, an AHC sensor (ForteBio, Cat# 18-5060) was placed in Running Buffer (1X PBS Hyclone, Cat# SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate as follows: a) equilibrate the baseline with Running Buffer for 100 seconds; b) add the bispecific antibody diluted in Running Buffer to a final concentration of 5 μg / mL and allow to cure for 200 seconds; c) equilibrate the baseline with Running Buffer for 300 seconds; d) add human and cynomolgus macaque FGFR2IIIB proteins diluted in Running Buffer to each well at the following concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM; allow 200 seconds for association and 600 seconds for dissociation. Data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0177] The affinities of B4-IgG and 1B4-IgG antibodies to human FGFR2IIIB and cynomolgus monkey FGFR2IIIB are both around nM, as shown in Table 1.

[0178] Table 1: Affinity of B4-IgG and 1B4-IgG antibodies binding to FGFR2IIIB

[0179] Example 7. Specific hybrid antibody binding to cells expressing cynomolgus monkey FGFR2IIIB

[0180] A cell binding experiment was performed to determine whether the anti-FGFR2IIIB-specific hybrid antibodies B4-IgG and 1B4-IgG of the present invention can bind to the cynomolgus monkey FGFR2IIIB protein stably expressed on the surface of CHO-K1 cells (Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences, Cat# SCSP-507).

[0181] First, use Lipofectamine TM 2000 (Invitrogen, Cat#11668019) transfection reagent was used according to the reagent instructions. A eukaryotic expression vector encoding the cynomolgus macaque FGFR2IIIB protein (Accession#P21802-3, codon-optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd.) was transfected into CHO-K1 cells. 48 hours after transfection, 4 μg / mL puromycin dihydrochloride (Gibco, Cat#A1113802) was used for selection for 3-5 days to obtain CHO-K1 cells that highly expressed the cynomolgus macaque FGFR2IIIB protein.

[0182] The binding of B4-IgG antibody and 1B4-IgG antibody to CHO-K1 cells expressing cynomolgus monkey FGFR2IIIB was detected by flow cytometry. For specific methods, see Example 4. The test antibody was diluted to 10 and 1 μg / mL, and the cells were resuspended and incubated at 4°C for 30 minutes. After washing the cells twice with PBS, the cell pellet was resuspended with 1:150 diluted R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific and incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS, the cells were resuspended and the PE channel fluorescence signal was detected using a Beckman CytoFlex (Beckman) flow cytometer.

[0183] As shown in the results in FIG2 , the tested antibodies can bind to the cynomolgus monkey FGFR2IIB protein expressed on CHO-K1 cells, and both the B4-IgG antibody and the 1B4-IgG antibody clones bind well.

[0184] Example 8. Construction of a bispecific antibody targeting FGFR2IIIB & VEGFR2

[0185] 1. Construction of bispecific antibodies

[0186] The nucleotide sequences encoding the heavy chain variable regions of the B4 and 1B4 scFv clones screened in Example 3 were constructed into an expression vector containing a nucleotide sequence encoding an IgG1 (knob) heavy chain constant region comprising a KIH mutation (amino acid sequence SEQ ID NO: 60). The nucleotide sequence encoding the ramucirumab heavy chain variable region was constructed into another eukaryotic expression vector containing a nucleotide sequence encoding an IgG1 (hole) heavy chain constant region comprising a KIH mutation (amino acid sequence SEQ ID NO: 61). The constructed anti-FGFR2IIIB heavy chain-expressing vector, the ramucirumab heavy chain-expressing vector, and the Rmab-VL vector expressing the ramucirumab light chain were co-transfected into cells to express the bispecific antibody. See Table 2 for specific combinations.

[0187] Table 2: Names and compositions of constructed bispecific antibodies

[0188] 2. Expression and Purification of Bispecific Antibodies

[0189] The purity of the obtained bispecific antibodies, B4Ram-KH and 1B4Ram-KH, was confirmed by size exclusion chromatography. Specifically, 20 μg of sample was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na₂SO₄ (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data analyzed using OpenLAB software.

[0190] The results are shown in Table 3: The main peak of the bispecific antibodies B4Ram-KH and 1B4Ram-KH in SEC is higher than 90%, which indicates that the bispecific antibodies obtained according to the method disclosed in this application have higher purity and integrity. The SEC test results are shown in Figure 5.

[0191] Table 3. Purity of bispecific antibodies

[0192] Example 9. Physicochemical Analysis and Affinity Testing of Bispecific Antibodies

[0193] Differential scanning fluorimetry (DSF) is used to assess the thermal stability of proteins by detecting the amount of fluorescent dye bound to proteins whose structures change during temperature increases. Specifically, it is used to measure the midpoint of the thermal transition (Tm), which is an indicator of the relative stability of proteins in liquids. Antibodies B4Ram-KH and 1B4Ram-KH were diluted to 2 μM with PBS buffer, and the fluorescent dye was diluted 150 times. After thorough mixing with the antibody, the sample was transferred to a 96-well plate and placed in a fluorescent quantitative PCR instrument for detection. The sample volume per detection well was 25 μl. The temperature range was 25-95°C, the heating rate was 1°C / min, and the results were analyzed using Bio-Rad CFX Manager automated analysis software.

[0194] The results showed that B4Ram-KH and 1B4Ram-KH had good thermal stability, with the Tm1 value of B4Ram-KH being 66.2℃ and the Tm1 value of 1B4Ram-KH being 64.6℃.

[0195] The binding affinities of the bispecific anti-FGFR2IIIB & VEGFR2 antibodies B4Ram-KH and 1B4Ram-KH to human FGFR2IIIB and human VEGFR2 proteins (ACRO, Catalog No. KDR-H5227, sequence shown in SEQ ID No: 43), respectively, were tested using the ForteBio Octet RED96. For the assay method, see Example 6, and for the results, see Table 4. The results demonstrate that the constructed bispecific antibodies possess excellent binding affinities for their respective antigens.

[0196] Table 4. Binding affinity of bispecific antibodies to human FGFR2IIIB and VEGFR2 proteins

[0197] Example 10. Sandwich ELISA to detect the binding of bispecific antibodies to FGFR2IIIB & VEGFR2 proteins

[0198] Human FGFR2IIIB-mFc (SEQ ID NO: 17) was diluted to a concentration of 0.5 μg / mL with PBS buffer at pH 7.4, and added to a 96-well ELISA plate at a volume of 50 μL / well. The plate was placed at 4°C overnight. The next day, the liquid was discarded, and 200 μL / well of 1% skim milk blocking solution diluted with PBS was added. The plate was incubated at 37°C for 1 hour for blocking. After the blocking was completed, the blocking solution was discarded and the plate was washed with PBST buffer (pH 7.4 After washing the plate twice with PBS containing 0.005% tween-20, B4Ram-KH, 1B4Ram-KH, B4-IgG antibody, 1B4-IgG antibody, and Ramucirumab were diluted 3-fold starting from 200nM, and a total of 6 dilutions were added to the corresponding wells. The plate was incubated at 37°C for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed twice with PBST, and 50ul / well of diluted human VEGFR was added. 2-his (ACRO, Catalog No. KDR-H5227) at a concentration of 0.5ug / ml was placed in a 37°C incubator for 1 hour. After incubation, the reaction solution in the ELISA plate was discarded, and the plate was washed twice with PBST. HRP-labeled mouse anti-His tag secondary antibody (Convis Century, Catalog No. CW0285M) was added and incubated at 37°C for 1 hour. After washing the plate twice with PBST, 50ul / well TMB was added for color development for 15min, and then 50μL / well 1M H2SO4 was added to terminate the reaction. The absorbance value was read at a wavelength of 450nm using a microplate reader, and the data was analyzed using software.

[0199] As shown in Figures 3A-3B, the bispecific antibody can bind to both human FGFRIIIB protein and human VEGFR2 protein.

[0200] Example 11. Immunization of Animals Using the Antigen Human LILIRB4-his

[0201] The method of Example 1 was used, but the antigen was replaced with human LILIRB4-his (ACRO, catalog number LI4-H52H7) for corresponding immunization.

[0202] Example 12. Construction of a phagemid vector containing the humanized SP34 light chain variable region of the CD3 antibody

[0203] The nucleotide sequence encoding the light chain variable region of CD3 antibody SP34 was inserted into a phagemid vector modified to contain restriction sites, the insertion sites being BamHI and HindIII. The constructed phagemid was named pSid-CD3-VL. The heavy chain construction method was the same as in Example 2.

[0204] Example 13 Immune library construction and screening

[0205] Using the method disclosed in Example 3, a bacterial library containing the phagemid pSid-CD3-VL described in Example 12 was constructed. The library capacity was 2 × 10e8 CFU, and the library insertion rate was 100%. The primers used are shown in the following table:

[0206] The obtained library was then screened using the library screening method disclosed in Example 3, except that the antigen used was LILIRB4-his.

[0207] After two rounds of screening, all positive clones were sequenced and identified. Antibodies with different sequences were selected as candidates. After screening, the antibody scFv clones numbered 1B2, 1D5, and 1H4 were finally selected for further study. Their CDR and VH sequences are shown in Table C below.

[0208] Table C:

[0209] Example 14. Construction of specific hybrid antibodies against LILRB4

[0210] The nucleotide sequences encoding the heavy chain variable regions of the positive clones 1B2, 1D5, and 1H4 were constructed into a eukaryotic expression vector containing an IgG1 constant region (containing mutations L234A, L235A, D265A, and P329A that abolish ADCC effector function) (amino acid sequence SEQ ID NO: 57). This vector was then co-transfected with the sp34-LC vector encoding the CD3 antibody SP34 light chain into ExpiCHO-S cells to form specific hybrid antibodies containing the interacting CD3 antibody SP34 light chain and the anti-LILRB4 heavy chain: 1B2-IgG antibody, 1D5-IgG antibody, and 1H4-IgG antibody. Specific combinations are shown in the table below, and their sequences are shown in Table D below.

[0211] Table D:

[0212] Example 15. Physicochemical analysis and affinity detection of specific antibodies

[0213] The purity of the expressed and purified 1B2-IgG antibody, 1D5-IgG antibody, and 1H4-IgG antibody was tested according to the method of Example 9. The purity results are shown in Table 5. The main peak of the specific antibodies 1B2-IgG antibody, 1D5-IgG antibody, and 1H4-IgG antibody on SEC was higher than 90%, indicating that the bispecific antibodies obtained according to the method disclosed in this application have higher purity and integrity.

[0214] Table 5 Purity of specific antibodies

[0215] The binding affinities of specific antibodies 1B2-IgG, 1D5-IgG, and 1H4-IgG to human LILRB4 protein (SEQ ID NO: 44) were tested using a ForteBio Octet RED96. The assay method is described in Example 6, and the assay results are described in Table 6.

[0216] Table 6. Binding affinity of specific antibodies to human LILRB4 protein

[0217] Example 16. Construction of bispecific antibodies targeting LILRB4 & CD3

[0218] 1. Construction of bispecific antibodies

[0219] The nucleotide sequences encoding the heavy chain variable regions of the 1B2, 1D5, and 1H4 clones screened in Example 13 were constructed into an expression vector containing a nucleotide sequence encoding an IgG1 mut (hole) heavy chain constant region comprising a KIH mutation (comprising L234A, L235A, D265A, P329A, Y407V, T366S, L368A, and Y349C mutations) (amino acid sequence of SEQ ID NO: 59). The nucleotide sequence encoding the SP34 heavy chain variable region was constructed into another expression vector containing a nucleotide sequence encoding an IgG1 mut (knob) heavy chain constant region comprising a KIH mutation (comprising L234A, L235A, D265A, P329A, S354C, and T366W mutations) (amino acid sequence of SEQ ID NO: The constructed vector expressing the anti-LILRB4 heavy chain, the SP34 heavy chain, and the SP34-LC vector expressing the SP34 light chain were co-transfected into cells to express the bispecific antibody. Specific combinations are shown in Table 7.

[0220] Table 7: Names and compositions of constructed bispecific antibodies

[0221] 2. Expression and Purification of Bispecific Antibodies

[0222] Referring to the method disclosed in Example 8, the purity of the obtained bispecific antibody was confirmed by size exclusion chromatography. The SEC test results are shown in Figure 6.

[0223] The results are shown in Table 8: Bispecific Antibodies

[0224] Table 8. Bispecific Antibody Purity

[0225] Example 17. Sandwich ELISA to detect the binding of bispecific antibodies to human LILRB4 and human CD3 proteins

[0226] Human LILRB4-Fc (ACRO, Catalog No. LI4-HF254) was diluted to a concentration of 0.5 μg / mL with pH 7.4 PBS buffer and added to a 96-well ELISA plate at a volume of 50 μL / well. The plate was incubated at 4°C overnight. The next day, the liquid was discarded and 1% skim milk blocking solution diluted in PBS was added at 200 μL / well. The plates were incubated at 37°C for 1 hour for blocking. After blocking, the blocking solution was discarded, and the plate was washed twice with PBST buffer (pH 7.4 PBS containing 0.005% tween-20). The bispecific antibody was diluted 4-fold starting from 200 nM, and a total of 6 dilutions were added to the corresponding wells. The plate was incubated at 37°C for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed twice with PBST, and 50 μL / well of diluted human CD3-his (SinoBiological, Catalog No. 10977-H08H) at a concentration of 0.5 μg / ml was added. The plate was incubated at 37°C for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed twice with PBST, and HRP-labeled mouse anti-His tag secondary antibody (Comvison, Catalog No. CW0285M) was added. The plate was incubated at 37°C for 1 hour. After washing twice with PBST, 50 μL / well TMB was added for color development for 15 minutes, and then 50 μL / well 1M The reaction was terminated with H2SO4, the absorbance was read at a wavelength of 450 nm using a microplate reader, and the data were analyzed using software.

[0227] As shown in FIG4 , the bispecific antibody can bind to both human LILRB4 protein and human CD3 protein.

[0228] Sequence Listing

[0229] Ramucirumab-VL:

[0230] Ramucirumab-LC:

[0231] Ramucirumab-VH:

[0232] Ramucirumab-VH-IgG1(hole), containing mutations (Y407V, T366S, L368A, Y349C)

[0233] IgG1 antibody constant region

[0234] B4-VH-IgG1(knob), containing mutations (T366W, S354C)

[0235] 1B4-VH-IgG1(knob), containing mutations (T366W, S354C)

[0236] IgG1 (knob) with mutations (T366W, S354C)

[0237] IgG1(hole), with mutations (Y407V, T366S, L368A, Y349C)

[0238] IgG1 mut, with mutations (L234A L235A, D265A P329A)

[0239] 1B2-IgG1 mut(hole), with mutations (L234A, L235A, D265A, P329A, Y407V, T366S, L368A, Y349C)

[0240] SP34-IgG1 mut(knob), with mutations (L234A, L235A, D265A, P329A, S354C, T366W)

[0241] IgG1 mut(knob) with mutations (L234A, L235A, D265A, P329A, S354C, T366W)

[0242] SP34-VH

[0243] SP34-LC(Lambda):

[0244] SP34-VL

[0245] 1D5-IgG1 mut(hole), with mutations (L234A, L235A, D265A, P329A, Y407V, T366S, L368A, Y349C)

[0246] 1H4-IgG1 mut(hole), with mutations (L234A, L235A, D265A, P329A, Y407V, T366S, L368A, Y349C)

[0247] IgG1 mut(hole) with mutations (L234A, L235A, D265A, P329A, Y407V, T366S, L368A, Y349C)

[0248] Human FGFR2IIIB-His:

[0249] human FGFR2IIIB-mFc

[0250] Cynomolgus monkey FGFR2IIIB-His:

[0251] Human VEGFR2-His:

[0252] Human LILRB4-his:

Claims

1. A method for preparing a bispecific antibody, comprising: 1) obtaining all heavy chain variable regions of a library of corresponding camelid immunoglobulin antibodies (first antibodies) against a first antigen, and obtaining light chain variable regions of a second antibody against a second antigen; 2) co-expressing all heavy chain variable regions of the first antibody library with the light chain variable regions of the second antibody to obtain a hybrid library; 3) screening the hybrid library against the first antigen to obtain a heavy chain variable region that specifically recognizes the first antigen; 4) preparing the heavy chain variable region that recognizes the first antigen obtained in step 3) into a heavy chain; 5) co-expressing the heavy chain obtained in step 4) with the heavy chain and light chain of the second antibody to form a bispecific antibody that specifically binds to the first antigen and the second antigen and has the same light chain of the second antibody.

2. The method of claim 1, wherein the hybrid library is a hybrid scFv library or a hybrid Fab library, and optionally, the immunoglobulin in step 1) is IgG. In one embodiment, the second antibody is a human antibody.

3. The method of claim 1 or 2, wherein the camelid immunoglobulin antibody library in step 1) can be an immune library, a natural library or a synthetic antibody library (such as but not limited to a yeast library or a phage library).

4. The method of any one of claims 1 to 3, wherein the hybrid scFv library or hybrid Fab library is a yeast library or a phage library.

5. The method of any one of claims 1 to 4, wherein in step 4), the heavy chain is formed by combining the heavy chain variable region of the first antibody with an IgG antibody constant region, preferably, the IgG antibody constant region is an IgG1 antibody constant region, and optionally, the constant region has amino acid modifications, such as modifications that improve constant region interactions, modifications that improve ADCC activity, modifications that remove ADCC effector functions, etc.

6. The method of any one of claims 1 to 5, wherein the first antigen is FGFR2IIIB, the second antigen is VEGFR2, and the generated bispecific antibody against FGFR2IIIB and VEGFR2 comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively; or the first antigen is LILIRB4, the second antigen is CD3, and the generated bispecific antibody against LILIRB4 and CD3 comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively.

7. A bispecific antibody having identical antibody light chains prepared by the method of any one of claims 1 to 6, which consists of two different heavy chains and two identical light chains, wherein the first heavy chain is derived from an immunoglobulin antibody of camelids that recognizes a first antigen, the second heavy chain is derived from a human antibody that recognizes a second antigen, and the two identical light chains are derived from a human antibody that recognizes the second antigen, wherein the two heavy chains interact with the two light chains respectively to form binding arms that specifically bind to the first antigen and the second antigen, wherein The first antigen is FGFR2IIIB, the second antigen is VEGFR2, and the generated anti-FGFR2IIIB and VEGFR2 bispecific antibody comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein: 1) The antibody arm that binds to FGFR2IIIB comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 45, LCDR2 shown in SEQ ID NO: 46, and LCDR3 shown in SEQ ID NO: 47; wherein the antibody arm that binds to VEGFR2 comprises SEQ ID NO: 48 , HCDR1 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:50, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47; or 2) The antibody arm that binds to FGFR2IIIB comprises HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:9, HCDR3 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47; wherein the antibody arm that binds to VEGFR2 comprises HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:49, HCDR3 shown in SEQ ID NO:50, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47; or The first antigen is LILIRB4, the second antigen is CD3, and the generated bispecific antibody against LILIRB4 and CD3 comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein: 1) The antibody arm that binds to LILIRB4 comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:19, HCDR3 shown in SEQ ID NO:20, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, HCDR3 shown in SEQ ID NO:53, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; 2) the antibody arm that binds to LILIRB4 comprises HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, HCDR3 shown in SEQ ID NO:53, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; or 3) The antibody arm that binds to LILIRB4 comprises HCDR1 shown in SEQ ID NO:32, HCDR2 shown in SEQ ID NO:33, HCDR3 shown in SEQ ID NO:34, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; wherein the antibody arm that binds to CD3 comprises HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:52, HCDR3 shown in SEQ ID NO:53, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:

56.

8. The bispecific antibody of claim 7, wherein the first antigen is FGFR2IIIB, the second antigen is VEGFR2, and the resulting bispecific antibody against FGFR2IIIB and VEGFR2 comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein: 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 14 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; or 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 11 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; wherein the antibody arm that binds to VEGFR2 comprises a light chain variable region comprising the sequence shown in SEQ ID NO: 14 A heavy chain variable region having a sequence and a light chain variable region having a sequence shown in SEQ ID NO: 15; or The first antigen is LILIRB4, the second antigen is CD3, and the generated bispecific antibody against LILIRB4 and CD3 comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein: 1) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 39 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; 2) the antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 28 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 39 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; or 3) The antibody arm that binds to LILIRB4 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:35 and a light chain variable region comprising the sequence shown in SEQ ID NO:40; wherein the antibody arm that binds to CD3 comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:39 and a light chain variable region comprising the sequence shown in SEQ ID NO:

40.

9. The bispecific antibody of claim 7 or 8, wherein the first antigen is FGFR2IIIB and the second antigen is VEGFR2, and the resulting bispecific antibody against FGFR2IIIB and VEGFR2 comprises antibody arms that specifically bind to FGFR2IIIB and VEGFR2, respectively, wherein: 1) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO:7 and a light chain comprising the sequence shown in SEQ ID NO:6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO:16 and a light chain comprising the sequence shown in SEQ ID NO:6; or 2) The antibody arm that binds to FGFR2IIIB comprises a heavy chain comprising the sequence shown in SEQ ID NO: 13 and a light chain comprising the sequence shown in SEQ ID NO: 6; wherein the antibody arm that binds to VEGFR2 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 16 and a light chain comprising the sequence shown in SEQ ID NO: 6; or The first antigen is LILIRB4, the second antigen is CD3, and the generated bispecific antibody against LILIRB4 and CD3 comprises antibody arms that specifically bind to LILIRB4 and CD3, respectively, wherein: 1) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 24 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: 23; 2) the antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO:31 and a light chain comprising the sequence shown in SEQ ID NO:23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO:41 and a light chain comprising the sequence shown in SEQ ID NO:23; or 3) The antibody arm that binds to LILIRB4 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 38 and a light chain comprising the sequence shown in SEQ ID NO: 23; wherein the antibody arm that binds to CD3 comprises a heavy chain comprising the sequence shown in SEQ ID NO: 41 and a light chain comprising the sequence shown in SEQ ID NO: The light chain has the sequence shown in ID NO:

23.

10. An antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB, comprising: 1) HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47; or 2) HCDR1 shown in SEQ ID NO:8, HCDR2 shown in SEQ ID NO:9, HCDR3 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:45, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:

47.

11. The antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB according to claim 10, comprising: 1) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the sequence shown in SEQ ID NO: 15; or 2) A heavy chain variable region comprising the sequence shown in SEQ ID NO:11 and a light chain variable region comprising the sequence shown in SEQ ID NO:

15.

12. The antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB according to claim 10 or 11, comprising: 1) a heavy chain comprising the sequence shown in SEQ ID NO: 5 and a light chain comprising the sequence shown in SEQ ID NO: 6; or 2) A heavy chain comprising the sequence shown in SEQ ID NO: 12 and a light chain comprising the sequence shown in SEQ ID NO:

6.

13. An antibody or antigen-binding fragment thereof that specifically binds to LILIRB4, comprising: 1) HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:19, HCDR3 shown in SEQ ID NO:20, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; 2) HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:56; or 3) HCDR1 shown in SEQ ID NO:32, HCDR2 shown in SEQ ID NO:33, HCDR3 shown in SEQ ID NO:34, LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:55, and LCDR3 shown in SEQ ID NO:

56.

14. The antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 according to claim 13, comprising: 1) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; 2) a heavy chain variable region comprising the sequence shown in SEQ ID NO: 28 and a light chain variable region comprising the sequence shown in SEQ ID NO: 40; or 3) a heavy chain variable region comprising the sequence shown in SEQ ID NO:35 and a light chain variable region comprising the sequence shown in SEQ ID NO:

40.

15. The antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 according to claim 13 or 14, comprising: 1) a heavy chain comprising the sequence shown in SEQ ID NO: 22 and a light chain comprising the sequence shown in SEQ ID NO: 23; 2) a heavy chain comprising the sequence shown in SEQ ID NO: 29 and a light chain comprising the sequence shown in SEQ ID NO: 23; or 3) A heavy chain comprising the sequence shown in SEQ ID NO:36 and a light chain comprising the sequence shown in SEQ ID NO:

23.

16. An antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB according to any one of claims 10-12, or an antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 according to any one of claims 13-15, wherein the antigen-binding fragment is selected from Fab, Fab'-SH, Fv, scFv or (Fab')2 fragment.

17. A nucleic acid molecule encoding the bispecific antibody of any one of claims 7-9, encoding the antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB of any one of claims 10-12, or encoding the antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 of any one of claims 13-15.

18. A vector comprising the nucleic acid molecule of claim 17.

19. A host cell comprising the nucleic acid molecule of claim 17 or the vector of claim 18.

20. A pharmaceutical composition comprising the bispecific antibody of any one of claims 7-9, or the antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB of any one of claims 10-12, or the antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 of any one of claims 13-15, and a pharmaceutically acceptable carrier.

21. Use of the bispecific antibody according to any one of claims 7 to 9, or the antibody or antigen-binding fragment thereof that specifically binds to FGFR2IIIB according to any one of claims 10 to 12, or the antibody or antigen-binding fragment thereof that specifically binds to LILIRB4 according to any one of claims 13 to 15, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating a disease.

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