WO2019196522A1

WO2019196522A1 - Antibody and antibody modification method

Info

Publication number: WO2019196522A1
Application number: PCT/CN2019/070619
Authority: WO
Inventors: 张文军; 张兵; 刘嘉熙; 黄引娣; 邱小林
Original assignee: 广州爱思迈生物医药科技有限公司
Priority date: 2018-04-10
Filing date: 2019-01-07
Publication date: 2019-10-17
Also published as: CN110357960A

Abstract

Provided by the present invention is a method for modifying a Fab region of an antibody. In particular, provided by the present invention is a method for mutating particular sites of a CH1-hinge region and a CL region of an antibody so as to form an unnatural disulfide bond. Further provided by the present invention are an antibody produced by means of the described modification method and a use thereof.

Description

Antibody and antibody modification method

Technical field:

The invention belongs to the field of protein engineering and relates to a method for modifying a disulfide bond in or between molecules. In particular, the invention relates to methods of engineering a disulfide bond between the heavy and light chains of an antibody to alter or increase its binding specificity. The invention also relates to disulfide-modified antibodies, such as bispecific antibodies, methods for their preparation, and uses.

technical background:

In the field of biomedicine, antibody drugs have been widely used in the treatment of various diseases such as malignant tumors, autoimmune diseases, inflammatory infections, and cardiovascular diseases. Among them, Bispecific antibody (BsAb) is a kind of antibody that can bind at least two antigenic epitopes, and its safety and effectiveness are greatly improved compared with traditional single target antibodies. A research and development hotspot in the field of antibody engineering and tumor immunotherapy.

Bispecific antibodies are not naturally occurring antibodies of cells and can only be artificially prepared by cell fusion or DNA recombination techniques. In the preparation of bispecific antibodies, it is necessary to simultaneously express two different heavy chains and two different light chains, and a Fab that recognizes both antigens is formed by pairing of two pairs of HC-LCs. In this process, homologous heavy chain binding and mismatching problems resulting from heterologous light chain binding produce ten different products, only one of which is the correct product required. Thus the main problem to be solved in the preparation of such antibodies is the random assembly of different chains such as homologous heavy chain mismatches, light chain mismatches and contamination of other non-target products.

Although there have been some solutions to the mismatch problem of bispecific antibodies, there are still various problems such as low stability and solubility, complicated process, and low yield. Thus, there remains a need for new methods of engineering bispecific antibodies to reduce mismatches between their heavy and light chains and to increase the yield of the desired product.

Summary of invention

The present inventors conducted in-depth analysis of the interaction and spatial structure of the heavy and light chains of the antibody, and found that a specific site in the heavy chain constant region 1 (CH1)-light chain constant region (CL) interface of the antibody was introduced. Non-native disulfide bonds can alter or increase the interaction between the heavy and light chains of an antibody, based on the introduction of minimal site mutations and without affecting the structure and function of the antibody. The above methods can be used to engineer antibodies or antibody fragments.

Accordingly, in one aspect, the invention relates to a method of engineering a Fab region of an antibody, the method comprising the step of introducing one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL;

e) K218E or K218D mutations in the hinge region and E213R or E213K mutations in CL, wherein the antibody heavy and light chains are numbered according to the EU number.

In some embodiments of the above methods, one, two, three, four or five of the mutations a)-e) are introduced into the Fab region. In a preferred embodiment, one or two mutations in mutations a)-d) are introduced to the Fab region, and the optional mutation e).

In some embodiments, the above method further comprises disrupting the native disulfide bond between the hinge region-CL, ie, disrupting the cysteine (C220) at position 220 in the hinge region and the 214th half of the CL region. A disulfide bond formed between cystine (C214). Thus, in some embodiments, the methods of the invention further comprise mutating C220 in the hinge region to an amino acid other than cysteine or deleting C220, and/or mutating C214 in CL to cysteine. Other amino acids or C214 deletion. In a preferred embodiment, the amino acid other than cysteine is selected from the group consisting of serine, alanine or glycine.

In any of the above methods, the Fab region is derived from IgG, IgA, IgM, IgE or IgD, such as an IgGl, IgG2, IgG3 or IgG4 isotype.

In another aspect, the invention features a method of producing an antibody or antibody fragment having at least two different Fab regions, the method comprising the steps of:

1) introducing one or more mutations selected from the group consisting of: in the first Fab region of the antibody or antibody fragment:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, numbered according to the EU number,

2) cultivating a host cell containing a nucleic acid encoding the antibody or antibody fragment under conditions in which the antibody or antibody fragment is expressed, and

3) recovering the antibody or antibody fragment from the host cell culture.

In some embodiments, the above method further comprises introducing one or more mutations selected from the group consisting of: in the second Fab region of the antibody or antibody fragment:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, wherein the mutation introduced into the first Fab region and the mutation introduced into the second Fab region are not identical.

In some embodiments of the above methods, one, two, three, four or five of the mutations a)-e) are introduced in the first Fab region, introduced in the second Fab region One, two, three, four or five of the mutations a)-e), and wherein the mutation introduced into the first Fab region and the mutation introduced into the second Fab region are not identical. In a preferred embodiment, one or two of the mutations a)-d), and optionally the mutation e), are introduced in the first Fab region and/or the second Fab region.

In some embodiments, the above method further comprises disrupting a native disulfide bond between the first Fab region and/or the hinge region CL in the second Fab region, ie, the first Fab region and/or the second Fab region C220 in the hinge region is mutated to an amino acid other than cysteine or to C220, and/or C214 in CL of the first Fab region and/or the second Fab region and/or the second Fab region Mutated to an amino acid other than cysteine or deleted C214. In a preferred embodiment, the amino acid other than cysteine is selected from the group consisting of serine, alanine and glycine.

In some embodiments of the above methods, the first Fab and the second Fab region bind to different antigens.

In some embodiments of the above methods, the first Fab region and the second Fab region bind to two different epitopes of the same antigen.

In some embodiments of the above method of engineering a Fab region and a method of producing an antibody or antibody fragment, the Fab region, the first Fab region and the second Fab region bind to an antigen selected from the group consisting of CD2, CD3, CD3E , CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56 , CD64, CD80, CD147, GD3, IL-1α, IL-1β, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL -8, IL-9, IL-12, IL-13, IL-15, IL-17, IL-17R, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma; TNF-alpha , TNFβ, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin Beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β1, integrin β2, integrin α4/β7, integrin α2, integr Protein α3, integrin α4, integrin α5, integrin α6, integrin αv, whole Protein αVβ3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7 -1, B7-2, VNR integrin, TGFβ1, TGFβ2, eotaxin1, Blys (B lymphocyte stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, sticky Protein MUC1, MUC18, heparinase I, human cardiac myosin, tumor associated glycoprotein-72 (TAG-72), tumor associated antigen CA 125, prostate specific membrane antigen (PSM-A), high molecular weight melanoma associated Antigen (HMW-MAA), cancer-associated antigen, Gco protein Iib/IIIa (GPIIb/IIIa), tumor-associated antigen expressing Lewis Y-related carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV Gp 120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD Glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and Clostridium perfringens toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4 , KIR, LAG-3, TCRα, TCRβ, TCRγ, TCRδ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1 , GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, myostatin, PCSK9, CXCR4, MIF, PEG2. In a preferred embodiment, the Fab region, the first Fab region and the second Fab region bind to an antigen selected from the group consisting of CD3 and Her2.

In some embodiments, the above production methods are used to produce antibody fragments selected from the group consisting of Fab fragments, Fab' fragments, and F(ab') ₂ fragments.

In some embodiments, the above production method is for producing a bispecific antibody having a first heavy chain and a first light chain, and a second heavy chain and a second light chain, wherein the first The heavy chain and the first light chain form the first Fab region, and the second heavy chain and the second light chain form the second Fab region.

In some embodiments, the above method further comprises introducing P395K, P396K, and V397K to the first heavy chain, and introducing T394D, P395D, and P396D to the second heavy chain, or introducing T394D to the first heavy chain, P395D and P396D, and introducing P395K, P396K and V397K to the second heavy chain. The above mutations are capable of increasing the binding specificity between the first heavy chain and the second heavy chain to avoid the formation of heavy chain homodimers.

In some embodiments of the methods described above, the antibody or antibody fragment is derived from IgG, IgA, IgM, IgE or IgD, eg, an IgGl, IgG2, IgG3 or IgG4 isotype.

The invention also relates to antibodies or antibody fragments produced by the above methods.

In a further aspect, the invention relates to an antibody or antibody fragment having at least two different Fab regions, the first Fab region of the antibody or antibody fragment having one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in the CL,

It is numbered according to the EU number.

In some embodiments of the above antibodies or antibody fragments, the second Fab region of the antibody or antibody fragment has one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

The mutation in the second Fab region and the mutation in the first Fab region are not identical.

In some embodiments of the above antibodies or antibody fragments, the first Fab region has one, two, three, four, or five of the mutations a)-e), and the second Fab region has a mutation 1), 2, 3, 4 or 5 of a)-e), and wherein the mutation of the first Fab region and the mutation of the second Fab region are not identical. In a preferred embodiment, the first Fab region and/or the second Fab region has one or both of the mutations a)-d), and optionally the mutation e).

In some embodiments of the above antibody or antibody fragment, the first Fab region and/or the second Fab region further has a C220 mutation in the hinge region to an amino acid other than cysteine or a deletion C220, and/or C214 in CL is mutated to an amino acid other than cysteine or to C214. Among them, the amino acid other than cysteine is preferably selected from the group consisting of serine, alanine and glycine.

In some embodiments of the above antibodies and antibody fragments, the first Fab region and the second Fab region bind to different antigens.

In some embodiments of the above antibodies and antibody fragments, the first Fab region and the second Fab region bind to two different epitopes on the same antigen.

In some embodiments of the above antibodies and antibody fragments, the antigen is selected from the group consisting of CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1α, IL-1β, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL- 17R, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma; TNF-alpha, TNF beta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM , integrin β1, integrin β2, integrin α4/β7, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin αv, integrin Protein αVβ3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-I d, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, eotaxin1 , Blys (B lymphocyte stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline Phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, mucin MUC1, MUC18, heparinase I, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72) , tumor-associated antigen CA 125, prostate specific membrane antigen (PSM-A), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, Gco protein Iib/IIIa (GPIIb/IIIa), expression Lewis Tumor-associated antigens of Y-related carbohydrates, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and Clostridium perfringens ( Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCRα, TCRβ, TCRγ, TCRδ, VEGF, EGF, VEGFR, EGFR , EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1 , CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, myostatin , PCSK9, CXCR4, MIF, PEG2. In a preferred embodiment, the antigen is selected from the group consisting of CD3 and HER2.

In some embodiments, the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, and an F(ab') ₂ fragment.

In some embodiments, the antibody is a bispecific antibody having a first heavy chain and a first light chain, and a second heavy chain and a second light chain, wherein the first heavy chain and the first light chain are formed The first Fab region, the second heavy chain and the second light chain form the second Fab region. In a further embodiment, the first heavy chain has a P395K, P396K, and V397K mutation, and the second heavy chain has a T394D, P395D, and P396D mutation, or the first heavy chain has T394D, P395D, and P396D and a mutation, And the second heavy chain has P395K, P396K and V397K mutations.

In some embodiments, the antibody or antibody fragment is derived from IgG, IgA, IgM, IgE, or IgD, such as IgGl, IgG2, IgG3, or IgG4.

In another aspect, the invention relates to a method of engineering a bispecific antibody, wherein the bispecific antibody has a first heavy chain and a first light chain that bind to CD3, and a second heavy chain and a second that bind HER2 a light chain, the second heavy chain having the amino acid sequence of SEQ ID NO: 7, and the second light chain having the amino acid sequence of SEQ ID NO: 5, the method comprising the second heavy chain and the second The light chain introduces one or two mutations selected from the following a)-d):

a) F170C in heavy chain constant region 1 (CH1) and S162C in light chain constant region (CL);

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL; and

d) V173C in CH1 and Q160C in CL,

The antibody heavy and light chains are numbered according to the EU number.

In some embodiments of the above-described engineering methods, further comprising mutating C220 in the hinge region of the second heavy chain to an amino acid other than cysteine or deleting C220, and/or placing the second light chain The C214 in the CL is mutated to an amino acid other than cysteine or a step of deleting C214.

In some embodiments of the above-described engineering methods, further comprising introducing a K218E or K218D mutation into the first heavy chain and introducing an E213R or E213K mutation into the first light chain; or introducing K218E to the second heavy chain Or K218D is mutated and the E213R or E213K mutation is introduced into the second light chain.

In some embodiments of the above-described engineering methods, the first heavy chain has the amino acid sequence of SEQ ID NO: 3, and the first light chain has the amino acid sequence of SEQ ID NO: 1.

The invention also relates to bispecific antibodies obtained by the above described engineering methods.

In one aspect, the invention relates to an antibody or antibody fragment having a CL region selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19. SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27.

In one aspect, the invention relates to an antibody or antibody fragment having a CH1 region or a hinge region selected from the group consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43.

In one aspect, the invention relates to an antibody or antibody fragment having a CL region and a CH1 region selected from the group consisting of:

a) the CL region of SEQ ID NO: 11 and the CH1 region of SEQ ID NO: 33;

b) the CL region of SEQ ID NO: 13 and the CH1 region of SEQ ID NO: 29;

c) the CL region of SEQ ID NO: 15 and the CH1 region of SEQ ID NO: 35;

d) the CL region of SEQ ID NO: 17 and the CH1 region of SEQ ID NO: 33;

e) the CL region of SEQ ID NO:27 and the CH1 region of SEQ ID NO:43.

In another aspect, the invention relates to an antibody conjugate comprising an antibody or antibody fragment of the invention, or a bispecific antibody, and a portion coupled to the antibody or antibody fragment, or a bispecific antibody, wherein The moiety is selected from the group consisting of a cytotoxin, a radioisotope, a fluorescent label, a luminescent substance, a chromogenic substance, or an enzyme.

In some embodiments, the portion that is conjugated to an antibody or antibody fragment of the invention, or a bispecific antibody to form an antibody conjugate, is a cytotoxin. In some embodiments, the cytotoxin is selected from the group consisting of colchicine, emtansine, maytansinoid, auristatin, vindesine, tubulysin, and the like.

In some embodiments, the portion that is conjugated to an antibody or antibody fragment of the invention, or a bispecific antibody to form an antibody conjugate, is a radioisotope. In some embodiments, the radioisotope is selected from the group consisting of: At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , a radioisotope of P ³² , and the like.

In some embodiments, the moiety that is conjugated to an antibody or antibody fragment of the invention, or a bispecific antibody to form an antibody conjugate, is selected from the group consisting of a fluorescent label, a luminescent substance, and a chromogenic substance, eg, FITC, luciferase, HRP Wait.

In some embodiments, a portion that is conjugated to an antibody or antibody fragment of the invention, or a bispecific antibody to form an antibody conjugate, is an enzyme, such as an enzymatically active toxin of bacterial, fungal, plant or animal origin, including its activity. Fragments and/or variants.

In still another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antibody fragment of the invention, a bispecific antibody, or an antibody conjugate, and optionally one or more pharmaceutically acceptable carriers, surface Active agent and / or diluent.

In one aspect, the invention relates to the use of an antibody or antibody fragment, a bispecific antibody, or an antibody conjugate of the invention in the manufacture of a pharmaceutical composition for the treatment of a disease. In some embodiments, the disease is cancer.

In another aspect, the invention relates to a method of treating a disease comprising the step of using an antibody or antibody fragment, a bispecific antibody, an antibody conjugate or a pharmaceutical composition of the invention. In some embodiments, the disease is cancer.

In one aspect, the invention encompasses a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20. SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.

In another aspect, the invention includes a vector comprising a nucleic acid molecule of the invention.

In still another aspect, the invention encompasses a host cell comprising a nucleic acid molecule or vector of the invention.

DRAWINGS

Figure 1 shows a schematic representation of the structure of an antibody that can be engineered or produced using the methods of the invention. The antibodies may have different structures and may be bispecific, trispecific or tetraspecific antibodies. Among them, FIG. 1A shows a common "Y"-shaped antibody, which may be a bispecific antibody. Figures 1B-1H show the trispecific and tetraspecific antibody formats derived from Figure 1A.

Figure 2 shows the results of SDS-PAGE electrophoresis of anti-Her2× anti-CD3 bispecific antibodies MutB, MutC, MutD and MutE and control bispecific antibody WT modified by the method of the present invention after protein A affinity chromatography purification. . WT in the figure refers to a bispecific antibody encoded by the plasmids pFUSE-Her2-HC-OB-6His, pFUSE-C31-HC-OA, pCDNA3.1-C31-LC, and pCDNA3.1-Her2-LC, wherein WT (1:1:1:1) indicates that the above four plasmids were transfected at a ratio of 1:1:1:1; WT (2:1:1:1) indicated plasmid pFUSE-Her2-HC-OB-6His, The transfection ratio of pFUSE-C31-HC-OA, pCDNA3.1-C31-LC, and pCDNA3.1-Her2-LC was 2:1:1:1. Figure 2A shows the results of non-reducing SDS-PAGE electrophoresis, and Figure 2B shows the results of reductive SDS-PAGE electrophoresis.

Figure 3 is a graph showing the results of SDS-PAGE electrophoresis of the bispecific antibodies MutC-ER, MutC+ER and MutC-DeC which were further engineered on the basis of MutC after protein A affinity chromatography purification. Figure 3A shows the results of non-reducing SDS-PAGE electrophoresis and Figure 3B shows the results of reductive SDS-PAGE electrophoresis.

Figure 4 is a graph showing the peak shape of the bispecific antibody MutC and the SDS-PAGE electrophoresis results after purification by the cation exchange chromatography (CIEX) method. 4A is a CIEX peak shape of a bispecific antibody MutC sample; FIG. 4B is a non-reducing SDS-PAGE electrophoresis pattern, from left to right, lane 1 is a protein Marker; and lane 2 is purified by protein A affinity chromatography. Samples that have not been subjected to the CIEX method are labeled as Input; lanes 3-4 are samples corresponding to the A peak of CIEX; lanes 5-10 are samples corresponding to the B peak of CIEX. Figure 4C is a diagram of the reductive SDS-PAGE electrophoresis corresponding to Figure 4B.

Figure 5 is a graph showing the results of SDS-PAGE electrophoresis of the bispecific antibodies MutC-D+ER and MutC-D-DeC of the present invention after protein A affinity chromatography purification. 5A is a non-reducing SDS-PAGE electropherogram, and FIG. 5B is a reducing SDS-PAGE electropherogram.

Figure 6 shows the results of ELISA of anti-Her2 x anti-CD3 bispecific antibody binding to human Her2 antigen. Figure 6A shows the results of the bispecific antibodies MutC, MutC+ER and MutC-ER. Figure 6B shows the results of MutC-DeC, MutC-D+ER and MutC-D-DeC.

Figure 7 shows the results of ELISA of Her2 x anti-CD3 bispecific antibody binding to human CD3 antigen. Figure 7A shows the results of the bispecific antibodies MutC, MutC+ER and MutC-ER. Figure 7B shows the results of MutC-D+ER, MutC-D-DeC and MutC-DeC.

Figure 8 shows the results of detecting the binding of an antibody to two antigens using a HRP-labeled human Her2 antigen and a double antigen ELISA assay of human CD3 antigen adsorbed on an enzyme plate. Figure 8A shows the results of ELISA for binding the two antigens of the bispecific antibodies MutB, MutC and MutD of the invention compared to control WT and hIgG. Figure 8B shows the results of the bispecific antibody MutE.

Figure 9 shows the results of experiments with different concentrations of bispecific antibody mediated T cell activation and tumor cell killing (CTL). Figure 9A shows the results of the bispecific antibodies MutB, MutC and MutD compared to control WT and hIgG. Figure 9B shows the results of the bispecific antibodies MutC and MutE.

Figure 10 shows the results of experiments with different concentrations of bispecific antibody mediated T cell activation and tumor cell killing (CTL). Figure 10A shows the results of the bispecific antibodies MutC, MutC+ER and MutC-ER compared to control WT and hIgG. Figure 10B shows the results of the bispecific antibodies MutC-D+ER, and MutC-D-DeC and MutC-Dec.

Detailed description of the invention

Terms and abbreviations

Unless otherwise defined herein, scientific and technical terms and acronyms used in connection with the present application shall have the meaning as commonly understood by one of ordinary skill in the art. Some of the terms and abbreviations used in this article are listed below.

BsAb: bispecific antibody

HC: heavy chain

LC: light chain

VH: variable region of heavy chain

VL: variable region of light chain

CH: constant region of heavy chain

CL: constant region of light chain

CDR: complementarity determining region

scFv: single-chain variable fragment (single-chain variable fragment)

ADCC: antibody dependent cellular cytotoxicity

ELISA: enzyme-linked immunosorbent assay

The procedures for molecular cloning, cell culture, protein purification, immunological experiments of the present invention are routine steps that are widely used in the art. Unless otherwise indicated, the amino acid sequences of the present invention are arranged and written in the direction from the amino terminus to the carboxy terminus. The three-letter abbreviations and nucleotide single-letter abbreviations referred to in the present invention are generally accepted forms in the art, and the amino acid single letter abbreviations are in the form recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The term "amino acid" refers to any of the 20 naturally occurring amino acids or any non-natural analog that may be present at a particular position. "Amino acid mutation" as used herein refers to amino acid substitutions, additions, insertions and/or deletions in a polypeptide sequence. Preferred amino acid mutations herein are substitutions. As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid. For example, a substitution C220S refers to a variant polypeptide in which the cysteine at position 220 of the polypeptide has been substituted with a serine.

The term "antibody" refers to an immunoglobulin molecule comprising at least one antigen recognition site and capable of specifically binding an antigen. Here, the term "antigen" is a substance which induces an immune response in the body and specifically binds to an antibody, such as a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, a hapten or a combination thereof. . The binding of an antibody to an antigen is mediated by interactions formed between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region of the surface of the antigen that binds to the antibody is an "antigenic determinant" or "epitope". Generally, each antigen has multiple determinants.

The term "antibody" as referred to in the present invention is understood in its broadest sense and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, antibody fragments, and at least two different antigen binding domains. Multispecific antibodies (eg, bispecific antibodies). Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. The antibodies of the invention may be derived from any animal including, but not limited to, humans, non-human primates, mice, rats, cows, horses, chickens, camels, llamas, alpacas, An immunoglobulin molecule of the llama (Guanaco), llama (Vicunas) or shark. Antibodies may contain additional alterations such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modification to an antigen recognition site, so long as these antibodies exhibit the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen binding domain.

Human immunoglobulins can be divided into five classes based on the amino acid sequence of the heavy chain constant region of the antibody: IgA, IgD, IgE, IgG, and IgM, which can be further divided into different subclasses (isotypes), such as IgG1, IgG2. , IgG3, IgG4, IGA1, IGA2, and the like. Depending on the light chain amino acid sequence, the light chain can be classified as a lambda chain or a kappa chain. The antibody of the present invention may be of any kind (such as IgA, IgD, IgE, IgG, and IgM) or a subclass (such as IgG1, IgG2, IgG3, IgG4, IGA1, or IGA2).

The term "humanized antibody" refers to an antibody produced by a non-human species such as a rat, a mouse, or the like, which is modified to retain the affinity of the parent antibody for binding to the antigen while reducing the immunogenicity of the heterologous antibody in the human body. Generally, the amino acid sequence of the CDR regions of the humanized antibody is substantially identical to the parent antibody (non-human antibody), and the framework region and the constant region amino acid sequence are sequences of human immunoglobulin. The humanized antibody can be of any class (eg, IgA, IgD, IgE, IgG, and IgM) or subclass (eg, IgGl, IgG2, IgG3, IgG4, IGA1, or IGA2) of any class of immunoglobulins. The CDR regions or framework regions of the humanized antibody do not completely correspond to the sequence of the parent antibody, and residue insertions, deletions or substitutions can be made according to actual needs in order to allow the antibody to produce the desired properties. Humanization of non-human antibodies can be made by methods known in the art, including CDR grafting, surface recombination, molecular modeling, and the like.

The term "bispecific antibody" refers to an antibody molecule that is capable of binding two independent antigens or having binding specificity for different epitopes within the same antigen. For example, one arm of a bispecific antibody molecule binds to a tumor-associated antigen, and the other arm binds to an immune cell-associated antigen, which activates at a tumor cell and initiates a cellular immune-related mechanism. The term "multispecific antibody" refers to a bispecific, trispecific or tetraspecific antibody. Multispecific antibodies include two or more different antigen binding domains and are therefore capable of binding to two, three, four or more different antigens.

The term "antibody" also includes antibody fragments. "Antibody fragment" or "antigen binding fragments" include, but are not limited to: (i) Fab fragments, having a V _{_L,} C _L, V _H and C _H 1 domains; (ii) Fab 'fragment, which is in the C _H 1 C-terminal domain of the Fab fragment having one or more cysteine residues; (iii) a Fd fragment having the V _H and C _H 1 domains; (iv) Fd 'fragment having a V _H and C _H 1 domain and a C-terminus of the CH1 domain or more cysteine residues; (v) Fv fragments, in which a single arm of an antibody V _H and V _L domains; (vi) dAb fragment consisting of VH or VL domains Domain composition; (vii) F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments joined by a disulfide bond at the hinge region; (viii) a single chain variable fragment (scFv). As used herein, "antibody fragment" includes not only the above antibody fragments, but also antibodies engineered from whole antibodies and novel antibodies synthesized using recombinant DNA techniques.

The "variable region" of an antibody refers to the variable region of an antibody heavy or light chain, including individual variable regions, individually and in combination. The variable regions of the heavy and light chains are composed of three complementarity determining regions (CDRs, also called hypervariable regions) and four framework regions (FRs) located on the CDR flanks. The frame region supports the CDRs and defines the spatial relationship between the CDRs. The CDRs of the heavy or light chain are represented by the amino terminus as CDR1, CDR2, CDR3, respectively. The heavy and light chain variable regions are joined by a non-covalent bond, and the three CDRs of the heavy chain and the three CDRs of the light chain together constitute an antigen recognition site, and the partial amino acid residue is a subject in which the antibody participates in antigen binding, and constitutes The antibody recognizes the specificity of the antigen.

The term "Fc region of an antibody" or "human immunoglobulin Fc region" encompasses a constant region polypeptide other than the heavy chain constant region 1 (CH1) of the antibody, ie, the human immunoglobulin IgA, IgD, IgG heavy chain constant region carboxy terminus Two constant region domains CH2 and CH3, as well as three constant region domains CH2, CH3 and CH4 at the carboxy terminus of the human immunoglobulin IgE and IgM heavy chain constant regions, and also include flexible hinge regions at the amino terminus of these domains. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as a residue comprising from A231 to its carboxy terminus.

The immunoglobulin Fc region is a functional domain in which an antibody exerts an immune effect. The Fc of an IgG antibody is capable of interacting with a variety of receptors, the most important of which being the Fcy receptor family. The receptor family includes five activating receptors: FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, and one inhibitory receptor: FcγRIIb. Fc binds to the extracellular domain of FcγR to form an Fc/FcγR complex, resulting in the FcγR intracellular region ITAM (immunoreceptor tyrosine-based activation motifs) or ITIM (immunoreceptor tyrosine-based inhibitory motifs) Phosphorylation of the receptor tyrosine inhibitory motif activates the downstream signal transduction pathway and produces immune responses such as endocytosis, phagocytosis, cell killing, and the like. In addition, Fc can also bind to the complement protein C1q, resulting in a complement dependent cytotoxicity (CDC) effect.

The term "minibody" refers to an artificial antibody fragment consisting of the antibody fragment VL-VH-CH3.

The term "nanobody" refers to a camelid antibody fragment consisting of a single monomer variable antibody region.

The term "probody" is an artificial antibody molecule whose antigen binding site is masked until activation activates.

As used herein, "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. Most of the antigenic determinants are present on the surface of the antigenic material, and some are present in the interior of the antigenic material and must be exposed by enzymes or other means. Epitopes or antigenic determinants usually consist of chemically active surface groups of the molecule, such as amino acids, carbohydrates or sugar side chains, and typically have specific three dimensional structural characteristics as well as specific points and features. Epitopes can be "linear" or "conformational." In a linear epitope, the point of all interactions between a protein and an interacting molecule (such as an antibody) exists linearly along the primary amino acid sequence of the protein; in a conformational epitope, the point of interaction spans the protein amino acid residues separated from each other There is a foundation. A natural antigenic substance can have multiple and multiple determinants. In general, the larger the antigen molecule, the greater the number of determinants.

As used herein, "specifically binds" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) means that the antibody is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, Affinity (K _D ) of 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less binds to the antigen. In some embodiments of the invention, the term "targeting" refers to specific binding.

As used herein, "K _D" refers to a particular antibody - antigen interaction dissociation equilibrium constant, are used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody will have an equilibrium dissociation (K _{D ) of} less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less _. ) bind antigen.

The term "EC50" as used herein, ie, concentration for 50% of maximal effect, refers to the concentration of antibody that results in a 50% maximal effect.

As used herein, "Vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector enables expression of the protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell. Vectors are recognized by those skilled in the art, including but not limited to: (1) plasmids; (2) phagemids; (3) cosmids; (4) artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial labor Chromosome (BAC) or P1 derived artificial chromosome (PAC); (5) phage such as lambda phage or M13 phage and (6) animal virus, such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, spore Rash virus (such as simple herpes virus), pox virus, baculovirus. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes; in addition, the vector may also contain an origin of replication.

The antibodies of the invention, such as bispecific antibodies, can be extracted and purified from host cells using standard experimental methods. For example, antibodies can be purified using Protein A or Protein G affinity chromatography. Purification means include, but are not limited to, affinity chromatography, ion exchange, size exclusion chromatography, and protein ultrafiltration. The method for separating and purifying the bispecific antibody of the present invention also includes a combination of the above methods. As used herein, "purified" refers to the isolation and/or recovery of a component of interest from a cell, cell culture or other natural component. The antibodies described in the present invention are all purified antibodies unless otherwise specified. The term "isolated antibody" refers to an antibody that is substantially free of other molecules of a different structure or antigen specificity, and an "isolated bispecific antibody" is an antibody that is substantially free of other types of antibody molecules.

In the present invention, the numbering of residues in the immunoglobulin heavy chain is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The numbering of the EU index, which is also available on the World Wide Web, is expressly incorporated herein by reference in its entirety. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. As used herein, "Kabat sequence numbering" or "Kabat tag" refers to a sequence encoding a variable region, such as the EU index number in Kabat. For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2, and amino acid positions 95 to 102 of CDR3 according to Kabat numbering. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 of CDR1, amino acid positions 50 to 56 of CDR2, and amino acid positions 89 to 97 of CDR3 according to Kabat numbering.

Antibody modification method

An antibody is an immunoglobulin (Ig) secreted by a plasma cell and capable of specifically binding to an antigen after the antigen stimulates the body to generate an immune reaction. Natural antibodies are typically bivalent antibodies (other than the IgM class), ie one antibody molecule contains two antigen binding sites and each Fab arm contains one antigen binding site. The antibody light chain is covalently linked to the heavy chain by two disulfide bonds, and the heavy chain-light chain dimer is further composed of a disulfide bond formed between the heavy chains to form a Y-shaped antibody molecule. There may be differences in the number of disulfide bonds between the heavy chain of different classes of antibodies. The area between the two arms of the Y-shaped body and the trunk is a hinge area with a certain flexibility. Each antibody polypeptide chain includes a variable region as well as a constant region, which are formed by spatial folding to form different domain units. Wherein the amino terminus of the heavy chain of the antibody is a variable region, followed by three constant regions CH1, CH2, CH3; the amino terminus of the light chain is a variable region, followed by a constant region CL. The interaction between the heavy chain and the variable region of the light chain constitutes the region where the antigen recognizes binding, plus the interaction of CL with CH1 and the partial hinge region between CH1 and CH2 constitutes the Fab region of the antibody; the formation of CH2 and CH3 of the two heavy chains The homodimer is the Fc region of the IgG antibody; the disulfide bond formed by the hinge region between CH1 and CH2 further stabilizes the structure of the antibody.

The Fab region of an antibody comprises a variable region domain at the amino terminus of the heavy and light chain of the antibody, and a constant region domain and a portion of the hinge region located behind it. Wherein the first constant region domain (CH1) of the heavy chain binds to the constant region domain (CL) of the light chain, the variable region domain (VH) of the heavy chain and the variable region domain (VL) of the light chain Combine.

Compared with traditional chemotherapeutic drugs, antibodies have the advantages of less toxic side effects, high specificity and good cell killing effect, and are one of the research and development hotspots in the current pharmaceutical field. Since 1986, the first monoclonal antibody drug anti-CD3 monoclonal antibody Moromona (Muromonab OKT3) for clinical treatment has been approved by the FDA. Monoclonal antibodies are in malignant tumors, autoimmune diseases, inflammatory infections, cardiovascular diseases, etc. It has developed rapidly in the treatment of major diseases and is the highest compound growth rate in the biomedical field. By the end of 2016, there were 63 kinds of antibody drugs on the market, and the global sales of antibody drugs exceeded 100 billion US dollars, accounting for more than 20% of global drug sales. And six of the top 10 globally marketed drugs are antibody-based drugs, more than half of which are used in cancer treatment. The mechanism of action of monoclonal antibodies includes blocking growth signals, blocking tumor angiogenesis, inducing apoptosis, and activating immune cells to produce immune effects. Monoclonal antibodies play an important role mainly through Fc and immune cells (such as NK cells, mononuclear cells). Cells such as FcγR (Fcγ receptor) bind to activating cells to enhance ADCC, antibody-dependent cellular cytotoxicity), ADCP (antibody dependent cellular phagocytosis) or enhance CDC by binding of Fc to complement protein C1q to exert cell killing effect.

Although antibody drugs have made breakthroughs in the past decade, they have become the main direction of the development of the pharmaceutical industry in the future. However, in the practical application process, the monoclonal antibody drugs still face problems such as low overall effectiveness and easy emergence of drug resistance. FcγR is polymorphic in human population. For example, FcγRIIIa-V158 on NK cell surface has higher binding ability to Fc, while FcγRIIIa-F158 has weaker binding ability, which directly affects the response of some patients to drugs, and is also effective for monoclonal antibody. One of the lower reasons. In addition, monotherapy is not effective in the treatment of malignant tumors, especially solid tumors. Tumor heterogeneity, tumor stem cells, and multi-signal pathway regulation of tumor cells themselves make immunotherapy for single targets susceptible to drug resistance (Beck A, Et al. Nat Rev Immunol. 2010; 10(5): 345-52.).

Since T cells do not express FcγR, traditional monoclonal antibodies cannot directly activate killer T cells, so people try to modify the traditional monoclonal antibodies to achieve the purpose of activating T cells to kill tumors. Among them, bispecific antibodies (BsAb) are the most One of the methods of applying prospects. BsAb is a class of antibodies that bind to at least two epitopes. It is greatly improved in safety and efficacy compared to traditional single-target antibodies. It is a research and development in the field of antibody engineering and tumor immunotherapy. hot spot. In practical applications for the treatment of malignant tumors, BsAb usually binds to tumor cell surface antigens as well as immune cell surface antigens to kill tumor cells by activating the autoimmune system (Chames P, et al. Curr Opin Drug Discov Devel 2009, 12:276-283 .).

Bispecific antibodies are not naturally produced antibodies of cells and can only be artificially prepared by cell fusion or DNA recombination techniques. A variety of preparation methods are currently available. Early bispecific antibodies are mostly produced by chemical cross-linking of purified monoclonal antibodies or fusion of two different hybridoma cells, but the products produced by these methods have many problems such as product instability, low yield, and improper antibody modification. , immunogenicity, production and purification difficulties. With the advancement of genetic engineering technology in recent years, BsAb has been prepared by genetic engineering technology. There are more than 50 different bispecific antibody forms, which can be divided into two categories: no Fc and bispecific containing Fc region. Sex antibodies (Brinkmann U, et al. MAbs 2017, 9: 182-212.). The former is usually a small molecular weight antibody fragment and does not mediate Fc-related biological functions. The latter is usually in the form of IgG, which is similar to the native antibody form. The Y-shaped arms can bind to two different antigens respectively. The Fc region can mediate ADCC, ADCP and CDC, increase the half-life of the antibody in blood, Stability and solubility are easily prepared in large quantities by existing methods.

In the preparation of IgG-structured antibodies, it is necessary to simultaneously express two different heavy chains and two different light chains, and form a Fab that recognizes two antigens by pairing two pairs of HC-LCs. In this process, homologous heavy chains The mismatch problem created by binding and heterologous light chain binding produces ten different products, of which only one is the correct product required. Thus the main problem to be solved in the preparation of such antibodies is the random assembly of different chains such as homologous heavy chain mismatches, light chain mismatches and contamination of other non-target products.

One solution to the problem of homologous heavy chain mismatches is the IgG antibody "Knob Into Hole" chimeric technology ("KIH"), invented by Ridgeway et al., the basic principle of which is the two heavy chains of IgG antibodies. The Fc region introduces different amino acid mutations, introducing an amino acid having a large side chain group (T366Y) into the first heavy chain and introducing an amino acid having a small side chain group (Y407T) into the second heavy chain. When the two chains are expressed simultaneously in the cell, they tend to bind together to form a heterodimer rather than a homodimer due to the complementary spatial structure, in order to overcome the instability caused by the mutation, Furthermore, phage display technology was used to screen random mutations to construct a more stable structure, namely: raised structure-T366W, depressed structure-T366S, L368A, Y407V. In theory, any two different antibodies can be formed into a heterodimer by this method, but there are still 5% homodimers, and the problem of light chain mismatch cannot be solved (US 5,731,168 A, US 5,731,168 A) Ridgway JB, et al. Protein Eng 1996, 9: 617-621.; Atwell S, et al. J Mol Biol 1997, 270: 26-35.; Merchant AM, et al. Nat Biotechnol 1998, 16:677-681 .).

In addition to overcoming the formation of homodimers using hydrophobic interactions formed by steric hindrance, another solution is to use ionic bonds to artificially introduce amino acids with opposite charge properties in the two heavy chains, thus repelling by the same charge. Inhibition of the formation of homodimers, the related methods are described in detail in the patents and literature (US8592562 B2, US 20170058054 A1, WO 2014084607 A1; Gunasekaran K, et al. J Biol Chem 2010, 285: 19637-19646; Strop P, et al. J Mol Biol 2012, 420: 204-219; Choi HJ, et al. Mol Immunol 2015, 65: 377-383).

In the present inventor's prior patent application (Publication No. WO 2017034770 A1), a method of modifying the heavy chain portion of an antibody to increase the binding activity and specificity between the two heavy chains is disclosed. Specifically, the method successfully solves the problem of heavy chain mismatch in the preparation of bispecific antibodies by changing the charge properties of amino acids at the interaction interface of the CH3 region, reducing homodimers, and promoting the formation of heterodimers. problem.

For the mismatch between heavy and light chains, there are currently several solutions: 1) screening and using common light chains; 2) merging light chains to heavy chains; 3) modifying light and heavy chains Make it possible to specifically pair.

The most straightforward way to solve the light chain mismatch is to use a common light chain, ie the two heavy chains share the same light chain. In general, the variable regions of the antibody light chain are different, and the diverse variable regions also determine the specificity of the antibody for antigen recognition. In practical applications, the acquisition of common light chains is difficult, requires a lot of manpower and time to screen, and does not guarantee the screening of suitable common light chains; in addition, the use of common light chains may reduce the specificity and affinity for antigens, Therefore the common light chain is not suitable for all bispecific antibodies.

Another solution is to fused the light and heavy chains by engineering, for example, by joining a heavy chain variable region and a light chain variable region with a non-native adaptor sequence to generate a single chain variable fragment (scFv), Or the light chain is linked to the variable region of the heavy chain to form a single chain Fab antibody fragment (scFab), but the introduced adaptor sequence brings stability, solubility and the like, and is easy to form antibody polymerization. , may cause immunogenicity problems (Gunasekaran K, et al. J Biol Chem 2010, 285: 19637-19646.; Muda M, et al. Protein Eng Des Sel 2011, 24: 447-454.; Wranik BJ, et al .J Biol Chem 2012, 287:43331-43339.).

For bispecific antibodies with two light chains, the two antibodies can be expressed separately to avoid light chain mismatches, such as controlled Fab-arm exchange (cFAE), chemical cross-linking, and the like. The cFAE method assembles two specific antibodies together to form a bispecific antibody resembling a native IgG structure by redox expression in vitro by separately expressing and purifying two specific monoclonal antibodies. The principle draws on the physiological process of Fab arm exchange in human IgG4 molecule. In this process, the arginine (R409) at position 409 and the 228th serine (S228) at the IgG4CH3 region play a key role. . At present, this assembly method is complicated in process, requires secondary purification, and has low yield, which greatly increases production cost (WO 2008119353, WO2011131746 A2; Labrijn AF, et al. Proc Natl Acad Sci USA 2013, 110: 5145-5150. Cook AG, et al. J Immunol Methods 1994, 171:227-237.).

Another solution is described by Lindhofer et al. (Lindhofer H, et al. J Immunol 1995, 155: 219-225), which utilizes the characteristics that the rat heavy chain can naturally bind to the mouse heavy chain and will produce rat Two hybridoma cells of the source monoclonal antibody and the mouse-derived monoclonal antibody form a cell capable of producing a bispecific antibody by a technique of somatic fusion. The antibodies produced by this method overcome the light chain mismatch by species restriction and the mismatch rate can be reduced to 4-10%. The earliest marketed bispecific antibody, Catumaxomab (trade name: RemovabTM ⁾ , was produced by this technique for the treatment of malignant ascites. However, since the antibody is murine, it causes immunogenicity.

Although the above-described technical solutions for solving the problem of mismatch between the heavy chain and the light chain of the bispecific antibody have existed, there are still various problems such as complicated process, antigenicity, low yield of the target antibody, and unsuitability for all antibodies. Therefore, there is still a need for a new and effective method of solving the mismatch between heavy and light chains.

The present invention deeply analyzes the spatial structure of the interaction between human IgG1 CH1-CL and hinge region-CL, and finds that non-natural disulfide bonds are introduced at the CH1-CL interface, and the natural disulfide of the hinge region-CL region is optionally removed. The bond (HC-C220S, LC-C214S) is capable of forming a paired heavy chain-light chain Fab or whole antibody using an artificially engineered disulfide bond based on the introduction of minimal site mutations without affecting the structure and function of the antibody. .

In particular, the present invention identifies four amino acid pairs between the antibody heavy chain CH1 and the light chain CL (see Table 1 below) which are suitable for mutating to cysteine for heavy chain CH1 and light chain CL An unnatural disulfide bond is formed between them.

Table 1. Amino acid pairs that can form disulfide between the heavy chain CH1-light chain CL

Furthermore, the inventors analyzed the conservation of the above sites between different antibody isotypes. As shown in Table 2, the above four amino acid pairs were conserved among the IgG1, IgG2, IgG3 and IgG4 isotypes.

Table 2. Conservatives of amino acid pairs in IgG1, IgG2, IgG3, and IgG4

The present invention also provides a method of charge engineering by introducing a K218E or K218D mutation into the hinge region of an antibody and introducing an E213R or E213K mutation into the CL, which increases the binding specificity between the heavy chain and the light chain. This pair of charge engineering can be used alone or in combination with the disulfide-engineered methods of the invention for use in the construction of antibodies such as bispecific antibodies or multispecific antibodies to facilitate proper pairing of the heavy chain-light chain of the antibody.

Thus, in one aspect, the invention relates to a method of engineering a Fab region of an antibody, the method comprising the step of introducing one or more mutations selected from the group consisting of:

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL;

The antibody heavy and light chains are numbered according to the EU number.

One or more of the above mutation combinations a)-e) may be introduced into the Fab region of the antibody, such as 1, 2, 3, 4 or 5 terms. In a preferred embodiment, the Fab region of the antibody is introduced into one or two of the above-described mutated combinations a)-d) to form between the heavy and light chains of the antibody, or fragments of the heavy and light chains. One pair or two pairs of non-native disulfide bonds, and optionally introduced mutation combinations e) to further increase the binding specificity between the antibody heavy and light chains.

In some embodiments, the engineered method of the invention further comprises disrupting the destruction of the native disulfide bond between CH1-CL, ie, disrupting the cysteine at position 220 (C220) in the hinge region and the 214th in CL A disulfide bond formed between the cysteine (C214). It can be achieved by mutating the cysteine at position 214 of the light chain CL region to any non-cysteine, preferably serine, alanine and glycine, or deleting the cysteine at position 214; Or the cysteine at position 220 of the heavy chain hinge region is any non-cysteine; or the cysteine at position 214 of the light chain CL region is any non-cysteine, and the heavy chain hinge region is 220 The cysteine is any non-cysteine; or the cysteine at position 214 of the light chain CL region is deleted, while the cysteine at position 220 of the heavy chain hinge region is mutated to any non-cysteine.

In any of the above methods, the Fab region is derived from IgG, IgA, IgM, IgE or IgD, such as an IgGl, IgG2, IgG3 or IgG4 isotype. As shown in Table 2, the amino acid pairs identified in the present invention are conserved among the IgGl, IgG2, IgG3 and IgG4 isotypes. Thus the methods of the invention can be used in the above antibody isotypes.

It will be appreciated that the methods of the invention for engineering a Fab region can be applied to any antibody or antibody fragment having at least one Fab region, including, for example, monoclonal antibodies, bispecific antibodies or multispecific antibodies; murine antibodies, human sources Antibodies, chimeric antibodies, human antibodies, and antibodies of other origin; and any antibody fragment having a Fab region, such as a Fab fragment, a Fab' fragment, and an F(ab') ₂ fragment.

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL; and

d) V173C in CH1 and Q160C in CL,

The antibody heavy and light chains are numbered according to the EU number.

Antibody production method

In one aspect, the invention relates to a method of producing an antibody or antibody fragment having at least two different Fab regions, the method comprising the steps of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

3) recovering the antibody or antibody fragment from the host cell culture.

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

In some embodiments, the above method further comprises disrupting a native disulfide bond between the hinge region-CL region in the first Fab region and/or the second Fab region, ie, the first Fab region and/or the second C220 in the hinge region of the Fab region is mutated to an amino acid other than cysteine or to C220, and/or to CL in the first Fab region and/or the second Fab region and/or the second Fab region. The C214 mutation is an amino acid other than cysteine or a deletion C214. In a preferred embodiment, the amino acid other than cysteine is selected from the group consisting of serine, alanine and glycine.

In some embodiments of the above methods, the first Fab region and the second Fab region bind to different epitopes on the same antigen.

In some embodiments of the above methods, the first Fab region binds to a first antigen, the second Fab region binds to a second antigen, and the first antigen and the second antigen are different.

It will be appreciated that the methods described herein for producing antibodies or antibody fragments can be used with any antibody or antibody fragment having at least two different Fab regions, including bispecific antibodies and multispecific antibodies. In addition, different Fab regions can be selected as needed to construct bispecific antibodies or multispecific antibodies.

Modified antibody

In another aspect, the invention features an antibody or antibody fragment having at least two different Fab regions, the first Fab region of the antibody or antibody fragment having one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) K218E or K218D mutations in the hinge region and E213R or E213K mutations in CL, numbered according to EU numbering.

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, wherein the mutation in the second Fab region is not identical to the mutation in the first Fab region.

In some embodiments of the above antibodies and antibody fragments, the first Fab region and the second Fab region bind to different epitopes on the same antigen.

In some embodiments of the above antibodies and antibody fragments, the first Fab region binds to a first antigen, the second Fab region binds to the second antigen, and wherein the first antigen and the second antigen are different.

In any embodiment, the engineered Fab region, antibody or antibody fragment of the invention binds to a cancer antigen, a non-cancer protein associated with cancer development or invasiveness, or a virus-associated protein. Indeed, a Fab region, antibody or antibody fragment of the invention may bind to any antigen, including but not limited to the following proteins, subunits, domains, motifs and epitopes: CD2; CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL- 1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, Interferon alpha, interferon beta, interferon gamma; TNF-alpha, TNF beta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β1, integrin β2 , integrin α4/β7, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin αv, integrin αVβ3, FGFR-3, keratinocytes Growth factor, VLA-1, VLA-4, L-selection , anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, eosinophil chemotactic factor 1 ( Eotaxin1), Blys (B lymphocyte stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, VEGF, VEGFR, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, T cell receptor α/β, E-selectin, digoxin, placenta base Phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, mucin MUC1, MUC18, heparinase I, human cardiac myosin , tumor associated glycoprotein-72 (TAG-72), tumor associated antigen CA 125, prostate specific membrane antigen (PSMA), high molecular weight melanoma associated antigen (HMW-MAA), cancer associated antigen, Gco protein Iib /IIIa (GPIIb/IIIa), a tumor-associated antigen expressing Lewis Y-related carbohydrates, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, VNR Connexin, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB package Membrane glycoprotein and Clostridium perfringens toxin.

In some embodiments, the engineered Fab region, antibody or antibody fragment of the invention binds to an antigen selected from the group consisting of: PSMA, CD133, CD138, CD20, CD19, OX40, GITR, PD-1, PD-L1 or PD-L2 , CTLA-4, KIR, LAG-3, CD3, TCRα, TCRβ, TCRγ, TCRδ, CD40, CD40L, VEGF, EGF, VEGFR, EGFR, Her1, Her2, Her3, EpCAM, mesothelin, Glypicans, CD28, Erbl , Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2 , CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, CD64, CD32a, CD32b, CD16, integrin, RANK ligand, CEA, DLL-4, GM-CSFR, ADAMS, Myostatin, PCSK9, CXCR4, IL-1α, IL-1β, IL-12, IL-18, TNFα, IL-23, IL-13, MIF, IL-17, IL-17R, IL-15, IL -9, IL-5, IL-5R, IL-6, IL-25, PEG2, and the like. The antibodies of the invention may be specific for viral related targets, such as HIV proteins, HPV proteins, CMV proteins, influenza virus proteins or prion proteins.

In certain aspects, the engineered Fab regions, antibodies or antibody fragments of the invention are useful in diagnostic and therapeutic applications. More specifically, the methods of the invention can be used to generate bispecific or multispecific antibodies that can bind two or more target antigens, which can be selected from, but not limited to, the following targets: IL-1α , IL-1β, IL-12, IL-18, TNFα, IL-23, IL-13, MIF, IL-17, IL-17R, IL-15; VEGF, VEGFR, EGFR; IL-9, IL-5 , IL-5R, IL-6, IL-25, IL-13, ADAMS, PEG2, Her1, Her2 and Her3. In addition, those skilled in the art will be able to identify other possible targets.

The methods of the invention can be used to engineer bispecific antibodies that can be used, but are not limited to, to activate T cells to kill target cells carrying tumor antigens, wherein one antigen binding region consists of the Fab region of the monoclonal antibody, the C-terminus The N-terminus of the hinge region of one heavy chain is linked to the tumor cell antigen target or the signal pathway target on the immune effector cell; the other antigen binding region is composed of the Fab region of the monoclonal antibody, and the C-terminus and the second The N-terminus of the hinge region of the heavy chain is connected; similarly, it can also specifically bind to the antigen target of the tumor cell or the signal pathway target on the immune effector cell; likewise, the bispecific produced by the mutation according to the above technical scheme of the present invention Sex antibodies can also be used to block, antagonize or activate target antigens, such as antagonizing cytokines or cytokine receptors.

a) the CL region of SEQ ID NO: 11 and the CH1 region of SEQ ID NO: 33;

b) the CL region of SEQ ID NO: 13 and the CH1 region of SEQ ID NO: 29;

c) the CL region of SEQ ID NO: 15 and the CH1 region of SEQ ID NO: 35;

d) the CL region of SEQ ID NO: 17 and the CH1 region of SEQ ID NO: 33;

e) the CL region of SEQ ID NO:27 and the CH1 region of SEQ ID NO:43.

Nucleic acid sequence and host cell

The invention also relates to nucleotide sequences encoding the antibodies of the invention, as well as vectors comprising these nucleotide sequences. In the process of expressing an antibody, the nucleotide sequence is inserted into a suitable vector including, but not limited to, a plasmid, a phage expression vector, a cosmid, an artificial chromosome, a phage, and an animal virus. The expression vector contains elements for regulating expression including, but not limited to, a promoter, a transcription initiation sequence, an enhancer, a signal peptide sequence, and the like. Promoters include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β-actin promoter, the EF-1α promoter, the CMV promoter, and the SV40 promoter. Transfer of the expression vector into a host cell can be carried out using suitable methods known in the art including, but not limited to, calcium phosphate precipitation, polyethyleneimine transfection, lipofection, electroporation, PEI ( Polyethyleneimine) transfection method.

In still another aspect, the invention encompasses a host cell comprising a nucleic acid molecule or vector of the invention. The host cells include, but are not limited to, CHO cells (Chinese hamster ovary cells), HEK293 cells (Human embryonic kidney cells 293), myeloma cells, yeast or prokaryotic cells such as Escherichia coli. (Escherichia coli).

Antibody conjugate

Pharmaceutical composition and disease treatment method

In another aspect, the invention relates to a method of treating a disease comprising the step of using an antibody or antibody fragment, a bispecific antibody, an antibody conjugate or a pharmaceutical composition of the invention.

In some embodiments, the disease is cancer.

In one aspect, the invention relates to a pharmaceutical composition comprising an antibody or antibody fragment of the invention, a bispecific antibody or antibody conjugate, and optionally a pharmaceutically acceptable carrier, surfactant and/or diluent .

The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid ( For example, a lubricant, talc, calcium stearate or zinc or stearic acid or a solvent encapsulating material relates to maintaining the stability, solubility or activity of the LAP binding agent. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as Sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) shaping Agents such as cocoa butter and suppository wax; (8) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols such as propylene glycol; (10) polyols, Such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffers, such as magnesium hydroxide and hydrogen Alumina; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19) pH buffer solution; (20) polyester, polycarbonate and/or Polyanhydride; (21) fillers, such as polypeptides and amino acids (22) serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) used in pharmaceutical formulations Other non-toxic compatible substances. Release agents, coating agents, preservatives and antioxidants may also be present in the pharmaceutical formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

In some embodiments, in addition to an antibody, bispecific antibody or antibody conjugate of the invention, the pharmaceutical composition further comprises one or more additional therapeutic agents.

In some embodiments, the additional therapeutic agent includes, but is not limited to, a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, an agent for radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, and Other agents for the treatment of cancer, such as anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (eg tyrosine kinase inhibitors), HER1/EGFR inhibitors (eg erlotinib)

), Platelet derived growth factor inhibitors (e.g., GLEEVEC ^TM (imatinib mesylate (Imatinib Mesylate))), COX -2 inhibitors (e.g. celecoxib (, celecoxib)), interferons, cytokines An antagonist (eg, a neutralizing antibody) that binds to one or more of the following targets PD-1, PD-L1, PD-L2 (eg, pemrolizumab; nivolumab; MK-3475; AMP- 224; MPDL3280A; MEDI0680; MSB0010718C; and/or MEDI 4736); CTLA-4 (eg, tremelimumab (PFIZER) and ipilimumab); LAG-3 (eg BMS-986016); CD103; TIM-3 and/or other TIM series Members; CEACAM-1 and/or other CEACAM family members, ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA or VEGF receptors, TRAIL/Apo2 and other biological agents and organic chemicals, and the like. The methods described herein also specifically consider combinations thereof.

In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. Non-limiting examples of chemotherapeutic agents can include alkylating agents, such as thiotepa and

Cyclophosphamide, temozolomide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridine Aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methyl melamine Classes (methylamelamines), including altretamine, triethylenemelamine, trietylenephosphoramide (triethylenephosphoramide), triethithiothioamide (triethylenethiophosphoramide) and trishydroxymethyl honey Trimethylolomelamine; acetogenin (especially bullatacin and bullatacinone); camptothecin (including synthetic analogue topotecan (topotecan) ); bryostatin; callistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues); cryptosins ( Cry Ptophycins) (especially cryptocyanin 1 and cryptocin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin Sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, Isophorfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembibin, phenesterine, Prednimustine, trofosfamide, uracil mustard, nitrosoureas, such as carmustine, chlorozotocin, Fotemustine, lomustine, nimustine, and ramimustine; antibiotics, such as enediyne (such as california) Calicheamicin, especially calicheamicin γ1I And calicheamicin ωI1 (see, eg, Agnew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); anthracyclines (dynemicin), including dynemicin A; esperamicin; And neocarzinostatin chromophore and related chromoprotein diacetylene antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azo Azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, chromomycin, Actinomycin D (dactinomycin), daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine,

Doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolidol doxorubicin, and deoxydoxantine), epirubicin (epirubicin) ), esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, Nogalamycin, olivomycin, peplomycin, potfiromycin, potfiromycin, puromycin, quelamycin, arromycin Rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin (zorubicin); antimetabolites such as methotrexate, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin , trimetrexate; purine analogues such as fludarabine, mercaptopurine, thiophene (thia) Miprine), thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azuridine, carmofur, cytarabine , dideoxyuridine, dexifluridine, enocitabine, floxuridine; androgens, such as calustronone, talazetine propionate Dromostanolone propionate, epitiostolol, mepitiostane, testolactone; anti-adrenal, such as aminoglutethimide, mitotane, troose Trilostane; folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil ;amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elformithine; Elliptinium acetate Epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine And ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; Phenonamem; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;

Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran (sizofiran); spiral scorpion (spirogermanium); (tenuazonic acid); triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin) A, roridin A and anguidin); urethan; vindesine; dacarbazine; mannomustine; dibromomannose Mitocolitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa; Taxoids, for example

Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ),

Cremophor-free, albumin-modified nanoparticulate paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and

Doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil;

Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; Vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; Novartrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate Ibandronate); irinotecan (Camptosar, CPT-11) (including irinotecan and 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoride Methyl ornithine (DMFO); retinoids, such as retinoic acid, capecitabine; combretastatin; formyltetrahydrofolate (LV); Oxaliplatin, including oxaliplatin treatment (FOLFOX); lapatinib (TYKERB.); PKC-alpha, Raf, H-Ras VEGF-A An inhibitor; and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

The pharmaceutical compositions described herein may be specially formulated for administering a compound to a subject in solid, liquid or gel form, including those suitable for use in the following: (1) parenteral administration, for example by subcutaneous, intramuscular, intravenous Or epidural injection, for example as a sterile solution or suspension or sustained release formulation; (2) topical application, for example as a cream, ointment or controlled release patch or spray applied to the skin; (3) Intravaginal or rectal, for example as a pessary, cream or foam; (4) eye; (5) transdermal; (6) transmucosal; or (7) nasal.

In one aspect, the invention relates to the use of an antibody or antibody fragment, bispecific antibody or antibody conjugate of the invention in the manufacture of a pharmaceutical composition for the treatment of a disease. In some embodiments, the disease is cancer.

In another aspect, the invention relates to an antibody or antibody fragment, bispecific antibody, antibody conjugate or pharmaceutical composition of the invention for use in the treatment of a disease. In some embodiments, the disease is cancer.

In still another aspect, the invention relates to a method of treating a disease comprising the step of administering to a subject an antibody or antibody fragment, a bispecific antibody, an antibody conjugate or a pharmaceutical composition of the invention. In some embodiments, the disease is cancer.

Examples of cancer include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectal cancer; connective tissue cancer; Digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; liver cancer; intraepithelial neoplasm; kidney cancer; laryngeal cancer; leukemia; Lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; Blastoma; oral cancer (eg, lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; Squamous cell carcinoma; gastric cancer; teratocarcinoma; testicular cancer; thyroid cancer; uterus or endometrial cancer; urinary cancer; vulvar cancer; and other cancers and sarcomas; and post-transplant lymphoproliferative disorders (PTLD), and Spotty Survial configuration related abnormal vascular proliferation, edema (such as edema associated with brain tumors), and Meigs original tumor origin (Meigs)'s syndrome.

The antibody or antibody fragment, bispecific antibody, antibody conjugate or pharmaceutical composition of the invention can be administered to a subject or patient in need thereof. The terms "subject," "patient," and "individual" are used interchangeably herein and refer to an animal, such as a human. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. . The term "subject" also includes any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal such as a human or other mammal, such as a domesticated mammal, such as a dog, cat, horse, and the like. Production of mammals, such as cattle, sheep, pigs, etc., is also included in the term subject.

Specific implementation method

The contents of the present invention will be further described below in conjunction with the examples. It is to be understood that the following examples are merely illustrative and are not to be construed as limiting the scope of the invention.

Example 1: Design of a bispecific antibody and construction of an expression vector

(1) Design of bispecific antibodies

An exemplary bispecific antibody designed and constructed in this embodiment is a "Y" type antibody (see Figure 1A) comprising two intact heavy chains and two intact light chains that together form the Fab and Fc of the antibody Domain. The Fab arms specifically bind to human CD3 and human Her2, respectively. The portion in which human CD3 specifically binds is designated herein as C31, which comprises an antibody heavy chain and an antibody light chain, the sequence of which is derived from the antibody heavy and light chain derived from a murine monoclonal antibody (see Patent No. US 9587021). ). The portion that specifically binds to human Her2 comprises an antibody heavy chain and an antibody light chain, the sequence of which is derived from the sequence of the humanized monoclonal antibody Herceptin (see US Pat. No. 5,821,337). The Fc portion of the antibody was engineered according to the method of the inventor's prior patent publication WO2017034770A1, wherein the heavy chain binding to the human CD3 portion was mutated as follows: P395K, P396K, V397K, the mutation was labeled OA; the binding to the human Her2 portion The heavy chain was mutated as follows: T394D, P395D, P396D, and the mutation was labeled OB to form a heavy chain heterodimer. This bispecific antibody is referred to herein as the bispecific antibody WT.

Anti-CD3 (C31) light chain amino acid sequence (SEQ ID NO: 1)

Anti-CD3 (C31) heavy chain amino acid sequence (SEQ ID NO: 3)

Anti-Her2 (Herceptin) light chain amino acid sequence (SEQ ID NO: 5)

Anti-Her2 (Herceptin) heavy chain amino acid sequence (SEQ ID NO: 7)

(2) Molecular cloning of expression plasmid

A nucleotide sequence encoding the C31 light chain (SEQ ID NO: 2) and a nucleotide sequence encoding the C31 heavy chain (SEQ ID NO: 4) were synthesized. Nucleotide encoding the C31 light chain using a universal molecular cloning method (Sambrook JF, EF et al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012) The sequence was cloned into the engineered plasmid pCDNA3.1(+) (Invitrogen, Cat. No. V790-20), which was engineered to add a human interleukin-2 (IL-2) to the N-terminus of the multiple cloning site. The signal peptide sequences (SEQ ID NO: 9 and SEQ ID NO: 10) enable expression of secreted antibodies in cells (e.g., HEK293 cells) and the resulting expression plasmid was designated pCDNA3.1-C31-LC. The nucleotide sequence encoding the C31 heavy chain was cloned into the plasmid pFUSE-hIgG1-Fc2 (InvivoGene), and the resulting expression plasmid was named pFUSE-C31-HC-OA.

A nucleotide sequence encoding the anti-Her2 (Herceptin) light chain (SEQ ID NO: 6) and a nucleotide sequence encoding the anti-Her2 (Herceptin) heavy chain (SEQ ID NO: 8) were artificially synthesized. The nucleotide sequence encoding the anti-Her2 light chain was cloned into the above-described engineered plasmid pCDNA3.1(+) by a universal molecular cloning method to enable expression of the secreted antibody in cells (for example, HEK293 cells). The expression plasmid was named pCDNA3.1-Her2-LC; the nucleotide sequence encoding the anti-Her2 heavy chain was cloned into the plasmid pFUSE-hIgG1-Fc2 (InvivoGene), and the resulting expression plasmid was named pFUSE-Her2-HC-OB. .

In order to form four bands of the above bispecific antibody on the SDS-PAGE gel, a 6His tag was added to the C-terminus of the anti-Her2 heavy chain, and the constructed expression plasmid was named pFUSE-Her2-HC- OB-6His.

(3) Modification of bispecific antibodies

The antibody sequence is subjected to amino acid point mutation according to the method of the patent application WO2017034770A1 which has been published by the present inventors. Mutation of two non-cysteine residues of the amino acid pair represented by B, C, D or E in Table 2 to a cysteine residue, such that the heavy chain CH1 and the light chain CL are at the mutation site A disulfide bond is formed therebetween. At the same time, the natural disulfide bond between the original heavy chain hinge region and the light chain CL chain is destroyed, which can be achieved by mutating the cysteine at position 214 of the light chain CL region to any non-cysteine. Either delete the cysteine at position 214 and/or mutate the cysteine at position 220 of the heavy chain hinge region to any non-cysteine or delete the cysteine at position 220. In the case of mutating cysteine to non-cysteine, it is preferably mutated to serine, alanine or glycine. By the above mutation method, the present invention produced four exemplary anti-Her2 x anti-CD3 bispecific antibodies, designated MutE, MutB, MutC and MutD, respectively (see Table 3). Among them, MutE introduced C220S and V173C mutations in the anti-Her2 heavy chain sequence based on the bispecific antibody WT, and introduced C214S and Q160C mutations in the anti-Her2 light chain sequence; MutB introduced C220S in the anti-Her2 heavy chain sequence. And F170C mutation, and introduced C214S and S162C mutations in the anti-Her2 light chain sequence; MutC introduced C220S and L128C mutations in the anti-Her2 heavy chain sequence, and introduced C214S and F118C mutations in the anti-Her2 light chain sequence; MutD is The C220S and F126C mutations were introduced into the anti-Her2 heavy chain sequence and the C214S and Q124C mutations were introduced in the anti-Her2 light chain sequence.

Table 3 Anti-Her2× anti-CD3 bispecific antibody mutation combination I

To further increase the selectivity of the light chain to the heavy chain, the E213R mutation was introduced into one light chain of the bispecific antibody, and the K218E mutation was introduced into the corresponding heavy chain to obtain the anti-Her2×anti-CD3 bispecific shown in Table 4. Sex antibody mutations combine MutC+ER and MutC-ER. Among them, MutC+ER is based on MutC, introducing K218E mutation in anti-CD3 (C31) heavy chain sequence, and introducing E213R mutation in anti-CD3 light chain; MutC-ER is based on MutC, in anti-Her2 The K218E mutation was introduced into the heavy chain and the E213R mutation was introduced into the anti-Her2 light chain. In addition, a MutC-Dec construct was constructed in which the cysteine at position 214 of the anti-Her light chain was deleted (Del-C214).

Table 4 Anti-Her2×Anti-CD3 Bispecific Antibody Mutation Combination II

To simultaneously form two non-native disulfide in a bispecific antibody, the anti-Her2 x anti-CD3 bispecific antibody mutation combinations MutC-D+ER and MutC-D-DeC shown in Table 5 were constructed. Among them, MutC-D+ER introduced C220S, L128C and F126C mutations in the anti-Her2 heavy chain sequence based on the bispecific antibody WT, and introduced C214S, F118C and Q124C mutations in the anti-Her light chain sequence. The K218E mutation was introduced into the CD3 heavy chain sequence and the E213R mutation was introduced into the anti-CD3 light chain sequence. MutC-D-DeC introduced the C220S, L128C and F126C mutations in the anti-Her2 heavy chain sequence, and deleted the 214th cysteine in the anti-Her2 light chain sequence, and introduced the F118C and Q124C mutations.

Table 5 Anti-Her2× anti-CD3 bispecific antibody mutation combination III

Example 2: Bispecific antibody expression and purification

(1) Transient expression of anti-Her2× anti-CD3 bispecific antibody

A large amount of plasmid was extracted using an endotoxin-free plasmid (Endo-Free-Plasmid Maxi Kit (100), available from OMEGA, Inc., catalog number D6926-04), and the procedure was carried out according to the instructions provided in the kit. The HEK293 cells were cultured to a cell density of 2.0 to 3.0×10 ⁶ cells/mL, and the cell suspension was centrifuged for 5 minutes at a rotation speed of 1000 rpm. The old culture supernatant was discarded, and fresh medium (OPM-291CD03Medium, purchased from Shanghai Aopu) was used. Mai Biotech Co., Ltd., Item No. 81070-001) resuspend the cells to a density of 1.0×10 ⁶ /mL. The expression vector of the bispecific antibody WT constructed in Example 1 and the mutation combinations listed in Tables 4, 5 and 6 were used MutB, MutC, MutD, MutE, MutC-DeC, MutC+ER, MutC-ER MutC-D+ER and MutC-D-DeC were co-transfected with cells, respectively. The transfected cell suspension was placed in a culture shaker at 37 ° C, 5% CO ₂ , 120 rpm for 4-5 days in the dark.

(2) Purification of anti-Her2×anti-CD3 bispecific antibody

The expression supernatant was collected by centrifugation and the cell supernatant was filtered through a 0.22 μm filter. Been equilibrated using protein A affinity chromatography media (MabSelectSuRe ^TM, available from GE Healthcare Company, Cat. No. 17-5438-02) capture bispecific antibody protein in the supernatant using the equilibration buffer (137mMNaCl, 2.7 after _{_{mMKCl, 10mM Na 2 HPO 4,}} 1.8mM KH 2 PO 4) washing away non-specifically bound protein (about 10 column volumes). Subsequently, 5 column volumes were eluted using an elution buffer (100 mM glycine, pH 3.5), the eluate was collected, and the pH was adjusted to neutral with a neutralizing buffer (1 M Tris-HCl, pH 9.0). The eluted sample was analyzed by SDS-PAGE.

For the bispecific antibodies MutB, MutC, MutD and MutE, the purity of the protein of interest after purification by this step is shown in Figure 2. Among them, the control WT (1:1:1:1) refers to the plasmid pFUSE-Her2-HC-OB-6His, pFUSE-C31-HC-OA, pCDNA3.1-C31-LC, pCDNA3.1-Her2-LC The product obtained by transforming the cells in a ratio of 1:1:1:1. As shown by the results of the reductive SDS-PAGE in Figure 2B, the content of the anti-Her2 heavy chain in the WT (1:1:1:1) bispecific antibody was found to be low, so a control WT was designed (2:1:1). :1), wherein the transfection ratio of the plasmid pFUSE-Her2-HC-OB-6His encoding the anti-Her2 heavy chain to the other three plasmids was set to 2:1:1:1. However, as can be seen from Figure 2B, the proportion of the Her2 heavy chain in the control WT (2:1:1:1) was also low. This result indicates that the proportion of the Her2 heavy chain in the control bispecific antibody WT is not due to the low expression of the Her2 heavy chain, but is due to the incorrect pairing of the two heavy chains and the two light chains without modification. . The modified bispecific antibodies MutB, MutC, MutD and MutE, the ratio of the two heavy chains is close to 1:1, and the ratio of the two light chains is also close to 1:1.

After purification of the bispecific antibodies MutC+ER, MutC-ER and MutC-DeC by protein A affinity chromatography, the purity of the target protein is shown in Figure 3. The ratio of the two heavy chains is close to 1:1, and the two light chains The ratio is close to 1:1.

The Mut C eluate was further purified using an AKTA pure 25 L1 protein purification system. Wherein, the eluted sample to sample has been equilibrated cation exchange column (pre-column ^{Resource TM S, 1mL, GE Healthcare} , NO GE17-1178-01), using the equilibration buffer A (50mM sodium phosphate, pH 6.0 Wash away the non-specifically bound protein to a gentle UV absorption line, then linearly elute 30-50 column volumes from 5%-45% with elution buffer B (50 mM sodium phosphate, 1 M NaCl, pH 6.0), collect and wash De-peaking, Figure 4A shows the elution profile. The protein size and purity were analyzed by SDS-PAGE. After purification of the bispecific antibody MutC by this step, the purity of the target protein is shown in Figures 4B and 4C.

In a further experiment, the bispecific antibody was engineered to form a 2-pair disulfide bond (see Table 6). The bispecific antibody MutC-D+ER, MutC-D-DeC was expressed and purified by protein A, and the purity of the target protein was analyzed by SDS-PAGE. The results are shown in Figures 5A and 5B, wherein Figure 5A shows the results of non-reducing SDS-PAGE and Figure 5B shows the results of reducing SDS PAGE.

Example 3: Antigen binding assay of antibodies

In order to verify the antigen-binding ability of the bispecific antibody formed by the disulfide bond and charge modification disclosed by the present invention, the binding detection of the purified bispecific antibody to the antigen CD3 and the antigen Her2 was separately detected by ELISA. Specific steps are as follows.

1) The antigens used in the experiment were human Her2 antigen (purchased from Acrobiosystems) and human CD3 antigen (purchased from Sino Biological Inc.).

2) Coating: The antigen was diluted to 100 ng/mL with 1 x PBS. The diluted antigen was added to the microtiter plate, 200 μL was added to each well, and the reaction well was sealed with a sealing plate, and coated at room temperature for 1.5 hours or at 4 ° C for 16 hours. The plate was washed 5 times with 0.05% PBST.

3) Blocking: 3% M-PBS was prepared with skim milk powder and PBS Buffer. 300 μL of 3% M-PBS was added to each well of the plate, and the well was sealed with a sealing plate and incubated for 1 hour at room temperature. The plate was washed 5 times with 0.05% PBST.

4) Antibody binding: Antibodies diluted at a certain gradient concentration were added at 100 μL/well, and each antibody concentration was tested in 3 replicate wells. After the antibody was added, the well was sealed with a sealing plate and incubated at room temperature for 1.5 hours. The plate was washed 5 times with 0.05% PBST.

5) Secondary antibody: Diluted the secondary antibody, ie HRP-labeled goat anti-human IgG (purchased from Peprotech, catalogue number SA0001-17), in a 3% M-PBS solution at 1:2000, and the diluted secondary antibody at 50 μL. / Hole added to the hole. The wells were sealed with a sealing membrane and incubated for 1 hour at room temperature. The plate was washed 5 times with 0.05% PBST.

6) Color development: Add 100 μL/well of TMB coloring solution, incubate at room temperature for 10-20 min in the dark.

7) Termination: 100 μL/well of stop solution (2M HCl) was added, and the OD value at 450 nm was measured immediately after mixing.

8) Data analysis: Data processing was performed using GraphPad Prism 5 software. A curve is generated in which log (sample concentration) is plotted on the abscissa and OD is plotted on the ordinate, and data such as kd and R ² are obtained.

As shown in Figure 6, purified anti-Her2 x anti-CD3 bispecific antibodies MutC, MutC+ER, MutC-ER, MutC-D+ER, MutC-D-DeC and MutC-DeC showed strong resistance to Her2 antigen. Binding activity. The kd value of the anti-Her2 x anti-CD3 bispecific antibody binding to the Her2 antigen was calculated to be 0.8 nM. Furthermore, as shown in Figure 7, the anti-Her2 x anti-CD3 bispecific antibodies MutC, MutC+ER, MutC-ER, MutC-D+ER, MutC-D-DeC and MutC-DeC retained the ability to bind to CD3 antigen. The above results indicate that the bispecific antibody engineered by the method of the present invention retains the ability to bind antigen.

Example 4: Simultaneous binding assay of double antigen

In the process of antibody production, if the heavy and light chains of the bispecific antibody are mismatched, the bispecific antibody loses its binding ability and biological function to two different antigens. In order to further verify that the modification method of the present invention solves the above problems, a bispecific double antigen ELISA method is used to verify that the bispecific antibody assembled by the modification method of the present invention can simultaneously bind two antigens, and A bispecific antibody (WT) assembled from a natural heavy chain and two natural light chains was compared. In theory, only about 25% of the bispecific antibodies assembled by the unmodified two natural heavy chains and two natural light chains are correctly paired with the two light chains, so they can bind two at the same time. The antibody ratio of the antigen was 25%.

In this experiment, a bispecific double antigen ELISA method was used to adsorb the corresponding first antigen onto the surface of the solid phase carrier, and the bispecific antibody to be tested was added to react with the antigen on the surface of the solid phase carrier. Subsequently, the antigen-antibody complex formed on the solid phase carrier is washed, and the enzyme-labeled second antigen is further added to form a first antigen-antibody-second antigen complex. The reaction substrate of the enzyme is then added, which is catalyzed by the enzyme to a colored product. The amount of this product is directly proportional to the amount of bispecific antibody that is capable of specifically binding the two antigens.

The specific experimental procedure is as follows: the antigen CD3 is coated on a microplate (NUNC), coated at 4 ° C overnight, washed with a skim milk, and then added with a bispecific antibody and corresponding control antibody (WT and hIgG). Incubate for 1.5 hours at room temperature, wash the plate, add HRP-labeled human Her2 antigen (HRP-Her2), incubate for 2 hours at room temperature, wash the plate, add a luminescent substrate, and measure the luminescence value using a microplate reader (Synergy HTX, BioTeck). See Figure 8 for the results. Among them, FIG. 8A shows the results of the bispecific antibodies MutB, MutC and MutD compared to the control, and FIG. 8B shows the results of MutE. The above bispecific double antigen ELISA binding assay showed that the amounts of the bispecific antibodies MutB, MutC, MutD, MutE simultaneously binding to the Her2 antigen and the CD3 antigen were increased by about 2 fold, 4 fold, and 3, respectively, compared to the unmodified WT. Double, 2 times. The above results demonstrate that MutB, MutC, MutD, MutE designed and engineered by the present invention increase the correct pairing between the bispecific antibody heavy and light chains.

Example 5: T cell tumor killing assay (CTL)

In order to further verify the antigen-binding ability at the cellular level and the killing activity against tumors of the bispecific antibody formed by the disulfide bond and charge modification of the present invention, the purified bispecific antibody was subjected to CTL killing detection. Specific steps are as follows.

CTL killing assay: SKBR-3 cells are a Her2 high expression breast cancer cell line and serve as target cells in this experiment. Single cell suspensions were prepared by trypsinizing cells SKBR-3 cells. The cell density was adjusted to 0.20 × 10 ⁶ /mL with phenol red free 5% FBS-1640 medium, and added to the well of a 96-well plate at 50 μL/well, so that the final concentration of the cells was 1.0 × 10 ⁴ /well. In this experiment, effector cells PBMC were added 20 times (E: T = 20:1) to the target cell number, i.e., 1.50 × 10 ⁵ /well (1.50 × 10 ⁶ /mL, 100 μL / well). The antibody was diluted to 4 μg/mL with phenol red-free 5% FBS-RPMI 1640 medium, and then diluted by a ratio of 1:4 to obtain concentrations of 4000 ng/mL, 1000 ng/mL, 2500 ng/mL, and 625 ng, respectively. /mL, 156.25 ng/mL, 39.06 ng/mL, 9.77 ng/mL, 2.44 ng/mL, 0.61 ng/mL, 0.15 ng/mL of antibody. The antibody was added to the corresponding wells at 50 μL/well according to the experimental design. The cells and the antibody were mixed and cultured at 37 ° C in a 5% CO 2 incubator. After about 20 hours, the killing of the cells was detected by a lactate dehydrogenase cytotoxicity kit (purchased from Beyotime), which reflects the killing of the bispecific antibody. active. Calculate the kill rate according to the following formula:

Killing rate (%) = (OD _{sample -} S _spontaneous ) / (Max-S _spontaneous ) × 100%

Among them, S _spontaneous = OD _{spontaneous release hole (target cell + effector cell)} , Max = OD _{maximum release hole (target cell)} ;

As shown in Figures 9A and 9B, the bispecific antibodies WT (1:1:1:1) and WT (2:1:1:1) assembled without the two natural heavy chains and two natural light chains were engineered. Due to the large proportion of heavy-light chain mismatches (theoretically only 25% of the bispecific antibody heavy and light chains are correctly paired), the proportion of correctly paired bispecific antibodies with normal biological activity is The protein was lower in the EC50 concentration of 21.16 ng/ml and 41.65 ng/ml for the tumor cell SKBR-3 with high expression of Her2. In contrast, purified anti-Her2×anti-CD3 bispecific antibodies MutB, MutC, MutD and MutE have significant killing effect on Her2 high-expressing tumor cell SKBR3 with EC50 concentrations of 0.95 ng/ml, 1.28 ng/ml, and 0.71 ng, respectively. /ml and 0.13ng/ml.

In addition, Figure 10 shows that the purified bispecific antibodies MutC, MutC+ER, MutC-ER, MutC-DeC, MutC-D+ER and MutC-D-DeC have significant killing effects on SKBR3 cells, and their EC50 concentrations are 0.045 ng/ml, 0.055 ng/ml, 0.034 ng/ml, 0.078 ng/ml, 0.071 ng/ml, 0.063 ng/ml.

These data indicate that the bispecific antibody assembled by the disulfide bond and charge modification method of the present invention can recognize and bind to the CD3 antigen on the surface of the immune cell in an in vitro cytotoxicity assay, and simultaneously recognize and bind the Her2 antigen on the surface of the tumor cell SKBR3. And activate T cells to kill tumor cells SKBR3. The bispecific antibody engineered by the method of the present invention maintains antigen binding activity at the cellular level and exhibits enhanced cytotoxic activity (CTL effect) compared to unmodified antibody.

Claims

A method of engineering a Fab region of an antibody, the method comprising the step of introducing one or more mutations selected from the group consisting of:

a) F170C in heavy chain constant region 1 (CH1) and S162C in light chain constant region (CL);

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL;

e) K218E or K218D mutations in the hinge region and E213R or E213K mutations in CL, wherein the antibody heavy and light chains are numbered according to the EU number.
The method according to claim 1, wherein one, two, three, four or five of a) - e) are introduced to the Fab region.
The method of claim 1 wherein one or two of a)-d), and optionally e), are introduced to the Fab region.
The method according to any one of claims 1 to 3, wherein the method further comprises mutating C220 in the hinge region to an amino acid other than cysteine or deleting C220, and/or C214 in CL Mutated to an amino acid other than cysteine or deleted C214.
The method according to claim 4, wherein the amino acid other than cysteine is selected from the group consisting of serine, alanine and glycine.
The method according to any one of claims 1 to 5, wherein the Fab region binds to an antibody selected from the group consisting of CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20 , CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1α, IL-1β , IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL -15, IL-17, IL-17R, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma, TNF-alpha, TNF beta, TNF-R1, TNF-RII, FasL, CD27L, CD30L , 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, Integrin β1, Integrin β2, Integrin α4/β7, Integrin α2, Integrin α3, Integrin α4, Integrin α5, Integrin 66, integrin αv, integrin αVβ3, FGFR-3, keratinocyte growth factor, VLA-1, V LA-4, L-selectin, anti-Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, hobby Acid granulocyte chemotactic factor 1 (eotaxin1), Blys (B lymphocyte stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB- 2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, E-selectin, digoxin, placental alkaline phosphate Enzyme (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, mucin MUC1, MUC18, heparinase I, human cardiac myosin, tumor Related glycoprotein-72 (TAG-72), tumor associated antigen CA 125, prostate specific membrane antigen (PSM-A), high molecular weight melanoma associated antigen (HMW-MAA), cancer associated antigen, Gco protein Iib /IIIa(GPIIb/IIIa), a tumor-associated antigen expressing Lewis Y-related carbohydrates, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIVgp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, cytokeratin Tumor-associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and gas production Clostridium perfringens toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCRα, TCRβ, TCRγ, TCRδ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS , myostatin, PCSK9, CXCR4, MIF, PEG2.
The method of claim 6 wherein the antigen is CD3 or HER2.
The method of any of claims 1-7, wherein the antibody is of the IgG, IgA, IgM, IgE or IgD isotype.
The method according to any one of claims 1-8, wherein the antibody is of the IgG1, IgG2, IgG3 or IgG4 isotype.
A method of producing an antibody or antibody fragment having at least two different Fab regions, the method comprising the steps of:

1) introducing one or more mutations selected from the group consisting of: in the first Fab region of the antibody or antibody fragment:

a) F170C in heavy chain constant region 1 (CH1) and S162C in light chain constant region (CL);

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, numbered according to the EU number,

2) cultivating a host cell containing a nucleic acid encoding the antibody or antibody fragment under conditions in which the antibody or antibody fragment is expressed, and

3) recovering the antibody or antibody fragment from the host cell culture.
The method of claim 10, wherein the method further comprises introducing one or more mutations selected from the group consisting of: in the second Fab region of the antibody or antibody fragment:

a) F170C in heavy chain constant region 1 (CH1) and S162C in light chain constant region (CL);

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, wherein the mutation introduced into the first Fab region and the mutation introduced into the second Fab region are not identical.
The method according to claim 10 or 11, wherein one, two, three, four or five of the mutations a)-e) are introduced in the first Fab region, in the second Fab One, two, three, four or five of the mutations a)-e) are introduced in the region, and the mutation introduced into the first Fab region and the mutation introduced into the second Fab region are not identical .
The method of any one of claims 10 to 12, wherein the method further comprises mutating C220 in the hinge region of the first Fab region to an amino acid other than cysteine or a deletion C220, and / or mutating C214 in the CL of the first Fab region to an amino acid other than cysteine or a deletion C214.
The method of any one of claims 10-13, wherein the method further comprises mutating C220 in the hinge region of the second Fab region to an amino acid other than cysteine or a deletion C220, and / or mutating C214 in the CL of the second Fab region to an amino acid other than cysteine or a deletion C214.
The method according to claim 13 or 14, wherein the amino acid other than cysteine is selected from the group consisting of serine, alanine and glycine.
The method of any one of claims 10-15, wherein the first Fab region and the second Fab region bind to different antigens, or two different epitopes of the same antigen.
The method according to claim 16, wherein said antigen is selected from the group consisting of CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1α, IL-1β, IL-1R, IL-2, IL- 2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL-17R, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma, TNF-alpha, TNF beta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, whole Integrin β1, integrin β2, integrin α4/β7, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin αv, integrin αVβ3 , FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-Id, E-selection , HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, eotaxin1, Blys (B lymphocytes) Stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue Factor, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin Receptor, Carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin MUC1, MUC18, Heparinase I, Human cardiac myosin, Tumor-associated glycoprotein-72 (TAG-72), Tumor-associated antigen CA 125 , prostate specific membrane antigen (PSM-A), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, Gco protein Iib/IIIa (GPIIb/IIIa), tumors expressing Lewis Y-related carbohydrates Related antigens, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, C MV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and Clostridium perfringens Toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCRα, TCRβ, TCRγ, TCRδ, VEGF, EGF, VEGFR, EGFR, EpCAM, Mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, myostatin, PCSK9, CXCR4, MIF, PEG2.
The method of claim 17, wherein the first antigen and the second antigen are selected from the group consisting of CD3 and HER2.
The method according to any one of claims 10 to 18, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment and an F(ab') 2 fragment.
The method of any one of claims 10-18, wherein the antibody is a bispecific antibody having a first heavy chain and a first light chain, and a second heavy chain and a second light chain, wherein The first heavy chain and the first light chain form the first Fab region, and the second heavy chain and the second light chain form the second Fab region.
The method of claim 20, wherein the method further comprises introducing P395K, P396K, and V397K to the first heavy chain, and introducing T394D, P395D, and P396D to the second heavy chain, or to the first The heavy chain introduces T394D, P395D, and P396D, and P395K, P396K, and V397K are introduced to the second heavy chain.
The method of any one of claims 10-21, wherein the antibody or antibody fragment is derived from IgG, IgA, IgM, IgE or IgD.
The method according to any one of claims 10 to 22, wherein the antibody or antibody fragment is derived from IgG1, IgG2, IgG3 or IgG4.
An antibody or antibody fragment produced by the method of any of claims 10-23.
An antibody or antibody fragment having at least two different Fab regions, the first Fab region of the antibody or antibody fragment having one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) K218E or K218D mutations in the hinge region and E213R or E213K mutations in CL, numbered according to EU numbering.
The antibody or antibody fragment of claim 25, wherein the second Fab region of the antibody or antibody fragment has one or more mutations selected from the group consisting of:

a) F170C in CH1 and S162C in CL;

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL;

d) V173C in CH1 and Q160C in CL; and

e) a K218E or K218D mutation in the hinge region and an E213R or E213K mutation in CL, wherein the mutation in the second Fab region and the mutation in the first Fab region are not identical.
The antibody or antibody fragment according to claim 25 or 26, wherein said first Fab region has one, two, three, four or five of mutations a)-e), said second Fab The region has one, two, three, four or five of the mutations a)-e), and wherein the mutation of the first Fab region and the mutation of the second Fab region are not identical.
The antibody or antibody fragment according to any one of claims 25 to 27, wherein the first Fab region further has a C220 mutation in the hinge region to an amino acid other than cysteine or a deletion C220, and/or C214 in CL is mutated to an amino acid other than cysteine or to C214.
The antibody or antibody fragment of any one of claims 25 to 28, wherein the second Fab region further has a C220 mutation in the hinge region to an amino acid other than cysteine or a deletion C220, and/or C214 in CL is mutated to an amino acid other than cysteine or to C214.
The antibody or antibody fragment according to claim 28 or 29, wherein the amino acid other than cysteine is selected from the group consisting of serine, alanine and glycine.
The antibody or antibody fragment of any of claims 25-30, wherein the first Fab region and the second Fab region bind to a different antigen, or two different epitopes of the same antigen.
The antibody or antibody fragment according to claim 31, wherein the antigen is selected from the group consisting of CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28 , CD29, CD30, CD32a, CD32b, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1α, IL-1β, IL-1R, IL-2 , IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL -17R, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma, TNF-alpha, TNF beta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL , TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL receptor-1, adenosine receptor, lymphotoxin beta receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β1, integrin β2, integrin α4/β7, integrin α2, integrin α3, integrin α4, integrin α5, integrin α6, integrin αv, whole Connexin αVβ3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, Id, E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGFβ1, TGFβ2, eotaxin1 , Blys (B lymphocyte stimulating factor), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, hapten NP-cap or NIP-cap, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline Phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFG1, PEM, mucin MUC1, MUC18, heparinase I, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72) , tumor-associated antigen CA 125, prostate specific membrane antigen (PSM-A), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, Gco protein Iib/IIIa (GPIIb/IIIa), expression Lewis Tumor-associated antigens of Y-related carbohydrates, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, respiratory syncytial virus RSV F, RSVF Fgp, cytokeratin tumor-associated antigen, He p Bgp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 loop, respiratory syncytial virus (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMVgB envelope glycoprotein and Clostridium perfringens (Clostridium Perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCRα, TCRβ, TCRγ, TCRδ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosyl-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, myostatin, PCSK9, CXCR4, MIF, PEG2.
The antibody or antibody fragment of claim 29, wherein the antigen is selected from the group consisting of CD3 and HER2.
The antibody or antibody fragment of any one of claims 25-33, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, and an F(ab') 2 fragment.
The antibody or antibody fragment of any one of claims 25-33, wherein the antibody is a bispecific antibody having a first heavy chain and a first light chain, and a second heavy chain and a second light chain, Wherein the first heavy chain and the first light chain form the first Fab region, and the second heavy chain and the second light chain form the second Fab region.
The antibody or antibody fragment according to claim 35, wherein said first heavy chain has P395K, P396K and V397K mutations, and said second heavy chain has T394D, P395D and P396D mutations, or said first heavy chain has T394D, P395D and P396D and mutations, and the second heavy chain has P395K, P396K and V397K mutations.
The antibody or antibody fragment of any of claims 25-36, wherein the antibody or antibody fragment is derived from IgG, IgA, IgM, IgE or IgD.
The antibody or antibody fragment of any of claims 25-37, wherein the antibody or antibody fragment is derived from IgG1, IgG2, IgG3 or IgG4.
A method of engineering a bispecific antibody, wherein the bispecific antibody has a first heavy chain and a first light chain that bind to CD3, and a second heavy chain and a second light chain that bind to HER2, the second heavy The chain has the amino acid sequence of SEQ ID NO: 7, and the second light chain has the amino acid sequence of SEQ ID NO: 5, the method comprising introducing the second heavy chain and the second light chain from the following a) -d) 1 or 2 mutations:

a) F170C in heavy chain constant region 1 (CH1) and S162C in light chain constant region (CL);

b) L128C in CH1 and F118C in CL;

c) F126C in CH1 and Q124C in CL; and

d) V173C in CH1 and Q160C in CL,

The antibody heavy and light chains are numbered according to the EU number.
40. The method of claim 39, further comprising mutating C220 in the hinge region of the second heavy chain to an amino acid other than cysteine or deleting C220, and/or placing the second light chain The C214 in the CL is mutated to an amino acid other than cysteine or to C214.
A method according to claim 38 or 39, further comprising introducing a K218E or K218D mutation into said first heavy chain and introducing an E213R or E213K mutation into said first light chain, or to said second heavy chain A K218E or K218D mutation is introduced and an E213R or E213K mutation is introduced into the second light chain.
The method according to any one of claims 39-41, wherein the first heavy chain has the amino acid sequence of SEQ ID NO: 3 and the first light chain has the amino acid sequence of SEQ ID NO: 1.
A bispecific antibody produced by the method of any one of claims 39-42.
An antibody or antibody fragment having a CL region selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21. SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27.
An antibody or antibody fragment having a CH1 region or a hinge region selected from the group consisting of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43.
An antibody or antibody fragment having a CL region and a CH1 region selected from the group consisting of:

a) the CL region of SEQ ID NO: 11 and the CH1 region of SEQ ID NO: 33;

b) the CL region of SEQ ID NO: 13 and the CH1 region of SEQ ID NO: 29;

c) the CL region of SEQ ID NO: 15 and the CH1 region of SEQ ID NO: 35;

d) the CL region of SEQ ID NO: 17 and the CH1 region of SEQ ID NO: 33;

e) the CL region of SEQ ID NO:27 and the CH1 region of SEQ ID NO:43.
An antibody conjugate comprising the antibody or antibody fragment according to any one of claims 24-38 and 44-46, or the bispecific antibody according to claim 43, and the antibody or antibody fragment, or A bispecific antibody-conjugated moiety, wherein the moiety is selected from the group consisting of a cytotoxin, a radioisotope, a fluorescent label, a luminescent substance, a chromogenic substance, or an enzyme.
A pharmaceutical composition comprising the antibody or antibody fragment according to any one of claims 24-38 and 44-46, the bispecific antibody according to claim 43, or the method according to claim 47 An antibody conjugate, and one or more pharmaceutically acceptable carriers, surfactants and/or diluents.
The antibody or antibody fragment according to any one of claims 24-38 and 44-46, the bispecific antibody according to claim 43, or the antibody conjugate according to claim 47, which is prepared for the treatment of a disease Use in a pharmaceutical composition.
The use according to claim 49, wherein the disease is cancer.
A method of treating a disease comprising administering an antibody or antibody fragment according to any one of claims 24-38 and 44-46, a bispecific antibody according to claim 43, according to claim 47, to a subject in need thereof The antibody conjugate, or the pharmaceutical composition according to claim 48.
The method of claim 51 wherein the disease is cancer.
A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22. SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44.
A vector comprising the nucleic acid molecule of claim 53.
A host cell comprising the nucleic acid molecule of claim 53 or the vector of claim 54.