EA043006B1 - ANTI-PD-1/ANTI-HER2 HETERODIMERIC BI-SPECIFIC ANTIBODY WITH NATURAL ANTIBODY STRUCTURE AND METHOD FOR ITS PRODUCTION - Google Patents

Description

Technical field

The present description relates to an aHTu-PD-1/aHTu-HER2 bispecific heterodimeric antibody with a natural antibody structure and a method for producing the same. Specifically, the present disclosure provides a highly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibody having the characteristics of natural IgG and free from heavy chain-light chain mismatch, and a method for producing the same.

Prior Art

Monoclonal antibodies are highly specific antibodies that act on only one antigenic determinant and are widely used in cancer, inflammation, autoimmune diseases, infectious diseases, etc. However, such therapeutic molecules do not exhibit sufficient pharmacological effects when used alone. This is due to the complexity of the diseases. For example, cancer or inflammatory diseases are often associated with interactions between molecular pathways and signaling pathways that mediate various diseases. In this case, a molecule having a single target may not provide an optimal therapeutic effect, and the therapeutic effect can be improved by simultaneously blocking molecules located on several targets or at many sites on the same target. At the same time, in the case of dual-targeting therapies using multispecificity, such as bispecific molecules, it is possible to simplify the process of developing new drugs, since the bispecific molecule is a single molecule. Compared to using a combination of several monospecific molecules, this method is more convenient for both patients and healthcare professionals.

Many different types of bispecific antibodies or bifunctional molecules are known in the art. The first bispecific antibody was generated by combining two IgG molecules, Fab' or (Fab') ₂ fragments using a chemical method and a bifunctional coupling reagent. However, such a chemically coupled bispecific antibody has many limitations, including production intensity, purification of heterologous coupling products, difficulty in removing homologous coupling products, original monospecific antibodies or fragments, low efficiency, and so on.

Another method used in the production of bispecific antibodies is the use of hybridoma (quadroma) technology, which is a method for producing a bispecific antibody by somatically fusing two types of hybridoma cell lines that secrete different antibodies. Due to the random pairing of heavy and light chains of immunoglobulins, only one tenth of the antibody mixture is the necessary functional bispecific antibody, which complicates the purification process and reduces production yield.

WO 2013060867 describes a method for mass production of a heterodimeric bispecific antibody. In this method, a mixture of two types of homodimeric antibodies is first restored and asymmetric amino acid mutations are introduced into the CH3 domains of the two types of homodimeric antibodies, which facilitates the exchange of Fab fragments of different antibodies, and interchain disulfide bonds of the hinge region are oxidized to form a stable bispecific antibody.

WO 2009089004 describes a method for producing a heterodimeric protein. In this method, the amino acids at the CH3-CH3 interface are mutated into charged amino acids such that heterodimerization is electrostatically favorable and homodimer formation is electrostatically unfavorable.

US 5,731,168 describes a method for producing heterodimeric IgG using a protuberance-into-cavity strategy. In this method, protrusions are created by replacing small amino acids at the interface of the first chain CH3 domain with larger amino acids, and at the same time, cavities are created by replacing the corresponding large amino acids of the second chain CH3 domain with smaller amino acids. The interaction of the protrusion and cavity is favorable for the formation of heterodimeric IgG, but unfavorable for the formation of a homodimer.

WO 2012058768 describes a method for producing highly specific stable heterodimeric IgG. This method combines both negative and positive design strategies, as well as protein-based structural and computer modeling techniques, to mutate multiple amino acids in the CH3 structural domain of IgG1, thereby generating stable heterodimeric IgG with low levels of homodimeric contaminants.

Programmed death receptor-1 (PD-1) is an immune checkpoint that has received much attention recently and is mainly involved in controlling T-cell activation and regulating the strength and duration of immune responses. Under normal conditions, PD-1 mediates and maintains body tissue tolerance and prevents damage to autologous tissue caused by overactivation of the immune system during the inflammatory process, and therefore has a positive effect on the prevention of autoimmune diseases. In pathological conditions, PD-1 is involved in the emergence and development of tumor immunity and various autoimmune diseases (Anticancer Agents Med Chem. 2015; 15(3):307-13. Hematol Oncol

- 1 043006

Stem Cell Ther. March 2014; 7(1):1-17. Trends Mol Med. Jan 2015; 21(1):24-33. immunity. 2013 Jul 25;

39(1):61-73. J Clin Oncol. 2015 Jun 10; 33(17):1974-82.).

PD-1 belongs to the CD28 family, but unlike other members of the CD28 family such as CTLA4 etc., it can form covalent dimers via disulfide bonds and exists in a monomeric form. The structure of PD-1 mainly includes an immunoglobulin extracellular variable region as a structural domain, a hydrophobic transmembrane domain, and an intracellular domain. The intracellular domain includes two independent phosphorylation sites. Phosphorylation sites are the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM). PD-1 is inducibly expressed on the surface of activated T cells, as well as B cells, NK cells (natural killer cells), monocytes, and DC cells (dendritic cells). PD-1 ligands include programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), and these ligands belong to the B7 family. Of these, PD-L1 is inducibly expressed on the surface of various immune cells, including T cells, B cells, monocytes, macrophages, DC cells, endothelial cells, epidermal cells, etc., but PD-L2 is inducibly expressed only on some immune cells. cells such as macrophages, DC cells, B cells, etc. (Autoimmun Rev, 2013, 12(11):1091-1100. Front Immunol, 2013, 4:481. Nat Rev Cancer, 2012, 12(4 ): 252-264 Trends Mol Med 2015 Jan; 21(1): 24-33.).

In the 1980s Dennis Slamon first discovered overexpression of the human epidermal growth factor receptor 2 (HER2) gene in 30% of 189 primary breast cancers and found that HER2 was strongly associated with overall survival and recurrence time (Slamon DJ, et al., Science, 235: 177-182, 1985). According to recent studies, HER2 is overexpressed in approximately 25-30% of patients with breast cancer (Revillion F et al., Eur J Cancer, 34:791-808, 1998), which is associated with malignant tumor progression (Wright C et al., Cancer Res, 49: 2087-2090, 1989).

Trastuzumab is a humanized monoclonal antibody against the extracellular domain of HER2 (Carter P et al., PNAS, 89(10):4285-4289, 1992). However, the anticancer activity of trastuzumab in clinical studies is often lower than in preclinical studies, and therefore trastuzumab is usually used in combination with chemotherapeutic agents. (Slamon DJ et al., N Engl J Med, 344:783-792, 2001).

The creation of bifunctional antibodies capable of recruiting effector cells is an effective means of improving the efficiency of antibodies. So far, most research has been done to exploit the function of the CD3 molecule. The target tumor can be effectively removed by activating killer T cells with the CD3 molecule (Haas C et al., Immunobiology, 214:441-453, 2009). Among them, BiTE, which is a recombinant bifunctional T-cell stimulating antibody developed by Micromet, Inc., has shown great promise, but the biggest problem is that its serum half-life is very short, and its half-life in human is only 1 hour (Loffler A et al., Blood, 95:2098-2103). This is due to the structure of BiTE itself. BiTE consists of two single chain antibody fragments. Its molecular weight is only 60 kDa, and the Fc fragments, which play an important role in increasing the half-life in the antibody molecule, are removed.

Catumaxomab is another type of promising multifunctional antibody, and is a hetero-Ig molecule that targets CD3 and EpCAM (epithelial cell adhesion molecule). This product is currently approved for the treatment of malignant ascites (Jager M et al., Cancer Res, 72:24-32, 2012). Another multifunctional antibody in phase II clinical trials is ertumaxomab, which targets CD3 and HER2. The heavy and light chains of this heteroantibody are derived from rat IgG and target CD3; and the other heavy and light chains are derived from mouse IgG and target HER2. An accompanying problem is that these products are difficult to obtain. To obtain clones expressing bifunctional ertumaxomab, one hybridoma expressing anti-CD3 antibody and one hybridoma expressing anti-HER2 antibodies, respectively, are first prepared, and then these two hybridomas are hybridized to obtain a quadroma, which is a bifunctional antibody capable of expressing anti-CD3 and anti-HER2. Typically, only one hybridoma is needed to produce an antibody to a single target. In comparison, the process of making a bifunctional antibody is much more difficult, and it is also much more difficult to make a quadroma, which can lead to extremely high immunogenicity due to its rat origin.

In addition, the most obvious side effect of an anti-CD3 antibody is a transient spike in systemic cytokine release, also called a cytokine storm. Accordingly, there is a need for a new bifunctional antibody that recruits immune cells to the surface of tumor cells.

Description of embodiments technical problem

A first aspect of the present invention relates to a heterodimeric bispecific antibody comprising a first antigen-binding functional domain capable of specifically binding to PD-1 and a second antigen-binding functional domain capable of specifically binding to HER2. The bispecific antibody may include a first Fc chain and a second Fc chain that are connected to each other via one or more disulfide bonds, where the first Fc chain and the second Fc chain are respectively linked to the antigen-binding functional domain of PD-1 and the antigen-binding functional domain of HER2, and the first Fc -chain and second Fc chain include 5 amino acid substitutions at the following positions:

1) amino acid substitutions at positions 366 and 399 of the first Fc chain and amino acid substitutions at positions 351, 407 and 409 of the second Fc chain, or

2) amino acid substitutions at positions 366 and 409 of the first Fc chain and amino acid substitutions at positions 351, 399 and 407 of the second Fc chain, wherein the first and second Fc chains respectively comprising the above amino acid substitutions tend to undergo heterodimerization rather than homodimerization, and amino acid positions are numbered according to the KabatEU numbering system.

In one embodiment, the amino acid substitutions in the first Fc chain and the second Fc chain are as follows:

a) replacement of glycine, tyrosine, valine, proline, aspartic acid, glutamic acid, lysine or tryptophan at position 351;

b) replacement of leucine, proline, tryptophan or valine at position 366;

c) replacement of cysteine, asparagine, isoleucine, glycine, arginine, threonine or alanine at position 399;

d) replacement of leucine, alanine, proline, phenylalanine, threonine or histidine at position 407 and

e) replacement of cysteine, proline, serine, phenylalanine, valine, glutamine or arginine at position 409.

In one embodiment, amino acid substitutions may include:

a) substitutions T366L and D399R in the first Fc strand and substitutions L351E, Y407L and K409V in the second Fc strand;

b) T366L and D399C substitutions in the first Fc strand and L351G, Y407L and K4O9C substitutions in the second Fc strand;

c) substitutions T366L and D399C in the first Fc strand and substitutions L351Y, Y407A and K4O9P in the second Fc strand;

d) substitutions T366P and D399N in the first Fc strand and substitutions L351V, Y407P and K409S in the second Fc strand;

e) substitutions T366W and D399G in the first Fc strand and substitutions L351D, Y407P and K409S in the second Fc strand;

e) substitutions T366P and D399I in the first Fc strand and substitutions L351P, Y407F and K409F in the second Fc strand;

g) substitutions T366V and D399T in the first Fc strand and substitutions L351K, Y407T and K409Q in the second Fc strand;

h) substitutions T366L and D399A in the first Fc strand and substitutions L351W, Y407H and K409R in the second Fc strand.

In one embodiment, amino acid substitutions may include:

a) substitutions T366L and K409V in the first Fc strand and substitutions L351E, Y407L and D399R in the second Fc strand;

b) T366L and K4O9C substitutions in the first Fc strand and L351G, Y407L and D399C substitutions in the second Fc strand;

c) substitutions T366L and K4O9P in the first Fc strand and substitutions L351Y, Y407A and D399C in the second Fc strand;

d) substitutions T366P and K409S in the first Fc strand and substitutions L351V, Y407P and D399N in the second Fc strand;

e) substitutions T366W and K409S in the first Fc strand and substitutions L351D, Y407P and D399G in the second Fc strand;

e) substitutions T366P and K409F in the first Fc strand and substitutions L351P, Y407F and D399I in the second Fc strand;

g) substitutions T366V and K409Q in the first Fc strand and substitutions L351K, Y407T and D399T in the second Fc strand;

h) Substitutions T366L and K409R in the first Fc strand and substitutions L351W, Y407H and D399A in the second Fc strand.

In one embodiment, the first Fc chain has the amino acid substitutions T366L and D399R and the second Fc chain has the amino acid substitutions L351E, Y407L and K409V.

In one embodiment, the Fc chain is derived from IgG.

In one embodiment, the antigen-binding functional domains of PD-1 and HER2 are Fab fragments or scFv fragments.

In one embodiment, the antigen-binding functional domains of PD-1 and HER2 are all Fab fragments.

In one embodiment, one of the antigen-binding functional domains of PD-1 and HER is a Fab fragment and the other is an scFv fragment.

In one embodiment, the Fab fragment may include a first heavy chain variable region and a second heavy chain variable region that are different from each other, and a first light chain variable region and a second light chain variable region that are different from each other.

In one embodiment, the amino acid sequence of the bispecific antibody is selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

The second aspect of the present invention relates to an isolated polynucleotide encoding a heterodimeric bispecific antibody described in the first aspect.

In one embodiment, the polynucleotide sequence is selected from SEQ ID NOs: 1,3,6,7,9, 13, 15 and 17.

A third aspect of the present invention relates to a recombinant plasmid comprising the isolated polynucleotide described in the second aspect.

In one embodiment, the expression vector is the X0GC plasmid vector, which is obtained by modifying pcDNA.

A fourth aspect of the present invention relates to a host cell comprising the isolated polynucleotide described in the second aspect or the recombinant expression vector described in the third aspect.

In one embodiment, the host cell is selected from a human embryonic kidney cell HEK293 or HEK293T, HEK293F or HEK293E derived from a HEK293 cell; and Chinese Hamster Ovary CHO or CHO-S, CHO-dhfr-, CHO/DG44 or ExpiCHO cells derived from CHO cells.

A fifth aspect of the present invention relates to a composition comprising a heterodimeric bispecific antibody as described in the first aspect, an isolated polynucleotide as described in the second aspect, a recombinant expression vector as described in the third aspect, or a host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier.

The sixth aspect of the present invention relates to a method for producing a heterodimeric bispecific antibody described in the first aspect, including:

1) expressing the isolated polynucleotide described in the second aspect or the recombinant expression vector described in the third aspect in a host cell;

2) recovery of each protein expressed in the host cell and

3) mixing of reduced proteins and subsequent oxidation of the mixture.

In one embodiment, the host cell is selected from a human embryonic kidney cell HEK293 or HEK293T, HEK293F or HEK293E derived from a HEK293 cell; and Chinese Hamster Ovary CHO or CHO-S, CHO-dhfr-, CHO/DG44 or ExpiCHO cells derived from CHO cells.

In one embodiment, recovery may include:

1) adding a reducing agent, where the reducing agent is 2-mercaptoethylamine, dithiothreitol, tris-(2-carboxyethyl)-phosphine, other chemical derivatives, or combinations thereof,

2) carrying out the reduction reaction in the presence of dithiothreitol at a concentration of 0.1 mM or more at 4°C for at least 3 hours, and

3) removal of the reducing agent by desalination, etc.

In one embodiment, the oxidation may include:

1) oxidation in air or the addition of an oxidizing agent, where the oxidizing agent is selected from L-dehydroascorbic acid and other chemical derivatives, and

2) carrying out the oxidation reaction in the presence of L-dehydroascorbic acid at a concentration of 0.5 mM or more at 4°C for at least 5 hours.

In one embodiment, the method may further include isolation and purification.

A seventh aspect of the present invention relates to the use of a heterodimeric bispecific antibody as described in a first aspect and/or an isolated polynucleotide as described in a second aspect and/or a recombinant expression vector as described in a third aspect and/or a host cell as described in a fourth aspect, and /or the composition described in the fifth aspect, in the manufacture of a medicinal product for the prevention and/or treatment of a disease of the subject.

An eighth aspect of the present invention relates to the use of a heterodimeric bispecific antibody as described in a first aspect and/or an isolated polynucleotide as described in a second aspect and/or a recombinant expression vector as described in a third aspect and/or a host cell as described in a fourth aspect, and /or the composition described in the fifth aspect, in the prevention and/or treatment of a disease of the subject.

The ninth aspect of the present invention relates to a method for preventing and/or treating a disease, comprising the introduction of a heterodimeric bispecific antibody described in the first aspect, and/or an isolated polynucleotide described in the second aspect, and/or a recombinant expression vector described in the third aspect, and/ or a host cell described in a fourth aspect and/or a composition described in a fifth aspect to a subject in need thereof.

In one embodiment, the subject may be a mammal, or preferably may be a human.

In one embodiment, the disease may be selected from tumors such as leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, cancer liver, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma and melanoma.

Useful disclosure effects

In the present invention, a brand new anti-PD-1/anti-HER2 heterodimeric bispecific antibody with a natural antibody structure was constructed using PD-1 as an immune cell recruiting molecule and HER2 as a tumor cell target molecule. This bispecific antibody is a highly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibody with natural IgG characteristics and no heavy chain/light chain mismatch. A bispecific antibody can bind to both types of target molecules, PD-1 and HER-2, and thus may be more effective in treating

- 4 043006 complex diseases.

Brief description of graphic materials

In FIG. 1 shows the monomer elution peak of a heterodimeric antibody molecule;

In FIG. 2 shows the molecular structure of an anti-PD-1/anti-HER2 heterodimeric antibody;

In FIG. 3 shows the structure of the heavy chain and light chain of a semi-antibody molecule;

In FIG. 4 shows the results of analysis by size exclusion chromatography (SEC) of the heavy chain and light chain of the semi-antibody molecule, where A and B show the results of the analysis of the anti-HER2 semi-antibody molecule and the anti-PD-1 semi-antibody molecule, respectively;

In FIG. 5 shows the results of SDS-PAGE analysis of oxidized anti-PD-1 and anti-HER2 antibody semi-antibody molecules;

In FIG. 6 shows the elution peak of an anti-PD-1/anti-HER2 heterodimeric antibody molecule;

In FIG. 7 shows the results of an SDS-PAGE analysis of an anti-PD-1/anti-HER2 heterodimeric antibody molecule;

In FIG. 8 shows the results of a SEC analysis of an anti-PD-1/anti-HER2 heterodimer antibody molecule;

In FIG. 9 shows stability testing of an anti-PD-1/anti-HER2 heterodimeric antibody molecule;

In FIG. 10 shows stability testing of an anti-PD-1/anti-HER2 heterodimeric antibody molecule;

In FIG. 11 shows HER2 binding activity and PD-1 binding activity of an anti-PD-1/anti-HER2 heterodimer antibody molecule, where A and B show HER2 binding activity and PD-1 binding activity, respectively;

In FIG. 12 shows the co-binding of an anti-PD-1/anti-HER2 heterodimeric antibody molecule to PD-1 overexpressing CHO/PD-1 cells and HER2 overexpressing SK-BR3 cells, where A-D show co-binding of a HER2 monoclonal antibody, monoclonal PD-1 antibody, HER2 monoclonal antibody + PD-1 monoclonal antibody, and antiPD-1BJHM/anti-HER2, respectively;

In FIG. 13 shows the blocking activity of an anti-PD-1/anti-HER2 heterodimeric antibody molecule for PD-1/PD-L1 binding and PD-1/PD-L2 binding, where A and B show blocking activity against PD-1/PD -L1 and PD-1/PD-L2 binding, respectively;

In FIG. 14 shows secretion of the cytokine IL-2 stimulated by an anti-PD-1/anti-HER2 heterodimeric antibody molecule; And

In FIG. 15 shows the area under the curve (AUC) for an anti-PD-1/anti-HER2 heterodimer antibody molecule.

The best embodiment of the invention

Definitions:

A covalent bond means that two Fc chains or any one Fc chain and an antigen-binding functional domain associated therewith in a heterodimeric bispecific antibody are linked to each other via a covalent bond to form one molecule. Of these, the Fc chain includes a first antigennegative functional domain and a second antigennegative functional domain linked through one or more covalent bonds (or disulfide bond chains); each of the first Fc chain and the second Fc chain is linked to one antigen-binding functional domain via a covalent bond (imine bond or peptide bond); And

An antigen-binding functional domain is a domain in which a specific interaction with a target molecule, such as an antigen, occurs and its action is highly selective, and a sequence that recognizes one target molecule does not recognize sequences of other molecules. A typical antigen-binding functional domain includes an antibody variable region, a structural variant of an antibody variable region, a receptor-binding domain, a ligand-binding domain, or an enzyme-binding domain.

Communication through one or more chains of disulfide bonds refers to the formation of a heterodimeric fragment through the connection between the first chain of Fc and the second chain of Fc using one or more chains of disulfide bonds. In the present invention, one or more disulfide bonds may be formed when the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and its associated antigen-binding functional domains are synthesized in the same cell, or may be formed by reduction in vitro after the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains, are synthesized in different cells, respectively.

The first Fc chain and the second Fc chain form a linking moiety via a covalent bond, and the covalent bond includes a disulfide bond, and each chain includes at least a portion of an immunoglobulin heavy chain constant region; the first chain and the second chain differ from each other in their amino acid sequences and contain different amino acids in at least one position. In the first Fc chain and in the second Fc chain according to the present invention, there is

- 5 043006 strong repulsive force between identical chains, and between different chains there is an attractive force. Therefore, the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains, tend to form heterodimers when co-expressed in a cell. When the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains are expressed in two different host cells, respectively, the first Fc chains, or the first Fc chain and its associated antigen-binding functional domain do not tend to form homodimers, and the second Fc strands, or the second Fc strand and its associated antigen-binding functional domain, also do not tend to form homodimers. In the present disclosure, when the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains are expressed in two different host cells, respectively, and a reducing agent is present, the percentage of homodimers is less than 50%. , that is, the percentage of monomers (one Fc chain, or one Fc chain and one antigen-binding functional domain associated with it) is 50% or more.

Immunoglobulin has a symmetrical structure with four polypeptide chains; the two chains are identical heavy chains that are relatively long and have a relatively high molecular weight, each of these chains contains 450 to 550 amino acid residues and has a relative molecular weight of 55,000 to 70,000 Da; and the other two chains are identical light chains (L-chains) that are relatively short and have a relatively low molecular weight, each of these chains contains 210 amino acid residues and has a relative molecular weight of about 24,000 Da. Approximately 110 amino acid sequences near the N-terminus of the heavy and light chains of immunoglobulin are highly variable, and this region is called the variable region (V-region), and the remaining amino acid sequences near their C-terminus are relatively stable and are called the constant region (C-region). The variable region of the heavy chain _occupies approximately % of the length of the heavy chain, and the constant region occupies approximately ^3/4 of the length of the heavy chain. The 5 known types of immunoglobulins are IgG(y), IgA(a), IgD(δ), IgM(μ) and IgE(ε). Among them, the first three types of immunoglobulins have three constant regions consisting of CHI, CH2 and C3 in the H chain, and the last two types of immunoglobulins (IgM and IgE) have one VH domain and four constant domains, that is, CH1-CH4 in the H chain. region represents the backbone of the immunoglobulin molecule, and is also one of the regions that activate immune responses.

The constant region portion of the present invention includes at least a first Fc chain and a second Fc chain interaction region and, in the case of IgG, this region is located at any amino acid positions of the CH3 domain and includes at least GLN347, TYR349, THR 350, LEU 351, SER 354 ARG 355 ASP 356 GLU 357 LYS 360 SER 364 THR 366 LEU 368 LYS 370 ASN390 LYS392 THR394 PRO395 VAL 397 ASP399 SER400 PHE405 TYR407 LYS409, LYS439.

The first Fc chain and the second Fc chain, each associated with one antigen-binding functional domain via a covalent bond or linker, indicate the first Fc chain and the second Fc chain, each of which is associated with an antigen-binding fragment of a single antibody, or a single-chain antibody capable of recognizing an antigen, or another variant of the antibody fragment capable of recognizing an antigen, or a receptor capable of recognizing a ligand, or a ligand capable of recognizing the receptor via a covalent bond or linker. A covalent bond is a type of chemical bond in which two or more atoms share their outer electrons, in the ideal case of electron saturation, a relatively stable chemical structure is thus formed, or an interaction between atoms is formed through a common electron pair. Atoms of the same element, or atoms of different elements, can all be linked by a covalent bond. The covalent bond of the first Fc chain and the second Fc chain of the present invention may include an amide bond formed by dehydration between the amino group of an amino acid of one molecule and the carboxyl group of an amino acid of another molecule, or an amide bond between the aldehyde group of ethylene glycol or polyethylene glycol or another compound or polymer thereof and the amino group of an amino acid one molecule, but is not limited to them. A linker is one amino acid sequence, or one compound, or one multimer of one compound, capable of linking two polypeptide chains via a covalent bond. Of these, one amino acid sequence may include, but is not limited to, a small peptide such as GGGGSGGGGSGGGGS, and the amino acid sequence may link the first Fc chain or the second Fc chain and a single chain antibody capable of recognizing an antigen or other structural variant of an antibody fragment capable of recognizing an antigen by amide bond.

The first Fc chain and the second Fc chain tend to form heterodimers and do not tend to form homodimers; this means that in the first Fc chain and in the second Fc chain there is a strong repulsive force between the same polypeptide chains and there is an attractive force between different polypeptide chains, and therefore the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains have

- 6 043006 tendency to form heterodimers when co-expressed in the cell. When the first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional domains are expressed in two different host cells, respectively, the first Fc chains or the first Fc chain and its associated antigen-binding functional domain do not have tend to form homodimers, and the second Fc strands or the second Fc strand and its associated antigen-binding functional domain also do not tend to form homodimers.

The Kabat EU numbering system means assigning a number to each amino acid in an antibody sequence, and this method of assigning residue numbers is standard in the art. The Kabat method can be extended to other antibodies not included in its compendium by aligning the target antibody to one of the consensus sequences in Kabat, taking into account conserved amino acids.

The Fc region refers to the crystallizing fragment (Fc), corresponds to the CH2 and CH3 structural domains of Ig, and is the region where the interaction between the Ig and the effector molecule or cell occurs.

IgG is an abbreviation for immunoglobulin G (IgG) and is the main type of antibody in the blood serum. Human IgG has four subclasses IgG1, IgG2, IgG3 and IgG4 based on antigenic differences in the r-chains of the IgG molecule.

A semi-antibody molecule has a structure formed by one heavy chain and one light chain of an antibody, where the heavy chain and light chain can be linked through a covalent bond, or has the structure of a monovalent antigen-recognizing antibody, which can be formed without a covalent bond.

A Fab fragment is a molecule recognition sequence and an antigen binding fragment (Fab) and corresponds to two arms of an antibody molecule, each consisting of a complete light chain and the heavy chain VH and CH1 structural domains. scFv is a molecule recognition sequence and is a structural isomer of an antibody fragment obtained by genetic modification of the light chain variable region and the heavy chain variable region of an antibody. The extracellular domain of a membrane receptor is a molecule-recognizing sequence, and a membrane receptor typically includes an extracellular region that is located outside the cell and recognizes and binds to the corresponding antigen or ligand, a transmembrane region that anchors the receptor on the cell surface, and an intracellular region that has intracellular kinase activity or signaling. way. A cell membrane receptor ligand refers to a protein, small peptide, or compound that can be recognized and associated with the extracellular region of a membrane receptor. Cytokines are small molecular weight soluble proteins that are produced by various cell types induced by immunogens, mitogens or other stimulants and have various functions such as innate and adaptive immunity, hematopoiesis, cell growth, polyfunctional APSC cells (Adult Pluripotent Stem Cells) and restoration of damaged tissues, etc. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamilies, colony stimulating factors, chemotactic factors (chemokines), growth factors, and so on. A tag in protein expression means an amino acid sequence added at the N-terminus or C-terminus of a target protein and may be small peptides or long amino acids. The addition of a tag may be beneficial for proper protein folding, protein isolation and purification, and intracellular protein degradation. Frequently used tags may include, but are not limited to, HA, SUMO, His, GST, GFP, and Flag.

There are no restrictions on antibodies applicable to the heterodimeric bispecific antibody of the present invention. Antibodies already used in the art for the treatment and/or prevention of diseases can be applied to the present invention.

The heterodimeric bispecific antibody of the present invention may have one or more substitutions, deletions, additions and/or insertions. For example, despite the replacement of some amino acids in the protein structure, there is no significant loss of the ability to bind to other polypeptides (eg antigens) or cells. Since the binding capacity and properties of a protein determine the biological functional activity of a protein, substitutions of certain amino acids in a protein's sequence may not result in a significant loss of its biological utility or activity.

In many cases, polypeptide variants may include one or more conservative substitutions. A conservative substitution means that the amino acids in the variants of the polypeptide are replaced with other amino acids having similar properties, so that one skilled in the art of peptide chemistry would expect the secondary structure and hydrophilic nature of the polypeptide to be substantially unchanged.

Amino acid substitutions can generally be based on the relative similarity of properties of the substituents in the amino acid side chains, such as hydrophobicity, hydrophilicity, charge, size, and so on. Representative substitutions that take into account the various characteristics described above are well known to those skilled in the art and include arginine and lysine; glutamic acid and asparagine

- 7 043006 acid; serine and threonine; glutamine and asparagine; valine, leucine and isoleucine.

The term identity as used herein has a meaning well known in the art, and those skilled in the art are also familiar with the rules and criteria for determining the identity of different sequences, and identity refers to the percentage of homology between variant polynucleotide or polypeptide sequence residues and non-variant sequence residues after these sequences have been aligned. and the introduction of gaps (if necessary, to achieve the maximum % homology). In the present disclosure, while meeting the definition of identity, the resulting variant sequence is also required to have the biological activity that the parent sequence has. Methods and means for screening sequence variants using the above activities are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art from the information provided herein. In a specific embodiment, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99, 8% or 99.9% identity of the polynucleotide or polypeptide with the polynucleotide or polypeptide described here. Due to the redundancy of the genetic code, there are variants of these sequences encoding the same amino acid sequence.

In another aspect, a polynucleotide composition is provided that is capable of hybridizing to a polynucleotide sequence provided herein, or a fragment or complementary sequence thereof, under conditions of moderate to high stringency. Hybridization techniques are well known in the field of molecular biology. By way of explanation, suitable moderately stringent conditions for testing hybridization of a polynucleotide of the present invention with another polynucleotide may include pre-washing with a solution of 5xSSC (sodium chloride and citrate solution), 0.5% SDS (sodium dodecyl sulfate), 1.0 mM EDTA (pH 8 ,0); carrying out hybridization in 5xSSC at 50°C - 60°C during the night; and washing twice with 2x, 0.5x and 0.2x SSC solutions containing 0.1% SDS for 20 min at 65°C, respectively. Those skilled in the art will appreciate that the stringency of hybridization can be easily manipulated, for example by varying the salt content of the hybridization solution and/or the hybridization temperature. For example, in another embodiment, suitable very stringent hybridization conditions may include the conditions described above, with the exception of increasing the hybridization temperature, for example to 60°C, to 65°C, or to 65°C, to 70°C.

The host cell of the present invention may be any cell that can be used to express a foreign gene, and may include, but is not limited to, E. coli, yeast cells, insect cells, plant cells, and mammalian cells.

The vector of the present invention may be a vector that can replicate in any type of cell or organism and may include, for example, plasmids, bacteriophages, cosmids, and minichromosomes. In one embodiment, the vector comprising the polynucleotide of the present invention is a vector suitable for propagating or replicating the polynucleotide, or a vector suitable for expressing the polypeptide of the present invention. Such vectors are known in the art and are commercially available.

The vector may include a shuttle vector and an expression vector. Typically, the plasmid construct may also include an origin of replication (eg ColE1 origin of replication) and a selectable marker (eg ampicillin and tetracycline resistance) that are intended for replication and selection of the plasmid in bacteria, respectively. An expression vector refers to a vector containing a control sequence or regulatory element required for the expression of an antibody of the present invention, including antibody fragments, in bacterial or eukaryotic cells.

The vector of the present invention may be any vector used to express a foreign gene and may include, but is not limited to, a plasmid vector, wherein the plasmid vector includes at least an origin of replication, a promoter, a gene of interest, a multiple cloning site, a selectable the marker gene and vector of the present invention may include, but are not limited to, a plasmid vector derived from pcDNA, such as the X0GC vector.

The subject of the present disclosure may include birds, reptiles, mammals, and the like. The mammal may include rodents, primates. A primate may include a human.

The scope of diseases included in the present disclosure may include, without limitation, tumors. Tumors may include leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colon cancer, breast cancer , cancer

- 8 043006 ovaries, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, renal cell carcinoma, melanoma.

Pharmaceutically acceptable carriers mean pharmaceutical carriers which are commonly used in pharmaceuticals, such as diluents, excipients, water, etc., excipients such as starch, sucrose, lactose, microcrystalline cellulose, etc.; binding agents such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate, sodium hydrogen carbonate, etc.; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetanol, sodium lauryl sulfate, etc.; adsorption carriers such as kaolinite, bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, micronized silica gel, polyethylene glycol, etc. In addition, other additives such as flavors, sweeteners, etc. can be added to the composition.

The principle of the invention

Below, the present disclosure will be described in more detail with reference to the following non-limiting examples. Those skilled in the art will recognize that various modifications and changes may be made without departing from the spirit and scope of the present invention, and these modifications and changes are also included within the scope of the present invention.

The following experimental methods are common methods unless otherwise noted, and the substances used in the experiments can also be readily obtained from commercial companies unless otherwise noted. The various antibodies used in the following examples of this disclosure are all commercially available standard antibodies.

Example 1 Construction of the Vector Structure of an Anti-PD-1/Anti-HER2 Heterodimeric Antibody Molecule

X0GC expression vectors were constructed comprising the heavy chain and light chain of the human anti-PD-1(Pem) antibody, respectively. The antibody variable region sequence is from http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=9798. The nucleotide sequence of the light chain variable region is the same as in SEQ ID NO: 9, and its amino acid sequence is the same as in SEQ ID NO: 10; the nucleotide sequence of the light chain constant region is the same as in SEQ ID NO: 3, and its amino acid sequence is the same as in SEQ ID NO: 4; the nucleotide sequence of the heavy chain variable region is the same as in SEQ ID NO: 11, and its amino acid sequence is the same as in SEQ ID NO: 12; the nucleotide sequence of the heavy chain constant region is the same as in SEQ ID NO: 13, and its amino acid sequence is the same as in SEQ ID NO: 14. The light chain variable region and the light chain constant region, and the heavy chain variable region and the heavy chain constant region was amplified by PCR. All PCR reactions of the present specification used Phusion High-Fidelity DNA Polymerase (F-530L) from NEB, Inc. Primers for PCR were designed in the usual way in accordance with the principle of base complementation and the need for restriction enzyme sites. Reaction conditions were 8.9 µl H ₂ O, 4 µl 5x Phusion high fidelity DNA polymerase buffer, 4 µl 1 mM dNTP (deoxyribonucleoside triphosphate), 1 µl forward primer, 1 µl reverse primer, 0.1 µl Phusion high fidelity DNA polymerase, and 1 µl of matrix. The PCR products of the variable region and constant region were subjected to 1.5% agarose gel electrophoresis, and their corresponding fragments were extracted using a DNA extraction kit (Promega, A9282, same used hereinafter). The extracted variable region fragment and the constant region fragment were used as templates, and the variable region forward primer and the constant region reverse primer were used to perform PCR. Their respective fragments were recovered to give a full length heavy chain and light chain fragment. The X0GC vector and full-length fragment were digested with EcoRI (NEB, Cat. No. R3101L) and HindIII (NEB, Cat. No. R3104L). Restriction enzyme conditions were 2 µl 10xbuffer 3, 0.5 µl each of EcoRI and HindIII, 3 µl full-length fragment extracted from the gel, and 14.5 µl H ₂ O. Restriction enzymes were allowed to react at 37°C for 3 h. Restriction products were ligated using T4DNA ligase (NEB, Cat. No. M0202V) (same used hereinafter) and reaction conditions were 2 µl 10x ligase buffer, 0.5 µl ligase, 3 µl full length fragment extracted from the gel, 3 μl of the X0GC vector extracted from the gel, and 11.5 μl of H ₂ O, which were ligated at room temperature for 12 hours. The ligation product was transformed into E. coli DH5a competent cells (Tiangen, CB104, id. most used later). The antibody heavy and light chain X0GC expression vectors were generated in order to express the antibody heavy chain and light chain in eukaryotic cells, respectively.

Other X0GC expression vectors have also been constructed in the present invention,

- 9 043006 containing the heavy chain and light chain of antibodies against human PD-l (BJHM), respectively. The nucleotide sequence of the light chain variable region is the same as in SEQ ID NO: 15 and its amino acid sequence is the same as in SEQ ID NO: 16; the nucleotide sequence of the light chain constant region is the same as in SEQ ID NO: 3, and its amino acid sequence is the same as in SEQ ID NO: 4; the nucleotide sequence of the heavy chain variable region is the same as in SEQ ID NO: 17, and its amino acid sequence is the same as in SEQ ID NO: 18; the nucleotide sequence of the heavy chain constant region is the same as in SEQ ID NO: 13, and its amino acid sequence is the same as in SEQ ID NO: 14. The antibody heavy and light chain X0GC expression vectors were generated to express the antibody heavy and light chain in eukaryotic cells, respectively.

The present invention also constructed X0GC expression vectors comprising the anti-human HER2 antibody heavy and light chain, respectively. The antibody variable region sequence is from http://www.drugbank.ca/drugs/DB00072 and the heavy chain constant region is human IgG1(Fc ₂ ). The nucleotide sequence of the light chain variable region is the same as in SEQ ID NO: 1 and its amino acid sequence is the same as in SEQ ID NO: 2; the nucleotide sequence of the light chain constant region is the same as in SEQ ID NO: 3, and its amino acid sequence is the same as in SEQ ID NO: 4; the nucleotide sequence of the heavy chain variable region is the same as in SEQ ID NO: 5, and its amino acid sequence is the same as in SEQ ID NO: 6; the nucleotide sequence of the heavy chain constant region is the same as in SEQ ID NO: 7, and its amino acid sequence is the same as in SEQ ID NO: 8. The antibody heavy and light chain X0GC expression vectors were generated to express the antibody heavy and light chain in eukaryotic cells, respectively.

Example 2 Expression of an anti-PD-1/anti-HER2 heterodimeric antibody molecule

Expression vectors comprising the anti-human PD-1 heavy chain and light chain were transfected into 293F cells (FreeStyle™ 293-F Cells, Cat. No. R79007, Invitrogen), respectively, and expression vectors comprising the anti-human HER2 antibody heavy and light chain , were also transfected into 293F cells, respectively. The day before transfection, cells were seeded. On the day of transfection, cells were harvested by centrifugation and resuspended in fresh FreeStyle™ 293 expression medium (Cat. No. 12338001, Gibco) at a cell density of 200-10 cells/ml. The plasmid was added according to the volume of transfection, and when the final concentration was 36.67 μg/ml, light uniform mixing was performed. Then linear polyethyleneimine (PEI, linear, MM 25000, cat. no. 43896, Alfa Aesar) was added and when the final concentration was 55 μg/ml, gentle uniform mixing was performed. The mixture was then placed in an incubator and incubated with shaking at a speed of 120 rpm at 37°C for 1 hour. Then, 19 times the volume of the transfection volume of fresh medium was added thereto. Incubation was continuously carried out with shaking at a speed of 120 rpm at 37°C. Culture supernatants of transfected cells incubated for 5-6 days were collected by centrifugation.

The level of expression was determined using ELISA (enzyme-linked immunosorbent assay). Before purification by applying the culture supernatant to the chromatographic column, the precipitate was removed by filtration through a 0.2 μm filter. This procedure was performed at 4°C.

Example 3 Purification of the expression product of an anti-PD-1/anti-HER2 heterodimeric antibody molecule

Purification was performed at 4° C. using an AKTA explorer 100 protein purification system (GE Healthcare) and rProtein A Sepharose Fast Flow affinity chromatography (16 mm i.d., 22 ml, GE Healthcare). First, mobile phase A (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4) was used to equilibrate the chromatographic column. After the baseline stabilized, the supernatant of the above treated cells was loaded at a flow rate of 5 ml/min. After sample loading, equilibration was performed using mobile phase A. The sample was an anti-PD-1 expression product and an anti-HER2 expression product, respectively. Mobile phase B1 (mobile phase A containing 0.5 M arginine) was then used to elute 5 column volumes; 5 column volumes were washed with mobile phase B2 (100 mM citric acid, pH 3.0) to collect the elution peak, i.e. the peak of the protein of interest; the flush flow rate was 5 ml/min. The chromatogram of the anti-PD-1-Fc1 elution peak is shown in FIG. 1 and the anti-HER2-Fc2 elution peak was also similar (result not shown). The indicated elution peak was collected (grey area shown) and the pH was adjusted to 5.0 by dropwise addition of 1 M sodium acetate solution.

Example 4 Purification of the Anti-PD-1/Anti-HER2 Heterodimeric Antibody Molecule

The molecular structure of the anti-PD-1/anti-HER2 heterodimeric antibody is shown in FIG. 2.

Anti-PD-1 and anti-HER2 expression products prepared as described above with rProtein A Sepharose Fast Flow (16 mm i.d., 22 ml, GE Healthcare) were recombined in vitro to form a heterodimer. First, the purified and collected protein solutions were concentrated by ultra

- 10 043006 by filtering with an ultrafiltration concentration tube (nominal molecular weight cutoff 10 kDa) and the solution was replaced with phosphate buffered saline (PBS) (pH=7.4). The resulting anti-PD-1 and anti-HER2 expression products were adjusted to 1 mg/ml with PBS, and 1/200 times the final volume of 1 M DTT (dithiothreitol) was added so that the final concentration of DTT was 5 mM, respectively. The reduction was carried out at 4°C (3 to 8 hours) and the disulfide bonds were opened through the reduction process and the disulfide bonds of the hinge region of a small number of antibody homodimer molecules contained in anti-PD-1 and anti-HER2 expression products were also opened, thereby forming a molecule a semi-antibody containing one heavy chain and one light chain, the structures of which are shown in FIG. 3. The reconstituted sample was analyzed by SEC-HPLC (size exclusion high performance liquid chromatography) with 1 mM DTT reducing agent in mobile phase buffer. The results are shown in FIG. 4. The weight ratio of anti-PD-1 and anti-HER2 homodimers was less than 10%. In accordance with this, the mass ratio of the masses of the semi-antibody molecules was more than 90%.

Then, the reduced anti-PD-1 and anti-HER2 semi-antibodies were mixed according to the molar ratio, and the recombination reaction was carried out at 4° C. for 24 hours. anti-HER2 half-antibodies were formed through a non-covalent interaction between CH2 and CH3 of anti-PD-I and anti-HER2 half-antibodies. The protein solution was then concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal molecular weight cutoff was 10 kDa) and the solution was replaced with PBS (pH=7.4) to stop the reduction. The solution was oxidized in air or with an oxidizing agent to form disulfide bonds of the heterodimeric bispecific antibody. The oxidation conditions were as follows. 100 mM L-dehydroascorbic acid was added as an oxidizing agent, and when the final protein concentration was 1 mg/ml and the final concentration of oxidizing agent was 1 mM, oxidation was carried out at 4° C. for 24 hours. The sample obtained by the above oxidation was subjected to SDS- PAGE and the results are shown in FIG. 5.

Heterodimer molecules prepared by the above described reduction/oxidation of anti-PD-I and anti-HER2 semi-antibodies were concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal molecular weight cut-off was 10 kDa) and the solution was replaced with sodium phosphate buffer solution (pH=5, 8). Purification was performed at 4° C. using an AKTA explorer 100 protein purification system (GE Healthcare) and a Source 15S ion chromatography column (16 mm i.d., 17 ml, GE Healthcare). First, mobile phase A (10 mM sodium phosphate, pH 7.0) was used to equilibrate the chromatographic column. After the baseline stabilized, the protein solution treated above was loaded at a flow rate of 3 ml/min. After sample loading, equilibration was performed using mobile phase A. Thereafter, 20 column volumes (0% B-100% B, 170 min, flow rate 2 ml/min) were washed with a gradient from A (10 mM sodium phosphate, pH 5.8) to B (10 mM sodium phosphate, pH 5.8). This major elution peak was collected (see FIG. 6) and the collected protein solution was concentrated by ultrafiltration through an ultrafiltration concentration tube (nominal molecular weight cutoff was 10 kDa). The solution was replaced with a phosphate solution (PBS, pH=7.4), filtered and sterilized and then stored at 4°C. The purified product was analyzed by SDS-PAGE and the results are shown in FIG. 7. Based on purity analysis by SEC-HPLC, the purity was 97.3% (FIG. 8).

Example 5 Molecule Stability of Anti-PD-1/anti-HER2 Heterodimeric Antibody mg/mL fully sealed samples of PD-1/HER2 heterodimer were left in a constant climate chamber (BINDER KBF240) at 40°C and 20 µg of sample were taken in the corresponding time points (baseline (day 0), after 1 day, 3 days, 5 days, 7 days and 14 days) and separated by size exclusion chromatography (SEC-HPLC). The terms of the above SECHPLC were as follows:

(1) SEC column: TSKgel G3000SWx1 (Tosoh Bioscience), 5 µm, 7.8 mm x 30 cm;

(2) Mobile phase: 5 mM PBS, 150 mM NaCl, pH 6.7;

(3) Flow rate: 0.6ml/min;

(4) UV detection wavelength: 280nm;

(5) Collection time: 30 min.

The instrument used was an Agilent 1200 Infinity chromatography and the chromatogram was recorded using an Agilent ChemStation and the ratio of the remaining monomers was calculated. As shown in FIG. 9 (10 mg/mL) and 10 (1 mg/mL) the dimer did not aggregate significantly under experimental conditions at 40° C., and therefore the PD-1/HER2 heterodimer was considered to have excellent thermal stability.

Example 6 In vitro binding activity of an anti-PD-1/anti-HER2 molecule of a heterodimeric anti-body

The binding capacity of the heterodimeric PD-1/HER2 antibody for a single antigen was determined by enzyme immunoassay (ELISA).

The detailed procedure was as follows: recombinant human PD-1 (Beijing Yiqiao Shenzhou cat. no. 10377-H08H) or human HER2 (Beijing Yiyi) was coated on a 96-well high adsorption ELISA plate using carbonate buffer solution with pH 9.6, with a coating concentration of 1 µg/ml and a coating amount of 100 µl per well. The coating was performed at 4°C during the night. The plate was washed with PBST five times. The plate was blocked with 300 μl/well PBST containing 1% BSA, incubated for 1 hour at 25°C and washed with PBST five times. A heterodimeric antibody sample and a control serially diluted with PBST containing 1% BSA were added at 100 μl per well and incubated at 25° C. for 1 hour. The plate was washed with PBST five times. Horseradish peroxidase-labeled anti-human IgG (Chemicon, cat. no. AP309P) diluted 1:2000 with PBST containing 1% BSA was then added at 100 µl per well and incubated at 25°C for 1 hour. The plate was washed with PBST for five once. Colorimetric TMB (3,3,5,5-tetramethylbenzidine) substrate was added at 100 µl/well and developed for 10 min at room temperature. Color development was stopped by adding 100 μl/well of 1 M H ₂ SO ₄ . Absorbance at 450 nm was determined on a microplate reader.

As a result, as shown in FIG. 11, anti-PD-1 _pem /anti-HER2 and anti-PD-1 _BJHM /anti-HER2 all had high affinity for PD-1 and HER2, and the antigenic affinity activity of the bivalent monoclonal antibody was relatively well maintained. Among them, anti-PD-1 _BJHM /anti-HER2 had stronger affinity for PD-1 than anti-PD-1 _pem /anti-HER2.

Example 7 Simultaneous Binding Activity of an Anti-PD-1/Anti-HER2 Heterodimeric Antibody Molecule to Two Target Antigens

The simultaneous binding capacity of a PD-1/HER2 heterodimeric antibody to two target antigens was determined on PD-1 overexpressing CHO/PD-1 cells (GenScript Cat. No. M00529) and on HER2 overexpressing SK-BR-3 cells with using flow cytometry (FACS).

CHO/PD-1 cells were stained according to the instructions for the PKH26 kit (Sigma cat. no. SLBH4568V). Briefly, CHO/PD-1 cells were harvested and washed once with serum-free medium. CHO/PD-1 cells were prepared as a 2x10 ⁷ /ml cell suspension using diluent C from the PKH26 kit. The PKH26 dye was diluted to 4 μM. Then they were mixed in a ratio of 1:1. The mixed suspension had a cell density of 1 x 10 ⁷ /ml and a PKH26 concentration of 2 μM, and was incubated for 1 hour at room temperature, and then incubated with an equal volume of FBS for 1 minute, followed by cessation of staining. The suspension was centrifuged at 400g for 10 min, washed twice with complete medium and resuspended in complete medium for later use. SK-BR-3 cells were stained according to the instructions for the CFSE kit (Life technology, Cat. No. C34554). Briefly, CFSE was diluted with PBS to a working concentration of 0.5 μm and preheated to 37°C. SK-BR-3 cells were harvested by centrifugation at 1000 rpm for 5 min and resuspended in pre-warmed CFSE working solution followed by incubation at 37°C for 15 min. SK-BR-3 cells were harvested by centrifugation at 1000 rpm for 5 min and resuspended in complete medium followed by 30 min incubation. Cells were washed once with complete medium and resuspended in complete medium for later use.

The cells stained above were collected by centrifugation and washed once with cold PBS containing 2% FBS. Cells were resuspended in cold PBS containing 2% FBS at a cell density of 5 x 10 ⁶ /ml. SK-BR-3 and CHO/PD-1 were mixed in a ratio of 1:1, and then 100 µl were taken from each tube (i.e. 2.5x10 ⁵ SK-BR-3 and 2.5x10 ⁵ CHO/PD-1) . Then, 100 µl of heterodimeric antibody samples diluted with cold PBS containing 2% FBS, a control group, and an isotype control (human immunoglobulin, Jiangxi Boya Biopharmaceutical Co., Ltd., National Authorization No. S19993012) were added at a final concentration of 5 nM, respectively. The tube was incubated on ice for 30 min and washed twice with PBS containing 2% FBS.

As a result, as shown in Table. 1 and in FIG. 12, simultaneous binding of the heterodimeric antibody to both PD-1-overexpressing CHO/PD-1 cells and HER2-overexpressing SK-BR-3 cells was observed, suggesting that the heterodimeric PD-1/HER2 antibody is able to initiate close association between SK cells. -BR-3 and CHO/PD-1, which is the basis for T-cell mediated destruction of tumor cells.

- 12 043006

Table 1. Percentage of cells initiating tight association

Sample name % associated cells Isotype control 1.89 HER2 monoclonal antibody (100nM) 1.39 PD-1 monoclonal antibody _ret (JOM) 1.26 PD-1 monoclonal antibody hsm 1 AD (YUNM) PD-1 monoclonal antibody + HEK2 1 4^ monoclonal antibody (MLA) anti-RO-1 _ret / anti-NEK2 (YuonM) 39.71 anti-PD-1 hsm/anti-HEK2 (0.1 nM) 8.15 anti-PD-1 vshm/anti-HER2 (1nM) 32.79 anti-PD-1 hsm/anti-HER2 (JunM) 36.00 anti-PD-1 hsm/anti-HER2 (YuonM) 38.12

Example 8 Blocking activity of an anti-PD-1/anti-HEB2 molecule of a heterodimeric antibody against binding of PD-1 to a PD-L1 or PD-L2 ligand

Recombinant human PD-1-Fc was coated on a 96-well high adsorption ELISA plate using pH 9.6 phosphate buffer solution at a coating concentration of 1 μg/ml and a coating amount of 100 μl per well, and coating was performed at 4°C during the night. The plate was washed with PBST five times. The plate was blocked with 300 μl/well PBST containing 1% BSA, incubated for 1 hour at 25°C and washed with PBST five times. A heterodimeric antibody sample and a control were added, each serially diluted with PBST containing 1% BSA, while adding biotin-labeled PD-Ll-Fc at a final concentration of 1 μg/ml, or biotin-labeled PD-L2 at a final concentration of 4 μg/ml. ml, in the amount of 100 μl per well and incubated at 25°C for 1 hour. The plate was washed with PBST five times. Horseradish peroxidase-labeled streptavidin (BD, Cat # 554066) diluted 1:1000 with PBST containing 1% BSA was then added at 100 µl per well and incubated at 25° C. for 1 hour. The plate was washed with PBST five times. TMB colorimetric substrate was added at 100 μl/well and developed for 10 min at room temperature. Color development was stopped by adding 100 μl/well IM H ₂ SO ₄ . Absorbance at 450 nm was determined on a microplate reader.

As a result, as shown in FIG. 13, anti-PD-1 _ret /anti-HEB2 and anti-PD-1 _B shm/anti-HEB2 both have blocking activity against PD-1/PD-L1 and PD-1/PD-L2 binding, and blocking activity of bivalent monoclonal antibodies were relatively well preserved. Among them, anti-PD-1 _SN m/anti-HEB2 has stronger PD-1 blocking activity than PO-1 _ret /anti-HER2.

Example 9 T Cell Cytokine Secretion Enhancing Activity of Anti-PD-1/anti-HEB2 Heterodimeric Antibody Molecule

Human PBMCs (Lonza, Cat. No. CC-2702) were harvested. Human PBMCs were resuspended in complete medium (RPMI 1640 containing 10% FBS) at a cell density of 2 x 10 ⁶ /ml and 100 μl/well (2 x 10 ⁵ cells per well) of cells were plated into 96-well plates. A sample of PD-1/HER2 heterodimeric antibody and a control sample diluted with complete medium were added at 50 μl per well. RIA (Phaseolus vulgaris agglutinin) (Sigma, cat. no. L-2769) diluted with complete medium at a final concentration of 1 μg/ml was added in an amount of 50 μl per well. The plates were incubated in a carbon dioxide incubator at 37°C. After 3 days of incubation, 50 μl of the supernatant was taken and used for the detection of the cytokine IL-2 (Ray Biotech, Cat. No. ELH-IL2).

As shown in FIG. 14, human T cells upregulate IL-2 secretion upon PHA stimulation. The addition of the PD-1 antibody enhances T cell activation and stimulates cytokine secretion, while the PD-1/HER2 heterodimeric antibody has a similar effect to the PD-1 monoclonal antibody and stimulates the secretion of the cytokine IL-2 in a concentration-dependent manner.

Example 10 Pharmacokinetics of an anti-PD-1/anti-HEK2 heterodimeric antibody molecule in rats

Most SD rats aged 6-8 weeks purchased from Beijing Huafukang Biotechnology were used for the experiments. One week after the rats acclimatized to the environment, they were randomized into groups, each containing 3 rats. Each group was intravenously injected once with a monoclonal PD-1 antibody, a monoclonal HEK2 antibody, a heterodimeric RP-1/HEB2 antibody at a dose of 20 nmol/kg. Immediately after administration, after 5 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours, 264 hours, 312 hours, 360 hours, 408 hours and 480 hours after administration, 0.2 to 0.3 ml of blood was taken from the eyelid. The blood samples were left at room temperature for 30 minutes to 1 hour without the use of an anticoagulant, and after coagulation, the blood samples were centrifuged at 3000 rpm for 10 minutes. The resulting serum samples were frozen and stored at -80°C until testing.

Serum concentrations of PD-1 monoclonal antibody, HEK2 monoclonal antibody, and RP-1/HEB2 heterodimeric antibody were determined by ELISA. Briefly, recombinant human HER2 protein (Beijing Yiqiao Shenzhou, cat. no. 10004-H08H) or recombinant

-13043006 PD-1 protein (Beijing Yiqiao Shenzhou, cat. no. 10377-H08H) was coated on an overnight high adsorption ELISA plate using carbonate buffer pH 9.6 at 4°C. The plates were washed with PBST. To prevent non-specific binding, the plates were blocked with PBST containing 5% skim milk and washed with PBST. Then 10% mixed rat serum and a test serum sample diluted with PBST containing 1% BSA were added and incubated at 25° C. for 1 hour and the plates were washed with PBST. Horseradish peroxidase-labeled anti-human IgG (Chemicon Cat No. AP309P) diluted with PBST containing 5% skim milk was added and incubated at 25° C. for 1 hour. The plates were washed with PBST. Finally, color development was carried out using TMB colorimetric substrate for 10 minutes at room temperature. Color development was stopped by adding 1 M H ₂ SO ₄ . The absorbance at 450 nm was read on a microplate reader.

As a result, as shown in FIG. 15, 20 nmol/kg heterodimeric PD-1/HER2 antibody, which was used in a single intravenous injection, showed good pharmacokinetic characteristics in rats. The pharmacokinetic parameters of the heterodimeric anti-PD-1 _BJHM /anti-HER2 antibody are as follows: half-life (t _1/2 ) was 207 h; the area under the plasma drug concentration versus time curve (AUClast) was 33,448 nMh; Co was 534 nM; the observed volume of distribution (Vd) was 148 ml/kg; clearance (CL) was 0.50 ml/hour/kg; and the mean retention time (MRT _last ) was 159 h.

Claims (28)

1) oxidation in air or with the addition of an oxidizing agent, the oxidizing agent being selected from L-dehydroascorbic acid and other chemical derivatives, and 1) adding a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, tris(2carboxyethyl)phosphine, other chemical derivatives and combinations thereof, 1) expressing the isolated polynucleotide according to claim 9 or 10 or the recombinant expression vector according to claim 11 or 12 in a host cell; 1. A heterodimeric bispecific antibody comprising a first antigen-binding functional domain capable of specifically binding to PD-1 and a second antigen-binding functional domain capable of specifically binding to HER2, wherein the heterodimeric bispecific antibody comprises a first Fc chain and a second Fc chain that are connected to each other through one or more disulfide bonds, where the first Fc chain and the second Fc chain are respectively connected to the PD-1 antigen-binding functional domain and the HER2 antigen-binding functional domain via a covalent bond or linker, or the first Fc chain and the second Fc chain, respectively, are connected to the HER2 antigen-binding functional domain and an antigen-binding functional domain of PD-1 via a covalent bond or linker, and the first Fc strand and the second Fc strand contain 5 amino acid substitutions at the following positions: amino acid substitutions at positions T366 and D399 of the first Fc chain and amino acid substitutions at positions L351, Y407 and K409 of the second Fc chain; where the first Fc chain amino acid substitutions are T366L and D399R and the second Fc chain amino acid substitutions are L351E, Y407L and K409V, where the amino acid positions are numbered according to the Kabat EU numbering system. 2) carrying out the oxidation reaction in the presence of L-dehydroascorbic acid at a concentration of 0.5 mM or more at 4°C for 5 hours. 2) carrying out the reduction reaction in the presence of dithiothreitol at a concentration of 0.1 mM or more at 4°C for 3 hours and 2) purification and recovery of each protein expressed in the host cell; And 2. The heterodimeric bispecific antibody of claim 1, wherein the Fc chain is derived from IgG. 3) removal of the reducing agent by desalination, etc. 3) mixing of reduced proteins and subsequent oxidation of the mixture. 3. A heterodimeric bispecific antibody according to any one of claims 1 or 2, wherein the antigen-binding functional domains of PD-1 and HER2 are Fab fragments or scFv fragments. 4. The heterodimeric bispecific antibody of any one of claims 1 to 3, wherein the antigen-binding functional domains of PD-1 and HER2 are all Fab fragments. 5. A heterodimeric bispecific antibody according to any one of claims 1 to 3, wherein one of the antigen-binding functional domains of PD-1 and HER2 is a Fab fragment and the other is a scFv fragment. 6. The heterodimeric bispecific antibody of any one of claims 3 to 5, wherein the Fab fragment comprises a first heavy chain variable region and a second heavy chain variable region that are different from each other, as well as a first light chain variable region and a second light chain variable region which are different from each other. 7. The heterodimeric bispecific antibody of any one of claims 1 to 6, wherein when each of the first Fc chain covalently linked to the antigen-binding functional domain of PD-1 and the second Fc chain covalently linked to the antigen-binding functional domain of HER2, or each of the first the Fc chain covalently linked to the HER2 antigen-binding functional domain and the second Fc chain covalently linked to the PD-1 antigen-binding functional domain are present separately in the presence of a reducing agent, the weight ratio of the constituent homodimers is less than 50%. 8. A heterodimeric bispecific antibody according to any one of claims 1 to 7, wherein the amino acid sequence of the bispecific antibody comprises at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18. 9. An isolated polynucleotide encoding a heterodimeric bispecific antibody according to any one of claims 1-8. 10. 10. An isolated polynucleotide according to claim 9, having a sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17. 11. A recombinant expression vector containing the isolated polynucleotide according to claim 9 or 12. The recombinant expression vector according to claim 11, which is an X0GC plasmid vector obtained by modifying pcDNA. 13. A host cell containing an isolated polynucleotide according to claim 9 or 10 or a recombinant expression vector according to claim 11 or 12. 14. The host cell according to claim 13, which is selected from a human embryonic kidney cell HEK293 or HEK293T, HEK293E or HEK293F derived from a HEK293 cell; and Chinese Hamster Ovary CHO or CHO-S, CHO-dhfr-, CHO/DG44 or ExpiCHO cells derived from CHO cells. - 14 043006 - 15 043006 uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma and melanoma. 15. A composition for the prevention or treatment of cancer, comprising a heterodimeric bispecific antibody according to any one of claims 1 to 8, an isolated polynucleotide according to claim 9 or 10, a recombinant expression vector according to claim 11 or 12, or a host cell according to claim 13 or 14 and a pharmaceutically acceptable carrier. 16. The composition of claim 15, wherein the cancer is selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma and melanoma. 17. A method for producing a heterodimeric bispecific antibody according to any one of claims 1 to 8, including: 18. The method of claim 17, wherein the host cell is selected from a human embryonic kidney cell HEK293 or HEK293T, HEK293F or HEK293E derived from a HEK293 cell; and Chinese Hamster Ovary CHO or CHO-S, CHO-dhfr-, CHO/DG44 or ExpiCHO cells derived from CHO cells. 19. The method according to claim 17 or 18, where the recovery includes: 20. The method according to any one of claims 17-19, where the oxidation comprises: 21. The method according to any one of claims 17-20, further comprising isolation and purification. 22. The use of a heterodimeric bispecific antibody according to any one of claims 1 to 8, or an isolated polynucleotide according to claim 9 or 10, or a recombinant expression vector according to claim 11 or 12, or a host cell according to claim 13 or 14, or a composition according to 15 or 16 in the manufacture of a medicament for the prevention or treatment of cancer in a subject. 23. The use of a heterodimeric bispecific antibody according to any one of claims 1 to 8, or an isolated polynucleotide according to claim 9 or 10, or a recombinant expression vector according to claim 11 or 12, or a host cell according to claim 13 or 14, or a composition according to 15 or 16 for preventing or treating cancer in a subject. 24. Use according to claim 22 or 23, wherein the cancer is selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer bladder cancer, liver cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell cancer and melanoma. 25. A method for preventing or treating a malignant disease, comprising administering a heterodimeric bispecific antibody according to any one of claims 1 to 8, or an isolated polynucleotide according to claim 9 or claim 10, or a recombinant expression vector according to claim 11 or claim 12, or cells a host according to claim 13 or claim 14, or a composition according to claim 15 or claim 16 to a subject in need thereof. 26. The method of claim 25, wherein the cancer is selected from leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, 27. Use according to any one of claims 22-24, wherein the subject is a mammal, preferably a human. 28. The method according to claim 25 or 26, wherein the subject is a mammal, preferably a human.