CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No. PCT/CN2017/111310, filed on Nov. 16, 2017, which claims priority from China Patent Application No. 201611016435.0, filed on Nov. 18, 2016.

SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name: Sequence_Listing_As_Filed.txt; size: 21,488 bytes; and date of creation: Nov. 4, 2021, filed herewith, is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to an anti-PD-1/anti-HER2 natural antibody structural heterodimeric bispecific antibody and a method of preparing the same. Specifically, the present disclosure provides a highly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibody having characteristics of a natural IgG and having no mismatched heavy chain and light chain, and a method of preparing the same.

BACKGROUND ART

Monoclonal antibodies are highly specific antibodies that act only on a single antigenic determinant and have been widely used for cancer, inflammation, autoimmune diseases, infectious diseases, etc. However, such therapeutic molecules do not exhibit sufficient pharmacological effects when used alone. This is attributed 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 may be improved by simultaneously blocking molecules located on multiple targets or at many sites on a single target. At the same time, dual targeting therapy using multispecificity such as bispecific molecules may simplify the process of developing new drugs because a bispecific molecule is a single molecule. As compared with using a combination of several monospecific molecules, this method is a more convenient method for both patients and healthcare providers.

Many different types of bispecific antibodies or bifunctional molecules have been known in the art. The first bispecific antibody was obtained by coupling 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 the intensity of production work, purification of heterologous coupling products, complexity in the removal of homologous coupling products, original monospecific antibodies, or fragments, low efficiency, etc.

Another method used in the production of bispecific antibodies is to use a hybrid-hybridoma (quadroma) technology, which is a method of producing a bispecific antibody by somatic fusion of two kinds of hybridoma cell lines that secrete different antibodies. Due to arbitrary pairing of heavy and light chains of immunoglobulins, only one-tenth of the antibody mixture is the functional bispecific antibody required, thus complicating the purification process and reducing the production yield.

WO2013060867 describes a method of mass-producing a heterodimeric bispecific antibody. In this method, a mixture of two kinds of homodimeric antibodies is first reduced, and asymmetric amino acid mutations are introduced into CH3 domains of the two kinds of homodimeric antibodies to promote Fab arm exchange of the different antibodies, and inter-chain disulfide bonds of the hinge region are oxidized to form a stable bispecific antibody.

WO2009089004 describes a method of preparing a heterodimeric protein. In this method, amino acids at the CH3-CH3 interface are mutated into charged amino acids such that heterodimerization is electrostatically favorable but homodimer formation is electrostatically unfavorable.

U.S. Pat. No. 5,731,168 describes a method of preparing a heterodimeric IgG using a protuberance-into-cavity strategy. In this method, “protuberances” are constructed by replacing small amino acids at the interface of the CH3 domain of a first chain with larger amino acids, and at the same time, “cavities” are created by replacing corresponding large amino acids of the CH3 domain of a second chain with smaller amino acids. The protuberance and cavity interaction is favorable to heterodimeric IgG formation but unfavorable to homodimer formation.

WO2012058768 describes a method of preparing a highly specific stable heterodimeric IgG. This method combines both negative and positive design strategies along with structural and computational modeling guided protein engineering techniques to mutate a plurality of amino acids in the IgG1 CH3 structural domain, thereby forming a stable heterodimeric IgG with low homodimeric impurities.

Programmed death receptor-1 (PD-1) is an immune checkpoint that has recently attracted much attention, and is mainly involved in the control of T cell activation, and regulates strength and duration of immune responses. Under normal conditions, PD-1 mediates and maintains self-tolerance of body tissues and prevents damage of autologous tissue caused by excessive activation of the immune system during an inflammatory process, and therefore, has a positive effect on the prevention of autoimmune diseases. Under pathological conditions, PD-1 is involved in the occurrence and development of tumor immunity and various autoimmune diseases (Anticancer Agents Med Chem. 2015; 15(3):307-13. Hematol Oncol Stem Cell Ther. 2014 March; 7(1):1-17. Trends Mol Med. 2015 January; 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 may form covalent dimers via disulfide bonds, and exists in a monomeric form. The structure of PD-1 mainly includes an extracellular immunoglobulin variable region as a structural domain, a hydrophobic transmembrane domain, and an intracellular domain. The intracellular domain includes two independent phosphorylation sites. The phosphorylation sites are an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is inducibly expressed on the surface of activated T cells, and also on B cells, NK cells, monocytes, and DC 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 them, 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 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 January; 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 cancer cases, and revealed that HER2 is closely associated with overall survival rate and recurrence time (Salman D J, et al., Science, 235:177-182, 1985). According to recent studies, HER2 is overexpressed in approximately 25-30% of breast cancer patients (Revillion F et al., Eur J Cancer, 34:791-808, 1998), which is associated with malignant progression of tumors (Wright C et al., Cancer Res, 49: 2087-2090, 1989).

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

Designing bi-functional antibodies capable of recruiting effector cells is an effective means of improving the efficacy of antibodies. Until now, most studies have been conducted to exploit the function of the CD3 molecule. A target tumor can be effectively removed through activation of killer T cells by 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 the human body is only 1 hour (Loffler A et al., Blood, 95:2098-2103). This is attributed to the structure of BiTE itself. BiTE is composed of two single-chain antibody fragments. Its molecular weight is a mere 60 kDa, and Fc fragments which play an important role in half-life extension in an antibody molecule are removed.

Catumaxomab is another type of promising multi-functional antibody, and is a hetero Ig molecule targeting CD3 and EpCAM. Currently, this product is approved for the treatment of malignant ascites (Jager M et al., Cancer Res, 72:24-32, 2012). Still another multifunctional antibody under phase II clinical trial is ertumaxomab which targets CD3 and HER2. A heavy chain and a light chain of the heteroantibody is derived from rat IgG and targets CD3; and another heavy chain and light chain is derived from mouse IgG and targets HER2. A problem which accompanies this is that production of these products is very difficult. To acquire clones expressing bifunctional ertumaxomab, one hybridoma expressing a CD3 antibody and one hybridoma expressing a HER2 antibody are first prepared, respectively, and then the two hybridomas are hybridized to obtain a quadroma which is a bifunctional antibody capable of expressing anti-CD3 and HER2. Usually, in order to produce a single-target antibody, only one hybridoma is needed. In comparison, the production process of the bifunctional antibody is much more complicated, and it is also more difficult to obtain the quadroma, which may result in extremely high immunogenicity due to its rat origin.

Further, the most obvious side effect of the anti-CD3 antibody is a transient burst of systemic cytokine release, also called a ‘cytokine storm’. Accordingly, there is a demand 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 disclosure relates to a heterodimeric bispecific antibody including 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 which are linked to each other via one or more disulfide bonds, wherein the first Fc chain and the second Fc chain are respectively linked to the PD-1 antigen-binding functional domain and the HER2 antigen-binding functional domain, and the first Fc chain and the 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 including the above amino acid substitutions respectively have a tendency to undergo heterodimerization rather than homodimerization, and the amino acid positions are numbered according to the Kabat EU numbering system.

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

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

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

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

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

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

In an embodiment, the amino acid substitutions may include:

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

b) T366L and D399C substitutions in the first Fc chain and L351G, Y407L, and K409C substitutions in the second Fc chain;

c) T366L and D399C substitutions in the first Fc chain and L351Y, Y407A, and K409P substitutions in the second Fc chain;

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

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

f) T366P and D399I substitutions in the first Fc chain and L351P, Y407F, and K409F substitutions in the second Fc chain;

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

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

In an embodiment, the amino acid substitutions may include:

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

b) T366L and K409C substitutions in the first Fc chain and L351G, Y407L, and D399C substitutions in the second Fc chain;

c) T366L and K409P substitutions in the first Fc chain and L351Y, Y407A, and D399C substitutions in the second Fc chain;

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

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

f) T366P and K409F substitutions in the first Fc chain and L351P, Y407F, and D399I substitutions in the second Fc chain;

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

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

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

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

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

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

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

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

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

A second aspect of the present disclosure relates to an isolated polynucleotide encoding the heterodimeric bispecific antibody described in the first aspect.

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

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

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

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

In an embodiment, the host cell is selected from a human embryonic kidney cell HEK293, or HEK293T, HEK293F, or HEK293E derived from the HEK293 cell; and a Chinese hamster ovary cell CHO, or CHO-S, CHO-dhfr−, CHO/DG44, or ExpiCHO derived from CHO cell.

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

A sixth aspect of the present disclosure relates to a method of producing the heterodimeric bispecific antibody described in the first aspect, the method 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) reducing each protein expressed in the host cell; and

3) mixing the reduced proteins and then oxidizing the mixture.

In an embodiment, the host cell is selected from a human embryonic kidney cell HEK293, or HEK293T, HEK293F, or HEK293E derived from the HEK293 cell; and a Chinese hamster ovary cell CHO, or CHO-S, CHO-dhfr−, CHO/DG44, or ExpiCHO derived from CHO cell.

In an embodiment, the reducing may include 1) adding a reducing agent, wherein the reducing agent is 2-mercaptoethylamine, dithiothreitol, tri(2-carboxyethyl)phosphine, other chemical derivatives, or a combination thereof, 2) performing a 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) removing the reducing agent by desalting, etc.

In an embodiment, the oxidizing may include 1) oxidizing in air or adding an oxidizing agent, wherein the oxidizing agent is selected from L-dehydroascorbic acid and other chemical derivatives, and 2) performing an oxidization 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 an embodiment, the method may further include isolating and purifying.

A seventh aspect of the present disclosure relates to use of the heterodimeric bispecific antibody described in the first aspect, and/or the isolated polynucleotide described in the second aspect, and/or the recombinant expression vector described in the third aspect, and/or the host cell described in the fourth aspect, and/or the composition described in the fifth aspect, in the preparation of a drug for preventing and/or treating a disease of a subject.

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

A ninth aspect of the present disclosure relates to a method of preventing and/or treating a disease, the method including administering the heterodimeric bispecific antibody described in the first aspect, and/or the isolated polynucleotide described in the second aspect, and/or the recombinant expression vector described in the third aspect, and/or the host cell described in the fourth aspect, and/or the composition described in the fifth aspect to a subject in need thereof.

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

In an 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 carcinoma, esophageal cancer, stomach 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.

Advantageous Effects of Disclosure

In the present disclosure, a completely new anti-PD-1/anti-HER2 natural antibody structural heterodimeric bispecific antibody has been designed by employing PD-1 as a molecule recruiting immune cells and HER2 as a target molecule of tumor cells. The bispecific antibody is a highly stable anti-PD-1/anti-HER2 heterodimeric bispecific antibody having characteristics of a natural IgG and having no mismatched heavy chain and light chain. The bispecific antibody may bind to both of the two kinds of target molecules, PD-1 and HER-2, and thus may be more effective in treating complex diseases.

