WO2022271053A1

WO2022271053A1 - Isolated bispecific antibody that specifically binds to cd47 and pd-l1

Info

Publication number: WO2022271053A1
Application number: PCT/RU2022/050195
Authority: WO
Inventors: Alexey Konstantinovich MISORIN; Artur Khamidovich SABIROV; Aleksandra Dmitrievna AZARIAN; Tatyana Andreevna VODOPYANOVA; Sergey Aleksandrovich LEGOTSKY; Aleksandr Andreevich GORDEEV; Aleksandr Nikolaevich Doronin; Valery Vladimirovich SOLOVYEV; Dmitry Valentinovich MOROZOV
Original assignee: Joint Stock Company "Biocad"
Priority date: 2021-06-21
Filing date: 2022-06-21
Publication date: 2022-12-29
Also published as: CO2023018165A2; CR20230612A; AR126185A1; ECSP23097121A; TW202306998A; CL2023003854A1; CN117858904A; MX2024000266A; BR112023026982A2; EP4359445A1

Abstract

The present invention relates to the field of biotechnology and medicine, in particular to a bispecific antibody that specifically binds to CD47 and PD-L1. The invention further relates to a nucleic acid encoding said bispecific antibody, an expression vector, a host cell for producing said bispecific antibody and a method for producing said cell, pharmaceutical compositions comprising the bispecific antibody according to the invention, pharmaceutical compositions comprising the bispecific antibody according to the invention and other therapeutically active compounds, methods for treating diseases or disorders mediated by CD47 and PD-L1, use of the bispecific antibody or pharmaceutical compositions thereof for treating diseases or disorders mediated by CD47 and PD-L1, and the use of the bispecific antibody according to the invention and other therapeutically active compounds for treating diseases or disorders mediated by CD47 and PD-L1.

Description

Isolated hispecific antibody that specifically binds to CD47 and PD-L1

Field of the invention

Background of the invention

Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven to be useful as effective medicine for treating a number of disorders and diseases.

Naturally occurring human antibody molecules consist of two heavy chain homodimers, each of which forms a heterodimer in partnership with two identical light chain molecules. Conventional monoclonal antibodies in the form of whole molecules consist of bivalent ("two-armed") heterodimers of heavy and light chains.

Diseases are often caused as a result of several pathologies and are accompanied by many concomitant diseases. Bispecific antibodies are capable of binding and thereby neutralizing two different antigens per antibody molecule. The potential for a significant improvement in the therapeutic properties (and value) of medicinal products as compared to monoclonal antibodies has made bispecific antibodies an active area of research. Over the past twenty years, the literature has described many solutions regarding engineered versions of bispecific antibodies, as described in Brinkmann, U and RE Kontermann, 2017, The Making of Bispecific Antibodies, MAbs; 209 Feb/Mar; 9(2):182-212, doi: 10.1080/19420862.2016.1268307.

Therapeutic targeting of PD-1 and other molecules that transduce a signal through interaction with PD-1, such as PD-L1 and PD-L2, has received strong attention. Inhibition of PD-L 1 signals has been suggested as a means to increase T cell immunity (for example, antitumor immunity) for treating cancer and infection, including both acute and chronic infection. Inhibitors blocking the PD-Ll/PD-1 interaction are known, for example, from patent applications W02004004771, W02006121168, W02007005874, WO2008156712, W02010036959, W02010077634 and WO2011066389. CD47 expression and/or activity have been observed in a number of diseases and disorders. Accordingly, there exists a need for therapies that target CD47.

Antibodies inhibiting the interaction between CD47 and SIRPa ligand have been described in the following patent applications: WO2014123580, WO2013119714, WO2015191861,

WO201 1143624, WO2014093678, WO2017053423.

Bi specific antibodies that specifically bind to CD47 and PD-L1 with different formats of the first and second antigen-binding sites are known from patent application EA201791961 (W02019068302).

To date, no bispecific antibody that specifically binds to CD47 and PD-L1 and that is approved for therapeutic use exists in the world. In connection with the above, it is relevant to create novel bispecific antibodies that specifically bind to CD47 and PD-L1 and have high affinity for targets, good colloidal, thermal and aggregation stability values.

Description of the invention

The authors of the invention surprisingly found that the bispecific antibody that specifically binds to CD47 and PD-L1 and has a common (identical) light chain within the first and second antigen -binding fragments has higher colloidal, thermal and aggregation stability as compared to a bispecific antibody that specifically binds to CD47 and PD-L1 and has a different format of the first and second antigen binding fragments, for example, Fab and scFv format. The bispecific antibody that specifically binds to CD47 and PD-L1 and has a common light chain within the first and second antigen-binding fragments has a high affinity for targets.

The use of a common (identical) light chain in the first and second antigen-binding portions of a bispecific antibody has been described in Van Blarcom T ET AL., Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies, MAbs. 2018 Feb/Mar; 10(2):256-268. doi: 10.1080/19420862.2017.1406570. This format of a bispecific antibody makes it possible to solve the urgent problem of incorrect pairing of two different light chains to their cognate heavy chains for producing bispecific antibodies.

Brief description of the invention

In one aspect, the present invention relates to an isolated bispecific antibody that specifically binds to CD47 and PD-L1 and includes:

1) a first antigen-binding fragment that specifically binds to CD47 and includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3; and (b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6;

2) a second antigen-binding fragment that specifically binds to PD-L1 and includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 7,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 8,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 9; and

(b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6.

In some embodiments of the invention, the isolated bispecific antibody characterized in that the first antigen-binding fragment that specifically binds to CD47 is a Fab bound to an Fc fragment monomer.

In some embodiments of the invention, the isolated bispecific antibody characterized in that the second antigen-binding fragment that specifically binds to PD-L1 is a Fab bound to the Fc fragment monomer.

In some embodiments of the invention, the isolated bispecific antibody is characterized in that the antibody is a full-length IgG antibody.

In some embodiments of the invention, the isolated bispecific antibody is a full-length IgG antibody that is of human IgGl, IgG2, IgG3 or IgG4 isotype.

In some embodiments of the invention, the isolated bispecific antibody is a full-length IgG antibody that is of human IgGl isotype.

In some embodiments of the invention, the isolated bispecific antibody includes a heavy chain variable domain of the first antigen-binding fragment that specifically binds to CD47 and comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments of the invention, the isolated bispecific antibody includes a heavy chain variable domain of the second antigen-binding fragment that specifically binds to PD-L1 and comprises the amino acid sequence of SEQ ID NO: 24.

In some embodiments of the invention, the isolated bispecific antibody includes a variable domain of the common light chain for the first and second antigen-binding fragments, which comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments of the invention, the isolated bispecific antibody includes a first antigen binding fragment that specifically binds to CD47 and comprises:

(a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 22;

(b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23; and a second antigen-binding fragment that specifically binds to PD-L1 and comprises:

(a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 24,

(b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23.

In some embodiments of the invention, the isolated bispecific antibody includes the heavy chain of the first antigen-binding fragment that specifically binds to CD47 and comprises the amino acid sequence of SEQ ID NO: 25.

In some embodiments of the invention, the isolated bispecific antibody includes the heavy chain of the antigen-binding fragment that specifically binds to PD-L1 and comprises the amino acid sequence of SEQ ID NO: 27.

In some embodiments of the invention, the isolated bispecific antibody includes a common light chain for the first and second antigen-binding fragments, which comprises the amino acid sequence of SEQ ID NO: 26.

In some embodiments of the invention, the isolated bispecific antibody includes a first antigen binding fragment that specifically binds to CD47 and comprises:

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 25,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26; and a second antigen-binding fragment that specifically binds to PD-L1 and comprises:

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 27,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26.

In some embodiments of the invention, the isolated bispecific antibody is a bivalent antibody.

In one aspect, the present invention relates to an isolated nucleic acid that encodes any of the above bispecific antibodies.

In some embodiments of the invention, the nucleic acid is DNA.

In one aspect, the present invention relates to an expression vector comprising any of the above nucleic acids.

In one aspect, the present invention relates to a method for producing a host cell for producing any of the above bispecific antibodies and includes transformation of a cell with the above expression vector.

In one aspect, the present invention relates to a host cell for producing any of the above bispecific antibodies, the host cell comprises any of the above nucleic acids. In one aspect, the present invention relates to a method for producing any of the above bispecific antibodies, which comprises culturing the above host cell in a growth medium under conditions sufficient to produce said antibody, if necessary, followed by isolation and purification of the resulting antibody.

In one aspect, the present invention relates to a pharmaceutical composition used for treating a disease or disorder mediated by PD-L1 and CD47, which comprises any of the above bispecific antibodies in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by PD-L1 and CD47, the pharmaceutical combination comprising any of the above bispecific antibodies and at least one other therapeutically active compound.

In some embodiments of the pharmaceutical composition, the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof.

In some embodiments of the pharmaceutical composition, the chemotherapeutic agent is selected from the group that comprises: docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5- fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody that specifically binds to HER2 (human epidermal growth factor receptor 2).

In some embodiments of the pharmaceutical composition, the therapeutically active compound is trastuzumab.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is trastuzumab and a chemotherapeutic agent that is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

In one aspect, the present invention relates to a method for inhibiting the biological activity of PD-L1 and CD47 in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any of the above bispecific antibodies. In one aspect, the present invention relates to a method for treating a disease or disorder mediated by PD-L1 and CD47, which comprises administering to a subject in need of such treatment any of the above bispecific antibodies or any of the above pharmaceutical compositions in a therapeutically effective amount.

In one aspect, the present invention relates to a method for treating a disease or disorder mediated by PD-L1 and CD47, which comprises administering to a subject in need of such treatment any of the above bispecific antibodies and at least one other therapeutically active compound.

In some embodiments of the method of treatment, the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

In some embodiments of the method of treatment, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof.

In some embodiments of the method of treatment, the chemotherapeutic agent is selected from the group that comprises: docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5- fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.

In some embodiments of the method of treatment, the other therapeutically active compound is an antibody that specifically binds to HER2 (human epidermal growth factor receptor 2).

In some embodiments of the method of treatment, the other therapeutically active compound is trastuzumab.

In some embodiments of the method of treatment, the other therapeutically active compound is trastuzumab and a chemotherapeutic agent selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.

In one aspect, the present invention relates to the use of any of the above bi specific antibodies or any of the above pharmaceutical compositions for treating a disease or disorder mediated by PD-L1 and CD47 in a subject in need of such treatment.

In one aspect, the present invention relates to the use of any of the above bispecific antibodies and at least one other therapeutically active compound for treating a disease or disorder mediated by PD- L1 and CD47 in a subject in need of such treatment.

In some embodiments of the use, the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

In some embodiments of the use, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof.

In some embodiments of the use, the chemotherapeutic agent is selected from the group that comprises: docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.

In some embodiments of the use, the other therapeutically active compound is an antibody that specifically binds to HER2 (human epidermal growth factor receptor 2).

In some embodiments of the use, the other therapeutically active compound is trastuzumab.

In some embodiments of the use, the other therapeutically active compound is trastuzumab and a chemotherapeutic agent that is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

Brief description of drawings

Figure l is a map of the plasmid vector pEE_HCknobLALA_VH_CD47.

Figure 2 is a map of the plasmid vector pEE CLC.

Figure 3 is a map of the plasmid vector pEE HCholeLALA VH PD-Ll.

For Figures 1 to 3

Figure 4 is an electrophoregram of 09-001 in 7.5 % polyacrylamide gel under denaturing non reducing conditions following the first stage of purification on Protein A sorbent.

1 -molecular weight marker.

2- antibody 09-001 (10 pg).

3- antibody 09-001 (40 pg).

Figure 5 is an electrophoregram of 09-001 in 7.5 % polyacrylamide gel under denaturing non reducing conditions following purification on SP Sepharose HP sorbent.

1 -molecular weight marker.

2- antibody 09-001 (10 pg).

3- antibody 09-001 (40 pg).

Figure 6 is an electrophoregram of 09-001 in 12 % polyacrylamide gel under denaturing reducing conditions following purification on SP Sepharose HP sorbent.

1 -molecular weight marker.

2- antibody 09-001 (10 pg).

Figure 7 is a schematic representation of the format of the bispecific antibody with a common light chain, which specifically binds to CD47 and PD-L1.

Definitions and general methods

Unless defined otherwise herein, all technical and scientific terms used in connection with the present invention will have the same meaning as is commonly understood by those skilled in the art.

Furthermore, unless otherwise required by context, singular terms shall include plural terms, and the plural terms shall include the singular terms. Typically, the present classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and chemistry of protein and nucleic acids described herein are well known by those skilled and widely used in the art. Enzyme reactions and purification methods are performed according to the manufacturer's guidelines, as is common in the art, or as described herein.

The term "KD" in this description refers to the affinity constant (or equilibrium constant, or equilibrium dissociation constant), which is calculated from the ratio of Kd to Ka (i.e. Kd/Ka), and it is expressed as a molar concentration (M).

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, "binding affinity" refers to intrinsic (characteristic, true) binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity of a molecule X for its binding partner Y can generally be represented by the affinity constant (KD). The preferred Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, including those described in the present description. Low-affinity antibodies generally bind an antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind an antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

The term "Kd", "koff ' or "kdis" refers to the off rate constant of a particular interaction between a binding molecule and antigen. The off rate constant koff can be measured using bio-layer interferometry, for example, using Octet™ system.

The term "Ka", "kon" or "on-rate" refers to the association rate constant.

The term "Response" refers to the antibody-antigen binding signal.

As used in the present description and claims that follow, unless otherwise dictated by the context, the words "include" and "comprise," or variations thereof such as "includes", "including", "comprises," or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Detailed description of the invention

Bispecific antibody

The present invention relates to a bispecific antibody that specifically binds to CD47 and PD-L1.

The bispecific antibody according to the invention is a monoclonal antibody.

The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated by a separate clonal population of cells.

The bispecific antibody according to the invention is a recombinant antibody.

The term "recombinant antibody" refers to an antibody that is expressed in a cell or cell line comprising nucleotide sequence(s) encoding antibodies, wherein said nucleotide sequence(s) is (are) not associated with the cell in nature.

The bispecific antibody according to the invention is an isolated antibody.

The term "isolated" used to describe various antibodies according to this description refers to an antibody which has been identified and separated and/or regenerated from a cell or cell culture, in which the antibody is expressed. Impurities (contaminant components) from natural environment are materials which typically interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. The isolated polypeptide is typically prepared by at least one purification step.

The bispecific antibody according to the invention has a common light chain (CLC) for the first and second antigen-binding fragments.

Figure 7 shows a schematic representation of the format of the bispecific antibody with a common light chain, which specifically binds to CD47 and PD-L1.

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3; and

(b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6;

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 7,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 8,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 9; and

(b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6.

Amplification of the CD47 gene and/or overexpression of protein thereof have been observed in many cancers, for example, in any of the diseases from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

The term "antibody" or "immunoglobulin" (Ig), as used in the present description, includes whole antibodies. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated referred to in the present description as VET) and a heavy chain constant region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains g, a and d have a constant region composed of three constant domains CHI, CH2 and CH3 (in a line), and a hinge region for added flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, pp.89-99). In mammals, known are only two types of light chains denoted by lambda (l) and kappa (K). Each light chain consists of a light chain variable region (abbreviated referred to in the present description as VL) and light chain constant region. The approximate length of a light chain is 211 to 217 amino acids. Preferably, the light chain is a lambda (l) light chain, and the constant domain CL is preferably C lambda (l).

VL and VH regions may be further subdivided into hyper-variability regions called complementarity determining regions (CDRs), located between regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of antibodies may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of antibody or "antigen -binding fragment", as used in the present description, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of antibody can be performed by fragments of a full-length antibody. As used in the present invention, the term "antigen -binding fragment" means a Fab-fragment, i.e. a monovalent fragment, consisting of VL, VH, CL and CHI domains, which is linked with the Fc-fragment monomer.

The term "variable" refers to the fact that certain portions of the variable domains greatly differ in sequence among antibodies. The V domain mediates antigen binding and determines specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of invariant fragments termed framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability termed "hypervariable regions" or CDRs. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest. 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding of antibody to antigen, but exhibit various effector functions, such as participation of antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" according to the present description refers to the amino acid residues of antibody which are responsible for antigen binding. The hypervariable region typically comprises amino acid residues from a "complementarity determining region" or "CDR" and/or those residues from a "hypervariable loop".

“Kabat numbering scheme” or “numbering according to Kabat” as used in this application refers to the system for numbering of amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in variable regions of heavy and light chains of the antibody (Kabat et al. Ann. N.Y. Acad. Sci., 190:382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

The antibody of the present invention "which binds" a target antigen refers to an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting a protein or cell or tissue expressing the antigen, and slightly cross-reacts with other proteins. According to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA, in such embodiments, the degree of antibody binding to a non target protein is less than 10 % of antibody binding to a specific target protein. With regard to the binding of antibody to a target molecule, the term “specific binding” or “specifically binds to” or “is specific for” a particular polypeptide or an epitope on a particular target polypeptide means binding that is significantly (measurably) different from a non-specific interaction.

Specific binding may be measured, for example, by determining binding of a molecule as compared to binding of a control molecule. For example, specific binding may be determined by competition with another molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess of unlabeled target. As used in the present description, the term "specific binding" or phrases "specifically binds to" or " is specific for" a particular polypeptide or an epitope on a particular target polypeptide may be described by example of a molecule having a Kd for the target of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.

The term "bispecific antibody" refers to an antibody having antigen-binding fragments that are capable of specific binding to two distinct epitopes on a single biological molecule or capable of specific binding to epitopes on two distinct biological molecules. The bispecific antibody is also referred to herein as having "dual specificity" or as being a "dual specificity" antibody.

The fragment crystallizable region ("Fc region, Fc") of an immunoglobulin is the "tail" region of an immunoglobulin molecule that interacts with cell surface Fc-receptor, as well as some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains, respectively, of the two heavy chains.

The "Fc fragment monomer" refers to the Fc region from the second and third constant domains of any one of the two heavy chains (for IgG, IgA and IgD isotypes).

In some embodiments of the invention, the isolated bispecific antibody includes a variable domain of the common light chain for the first and second antigen-binding fragments, which comprises the amino acid sequence of SEQ ID NO: 23.

(b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23. In some embodiments of the invention, the bispecific antibody according to the invention includes a CH3 domain of one heavy chain, which is modified to form Knob, and a CH3 domain of another heavy chain, which is modified to form Hole, or vice versa.

"Knobs-into-holes" (interactions of the "knobs -into-holes" type) is an approach that enables to circumvent the problem associated with mispaired byproducts. This approach aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interfaces. On one chain, bulky amino acids were replaced by amino acids with short side chains to create a "hole". Conversely, amino acids with large side chains were introduced into the other CH3 domain to create a "knob". Co-expression of these two heavy chains produced a high yield of the heterodimer formation ("knob-hole") relative to the homodimer formation ("hole-hole" or "knob-knob") (WO9627011 and WO9850431, as well as Merchant AM ET ALL., An efficient route to human bispecific IgG, Nat Biotechnol. 1998 Jul;16(7):677-81).

In some embodiments of the invention, the bispecific antibody includes a CH3 domain of one heavy chain, which domain has amino acid substitutions S354C/T366W, to form Knob, and a CH3 domain of another heavy chain, which domain has amino acid substitutions Y349C/T366S/L368A/Y407V, to form Hole.

In some embodiments of the invention, the bispecific antibody includes a CH3 domain of one heavy chain, which domain has amino acid substitutions Y349C/T366S/L368A/Y407, to form Hole, and a CH3 domain of another heavy chain, which domain has amino acid substitutions S354C/T366W, to form Knob.

In some embodiments of the invention, the bispecific antibody includes an Fc fragment monomer, wherein LALA substitutions (L234A and L235A) are further introduced. These mutations are introduced to reduce the antibody effector function.

The above mutations in the Fc fragment are numbered according to EU numbering for amino acid chains of antibodies (Edelman, G.M., et ak, Proc. Natl. Acad. Sci. USA 63 (1969), pp. 78-85; Kabat, E.A., et ak, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

In some embodiments of the invention, the isolated bispecific antibody includes the heavy chain of the antigen-binding fragment that specifically binds to PD-L1 and comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments of the invention, the isolated bispecific antibody includes a common light chain for the first and second antigen-binding fragments, which comprises the amino acid sequence of SEQ ID NO: 26.

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 25,

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 27,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26.

In some embodiments of the invention, the isolated bispecific antibody that specifically binds to CD47 and PD-L1 is antibody 09-001.

The bispecific antibody 09-001 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 3; and

(b) a common light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 6; and

(a) a heavy chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 7,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 8,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 9; and

(b) a common light chain variable domain comprising:

(i) CDR1 (Kabat) with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 (Kabat) with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 (Kabat) with the amino acid sequence of SEQ ID NO: 6.

The bispecific antibody 09-001 includes:

1) a first antigen-binding fragment that specifically binds to CD47 and includes: (a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 10,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 11,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 12; and

(b) a common light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 13,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 14,

(iii) CDR3 (Chothia) with the amino acid sequence SEQ ID NO: 15; and

(a) a heavy chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 16,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 17,

(iii) CDR3 (Chothia) with the amino acid sequence of SEQ ID NO: 18; and

(b) a common light chain variable domain comprising:

(i) CDR1 (Chothia) with the amino acid sequence of SEQ ID NO: 13,

(ii) CDR2 (Chothia) with the amino acid sequence of SEQ ID NO: 14,

(iii) CDR3 (Chothia) with the amino acid sequence SEQ ID NO: 15.

The bispecific antibody 09-001 includes:

1) a first antigen-binding fragment that specifically binds to CD47 and includes a common light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 19,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 20,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 21; and

2) a second antigen-binding fragment that specifically binds to PD-L1 and includes a common light chain variable domain comprising:

(i) CDR1 (IMGT) with the amino acid sequence of SEQ ID NO: 19,

(ii) CDR2 (IMGT) with the amino acid sequence of SEQ ID NO: 20,

(iii) CDR3 (IMGT) with the amino acid sequence of SEQ ID NO: 21.

The bispecific antibody 09-001 includes a first antigen-binding fragment that specifically binds to CD47 and comprises:

(b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23; and a second antigen-binding fragment that specifically binds to PD-L1 and comprises: (a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 24,

The bispecific antibody 09-001 includes: the first Fc fragment monomer that comprises the second and third constant domains of the heavy chain (CH2 and CH3) with LALA mutations (L251A, L252A according to sequential numbering from the beginning of the chain (L234A, L235A according to EU or L247A, L248A according to kabat)) and mutations S371C, T383W according to sequential numbering from the beginning of the chain (S354C, T366W according to EU or S375C, T389W according to kabat) as compared to CH2 and CH3 of wild type human IgGl; and the second Fc fragment monomer that comprises the second and third constant domains of the heavy chain (CH2 and CH3) with LALA mutations (L240A, L241 A according to sequential numbering from the beginning of the chain (L234A, L235A according to EU or L247A, L248A according to kabat)) and mutations Y355C, T372S, L374A, Y413V according to sequential numbering from the beginning of the chain (Y349C, T366S, L368A, Y407V according to EU or Y370C, T389S, L391A, Y438V according to kabat) as compared to CH2 and CH3 of wild type human IgGl .

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 25,

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 27,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26.

The bispecific antibody 09-001 was developed on the basis of antibody BCD 106-02-001 from the patent document EA201791961A1 and therefore 09-001 is compared with BCD106-02-001 in the examples. Sequences of the antigen-binding fragment in the scFv format, which specifically binds to CD47, of antibody BCD106-02-001 were recloned into Fab format to produce 09-001. Further, for the bispecific antibody 09-001, we selected a new format with a common light chain (CLC) for the first and second antigen-binding fragments.

Antibody BCD106-02-001 from patent document EA201791961A1 includes a first antigen binding fragment that specifically binds to CD47 and is provided in the scFv format, and a second antigen-binding fragment that specifically binds to PD-L1 and is provided in the Fab format.

Antibody BCD106-02-001 from patent document EA201791961A1 includes a first antigen binding fragment that specifically binds to CD47 and comprises:

(a) a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 22; (b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 34; and a second antigen-binding fragment that specifically binds to PD-L1 and comprises:

Nucleic acid molecule

In one aspect, the present invention relates to a nucleic acid that encodes any of the above bispecific antibodies.

In any of the above embodiments, the nucleic acid molecules may be isolated.

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", used interchangeably in the present description, mean a precise sequence of nucleotides, modified or not, determining a fragment or a region of a nucleic acid, containing unnatural nucleotides or not, and being either a double-strand DNA or RNA, a single-strand DNA or RNA, or transcription products of said DNAs.

It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in a natural state. The sequences of the present invention have been isolated and/or purified, i.e., they were sampled directly or indirectly, for example by copying, their environment having been at least partially modified. Thus, isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by chemical synthesis should also be mentioned here.

A reference to a nucleotide sequence encompasses the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood as one which encompasses the complementary strand thereof with the complementary sequence thereof.

An "isolated" nucleic acid molecule is one which is identified and separated from at least one nucleic acid molecule-impurity, which the former is bound to in the natural source of antibody nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that exists in cells under natural conditions.

In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1-27. A nucleic acid molecule can also comprise any combination of said nucleotide sequences.

As would be appreciated by those skilled in the art, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequence of the light chain or heavy chain of the above bispecific antibody according to the invention or fragments thereof (VH, VL, CDR, etc.). It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same amino acid sequences. Such variant DNA sequences are within the scope of the present invention.

In some embodiments of the invention, the isolated nucleic acid is DNA.

A nucleic acid molecule of the invention may be isolated from any source that produces the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention. In certain embodiments of the invention, the nucleic acid molecule of the invention may be synthesized, rather than isolated.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the variable domain of the heavy chain of the first antigen-binding fragment, which specifically binds to CD47, of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 28.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the variable domain of the common light chain for the first and second antigen-binding fragments of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 29.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the variable domain of the heavy chain of the second antigen-binding fragment, which specifically binds to PD-LI, of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 30.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of the first antigen-binding fragment, which specifically binds to CD47, of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 31.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain for the first and second antigen-binding fragments of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 32.

In some embodiments of the invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain of the second anti gen -binding fragment, which specifically binds to PD-LI, of the bispecific antibody 09-001 and includes a nucleotide sequence with SEQ ID NO: 33.

The nucleic acid molecules may be used to express the bispecific antibodies according to the invention.

Expression vector

In one aspect, the present invention relates to an expression vector comprising any of the above nucleic acids. The present invention relates to a vector suitable for the expression of any of nucleotide sequences described herein. The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments of the invention, the vector is a plasmid, i.e. a circular double stranded piece of DNA into which additional DNA segments may be ligated. In some embodiments of the invention, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments of the invention, vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin site of replication and episomal mammalian vectors). In further embodiments of the invention, vectors (e.g. non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into a host cell, and thereby are replicated along with the host gene. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

The present invention relates to vectors comprising the above nucleic acid molecules that encode the above bispecific antibody, or structural portions thereof selected from: a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 22, a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23; a heavy chain variable domain with the amino acid sequence of SEQ ID NO: 24, a heavy chain with the amino acid sequence of SEQ ID NO: 25, a common light chain with the amino acid sequence of SEQ ID NO: 26; a heavy chain with the amino acid sequence of SEQ ID NO: 27, as described herein.

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV derived episomes, and the like. DNA molecules may be ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the DNA. An expression vector and expression control sequences may be chosen to be compatible with the expression host cell used. DNA molecules partially or fully encoding the sequences of first and second binding domains (for example, heavy and light chain sequences where a binding domain comprises a heavy and light chain sequence) can be introduced into individual vectors. In one embodiment, any combination of said DNA molecules is introduced into the same expression vector. DNA molecules may be introduced into an expression vector by standard methods (e.g. ligation of complementary restriction sites on an antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

In some embodiments of the invention, a suitable vector is one that includes restriction sites such that any VH or VL sequence can easily be inserted and expressed, as described above. Polyadenylation and transcription termination may occur at a native chromosomal site downstream of coding regions. A recombinant expression vector can also encode a signal peptide that facilitates secretion of an antibody chain from a host cell. An antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of an immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non immunoglobulin protein).

In some embodiments of the invention, in addition to antibody chain genes, the recombinant vector expression of the invention can carry regulatory sequences that control the expression of antibody chain genes in a host cell. It will be understood by those skilled in the art that the design of an expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of a host cell to be transformed, the level of expression of a desired protein, and so forth. Preferred control sequences for an expression host cell in mammals include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from a retroviral LTR, cytomegalovirus (CMV) (such as a CMV promoter/enhancer), simian virus 40 (SV40) (such as a SV40 promoter/enhancer), adenovirus, (e.g. the major late promoter adenovirus (AdMLP)), polyomavirus and strong mammalian promoters such as native immunoglobulin promoter or actin promoter. Methods for expressing polypeptides in bacterial cells or fungal cells, e.g. yeast cells, are also well known in the art.

In some embodiments of the invention, in addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of a vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which a vector has been introduced.

In some embodiments of the invention, the vector may include an expression control sequence. The term "expression control sequence" as used in the present description refers to polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include the promoter of ribosome binding site, and transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences. The term "control sequences" includes at least all components, the presence of which is essential for expression and processing, and can also include additional components, the presence of which is advantageous, for example, leader sequences and fusion partner sequences.

Host cell

In one aspect, the present invention relates to a host cell for producing any of the above bi specific antibodies, the host cell comprises any of the above nucleic acids.

The term "recombinant host cell" (or simply "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector according to the invention described above.

The present invention further relates to host cells that include, for example, a nucleotide sequence encoding the first heavy chain of the bispecific antibody according to the invention, a nucleotide sequence encoding the common light chain of the bispecific antibody according to the invention, or a nucleotide sequence encoding the second heavy chain of the bispecific antibody according to the invention, or all of the above three sequences. It should be understood that "recombinant host cell" and "host cell" refer not only to a particular subject cell but to the progeny of such a cell as well. Since modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to a parental cell; however, such cells are still included within the scope of the term "host cell" as used herein.

Nucleic acid molecules comprising a nucleotide sequence that encodes the amino acid sequence of the light chain or heavy chain of the above bispecific antibody, selected from: a heavy chain with the amino acid sequence of SEQ ID NO: 25, a common light chain with the amino acid sequence of SEQ ID NO: 26; a heavy chain with the amino acid sequence of SEQ ID NO: 27.

Mammalian cell lines used as hosts for transformation are well known in the art and include a plurality of immortalized cell lines available. These include, e.g., Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide for necessary characteristics of the protein produced. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells. When the recombinant expression vectors encoding the above bispecific antibody or a portion thereof are introduced into mammalian host cells, the above bispecific antibody is produced by culturing the host cells for a period of time sufficient to express the above bispecific antibody or a portion thereof according to the invention in the host cells, or, more preferably, secrete the above bispecific antibody into the culture medium in which the host cells are cultured. The above bispecific antibody may be isolated from culture medium using standard protein purification techniques. Plant host cells include e.g. Nicotiana , Arabidopsis , duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyce pombe , Saccharomyces cerevisiae and Pichia pastoris.

Furthermore, level of production of the bispecific antibody of the invention from a producing cell line may be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with EP Nos. 0216846, 0256055, 0323997 and 0338841.

It is likely that the bispecific antibody of the invention in different cell lines or host cells will have different glycosylation patterns from each other. However, the bispecific antibody disclosed herein is part of this invention, regardless of the state of glycosylation of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

The above host cell does not refer to a host cell produced using human embryos.

The above host cell does not refer to a host cell produced by modifying the genetic integrity of human germline cells.

Method of producing the antibody

In one aspect, the present invention relates to a method for producing any of the above bispecific antibodies, which comprises culturing the above host cell in a growth medium under conditions sufficient to produce said antibody, if necessary, followed by isolation and purification of the resulting antibody.

The present invention relates to methods for producing the bispecific antibodies of the present invention. One embodiment of the invention relates to a method for producing bispecific antibodies as defined herein, comprising producing a recombinant host cell capable of expressing the bispecific antibody, culturing said host cells under conditions suitable for expression of the bispecific antibodies, and isolating the resulting bispecific antibodies. The bispecific antibody produced by such expression in such recombinant host cells is referred to herein as "bispecific antibody".

Pharmaceutical compositions In one aspect, the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by PD-L1 and CD47, the pharmaceutical combination comprising any of the above bispecific antibodies.

"Pharmaceutical composition" refers to a composition comprising an antibody of the present invention and at least one of components selected from the group comprising pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, auxiliary, distributing and sensing agents, delivery agents, such as preservatives, stabilizers, filler, disintegrators, moisteners, emulsifiers, suspending agents, thickeners, sweeteners, flavouring agents, aromatizing agents, antibacterial agents, fungicides, lubricants, and prolonged delivery controllers, the choice and suitable proportions of which depend on the type and way of administration and dosage. Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacant and their mixtures as well. Protection against action of microorganisms can be provided by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanole, sorbic acid, and similar compounds. The composition may also contain isotonic agents, such as, for example, sugars, polyols, sodium chloride, and the like. Prolonged action of the composition may be achieved by agents slowing down absorption of active ingredient, for example, aluminum monostearate and gelatine. Examples of suitable carriers, solvents, diluents and delivery agents are water, ethanol, polyalcohols and their mixtures, natural oils (such as olive oil) and organic esters (such as ethyl oleate) for injections. Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Examples of disintegrators and distributors are starch, alginic acid and its salts, silicates and the like. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and polyethylene glycol of high molecular weight as well. The pharmaceutical composition for peroral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, local or rectal administration of active ingredient, alone or in combination with another active compound may be administered to human and animals in a standard administration form, in a mixture with traditional pharmaceutical carriers. Suitable standard administration forms include peroral forms such as tablets, gelatin capsules, pills, powders, granules, chewing-gums and peroral solutions or suspensions; sublingual and transbuccal administration forms; aerosols; implants; local, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms. The term "excipient" is used herein to describe any ingredient other than the antibody of the present invention. These are substances of inorganic or organic nature which are used in the pharmaceutical production/manufacturing in order to give drug products the necessary physicochemical properties.

In some embodiments, the compositions are intended to improve, prevent, or treat disorders that may be associated with CD47 and/or PD-L1.

The term "disease or disorder mediated by CD47 and PD-L1" refers to any disease or disorder that is either directly, or indirectly associated with CD47 and/or PD-L1, including etiology, development, progression, persistence or pathology of a disease or disorder.

“Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment.

In one aspect, the subject of treatment, or patient, is a mammal, preferably a human subject. Said subject may be either male or female, of any age.

The term "disorder" means any condition that would benefit from treatment with the compound of the present invention. The definition includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.

“Therapeutically effective amount” refers to that amount of the therapeutic agent being administered during treatment which will relieve to some extent one or more of the symptoms of the disease being treated.

The pharmaceutical compositions of the present invention and methods of preparation thereof will be undoubtedly apparent to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in compliance with the GMP (Good Manufacturing Practice) requirements. The composition may comprise a buffer composition, tonicity agents, stabilizers and solubilizers. Prolonged action of composition may be achieved by agents slowing down absorption of active pharmaceutical ingredient, for example, aluminum monostearate and gelatine. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyalcohols and their mixtures, oils, and organic esters for injections.

The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably a human.

The terms "buffer", "buffer composition", "buffering agent" refers to a solution, which is capable of resisting changes in pH by the action of its acid-base conjugate components, and which allows the product of bispecific antibody that specifically binds to CD47 and PD-L1 according to the present invention to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range from 4.0 to 8.0. Examples of buffers used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, etc. buffer solutions.

The terms "tonic agent", "osmolyte" or "osmotic agent", as used herein, refer to an excipient that can increase the osmotic pressure of a liquid antibody formulation. "Isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic drugs typically have an osmotic pressure from about 250 to 350 mOsm/kg. Isotonic agents used include, but are not limited to, polyols, saccharides and sucrose, amino acids, metal salts, for example, sodium chloride, etc.

"Stabilizer" refers to an excipient or a mixture of two or more excipients that provide the physical and/or chemical stability of the active agent. Stabilizers may be amino acids, for example, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, for example, but not limited to, polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene- polypropylene glycol and copolymers thereof (trade names: Poloxamer, Pluronic, sodium dodecyl sulfate (SDS); antioxidants, for example, but not limited to, methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfurous acid salts, etc.; chelating agents, for example, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), sodium citrate, etc.

The pharmaceutical composition according to the invention is a stable composition.

The pharmaceutical composition is "stable" if the active agent retains physical stability and/or chemical stability and/or biological activity thereof during the specified shelf life at storage temperature, for example, of 2-8 °C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. Storage period is adjusted based on the results of stability test in accelerated or natural aging conditions.

A pharmaceutical composition according to the invention may be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready formulation. The term "single unit dose" as used herein refers to discrete quantity of a pharmaceutical composition containing a predetermined quantity of an active ingredient. The quantity of the active ingredient typically equals the dose of the active ingredient to be administered in a subject, or a convenient portion of such dose, for example, half or a third of such dose.

The pharmaceutical compositions according to the present invention are typically suitable for parenteral administration as sterile formulations intended for administration in a human body through the breach in skin or mucosal barriers, bypassing the gastrointestinal tract by virtue of injection, infusion and implantation. In particular, it is contemplated that parenteral administration includes, inter alia, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial, transdermal injection or infusion, and kidney dialytic infusion techniques. Intra-tumor delivery, for example, intra-tumor injection, may also be employed. Regional perfusion is also contemplated. Preferred embodiments include intravenous and subcutaneous routes.

Any method for administering peptides, proteins or antibodies which is accepted in the art may be suitably employed for the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention.

Injectable formulations may be prepared, packaged, or sold, without limitation, in unit dosage form, such as in ampoules, vials, in plastic containers, pre-filled syringes, autoinjection devices. Formulations for parenteral administration include, inter alia, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

In another embodiment, the invention provides a composition for parenteral administration comprising a pharmaceutical composition which is provided in dry (i.e. powder or granular) form for reconstitution with a suitable base (e.g. sterile pyrogen-free water) prior to administration. Such medicinal formulation may be prepared by, for example, lyophilization, i.e. a process, which is known in the art as freeze drying, and which involves freezing a product followed by removal of solvent from frozen material.

The bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention can also be administered intranasally or by inhalation, either alone, as a mixture with a suitable pharmaceutically acceptable excipient from an inhaler, such as a pressurised aerosol container, pump, spray, atomiser, or nebuliser, wherein a suitable propellant is used or not used, or as nasal drops, or spray.

Medicinal formulations for parenteral administration may be formulated to be immediate or modified release. Modified release medicinal formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

In some embodiments of the pharmaceutical composition, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof. In some embodiments of the pharmaceutical composition, the chemotherapeutic agent is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5- fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

Therapeutic use of the bispecific antibody that specifically binds to CD 47 and PD-L1

In one aspect, the bispecific antibody that specifically binds to CD47 and PD-L1 is used in the treatment of disorders mediated by CD47 and PD-L1 activity.

In the case of a tumor (for example, cancer), the therapeutically effective amount of the bispecific antibody that specifically binds to CD47 and PD-L1 may reduce the number of cancer cells; reduce the initial tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. The bispecific antibody of the invention may to some extent prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can, for example, be measured by assessing survival, time to tumor progression (TTP), tumor response rate to treatment (RR), duration of response and/or quality of life.

The uses or methods used herein relating to the bispecific antibody that specifically binds to CD47 and PD-L1 with one or more other therapeutic agents are contemplated to mean, refer to and include the following:

1) simultaneous administration of such combination of the bispecific antibody that specifically binds to CD47 and PD-L1 and a therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said patient, 2) simultaneous administration of such combination of the bispecific antibody that specifically binds to CD47 and PD-L1 and a therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said patient, whereupon said components are released at substantially the same time to said patient,

3) sequential administration of such combination of the bispecific antibody that specifically binds to CD47 and PD-L1 and a therapeutic agent to a patient in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said patient with a significant time interval between each administration, whereupon said components are released at substantially different times to said patient; and

4) sequential administration of such combination of the bispecific antibody that specifically binds to CD47 and PD-L1 and a therapeutic agent to a patient in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner, whereupon they are concurrently, consecutively, or jointly released at the same and/or different times to said patient, where each portion may be administered by either the same or different routes.

The bispecific antibody that specifically binds to CD47 and PD-L1 may be administered without further therapeutic treatment, i.e., as an independent therapy.

In one aspect, the present invention relates to a method for inhibiting the biological activity of PD-L1 and CD47 in a subject in need of such inhibition, which comprises administering to the subject an effective amount of any of the above bispecific antibodies.

In one aspect, the present invention relates to a method for treating a disease or disorder mediated by PD-L1 and CD47, which comprises administering to a subject in need of such treatment any of the above bispecific antibodies or any of the above pharmaceutical compositions in a therapeutically effective amount.

In some embodiments of the method of treatment, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof. A "chemotherapeutic agent" is a chemical compound useful in the treatment of a malignant neoplasm. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylmelamine; acetogenins (e.g. bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (e.g. cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, e.g. calicheamicin gamma II and calicheamicin omega II (see, e.g. Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXOL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vinca alkaloids, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); biphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE- 58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAJX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, e.g. those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN®); rrnRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovovin.

Hormonal agents are agents that act to regulate or inhibit hormone action on tumors. Examples of such agents are anti -estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs), such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrazole (AREVIIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, imidazole; lutenizing hormone-releasing hormone agonists, including leuprolide (LEIPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines, such as megestrol acetate and medroxyprogesterone acetate, estrogens, such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti- progesterones; estrogen receptor down-regulators (ERDs); anti-androgens, such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

In some embodiments of the method of treatment, the chemotherapeutic agent is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

In some embodiments of the method of treatment, the other therapeutically active compound is trastuzumab and a chemotherapeutic agent that is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

In some embodiments of the use, the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer. In some embodiments of the use, the other therapeutically active compound is an antibody, chemotherapeutic agent, hormone therapy agent, or any combination thereof.

In some embodiments of the use, the chemotherapeutic agent is selected from the group comprising docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel or any combination thereof.

In some embodiments of the method of treatment, the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is used in combination with paclitaxel or docetaxel or doxorubicin or variants thereof or with carboplatin for treating locally advanced or metastatic triple negative breast cancer (TNBC) with tumors that express PD-L1 or CD47, as first-line therapy.

In some embodiments of the method of treatment, the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is used as monotherapy or in combination with chemotherapy including a platinum agent and 5-fluorouracil (5-FU) for treating locally advanced or metastatic head and neck cancer (HNC), as first-line therapy.

In some embodiments of the method of treatment, the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is used in combination with standard chemotherapy and/or trastuzumab for treating locally advanced or metastatic gastric cancer (GS) or esophagogastric junction cancer with tumors that express HER2 or PD-L1, following 2 or more lines of standard therapy.

In some embodiments of the method of treatment, the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is used in combination with tyrosine kinase inhibitors (for example, axitinib or another product) for treating locally advanced or metastatic renal cell carcinoma with tumors that express CD47, as first-line therapy.

In some embodiments of the method of treatment, the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is used in combination with chemotherapy including a platinum agent and paclitaxel or variants thereof for treating locally advanced or metastatic cervical cancer with tumors that express CD47, as first-line therapy.

Doses and routes of administration The bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention will be administered in an amount that is effective in treatment of the condition in question, i.e. in doses and during the periods of time required to achieve the desired result. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex and weight of the patient, and whether the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention is being administered as a stand-alone treatment or in combination with one or more additional drugs or treatments.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in a unit dosage form for ease of administration and uniformity of dosage. A unit dosage form as used herein refers to physically discrete units suited as unitary dosages for patients/subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier. Specification for the unit dosage forms of the invention is typically dictated by and directly dependent on (a) the unique characteristics of a therapeutic agent and particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in subjects.

Thus, a skilled artisan would appreciate, based upon the disclosure provided herein, that the doses and dosage regimen are adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic effect to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic effect to a patient. Thus, while certain doses and administration regimens are exemplified herein, these examples in no way limit the doses and administration regimens that may be provided to a patient in practicing the embodiments of the invention.

It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. Furthermore, it is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the judgment of a medical professional administering or supervising the administration of the bispecific antibody, and that dosage ranges set forth in the present description are exemplary only and are not intended to limit the scope or practice of the claimed bispecific antibodies. Furthermore, the dosage regimen with the compositions of the present invention can be based on various factors, including the type of a disease, age, weight, gender, patient's health condition, severity of a condition, route of administration of the bispecific antibody that specifically binds to CD47 and PD-L1 according to this invention. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention encompasses intra-patient dose-escalation as determined by one skilled in the art. Methods for determining appropriate dosage and regimen are well-known in the art and would be understood by a skilled artisan once provided the ideas disclosed herein.

Examples of suitable administration methods are provided above.

It is believed that a suitable dose of the bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention will be in the range of 0.1-200 mg/kg, preferably 0.1-100 mg/kg, including about 0.5-50 mg/kg, for example about 1-20 mg/kg. The bispecific antibody that specifically binds to CD47 and PD-L1 according to the invention may be administered, e.g. in a dose of at least 0.25 mg/kg, such as at least 0.5 mg/kg, including at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2 mg/kg, e.g. at least 3 mg/kg, including at least 4 mg/kg, e.g. at least 5 mg/kg; and for example up to a maximum of 50 mg/kg, including up to a maximum of 30 mg/kg, e.g. up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. The administration will typically be repeated in appropriate time intervals, such as once a week, once every two weeks, once every three weeks or once every four weeks, and for as long as deemed appropriate by a responsible physician, who may, in some cases, increase or reduce the dose if necessary.

Examples

The following examples are provided for better understanding of the invention. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference. Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

Materials and general methods

General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A., et ah, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et ah, Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer protocols.

Gene synthesis

Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The gene segments of 300-1400 bp long, which were flanked by singular restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned via the restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

DNA sequence determination

DNA sequences were determined by Sanger sequencing.

DNA and protein sequence analysis and sequence data management

The Unipro's UGENE suite version 1.29 and SnapGene Viewer were used for sequence creation, mapping, analysis, annotation and illustration.

Expression vectors

For the expression of the antibodies described in the application materials, variants of expression plasmids intended for expression of antibodies in prokaryotic cells (E.coli), transient expression in eukaryotic cells (e.g., in CHO cells) were applied. Beside the antibody expression cassette, the vectors contained: an origin of replication which allows replication of said plasmid in E. coli , genes which confer resistance in E. coli to various antibiotics (e.g. to ampicillin, kanamycin).

The fusion genes comprising the described antibody chains as described below were generated by PCR and/or gene synthesis and assembled with known recombinant methods and techniques by connection of the according nucleic acid segments, e.g. using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections, larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures.

Example 1. Synthesis of genetic constructs

Sequences of an anti gen -binding fragment in scFv format from candidate BCD 106-02-001 (EA201791961A1) were recloned into Fab.

To this end, we generated PCR products comprising the genes of heavy chain variable domains of the antibody, with primers comprising restriction sites. The resulting heavy chain variable domains were cloned into the vector pEE HCknobLALA at Sal I / Nhe I restriction sites.

As a result, the plasmid pEE_HCknobLALA_VH_CD47 (Figure 1) was produced.

The plasmid pEE HCholeLALA VH PD-Ll was produced by recloning the variable fragment in pEE HCholeLALA at Sal I / Nhe I restriction sites (Figure 3).

The common light chain for the first and second antigen-binding fragments was produced using the plasmid pEE CLC (Figure 2).

Antibody 09-001 was produced using plasmids shown in Table 1.

Table 1. Plasmids for producing antibody 09-001.

Example 2. Production and purification of antibody 09-001

Full-length antibodies 09-001 were produced in established cell line cells obtained from Chinese hamster ovary cells (CHO-T line). The cells were cultured in a serum-free medium HyCell TransFx-C by HyClone supplemented with 8 mM L-Glutamine and 1 g/1 Pluronic 68 in baffled flasks on orbital incubator shakers at a temperature of +37°C, humidity of 70% and in the presence of 5% CO2 at 150 rpm. The cells were passaged every 3-4 days at a density of 0.3*10⁶ cells/ml.

The day before transfection, cells from day 3 were inoculated at a density of 0.9* 10⁶ cells/ml. Transient transfection was performed at a concentration of 1.9-2.1*10⁶ cells/ml. RPMI-1640 medium was employed to prepare the transfection mixture. In 5% of the total volume of the RPMI-1640 seed material, we diluted separately plasmids at a load of 0.75 pg/ml and transfection reagent polyethylenimine (PEI), at DNA:PEI ratio of 1:7 by weight. The mixture was prepared using the cotransfection vector E2.60 encoding the protein production enhancer XBP-1S and the cotransfection vector E2.13 encoding RFP, 5% of the total DNA load per reaction. Target plasmids were used in an equal ratio. Plasmids and PEI diluted in RPMI-1640 were combined and incubated for 10 minutes, thereafter the transfection mixture was introduced to the cells. Suspension cultivation was carried out in 1000 ml baffled flasks on orbital incubator shakers at +37⁰ C, in the presence of 5% CO2 and humidity of 70%, at 150 rpm. The next day following transfection, 30% Trypton up to 1% concentration and 200 mM L-Glutamine up to 4 mM were added to the cells and fluorescence parameters were measured on a Guava flow cytometer to assess the transfection efficiency by the expression of fluorescent protein. On day 2 following transfection, the culture fluid was transferred to 2000 ml baffled flasks, and then 12% Boost 4 to 0.4% concentration and BalanCD Growth A in a volume equal to inoculant were added. The resulting product for antibody 09-001 was removed on day 10 day of culturing. The culture liquid was filtered through a filter module with 0.22 pm pore size, and then the protein concentration was measured on ForteBio.

Antibody 09-001 was isolated from the culture fluid and purified using a column with Protein A affinity chromatography sorbent. Cleared culture liquid was passed through a column at a load of 10-20 mg of protein per 1 ml of sorbent, which was pre-equilibrated with phosphate buffered saline (PBS, pH 7.4). The column was then washed with PBS to remove non-specifically binding components. Bound antibodies were eluted using 0.1 M glycine buffer (pH 3.5). Collected eluate was exposed to acidic pH for 30 min for the purpose of viral inactivation, and then neutralized with 1M Tris-HCl solution to pH 7.0. The protein was then transferred to 20 mM acetate buffer pH 5.0 using dialysis cassettes to conduct additional purification on SP Sepharose HP cation-exchange sorbent in order to remove aggregates and fragments of antibodies. The column with sorbent was equilibrated with 20 mM acetate buffer pH 5.0. The antibodies were then applied onto the column at a load of 10-20 mg of protein per 1 ml of sorbent. The antibodies bound to sorbent were eluted in a salt gradient using a solution of 20 mM NaAcO + 1 M NaCl pH 5.0. Following purification, the protein was transferred to a 20 mM acetate buffer (pH 5.0) supplemented with 100 mM trehalose using dialysis cassettes, filtered through 0.22 pm Millex GP, transferred to test tubes and stored at -70 °C.

Polyacrylamide gel electrophoresis and size-exclusion HPLC were used to monitor the purity of the candidates. Electrophoresis was performed in 7.5% polyacrylamide gel under denaturing non-reducing conditions (Figure 4, Figure 5) and in 12% polyacrylamide gel under denaturing reducing conditions (Figure 6). The protein purity was determined by the intensity of band staining at a protein load of 40 pg per lane. Size-exclusion HPLC was performed in a mobile phase of 0.05M NaHiPCri, 0.3 M NaCl pH=7.0. Results for antibody 09-001 production are shown in Table 2.

Table 2. Summary table for antibody 09-001 production.

Example 3. Determination of affinity of antibody 09-001 on Forte Bio Octet RED 384 to human CD47 antigen

The affinity of the interaction between antibody 09-001 and the human CD47 antigen was studied by bio-layer interferometry on the Octet Red384 instrument (ForteBio). The CD47-FcLama antigen was used for the study.

The study was conducted using covalent immobilization of protein (antigens) on AR2G biosensors (Amine Reactive Second-Generation (AR2G) Biosensors, ForteBio). The kinetic constants experiment consisted of the following main steps: activating sensors, loading protein onto sensors, quenching unreacted activated groups, recording baselines, recording analyte association, recording dissociation. The sensors were activated in an aqueous solution comprising 20 mM EDC and 10 mM sNHS for 300 s. The antigen was loaded onto the surface of biosensors in a sodium -acetate buffer with a pH 5.0 for 300 s. The loading protein concentration was 20 pg/ml for the human CD47 antigen (CD47- FcLama). Unreacted active centers on the sensor surfaces were quenched in 1M aqueous solution of ethanolamine with pH 8.5 for 300 s. To check the nonspecific interaction between the analyte and the sensors, we used an antigen-free sensor (at the loading step, the sensor was immersed in sodium acetate buffer with pH 5.0; all other steps are similar to those used for the sensor loaded with antigen). The baseline and all subsequent steps of the experiment were carried out in a kinetic buffer. At the association step (step duration was 300 s), sensors with loaded protein were immersed into wells with a solution of analyte (antibody 09-001) prepared in a kinetic buffer. 3 concentrations of antibodies were selected for analysis as follows: 68.9 nM, 34.4 nM, 17.2 nM. At the dissociation step (step duration was 600 s), the sensors were immersed into wells with a kinetic buffer, in which the baseline was recorded. The measurements were carried out at a temperature of 30 °C; we employed tangential stirring at 1000 rpm in all steps. The reference sensors went through all the steps as the sensors used to record analyte sensograms did, with the exception of the association step - at the association step, the sensors were immersed in a kinetic buffer without analyte (the reference sensor signals were measured in parallel with the recording of the main sensograms). The reference signal was subtracted from the signal received on sensors interacting with the analyte during the processing of sensograms.

Following measurement, the sensors were regenerated in 10 mM glycine solution with HC1 (pH 1.8) to remove bound antibodies (3 regeneration cycles - 5 s regeneration, followed by 5 s neutralization in kinetic buffer). Then the baseline, association, dissociation and regeneration steps were repeated. A total of 4 sets of sensograms were produced as follows: 1 set before regeneration and 3 sets following regeneration. No significant effect of regeneration on the constant values was revealed, therefore, we obtained average values for three measurements following regeneration.

To obtain numerical values of kinetic constants (kon is the on/association rate constant, kdis is the dissociation rate constant, KD is the equilibrium dissociation constant), the resulting sensograms were processed according to the 1 : 1 interaction model using Global Fit (selection of one set of kon, kdis, KD constants to analyze several sensograms of different concentrations) of the ForteBio Octet Data Analysis 9.0 software. The results are shown in Table 3.

Table 3. Results of kinetic analysis of final candidates in interaction with human CD47 antigen.

Conclusion

Antibody 09-001 interacts with the human CD47 antigen, the KD value is 83 nM.

Example 4. Determination of affinity of antibody 09-001 on Forte Bio Octet RED 384 to human CD47 antigen

The study was conducted similarly to that described in Example 3. 3 sets of sensograms were obtained. The final kon, kdis, KD values were obtained by averaging these values from three measurements. The results are shown in Table 4.

Table 4. Results of kinetic analysis of antibody 09-001 in interaction with human PD-L1 antigen.

Conclusion

Antibody 09-001 interacts with the human PD-L1 antigen with an affinity of approximately 3 nM.

Example 5. Determination of affinity of antibody 09-001 on Forte Bio Octert RED 384 to cynomolgus monkey CD47 antigen

The affinity of the interaction between antibody 09-001 and the cynomolgus CD47 antigen was studied by bio-layer interferometry on the Octet Red384 instrument (ForteBio). The cynomolgus CD47 antigen (cynoCD47(ARCO), ARCO BioSystems, Cat. No.: CD7-C5252; Cynomolgus / Rhesus macaque CD47 Protein, Fc Tag) was employed for the study.

The study was conducted similarly to that described in Example 3. 3 sets of sensograms were obtained. The final kon, kdis, KD values were obtained by averaging these values from three measurements. The results are shown in Table 5. Table 5. Results of kinetic analysis of final candidates in interaction with cynomolgus CD47 antigen.

Conclusion

Antibody 09-001 interacts with the cynomolgus CD47 antigen (cynoCD47(ARCO)), the KD value is 57 nM.

Example 6. Test of simultaneous interaction between two PD-L1 and CD47 antigens and antibody 09-001 on Forte Bio Octert RED 384

Simultaneous interaction between two different antigens (PD-L1 and CD47) and the 09-001 antibody was studied by bio-layer interferometry on the Octet Red384 instrument (ForteBio). Proprietary PD-L1 and CD47 antigens were used for the study.

The study was conducted using covalent immobilization of protein (antigens) on AR2G biosensors (Amine Reactive Second-Generation (AR2G) Biosensors, ForteBio). The experiment consisted of the following main steps: loading PD-L1 onto sensors, loading antibody 09-001 onto sensors with PD-L1, interacting with CD47 (analyte). Thus, the interaction signal at the last step of the experiment is detected in the presence of a bispecific antibody capable of simultaneously binding to PD- L1 present on sensors and CD47 present in solution. Table 6 shows the complete list of steps (stages).

Table 6. Steps of the experiment to test the simultaneous interaction between antibody 09-001 and two different antigens (PD-L1 and CD47).

The sensors were activated in an aqueous solution comprising 20 mM EDC and 10 mM sNHS for 300 s. The PD-L1 antigen was loaded onto the surface of biosensors in a sodium-acetate buffer with a pH 5.0 for 300 s. The concentration of PD-L1 protein for loading was 20 pg/ml. Unreacted active centers on the sensor surfaces were quenched in 1M aqueous solution of ethanolamine with pH 8.5 for 300 s. All steps of the experiment following the quenching step were carried out in the kinetic buffer. Antibody 09-001 was loaded onto sensors with immobilized PD-L1 for 300 s, antibody concentration was 20 pg/ml (137.7 nM). At the association stage (interaction with CD47), the sensors were immersed in a solution with CD47 concentration of 100 pg/ml (1280 nM). To test nonspecific interaction of CD47 analyte (negative control), a sensor with immobilized PD-L1 not loaded with antibody 09-001 was used (at the antibody loading step, the sensor was immersed in a kinetic buffer, all other steps are similar to those performed with the sensor being analyzed). Sensograms were processed using ForteBio Octet Data Analysisn 9.0 software. Simultaneous interaction with two different antigens was determined by analyzing the signal level (response parameter) at the end of the CD47 interaction step. The results are shown in Table 7.

Table 7. Results of the experiment to test the simultaneous interaction between antibody 09-001 and two antigens, PD-L1 and CD47.

At the last step of the experiment (CD47 association), we observed an interaction signal of 0.6088 nm for antibody 09-001, whereas no nonspecific signal of CD47 binding to sensors not loaded with the antibody was detected.

Conclusion

Antibody 09-001 demonstrates simultaneous binding to two different antigens, PD-L1 and

CD47.

Example 7. Test of blocking of interaction between CD47 and SIRPa by antibody 09-001 on Forte Bio Octert RED 384

Blocking of interaction between CD47 and SIRPa (Signal-regulatory protein alpha) by antibodies 09-001 was studied by bio-layer interferometry on the Octet Red384 instrument (ForteBio). Proprietary human CD47 and SIRPa antigens were used for the study.

The study was conducted using covalent immobilization of protein (antigens) on AR2G biosensors (Amine Reactive Second-Generation (AR2G) Biosensors, ForteBio). The experiment consisted of the following main steps: loading the SIRPa antigen onto sensors, testing the binding of SIRPa antigen to CD47/antibodies 09-001 mixtures (premix). CD47 solution without antibodies served as a positive control,. Blocking of interaction was determing by comparing the premix signal (response) and positive control signal. Table 8 shows the list of experiment steps.

Table 8. Steps of experiment to test the blocking of interaction between CD47 and SIRPa by antibodies

09-001.

The sensors were activated in an aqueous solution comprising 20 mM EDC and 10 mM sNHS for 300 s. The SIRPa antigen was loaded onto the surface of biosensors in a sodium -acetate buffer with a pH 5.0 for 600 s. The concentration of SIRPa protein for loading was 20 pg/ml. To check the nonspecific interaction between the analyte and the sensors, we used an antigen-free sensor (at the loading step, the sensor was immersed in sodium acetate buffer with pH 5.0; all other steps are similar to those used for the sensor loaded with antigen). Unreacted active centers on the sensor surfaces were quenched in 1M aqueous solution of ethanolamine with pH 8.5 for 300 s. All steps of the experiment following the quenching step were carried out in the kinetic buffer. At the association step (step duration was 1200 s), sensors with loaded protein were immersed into wells with an analyte solution. A solution containing 250 nM antibody 09-001 and 50 nM CD47 in a kinetic buffer was used as an analyte at the association step. A solution (analyte) with CD47 concentration of 50 nM served as a positive control. The measurements were carried out at a temperature of 30 °C; we employed tangential stirring at 1000 rpm in all steps. The reference sensors went through all the steps as the sensors used to record analyte sensograms did, with the exception of the association step - at the association step, the sensors were immersed in a kinetic buffer without analyte (the reference sensor signals were measured in parallel with the recording of the main sensograms). The reference signal was subtracted from the signal received on sensors interacting with the analyte during the processing of sensograms. Sensograms were processed using ForteBio Octet Data Analysisn 9.0 software. The results of the experiment are shown in Table 9.

At the association step, positive control showed a signal of binding to SIRPa of 0.297 nm, whereas the analyte containing CD47 and antibodies 09-001 showed no signal of binding to SIRPa. Table 9. Results of experiment to test the blocking of interaction between CD47 and SIRPa by antibody

09-001.

Conclusion

Antibody 09-001 blocks the interaction between CD47 and SIRPa.

Example 8. Testing of blocking of interaction between PD-1 and PD-L1 by antibody 09-001 on Forte Bio Octert RED 384

Blocking of interaction between PD-1 and PD-L1 by antibody 09-001 was studied by bio-layer interferometry on the Octet Red384 instrument (ForteBio). Human PD-1 and PD-L1 antigens were used for the study.

The study was conducted using covalent immobilization of protein (antigens) on AR2G biosensors (Amine Reactive Second-Generation (AR2G) Biosensors, ForteBio). The experiment consisted of the following main steps: loading the PD-1 antigen onto sensors, testing the binding of the PD-1 antigen to PD-Ll/antibody 09-001 mixture (premix). PD-L1 solution without antibodies served as a positive control. Blocking of interaction was determined by comparing the premix signal (response) and positive control signal. Table 10 shows the list of experiment steps. Table 10. Steps of experiment to test the blocking of interaction between PD-1 and PD-L1 by antibody

09-001.

The sensors were activated in an aqueous solution comprising 20 mM EDC and 10 mM sNHS for 300 s. The PD-1 antigen was loaded onto the surface of biosensors in a sodium-acetate buffer with a pH 5.0 for 300 s. The concentration of PD-1 protein for loading was 20 pg/ml. To check the nonspecific interaction between the analyte and the sensors, we used an antigen-free sensor (at the loading step, the sensor was immersed in sodium acetate buffer with pH 5.0; all other steps are similar to those used for the sensor loaded with antigen). Unreacted active centers on the sensor surfaces were quenched in 1M aqueous solution of ethanolamine with pH 8.5 for 300 s. All steps of the experiment following the quenching step were carried out in the kinetic buffer. At the association step (step duration was 300 s), sensors with loaded protein were immersed into wells with an analyte solution. A solution containing 250 nM antibody 09-001 and 50 nM PD-L1 in a kinetic buffer was used as an analyte at the association step. A solution (analyte) with PD-L1 at a concentration of 50 nM served as a positive control.

The measurements were carried out at a temperature of 30 °C; we employed tangential stirring at 1000 rpm in all steps. The reference sensors went through all the steps as the sensors used to record analyte sensograms did, with the exception of the association step - at the association step, the sensors were immersed in a kinetic buffer without analyte (the reference sensor signals were measured in parallel with the recording of the main sensograms). The reference signal was subtracted from the signal received on sensors interacting with the analyte during the processing of sensograms. Sensograms were processed using ForteBio Octet Data Analysisn 9.0 software. The results of the experiment are shown in Table .

At the association step, positive control shows a signal of analyte binding to PD-1 (1,429 nm), whereas no signal ofbindingtoPD-1 (0.007 nm, background signal value) was detected for the analyte containing PD-L1 and antibodies 09-001.

Table 11. Results of experiment to test the blocking of interaction between PD1 and PD-L1 by antibodies 09-001.

Conclusion

Antibody 09-001 blocks interaction between PD-1 and PD-L1.

Example 9. Determination of thermal stability under thermal stress at 50°C

The test samples were placed in a thermostated air bath and thermostated at 50°C for 48 hours. After heating, intact and stressed samples were transferred for analysis by size-exclusion HPLC with a UV detector. Chromatography was performed on the Agilent 1100 HPLC system, detection was performed at a wavelength of 220 nm and 280 nm.

The resulting stability data for 09-001 under incubation at 50 °C are shown in Table 12.

Overall conclusion: the samples show high level of thermal stability.

Table 12. Results of determination of thermal stability for antibody 09-001 by SE HPLC.

Example 10. Determination of colloidal and thermal stability by protein aggregation point using dynamic light scattering

In order to determine the aggregation temperature of the samples under study by dynamic light scattering (DLS), dependence of particle size in the medium on temperature was obtained using DynaPro® Plate Reader II with gradual heating from 25 to 80 °C. The results are shown in Table 13. This analysis studied antibody 09-001 and antibody BCD106-02-001 from EA201791961A1.

Table 13. Aggregation point of BCD 106-02-001 and 09-001.

It can be concluded that 09-001 shows a high level of therm ocolloidal stability (aggregation point in20mM sodium-acetate buffer pH =5.0 is above 60.0 °C). Antibody BCD106-02-001 shows a lower level of thermocolloidal stability as compared to 09-001.

Example 11. Comparison of initial bispecific antibody in scFv-Fab format and bispecific antibody in CLC format (bispecific antibody with common light chain). Table 14 shows data for antibodies BCD106-02-001 and 09-001 by way of comparison.

Table 14. Results of determination of aggregation stability for antibodies BCD 106-02-001 and 09-001 by SE HPLC.

These results show that antibody 09-001 has a higher aggregation stability as compared to antibody BCD 106-02-001 according to the purity study by SE HPLC. Further, antibody 09-001 has a higher aggregation temperature as compared to BCD 106-02-001, which fact indicates a greater thermocolloidal stability relative to antibody BCD 106-02-001.

Claims

1. An isolated bispecifie antibody that specifically binds to CD47 and PD-L1 and includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3; and

(b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6;

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 7,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 8,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 9; and

(b) a common light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 4,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 5,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 6.

2. The isolated bispecific antibody according to claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 is a Fab bound to an Fc fragment monomer.

3. The isolated bispecific antibody according to claim 3L characterized in that the second antigen-binding fragment that specifically binds to PD-L1 is a Fab bound to an Fc fragment monomer.

4. The isolated bi specific antibody according to claim 1, characterized in that the antibody is a full-length IgG antibody.

5. The isolated bi specific antibody according to claim 4, wherein the full-length IgG antibody is of human IgGl, IgG2, IgG3 or IgG4 isotype.

6. The isolated bi specific antibody according to claim 1, wherein the heavy chain variable domain of the first antigen-binding fragment that specifically binds to CD47 comprises the amino acid sequence of SEQ ID NO: 22.

7. The isolated b specific antibody according to claim 1, wherein the heavy chain variable domain of the second antigen-binding fragment that specifically binds to PD-L1 comprises the amino acid sequence of SEQ ID NO: 24.

8. The isolated bispecific antibody according to claim 1, wherein the variable domain of the common light chain for the first and second antigen-binding fragments comprises the amino acid sequence of SEQ ID NO: 23.

9. The isolated bispecific antibody according to claim 1, wherein the first antigen-binding fragment that specifically binds to CD47 includes:

(b) a common light chain variable domain with the amino acid sequence of SEQ ID NO: 23; wherein the second antigen-binding fragment that specifically binds to PD-L1 includes:

10. The isolated bispecific antibody according to claim 1, wherein the heavy chain of the first antigen-binding fragment that specifically binds to CD47 comprises the amino acid sequence of SEQ ID NO: 25.

11. The isolated bispecific antibody according to claim 1, wherein the heavy chain of the second antigen-binding fragment that specifically binds to PD-L1 comprises the amino acid sequence of SEQ ID NO: 27.

12. The isolated bispecific antibody according to claim 1, wherein the common light chain for the first and second antigen-binding fragments comprises the amino acid sequence of SEQ ID NO: 26.

13. The isolated bispecific antibody according to claim 1, wherein the first antigen-binding fragment that specifically binds to CD47 includes:

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 25,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26; wherein the second antigen-binding fragment that specifically binds to PD-L1 includes:

(a) a heavy chain with the amino acid sequence of SEQ ID NO: 27,

(b) a common light chain with the amino acid sequence of SEQ ID NO: 26.

14. The isolated bispecific antibody according to claim 1, which is a bivalent antibody.

15. An isolated nucleic acid that encodes the bispecific antibody according to any of claims 1 to 14.

16. The isolated nucleic acid according to claim 15, wherein the nucleic acid is DNA.

17. An expression vector comprising the nucleic acid according to any of claims 15 to 16.

18. A method for producing a host cell to produce the bispecific antibody according to any of claims 1 to 14, comprising transformation of the cell by the expression vector according to claim 17.

19. A host cell for producing the bispecific antibody according to any of claims 1 to 14, comprising the nucleic acid according to any of claims 15 to 16.

20. A method for producing the bispecific antibody according to any of claims 1 to 14, comprising culturing the host cell according to claim 19 in a culture medium under conditions sufficient to produce said antibody, if necessary, followed by isolating and purifying the resulting antibody.

21. A pharmaceutical composition comprising the bispecific antibody according to any of claims 1 to 14 in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

22. A pharmaceutical composition comprising the bispecific antibody according to any of claims 1 to 14 and at least one other therapeutically active compound.

23. The pharmaceutical composition according to claim 22, wherein the other therapeutically active compound is an antibody, a chemotherapeutic agent, a hormone therapy agent or any combination thereof.

24. The pharmaceutical composition according to claim 23, wherein the chemotherapeutic agent is selected from a group that includes: docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof; wherein the antibody is trastuzumab; wherein the combination is trastuzumab and a chemotherapeutic agent selected from the group including docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5-fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.

25. A method for inhibiting biological activity of PD-L1 and CD47 in a subject in need of such inhibition, including administering to the subject an effective amount of the bispecific antibody according to any of claims 1 to 14.

26. The bispecific antibody according to any of claims 1 to 14 or the pharmaceutical composition according to any of claims 22, 23, 24 for use in the treatment of a disease or disorder mediated by PD-L1 and CD47.

27. The bispecific antibody according to any of claims 1 to 14 or the pharmaceutical composition according to any of claims 22, 23, 24 for use according to claim 30, wherein the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

28. Use of the bispecific antibody according to any of claims 1 to 14 or the pharmaceutical composition according to any of claims 22, 23, 24 for treating a disease or disorder mediated by PD-L1 and CD47.

29. The use according to claim 28, wherein the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

30. Use of the bispecific antibody according to any of claims 1 to 14 and at least one other therapeutically active compound for treating a disease or disorder mediated by PD-L1 and CD47.

31. The use according to claim 30, wherein the disease or disorder mediated by PD-L1 and CD47 is selected from the group: triple-negative breast cancer (TNBC), head and neck cancer (HNC), gastric cancer (GC), esophagogastric junction cancer, lung adenocarcinoma, non small cell lung cancer, small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, high microsatellite instability malignant neoplasm, multiple myeloma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer.

32. The use according to any of claims 30 to 31, wherein the other therapeutically active compound is an antibody, a chemotherapeutic agent, a hormone therapy agent or any combination thereof.

33. The use according to claim 32, wherein the chemotherapeutic agent is selected from a group that includes: docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5- fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof; wherein the antibody is trastuzumab; wherein the combination is trastuzumab and a chemotherapeutic agent, which is selected from a group including docetaxel, paclitaxel, doxorubicin, platinum agents, carboplatin, 5- fluorouracil, tyrosine kinase inhibitors, axitinib, liposomal doxorubicin, liposomal paclitaxel, or any combination thereof.