WO2019129054A1 - Triabody, preparation method and use thereof - Google Patents

Abstract

Provided is a novel and artificially designed triabody comprising three polypeptide chains, wherein the first polypeptide chain comprises a first heavy chain variable domain, the second polypeptide chain comprises a first light chain variable domain, the first heavy chain variable domain is paired with the first light chain variable domain to form a first antigen binding site; and the third polypeptide chain comprises a second single domain antigen binding site and a third single domain antigen binding site. Provided are also a polynucleotide encoding the triabody, a vector comprising the polynucleotide, host cells comprising the polynucleotide or the vector, an immunoconjugate comprising the triabody, a pharmaceutical composition comprising the triabody or the immunoconjugate thereof, and use of the triabody in immunotherapy, prevention and/or diagnosis of a disease.

Description

Triple-chain antibody, preparation method thereof and use thereof Field of invention

The present invention generally relates to the field of immunology and antibody engineering. In particular, the invention relates to a novel artificially designed triplex antibody, a polynucleotide encoding the same, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector , an immunoconjugate comprising the tri-chain antibody, and a pharmaceutical composition comprising the tri-chain antibody or immunoconjugate thereof, and use of the tri-chain antibody for immunotherapy, prevention and/or diagnosis of a disease .

Background of the invention

Antibody molecules are capable of targeted and specific binding to their corresponding antigens, and are increasingly becoming important therapeutic and prophylactic agents for various diseases (eg, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.). / or diagnostic agent. However, monospecific antibodies directed against only one target have some limitations in clinical applications. Patients may develop resistance or no response after receiving monospecific antibody therapy. With the study of cancer and many other diseases, it is recognized that a variety of signal transduction pathways are often involved in the occurrence and development of diseases, and single-target immunotherapy is usually not sufficient to treat diseases in many diseases.

Since multispecific antibodies (eg, bispecific antibodies) are capable of specifically binding to different antigens, when one antigen is located on a particular immune cell and the other antigen is on a disease cell, a multispecific antibody (eg, bispecific) Sexual antibodies) can redirect specific immune cells to diseased cells to enhance the lethality of immune cells to diseased cells. In addition, multispecific antibodies (eg, bispecific antibodies) can also be designed to act simultaneously on signal transduction pathways of two or more different media. These advantageous properties open up broad application prospects for multispecific antibodies (eg, bispecific antibodies).

)于2009年4月在欧洲获准用于治疗由EpCAM阳性上皮源性转移瘤所引起的恶性腹水。 Several multispecific antibody (eg, bispecific antibodies) formats have been developed by antibody engineering and their applicability in disease applications has been investigated. Currently, the two bispecific antibody products approved for marketing are Blinatumomab developed by Micromet and Amgen, and Catumaxomab developed by Trion Pharma. Blinatumomab is the first single-chain bispecific antibody with a molecular weight of approximately 55 kDa for the treatment of B-cell non-Hodgkin's lymphoma (NHL) and B precursor acute lymphoblastic leukemia (ALL). The two single-chain Fv molecules directed against the CD19 molecule and against the CD3 molecule are fused by a flexible linker peptide, which utilizes CD19 expressed in almost all B lymphocyte tumors and CD3 expressed on T cells, making T The cells are tightly linked to the target cells (tumor cells), which release perforin and telomerase into the synaptic cleft, causing a series of chemical reactions in the tumor cells, thereby destroying the tumor cells (Nagorsen D. and Baeuerle PA, Immunomodulatory) Therapy of cancer with T cell-engaging BiTE antibody blinatumomab, Exp Cell Res, 2011, 317: 1255-1260). Catumaxomab is a chimera consisting of two half-antibodies derived from the parental mouse IgG2a isotype and the rat IgG2b isotype, each having a light chain and a heavy chain, anti-CD3 rat IgG2b half. Antibodies for T cell recognition, mouse IgG2a half antibodies against tumor cell surface antigen EpCAM (epithelial adhesion molecule) for tumor cell recognition (Chelius D et al, Structural and functional characterization of the trifunctional antibody catumaxomab, MAbs, 2010, 2:309-319). Catumaxomab

) was approved in Europe in April 2009 for the treatment of malignant ascites caused by EpCAM-positive epithelial-derived metastases.

Multispecific antibodies (eg, bispecific antibodies) can be divided into many classes depending on the components and the manner in which they are constructed. For example, according to the left-right symmetry of the multi-specific antibody structure, it can be divided into a symmetric structure and an asymmetric structure; according to the Fc region of the multi-specific antibody with or without IgG, it is divided into an intact antibody and an antibody fragment; according to the antigen in the multispecific antibody The number of binding sites is divided into bivalent, trivalent, tetravalent or higher valence antibodies.

Different multi-specific antibody designs have their own advantages and disadvantages. For example, although Blinatumomab can be produced by large-scale culture of recombinant Chinese hamster ovary (CHO) cells, it is easy to form aggregates, has a short half-life in vivo, and requires additional equipment for practical use. Continuous infusion device; Catumaxomab production process is complex and murine heterologous antibodies are more prone to immunogenicity in the human body. Furthermore, unwanted pairing of unrelated heavy and light chains in a four-chain immunoglobulin (Ig)-like multispecific antibody results in the formation of an inactive antigen binding site and/or other non-functional unwanted byproducts, which It is also a problem in clinical scale production and therapeutic applications of antibodies (Klein, C. et al., Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies, mAbs, 2012, 4: 653-663). In theory, two heavy chains can be associated in four different combinations, and each of these heavy chains can be associated with the light chain in a random manner, yielding 2 ⁴ (= total 16) possible chain combinations . Of the 16 theoretically possible combinations, 10 combinations were actually found, but only one of them corresponds to a multispecific antibody of the desired functionality. It is difficult to isolate a multi-specific antibody required from a complex mixture and theoretically an intrinsic poor yield of up to 12.5%, making it difficult to produce a four-chain Ig-like multispecific antibody in a cell expression system.

The novel antibody pattern of the present invention overcomes the above disadvantages. The present invention provides a novel multispecific antibody pattern that is readily produced in increased yield due to proper coupling or pairing between the individual strands, and is readily expressed in cultured cells in vitro, without the need for Complex production process. Furthermore, the multispecific antibody pattern of the present invention is capable of maintaining the affinity of each antigen binding site in the multispecific antibody for binding to a corresponding different epitope, and does not create a spatial position when binding different epitopes. Resistance to interference, with good drug-forming properties. Further, the multispecific antibody format of the invention is physically stable and biologically stable, which allows the antibody to be more productive and developable.

Summary of invention

Disclosed herein is a novel triple-chain antibody comprising three polypeptide chains constructed by antibody engineering methods. The tri-chain antibody is capable of binding to one or more antigens with high affinity and high specificity, preferably, to two or more antigens. The invention also provides nucleic acid molecules encoding the three-chain antibodies, expression vectors, host cells and methods for producing the tri-chain antibodies. The invention also provides an immunoconjugate comprising the tri-chain antibody of the invention and a pharmaceutical composition comprising the tri-chain antibody or immunoconjugate thereof. The three-chain antibodies disclosed herein can be used alone or in combination with other drugs or other therapeutic modalities for the treatment, prevention, and/or diagnosis of diseases such as autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases (eg, chronic infectious diseases or Septicemia, tumors, etc.

Thus, in one aspect, the invention provides a triple chain antibody having one or more of the following properties:

(a) with a high affinity, for example with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably about 10 ⁹ M ^-1 or more, with one or more antigen specificities Combine

(b) easy to express in cultured cells in vitro, and the three strands can be correctly coupled or paired;

(c) having good physical stability, in particular, having good long-term thermal stability; and maintaining biological activity for a long period of time;

(d) exerting a biological function by specifically interacting with one or more antigens by acting on a signaling pathway in which each antigen is involved;

The tri-chain antibody of the present invention is at least a trivalent antibody (i.e., has at least three antigen-binding sites). In one embodiment, the tri-chain antibody is a (1+2 style) trivalent antibody, the first polypeptide chain comprising a first heavy chain variable domain and the second polypeptide chain comprising a first light chain a variable domain, the first heavy chain variable domain paired with a first light chain variable domain (hereinafter abbreviated as VH1/VL1 pair) forms a first antigen binding site; and the third polypeptide chain comprises a single Domain second antigen binding site and single domain third antigen binding site.

In one embodiment, a three-chain antibody of the invention comprises one antigen binding site that targets a first antigen, and the other two binding sites each target the same or a different epitope of a second antigen, respectively. In one embodiment, the three-chain antibody of the invention comprises three antigen binding sites that target three different antigens. In one embodiment, the three-chain antibody of the invention comprises three antigen-binding sites that target the same antigen.

In one embodiment, the invention provides a triplex antibody comprising three polypeptide chains, wherein the first polypeptide chain comprises a first heavy chain variable domain and an immunoglobulin CH1 domain, and the second polypeptide chain comprises a first light chain variable domain and an immunoglobulin CL domain, the first heavy chain variable domain paired with a first light chain variable domain to form a first antigen binding site; and a third polypeptide chain A single domain second antigen binding site and a single domain third antigen binding site, wherein the single domain second antigen binding site of the third polypeptide chain and the single domain third antigen binding site With or without a linker peptide.

In one embodiment, the single domain second antigen binding site and the single domain third antigen binding site of the third polypeptide chain have a linkage comprising a glycine (G) and a serine (S) residue. The peptide, for example, a linker peptide comprising 1-7 GGGGS repeats, preferably a linker peptide comprising 4 GGGGS repeats.

In a preferred embodiment, the third polypeptide chain in the tri-chain antibody of the invention does not comprise an immunoglobulin CH1 domain; the single domain second antigen binding site and the single domain third antigen are combined The locus is selected from the heavy chain variable domain of an antibody naturally lacking a light chain (such as the heavy chain variable domain of a naturally occurring heavy chain antibody in Camelidae species), and is referred to as a novel antigen in fish. Single domain antigen binding sites in immunoglobulins of new antigen receptors (NARs), such as naturally occurring IgNARs in shark serum, and recombinant single domain antigen binding sites derived therefrom. The heavy chain variable domain derived from a heavy chain antibody that naturally lacks a light chain is referred to herein as VHH to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas and guanaco. Other species other than camelids can also produce heavy chain antibodies that naturally lack light chains, and such VHHs are also within the scope of the invention.

In one embodiment, in the third polypeptide chain of the tri-chain antibody of the invention, the single domain second antigen binding site and the single domain third antigen binding site are the first VHH and the first The two VHHs, the sequences of the first VHH and the second VHH are the same or different, and bind to the same or different antigens, or bind to different antigenic epitopes on the same antigen.

In yet another embodiment, the invention provides a triplex antibody comprising three polypeptide chains, wherein the first polypeptide chain comprises a first heavy chain variable domain, an immunoglobulin CH1 domain from N-terminus to C-terminus And an Fc domain; the second polypeptide chain comprises a first light chain variable domain and an immunoglobulin CL domain from the N-terminus to the C-terminus; and the third polypeptide chain comprises a single domain from the N-terminus to the C-terminus a second antigen binding site, a single domain third antigen binding site, and an Fc domain, preferably, the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably, the immunoglobulin is a human IgG1 Immunoglobulin.

Among the three-chain antibodies comprising three polypeptide chains provided by the present invention, the inventors have also designed amino acid residues capable of stabilizing the structure of the triple-chain antibody and facilitating proper coupling or pairing between the respective chains. For example, a hinge region having a "CPPC" amino acid residue is included in the Fc domain of the first polypeptide chain and the third polypeptide chain of the triple-chain antibody such that the first polypeptide chain and the third polypeptide chain are each other The disulfide bond formed between the amino acid residues at the hinge region is stably associated. In one embodiment, the first polypeptide chain and the third polypeptide chain of the triplex antibody of the invention comprise Y349C and S354C, respectively, in the respective Fc domains or S354C and Y349C, respectively (according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), the EU index is numbered, hereinafter referred to as "EU number"), whereby the first polypeptide chain and the third largest The peptide chain further forms an interchain disulfide bond in the Fc region to stabilize the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain and/or the third polypeptide chain of a triplex antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the amino acid mutation is present in the CH2 domain of the Fc region, eg, the triplex antibody is comprised at positions 234 and 235 of the first polypeptide chain and/or the third polypeptide chain (EU numbering) Amino acid substitution at the place. In a specific embodiment, the amino acid substitutions are L234A and L235A (hereinafter referred to as "LALA mutations").

In one embodiment, the Fc domain of each of the first polypeptide chain and the third polypeptide chain of the triplex antibody of the invention comprises a bulge ("knob") or a hole ("hole", respectively. "), and the protrusions or holes in the Fc domain of the first polypeptide chain can be respectively placed in the holes or protrusions in the Fc domain of the third polypeptide chain, whereby the first The polypeptide chain and the third polypeptide chain form a "knob-in-hole" stable association with each other. In one embodiment, an amino acid substitution T366W is included in one of the first polypeptide chain and the third polypeptide chain, and is included in the other of the first polypeptide chain and the third polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering). Thus the protrusions in one strand can be placed in the cavities in the other strand, facilitating the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain of the triaborating antibody of the invention and the immunoglobulin CH1 domain and the CL domain of the second polypeptide chain comprise a bulge or a hole, respectively, and a region in the CH1 domain The protrusions or holes may be respectively placed in the holes or protrusions in the CL domain, such that the first polypeptide chain and the second polypeptide chain also form a stable association of "binding buckles" with each other. .

The first antigen binding site, the second antigen binding site, and the third antigen binding site in the triplex antibody of the present invention may bind to the same antigen or an epitope on a different antigen. For example, the first antigen binding site binds to an epitope of a first antigen, and the second antigen binding site and a third antigen binding site bind to the same or different epitopes on the second antigen, thereby A tri-chain antibody is a bispecific antibody directed against a first antigen and a second antigen. When the first antigen binding site binds to an epitope of the first antigen, the second antigen binding site and the third antigen binding site respectively bind to an epitope of the second antigen and an epitope of the third antigen, respectively The tri-chain antibody is a trispecific antibody. The antigen type to which the triple-chain antibody of the present invention specifically binds is not particularly limited, and the antigen may be, for example, a cytokine, a growth factor, a hormone, a signaling protein, an inflammatory mediator, a ligand, a cell surface receptor or a fragment thereof.

In one embodiment, the antigen to which the tri-chain antibody of the present invention specifically binds is selected from the group consisting of a tumor-associated antigen, an immunological checkpoint molecule, a costimulatory molecule in the immune system, and ligands and/or receptors of these molecules, for example, CD47, PD1, PD-L1, PD-L2, LAG-3, and 4-1BB (CD137).

The present invention exemplifies several triple-stranded bispecific antibodies as described below.

i) In one embodiment, the tri-chain antibody of the invention is an anti-CD47/PD-L1 bispecific antibody capable of being at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably Affinity constant of about 10 ⁹ M ^-1 or stronger binds to CD47 on the surface of tumor cells, thereby blocking the binding of CD47 to SIRPα on the surface of macrophages, and promoting the phagocytosis of tumor cells by macrophages in tumor tissue infiltration area. And binding to PD-L1 on the surface of tumor cells with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably about 10 ⁹ M ^-1 or more, thereby inhibiting T cells The binding of PD-1 to PD-L1 on the surface of tumor cells induces T cell activation and exerts an anti-tumor effect.

In one embodiment, an anti-CD47/PD-L1 bispecific antibody of the invention comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to CD47 on a first polypeptide chain and a second polypeptide chain And a first VHH and a second VHH on the third polypeptide chain that specifically bind to PD-L1.

In one embodiment, the first antigen binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises GSIIEYYWS derived from the anti-CD47 antibody ADI-29341 ( VH CDR2, YHYYTGSTNYNPSLKS (SEQ ID NO: 4) shown in SEQ ID NO: 3), VH CDR3, RASQGISRWLA (SEQ ID NO: 10) shown by ARGKTGSAA (SEQ ID NO: 5) VL CDR2 represented by VL CDR1, AASSLQS (SEQ ID NO: 11) and VL CDR3 represented by QQTVSFPIT (SEQ ID NO: 12), or one or two with one or more of the 6 CDRs Sequence of three, four, or five amino acid changes (eg, amino acid substitutions or deletions). In yet another embodiment, the first antigen binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises a SEQ derived from the anti-CD47 antibody ADI-29341 ID NO: a paired heavy chain variable region sequence/light chain variable region sequence of 2/9, or at least 90%, 91% with the paired heavy chain variable region sequence/light chain variable region sequence, Sequence of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

In one embodiment, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a first polypeptide chain set forth in SEQ ID NO: 1, a second polypeptide chain set forth in SEQ ID NO: 8, And the third polypeptide chain of SEQ ID NO: 14 or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, Sequence of 99% or higher identical).

In yet another embodiment, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain of SEQ ID NO: 1 and the second polypeptide chain of SEQ ID NO: And the third polypeptide chain of SEQ ID NO: 22, or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%) , 99% or higher of the same sequence.

Ii) In one embodiment, the tri-chain antibody of the invention is an anti-4-1BB/PD-L1 bispecific antibody capable of being at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and More preferably, an affinity constant of about 10 ⁹ M ^-1 or more binds to 4-1BB on the surface of the T cell, activates a costimulatory signaling pathway of the 4-1BB/4-1BB ligand, induces activation and proliferation of T cells, and Anti-apoptosis; and binds to PD-L1 on the surface of tumor cells with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably about 10 ⁹ M ^-1 or more, thereby It inhibits the binding of PD-1 on T cells to PD-L1 on the surface of tumor cells, induces T cell activation and exerts anti-tumor effects.

In one embodiment, the anti-4-1BB/PD-L1 bispecific antibody of the invention comprises a first VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds 4-1BB An antigen binding site, and a first VHH and a second VHH on the third polypeptide chain that specifically bind to PD-L1.

In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a BMS-derived from an anti-4-1BB antibody. All 6 heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 26/28 of 663513, or One or more of the CDRs of the CDRs have one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions). In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a BMS-derived from an anti-4-1BB antibody. The paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 26/28 of 663513, or at least 90% with the paired heavy chain variable region sequence/light chain variable region sequence, Sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

In one embodiment, the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO: 25, and the second polypeptide set forth in SEQ ID NO: a strand, and a third polypeptide chain set forth in SEQ ID NO: 14, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98) Sequence of %, 99% or higher identical).

In yet another embodiment, the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO: 25, and the second more represented by SEQ ID NO: a peptide chain, and a third polypeptide chain set forth in SEQ ID NO: 22, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or higher identical).

Iii) In one embodiment, the tri-chain antibody of the invention is an anti-LAG-3/PD-L1 bispecific antibody capable of being at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and More preferably, the affinity constant of about 10 ⁹ M ^-1 or more binds to LAG-3 on the surface of T cells, inhibits the LAG-3 inhibitory signaling pathway in T cells, thereby restoring CD8+ effector T cells and reducing a Treg population; and binds to PD-L1 on the surface of tumor cells with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably about 10 ⁹ M ^-1 or more, thereby inhibiting The binding of PD-1 on T cells to PD-L1 on the surface of tumor cells induces T cell activation and exerts an anti-tumor effect.

In one embodiment, an anti-LAG-3/PD-L1 bispecific antibody of the invention comprises a first VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to LAG-3 An antigen binding site, and a first VHH and a second VHH on the third polypeptide chain that specifically bind to PD-L1.

In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from an anti-LAG-3 antibody ADI- VH CDR2, VIV CDR3, QVRQDISNYLN (SEQ ID NO: 33), VH CDR2, QVRQDANYLN (SEQ ID NO: 33), GSI CDR1, SIVYSGYTYYNPSLKS (SEQ ID NO: 32), SEQ ID NO: 31 36) VL CDR2 represented by VL CDR1, DASNLET (SEQ ID NO: 37) and VL CDR3 represented by QQVLELPPWT (SEQ ID NO: 38), or one or more CDRs of the 6 CDRs A sequence having one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions). In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from an anti-LAG-3 antibody ADI- The paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 30/35 of 31853, or at least 90% with the paired heavy chain variable region sequence/light chain variable region sequence, Sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO:29, and the second polypeptide set forth in SEQ ID NO:34 a strand, and a third polypeptide chain set forth in SEQ ID NO: 14, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98) Sequence of %, 99% or higher identical).

In yet another embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO:29, and the second more represented by SEQ ID NO:34 a peptide chain, and a third polypeptide chain set forth in SEQ ID NO: 22, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or higher of the same).

In a second aspect, the invention provides a polynucleotide encoding a first polypeptide chain, a second polypeptide chain and/or a third polypeptide chain in a three chain antibody of the invention.

In a third aspect, the invention provides a vector, preferably an expression vector, comprising a polynucleotide encoding a first polypeptide chain, a second polypeptide chain and/or a third polypeptide chain of a triplex antibody of the invention.

In a fourth aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. For example, the host cell is a mammalian cell, preferably a CHO cell, a HEK293 cell; the host cell is a prokaryotic cell, preferably an E. coli cell.

In a fifth aspect, the invention provides a method for producing a tri-chain antibody of the invention, the method comprising the steps of (i) cultivating a host cell of the invention under conditions suitable for expression of the tri-chain antibody, and (ii) The tri-chain antibody is recovered from the host cell or the culture medium.

In a sixth aspect, the invention provides an immunoconjugate comprising a triplex antibody of the invention and a pharmaceutical composition comprising the triplex antibody or immunoconjugate thereof. The three-chain antibodies disclosed herein can be used alone or in combination with other drugs or other therapeutic modalities for the treatment, prevention, and/or diagnosis of diseases such as autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases (eg, chronic infectious diseases or Septicemia, tumors, etc.

In a seventh aspect, the present invention provides the use of a tri-chain antibody, immunoconjugate, and pharmaceutical composition of the present invention, as a drug for treating and/or preventing a disease in an individual, or as a diagnostic tool for a disease, or Hematopoietic stem cell implantation is increased in subjects in need. Preferably, the individual is a mammal, more preferably a human. In one embodiment, the disease is an autoimmune disease, an acute and chronic inflammatory disease, an infectious disease (eg, a chronic infectious disease or sepsis), a tumor.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention, which are described in detail below, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the presently preferred embodiments are shown. However, it is understood that the invention is not limited to the precise arrangements and means of the embodiments shown.

Figures 1A-1C illustrate the structure of a triple-chain antibody of the invention. 1A illustrates a triplex antibody of the present invention, wherein a first polypeptide chain is paired with a second polypeptide chain to form a first antigen binding site, and a third polypeptide chain comprises a single domain second antigen binding site and A single domain third antigen binding site and a flexible linker peptide between the single domain second antigen binding site of the third polypeptide chain and the single domain third antigen binding site. 1B illustrates another triple-chain antibody of the present invention, wherein a first polypeptide chain is paired with a second polypeptide chain to form a first antigen binding site, and a third polypeptide chain comprises a single domain second antigen binding site. And a single domain third antigen binding site, and no flexible linker peptide between the single domain second antigen binding site and the single domain third antigen binding site of the third polypeptide chain. 1C illustrates the structure of each peptide chain of the tri-chain antibody of the present invention from the N-terminus to the C-terminus, wherein the flexible linker peptide in the third polypeptide chain indicated by the dotted arrow is as needed (for example, according to an epitope bound to specificity) The affinity and steric hindrance may or may not be present in the tri-chain antibody of the present invention.

2A and 2B show the purity of two anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL prepared by the present invention by size exclusion chromatography (SEC), respectively. .

Figure 3 shows the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention obtained by a kinetic binding assay, and the anti-PD-L1 humanized Nb-Fc antibody as a control, kinetics of the anti-CD47 antibody ADI 29341 binding curve, and k _on, k _dis, and K _D data.

Figure 4A shows anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL detected by FACS, and anti-PD-L1 humanized Nb-Fc antibody as a control and overexpressing PD-L1 Binding of CHO-S cells. Figure 4B shows anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL detected by FACS, and anti-CD47 antibody ADI 29341 as a positive control, IgG1 antibody as a negative control and overexpression of CD47 Binding of CHO-S cells. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the mean fluorescence intensity (MFI).

Figure 5 shows the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL as determined by PEG precipitation, and the antibody Humira (also known as adalimumab, as a positive control). It is a physical stability and solubility of a humanized monoclonal antibody against TNFα.

Figure 6 shows the long-term thermostability of the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL. After the antibody was allowed to stand at 40 ° C for 0, 1, 3, 7, 10, 20, and 30 days, the antibody purity was determined by SEC-HPLC.

Figure 7A shows the binding of anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL to CHO-S cells overexpressing PD-L1, respectively, on day 0 and at 40 °C for 30 days. Figure 7B shows the binding of anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL to CHO-S cells overexpressing CD47, respectively, on day 0 and at 40 °C for 30 days. The horizontal axis represents the antibody concentration and the vertical axis represents the mean fluorescence intensity (MFI).

Figure 8 shows the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention detected by the MOA method, and the anti-PD-L1 humanized Nb-Fc antibody as a positive control, as a negative Effect of control IgG1 antibody on PD-1/PD-L1 signaling pathway.

Figure 9 shows the ability of the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL, and the anti-CD47 antibody ADI 29341 as a positive control to promote macrophage phagocytosis of tumor cells.

Figure 10 shows the effect of anti-CD47/PD-L1 bispecific antibody Kh2NF-PC, anti-CD47 antibody Hu5F9 (corresponding to "5F9" antibody in US2015/0183874 A1) and IgG1 control antibody on erythrocyte agglutination.

Figure 11 shows the ratio of anti-CD47/PD-L1 bispecific antibody Kh2NF-PC and Kh2NF-PC-NL selectively bound to the surface of tumor cells when tumor cells and human erythrocytes were co-incubated.

Figure 12 shows the purity of the anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 prepared by the present invention detected by SEC.

Figure 13A shows the binding of the anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 detected by FACS, and the anti-4-1BB antibody BMS-663513 as a positive control to CHO-S cells overexpressing 4-1BB. . Figure 13B shows anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 detected by FACS, and anti-PD-L1 humanized Nb-Fc antibody as a positive control and CHO-S overexpressing PD-L1 Cell binding. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the mean fluorescence intensity (MFI).

Figure 14 shows the purity of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL prepared by the present invention detected by SEC.

Figure 15A shows anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL detected by FACS, and anti-PD-L1 humanized Nb-Fc antibody as a positive control and CHO-S overexpressing PD-L1 Cell binding. Figure 15B shows the binding of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL detected by FACS, and the anti-LAG-3 antibody ADI-31853 as a positive control to HEK293 cells overexpressing LAG-3. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the mean fluorescence intensity (MFI).

Figure 16 shows anti-LAG-3 antibody ADI-31853, anti-PD-L1 humanized Nb-Fc antibody, anti-LAG-3 antibody ADI-31853+ anti-PD-L1 humanized Nb-Fc antibody, IgG4 control antibody To compare the effect of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL on the activation of T cells in vitro.

Figure 17 shows anti-PD-L1 humanized Nb-Fc antibody, anti-CD47 antibody ADI 29341, anti-PD-L1 humanized Nb-Fc antibody and anti-CD47 antibody ADI 29341 in combination with h-IgG, anti-drug Tumor Suppressive Activity of CD47/PD-L1 Bispecific Antibody Kh2NF-PC in a Raji-PD-L1/NOD-SCID Mouse Model.

Detailed description of the invention

I. Definition

The term "about" when used in connection with a numerical value is meant to encompass a numerical value within the range of the lower limit of 5% less than the specified numerical value and the upper limit of 5% greater than the specified numerical value.

The term "comprising" or "including", as used herein, is intended to include the recited elements, integers or steps, but does not exclude any other elements, integers or steps.

The term "antibody" is used herein in its broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), single chain antibodies, triple chain antibodies, intact antibodies, and antibody fragments.

The terms "full antibody", "full length antibody", "complete antibody" and "intact antibody" are used interchangeably herein to refer to the inclusion of at least two heavy chains (H) and two inter-connected by disulfide bonds. Light chain (L) glycoprotein. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (which are complementarity determining regions (CDRs) with more conserved regions interposed (framework regions (FR)). Each VH and VL consists of three CDRs and four The FR composition is arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions.

A "complementarity determining region" or "CDR region" or "CDR" is a sequence that is hypervariable in an antibody variable domain and that forms a structurally defined loop ("hypervariable loop") and/or contains an antigen contact residue ( The area of the "antigen contact point"). The CDR is primarily responsible for binding to the epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as VH CDR1, VH CDR2 and VH CDR3, while the CDRs located within the antibody light chain variable domain are referred to as VL CDR1, VL CDR2 and VL CDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr/ on the World Wide Web), and based on affinity propagation clustering using a large number of crystal structures North CDR definition.

However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, where an antibody is defined by a particular CDR sequence as defined by the present invention, the scope of the antibody also encompasses an antibody whose variable region sequence comprises the particular CDR sequence, but due to the application of a different protocol (eg Different assignment system rules or combinations result in different claimed CDR boundaries than the specific CDR boundaries defined by the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although the CDRs differ between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimal overlap region can be determined to provide a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As will be apparent to those skilled in the art, residues of the remainder of the CDR sequences can be determined by the structure of the antibody and protein folding. Accordingly, the invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.

The term "antigen-binding fragment" is a portion or portion of an intact or complete antibody that is less than the number of amino acid residues of an intact or fully antibody, which is capable of binding to an antigen or competing with an intact antibody (ie, an intact antibody derived from an antigen-binding fragment). Binding antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, single chain Fv, diabody, single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, for example, a Fab fragment can be obtained by digestion of a complete antibody by papain. In addition, F(ab') ₂ , which is a dimer of Fab', is a divalent antibody fragment by digesting a complete antibody under the disulfide bond of the hinge region by pepsin. F(ab') ₂ can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, thus converting the F(ab') ₂ dimer to a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of one arm of the antibody. In addition, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined by a synthetic linker capable of causing the two domains to be produced as a single protein chain using recombinant methods, The VL region and the VH region in a single protein chain are paired to form a single chain Fv. The antibody fragment can be obtained by chemical methods, recombinant DNA methods or protease digestion.

The term "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (eg, a heavy chain variable domain (VH) or a light chain can be The variable domain (VL), the heavy chain variable domain derived from the camelid heavy chain antibody, and the VH-like single domain (v-NAR) derived from the fish IgNAR confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include single domain antibodies derived from camelids (llamas and camels) and cartilage fish (eg, nurse sharks) (WO 2005/035572).

The term "camelized human VH domain" means that the transfer of a key element derived from Camelidae VHH to a human VH domain results in the human VH domain no longer needing to be paired with the VL domain to recognize the target antigen, which is camelized. The human VH domain alone confers antigen binding specificity.

The term "binding site" or "antigen binding site" as used herein denotes a region of an antibody molecule that actually binds to an antigen, including by an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). a VH/VL pair consisting of a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) from a shark family IgNAR, a camelized human VH domain, a human A derived camelid antibody heavy chain variable domain. In an embodiment of the invention, the tri-chain antibody of the invention comprises at least three antigen binding sites, for example, comprising a first antigen binding site (also referred to as a "first antigen binding site"), a second An antigen binding site (also referred to as a "second antigen binding site"), a third antigen binding site (also referred to as a "third antigen binding site").

The term "single domain antigen binding site" denotes a single variable domain of an antibody molecule (eg, a heavy chain variable domain (VH), a light chain variable domain (VL), derived from a camelid heavy chain antibody Heavy chain variable domain, v-NAR from IgNAR of sharks, camelized human VH domain, humanized camelid antibody heavy chain variable domain, and their recombined single domain ) the area that actually binds to the antigen. In one embodiment of the invention, the triplex antibody of the invention comprises two single domain antigen binding sites, referred to as "single domain second antigen binding site" and "single domain third antigen binding site, respectively" point".

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.

As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of the at least two antigen binding sites being different or different from the same epitope of the same antigen. Different epitopes of the antigen bind. The antibodies provided herein are typically multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In one embodiment, provided herein is a bispecific antibody having binding specificity for a first antigen and a second antigen.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an IgG-like immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3) ). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also referred to as a light chain variable domain, followed by a light chain constant domain (CL). The heavy chain of immunoglobulin can belong to one of five categories, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into sub- Classes such as γ ₁ (IgG1), γ ₂ (IgG2), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). The light chain of an immunoglobulin can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain. Immunoglobulins consist essentially of two Fab molecules and one Fc domain joined by an immunoglobulin hinge region.

The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises second and third constant domains (CH2 domain and CH3 domain) derived from two heavy chains of IgG, IgA and IgD class antibodies; or a source comprising The second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of the two heavy chains of the IgM and IgE class antibodies. Unless otherwise stated herein, the amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system (also known as the EU index) described in 1991 is numbered.

The term "effector function" refers to those biological activities attributed to the immunoglobulin Fc region that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) Cytokine secretion, immune complex-mediated uptake by antigen-presenting cells, down-regulation of cell surface receptors (eg, B cell receptors), and B cell activation.

The term "chimeric antibody" is an antibody molecule in which (a) changes, replaces or exchanges a constant region or a portion thereof, such that the antigen binding site is different from a different or altered class, effector function and/or species. a region or a completely different molecule (eg, an enzyme, a toxin, a hormone, a growth factor, a drug) that confers new properties to a chimeric antibody; or (b) a variable region or a portion thereof with a different or altered antigen specificity The variable region is changed, replaced or exchanged. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulin. Due to the replacement into the human constant region, the chimeric antibody retains its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original mouse antibody.

A "humanized antibody" is an antibody which retains antigen-specific reactivity of a non-human antibody (for example, a mouse monoclonal antibody) and which is less immunogenic when administered to a human as a therapeutic drug. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterpart (ie, the constant region and the portion of the variable region that is not involved in binding is the corresponding portion of the human antibody). See, for example, Padlan, Anatomy of the antibody molecule, Mol. Immun., 1994, 31: 169-217. Other examples of human antibody engineering techniques include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

The term "...valent" antibody refers to the number of antigen binding sites present in an antibody molecule. "Bivalent", "trivalent" and "tetravalent" antibodies refer to the presence of two antigen binding sites, three binding sites and four binding sites, respectively, in the antibody molecule. In one embodiment, the bispecific antibodies reported herein are "trivalent."

The term "flexible linker peptide" or "linker peptide" refers to a linker peptide consisting of amino acids, such as glycine and/or serine residues, used alone or in combination, to link various variable domains in an antibody. In one embodiment, the flexible linker peptide is a Gly/Ser linker peptide comprising an amino acid sequence (Gly ₄ Ser)n, wherein n is a positive integer equal to or greater than 1, eg, n is a positive integer from 1-7. In one embodiment, the flexible linker peptide is (Gly ₄ Ser) 4 (SEQ ID NO: 20). Also included within the scope of the invention is the linker peptide described in WO2012/138475, which is incorporated herein by reference.

As used herein, the term "binding" or "specifically binds" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. Was affinity molecule X for its partner Y can generally dissociation constant (K _D) is represented by the solution, the dissociation constant is the ratio of the dissociation rate constant and association rate constant (k _dis, respectively and k _on) of. Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.

The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. In some embodiments herein, the first antigen, the second antigen, and the third antigen are three different antigens.

The term "tumor-associated antigen" or "cancer antigen" refers interchangeably to a molecule (usually a protein, carbohydrate or lipid) that is expressed completely or as a fragment (eg, MHC/peptide) on the surface of a cancer cell, as compared to normal cells. And the molecule can be used in the preferential targeting of the agent to cancer cells. In some embodiments, the tumor associated antigen is a cell surface molecule that is overexpressed in tumor cells as compared to normal cells, eg, 1 fold overexpression, 2 fold overexpression, 3 fold overexpression or more than normal cells Overexpression. In some embodiments, the tumor associated antigen is a cell surface molecule that is improperly synthesized in tumor cells, such as a molecule that contains a deletion, addition, or mutation compared to a molecule expressed on a normal cell. In some embodiments, the tumor associated antigen is only expressed intact or as a fragment on the cell surface of the tumor cell and is not synthesized or expressed on the surface of normal cells. A number of tumor-associated antigens are disclosed in the prior art, for example, epidermal growth factor receptor variant III (EGFRvIII), tumor associated glycoprotein 72 (TAG72), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM). , interleukin 11 receptor alpha (IL-11Ra), vascular endothelial growth factor receptor 2 (VEGFR2), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), insulin-like growth factor 1 receptor (IGF) -I receptor), melanoma-associated antigen 1 (MAGE-A1), CD72, CD47, and the like.

The term "immunization checkpoint" means a class of inhibitory signaling molecules present in the immune system that protects against tissue damage by modulating the persistence and strength of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). The study found that one of the reasons that tumor cells can escape the immune system in the body and lose control of proliferation is to use the inhibitory signaling pathway of the immune checkpoint, thereby inhibiting the activity of T lymphocytes, so that T lymphocytes can not effectively exert the killing effect on tumors ( Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146). Immunological checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), and LAG-3, which directly inhibit immune cells. Immunological checkpoint molecules, such as PD-L1 and LAG-3, can modulate (e.g., synergistically modulate) T cell function to promote tumor immune evasion.

The term "costimulatory molecule" refers to a corresponding binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response to T cells, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to antigen receptors or their ligands. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, 4-1BB (ie CD137), CD27 and CD28. In some embodiments, the "costimulatory molecule" is 4-1BB (ie, CD137), CD27, and/or CD28.

The term "cytokine" is a generic term for a protein that is released by one cell population and acts as an intercellular medium on another cell. Examples of such cytokines are lymphokines, mononuclear factors, interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and Γ-interferon. As used herein, the term cytokine includes biologically active equivalents of proteins and natural sequence cytokines from natural sources or from recombinant cell cultures, including small molecule entities produced by artificial synthesis, and their pharmaceutically acceptable Derivatives and salts.

An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ^{212 ,} and Lu); Or drugs (eg, methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, phenylbutyric acid Nitrogen mustard, Zoorubicin or other intercalating agents; growth inhibitors; enzymes and fragments thereof such as lysozyme; antibiotics; small toxins or enzymatically active toxins such as toxins such as bacterial, fungal, plant or animal sources, Included are fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

The "percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in the present specification and, if necessary, the vacancy is introduced to achieve the maximum percent sequence identity, and no consideration is given. The percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the particular amino acid sequence shown in this specification when the conservative substitution is part of the sequence identity.

For polypeptide sequences, "conservative modifications" include substitutions, deletions or additions to polypeptide sequences which result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are additive relative to the polymorphic variants, interspecies homologs and alleles of the invention and do not exclude them. The following 8 groups contain amino acids that are conservatively substituted: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), guanidine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising an amino acid sequence.

The term "N-terminus" refers to the last amino acid at the N-terminus, and the term "C-terminus" refers to the last amino acid at the C-terminus.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. A host cell is any type of cellular system that can be used to produce a three-chain antibody of the invention. Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells within animal tissues.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids incorporated into recombinant polynucleotides, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, glands) Virus and adeno-associated virus).

The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse). In particular, the individual is a human.

The term "treatment" refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis. In some embodiments, the triple chain antibodies of the invention are used to delay the progression of the disease or to slow the progression of the disease.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer. , hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, Malignant freckle-like melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as with phagomatoses, edema (such as those associated with brain tumors), and Meigs (Meigs) Significant vascular proliferation, brain tumors and brain Cancer, as well as head and neck cancer, and related metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, including those metastatic forms of those cancers.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

The term "infectious disease" refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a parasitic infection, or a fungal infection.

II. Three-chain antibody of the present invention

The present invention provides a novel tri-chain antibody which can be used for immunotherapy, prevention and/or diagnosis of various diseases. The tri-chain antibody of the present invention comprises at least three antigen-binding sites capable of functioning as a monospecific antibody or a multispecific (e.g., bispecific) antibody, preferably, which is capable of multispecificity (e.g., bispecificity) ) Antibodies work.

In the production of monospecific or multispecific (e.g., bispecific) antibodies having multiple polypeptide chains, problems such as undesired interchain mismatch, decreased antibody affinity, and reduced stability typically occur. The triple-chain antibodies constructed in this application are capable of avoiding these common problems.

The triplex antibody platform constructed by the present application is a three-chain antibody comprising three polypeptide chains, wherein the first polypeptide chain comprises a first heavy chain variable domain and the second polypeptide chain comprises a first light chain variable domain, The first heavy chain variable domain is paired with a first light chain variable domain (VH1/VL1 pair) to form a first antigen binding site; and the third polypeptide chain comprises a single domain second antigen binding site And a single domain third antigen binding site.

In one embodiment, the single domain second antigen binding site and the single domain third antigen binding site in the third polypeptide chain of the triplex antibody of the invention have no linker peptide.

In another embodiment, a single domain second antigen binding site and a single domain third antigen binding site in a third polypeptide chain of a triplex antibody of the invention has a linker peptide. The type of the linker peptide is not particularly limited. In an embodiment, the linker peptide is a peptide having an amino acid sequence of from 1 to 100, in particular from 1 to 50, more particularly from 1 to 20 amino acids in length. In some embodiments, the peptide linker peptide is (GxS)n or (GxS)nGm, wherein G=glycine, S=serine and any integer of x=1-4, any of n=1-7 An integer, and any integer of m=0-3. In a specific embodiment, the linker peptide is (G ₄ S) ₄ (SEQ ID: NO: 20).

A single domain antigen binding site in a third polypeptide chain of a triplex antibody of the invention is a single variable domain capable of specifically binding a target antigen epitope with higher affinity, eg, a heavy chain variable domain (VH) a light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, a v-NAR from a shark family IgNAR, a camelized human VH domain, humanized Camelidae antibody heavy chain variable domains, and their recombined single domains. In one embodiment, the two single domain antigen binding sites in the third polypeptide chain of a triplex antibody of the invention are heavy chain variable domains derived from camelid heavy chain antibodies, camelized human VH structures Domain and/or humanized camelid antibody heavy chain variable domain.

The size, structure and antigenicity of human antibody proteins obtained from camelid species such as camelids, alpaca, dromedary, llama and guanaco have been characterized in the prior art. Certain IgG antibodies from the camelid family of mammals in nature lack light chains and are therefore structurally distinguished from common four-chain antibody structures with two heavy chains and two light chains from other animals. See PCT/EP 93/02214 (WO 94/04678, published March 3, 1994).

A heavy chain variable domain of a camelid heavy chain antibody having high affinity for a target antigen (this region is also referred to as VHH) can be obtained by a genetic engineering method. See U.S. Patent No. 5,759,808, issued June 2, 1998. Like other non-human antibody fragments, the amino acid sequence of Camelidae VHH can be recombinantly altered to obtain sequences that more realistically mimic human sequences, ie, "humanized," thereby reducing the antigenicity of Camelidae VHH to humans. Alternatively, key elements derived from Camelidae VHH can be transferred to the human VH domain to obtain a camelized human VH domain. In one embodiment of the invention, the single domain antigen binding site in the third polypeptide chain of a triplex antibody of the invention is a humanized VHH having the amino acid sequence set forth in SEQ ID NO:16.

The molecular weight of VHH is one tenth of the molecular weight of a human IgG molecule and has a physical diameter of only a few nanometers. VHH itself has extremely high thermal stability, is stable to extreme pH and proteolytic digestion, and has low antigenicity. Therefore, the structure contributes to the stability of the tri-chain antibody of the present invention and to the low antigenicity of human subjects. .

In one embodiment, the first polypeptide chain of a triplex antibody of the invention comprises a first heavy chain variable domain and an immunoglobulin CH1 domain, the second polypeptide chain comprises a first light chain variable domain and immunized a globin CL domain; and the third polypeptide chain comprises a single domain second antigen binding site and a single domain third antigen binding site. The classes, subclasses, forms and subtypes of immunoglobulins from which the CH1 domain and the CL domain are derived are not particularly limited. Preferably, the CH1 domain, the CL domain are all derived from or have substantially the same portion of a human immunoglobulin (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or more of the same).

In one embodiment, the tri-chain antibody of the invention further comprises a component having an extended half-life in vivo. Many factors may affect the in vivo half-life of the protein. For example, renal filtration, metabolism in the liver, degradation by proteolytic enzymes (protease), and immunogenic reactions (eg, protein neutralization of antibodies and uptake by macrophages and dendritic cells). A variety of strategies are available to extend the half-life of the triple-chain antibodies of the invention. For example, by chemical connection of polyethylene glycol (PEG), polysialic acid (PSA), hydroxyethyl starch (HES), conjugated albumin, immunoglobulin Fc, and the like.

In order to PEGylate an antibody, conditions are generally employed in which the tri-chain antibody is linked to an active ester or aldehyde derivative of polyethylene glycol (PEG), such as PEG, in which one or more PEG groups become attached to the triple-chain antibody. The next reaction. The PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" includes any form of PEG that has been used to derivatize other proteins, such as mono (C ₁ -C ₁₀ ) alkoxy or aryloxy-polyethylene glycol or polyethylene. Alcohol-maleimide. In certain embodiments, the antibody to be PEGylated is an aglycosylated antibody. Linear or branched PEG derivatization resulting in minimal loss of biological activity of the antibody is used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion chromatography or by ion exchange chromatography. The binding activity of PEG-derived antibodies can be tested using methods well known to those skilled in the art. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, Nishimura et al., EP 0 154 316.

Polysialylation is another technique that uses natural polymer polysialic acid (PSA) to extend the useful life of therapeutic peptides and proteins and improve their stability. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment to the conjugate. This increases the useful life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system.

Hydroxyethyl starch ("HES") of the antibody also extends the circulating half-life of the antibody, resulting in increased biological activity. A wide variety of HES immunoconjugates can be prepared by varying different parameters, such as the molecular weight of HES.

An IgG constant domain or an immunoglobulin Fc domain can also be introduced into a triple chain antibody of the invention to produce an antibody having an increased in vivo half-life. See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375.

Further, the tri-chain antibody of the present invention can be conjugated to albumin (for example, human serum albumin; HSA) to make the antibody more stable in vivo or have a longer in vivo half-life. These techniques are well known in the art, see, for example, International Publication No. WO 01/77137; and European Patent No. EP 413,622.

In one embodiment, a triplex antibody of the invention comprises an Fc region to extend the in vivo half-life of an antibody of the invention.

In yet another specific embodiment, the hinge region having a "CPPC" amino acid residue is contained in the Fc domain of the first polypeptide chain and the third polypeptide chain of the triplex antibody of the invention, respectively, and/or comprises Y349C, respectively. And S354C (according to Kabat's "EU numbering"), whereby the first polypeptide chain and the third polypeptide chain form an interchain disulfide bond in the Fc region, which also contributes to the first multi-chain antibody of the present invention. Correct pairing of the peptide chain and the third polypeptide chain.

In one embodiment, the triplex antibodies of the invention employ a "binding" technique (see, for example, John BBRidgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996.9 ( 7): p. 617-21; Shane Atwell et al, Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol, 1997. 270: p. 26-35), the technique can be used in The interface between the different strands of the inventive tri-chain antibody is engineered to facilitate proper association of the individual strands of the triple-chain antibody of the invention. Typically, this technique involves introducing a "bump" at the interface of one strand, introducing a corresponding "hole" at the interface of the other strand to be paired with, such that the projection can be placed in the void. The first preferred interface comprises the CH3 domain of the heavy chain constant domain of one strand and the CH3 domain of the heavy chain constant domain of the other strand to be paired with. The bulges can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one strand with a larger side chain (eg, tyrosine or tryptophan). By replacing a large amino acid side chain with a smaller side chain (eg, alanine or threonine), the interface of the CH3 domain of the heavy chain constant domain of another strand to be paired is identical or similar to the bulge Compensatory holes of size. A second preferred interface comprises the CL domain of the light chain and the CH1 domain of the heavy chain, where a bulge-hole interaction can be constructed as described above.

In one embodiment, the Fc region of a triplex antibody of the invention comprises a modification to the binding affinity of an Fc receptor. In one embodiment, the Fc receptor is an Fc gamma receptor, in particular a human Fc gamma receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In one embodiment, the modification reduces the effector function of the triplex antibody of the invention. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the modification is in the Fc region of the immunoglobulin molecule, particularly in its CH2 region. In one embodiment, the immunoglobulin molecule comprises an amino acid substitution at position 329 (EU numbering) of the immunoglobulin heavy chain. In a specific embodiment, the amino acid substitution is P329G. In one embodiment, a triplex antibody of the invention comprises an amino acid substitution at positions 234 and 235 (EU numbering) of the immunoglobulin heavy chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA mutations) (Armour KL et al, Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol, 1999. 29(8): 2613 twenty four). In one embodiment, a triplex antibody of the invention comprises an amino acid substitution at positions 234, 235 and 329 of the immunoglobulin heavy chain (EU numbering). In a specific embodiment, the immunoglobulin molecule comprises amino acid substitutions L234A, L235A and P329G (EU numbering) in the immunoglobulin heavy chain.

As shown in the schematic diagram in Figure 1A, an exemplary triple-chain antibody of the invention is a trivalent triple-chain antibody, wherein the first heavy chain variable domain in the first polypeptide chain and the first in the second polypeptide chain The light chain variable domain pair forms a first antigen binding site; the third polypeptide chain comprises a single domain second antigen binding site and a single domain third antigen binding site, and in the single domain second There is a linker peptide between the antigen binding site and the single domain third antigen binding site.

As shown in the schematic diagram in Figure IB, an exemplary triple-chain antibody of the invention is a trivalent triple-chain antibody, wherein the first heavy chain variable domain in the first polypeptide chain and the first in the second polypeptide chain The light chain variable domain pair forms a first antigen binding site; the third polypeptide chain comprises a single domain second antigen binding site and a single domain third antigen binding site, and in the single domain second There is no linker peptide between the antigen binding site and the single domain third antigen binding site.

i) In one embodiment, the tri-chain antibody is an anti-CD47/PD-L1 bispecific antibody or a multispecific antibody.

PD-L1 (also known as differentiation antigen cluster 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40 kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1 present on activated T cells, downregulating T cell activation (Latchman et al, 2001 Nat Immunol 2: 261-8; Carter et al, 2002 Eur J Immunol 32: 634-43) . PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer, colon cancer, and various myeloma, and PD-L1 expression is often associated with poor prognosis of cancer (Iwai et al. (2002) PNAS 99: 12293-7; Ohigashi et al. (2005) Clin Cancer Res 11: 2947-53; Okazaki et al. (2007) Intern. Immun. 19: 813-24; Thompson et al. (2006) Cancer Res. 66: 3381-5) . It has been suggested that immunosuppression can be reversed in a subset of tumor patients by inhibiting the local interaction of PD1 with PD-L1.

Roche's anti-PD-L1 antibody Atezolizumab, Germany's Merck KGaA and Pfizer's anti-PD-L1 antibody Avelumab, AstraZeneca's Durvalumab showed treatment for some cancer patients effect. Other anti-PD-L1 antibodies include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 of WO2010/077634) and anti-PD-L1 antibodies disclosed in WO2007/005874, and the like.

CD47 was first identified as an integrin-associated protein (IAP) (Brown EJ et al, Integrin-associated protein (CD47) and its ligands, Trends Cell Biol., 2001, 11(3): 130-135 ) is a tumor-associated antigen expressed on the surface of cells. CD47 interacts with a cell surface immunoglobulin SIRPα, which is expressed primarily by macrophages and dendritic cells, as a ligand, resulting in a cascade of cascades that inhibit macrophage and dendritic cell expression. Uptake and phagocytosis of cells of CD47.

CD47 expression is prevalent in normal tissues, for example, CD47 is expressed on the surface of viable red blood cells. A part of the function of CD47 is to protect viable red blood cells from phagocytosis (Oldenborg P.A. et al., Role of CD47as a marker of self on red blood cells, Science, 2000, 288 (5473): 2051-2054). In addition, overexpression of CD47 has been observed in tumors. The expressed CD47 binds to SIRPα on the surface of macrophages, releasing a signal of “don't eat me”, thereby inhibiting the phagocytosis of tumor cells by macrophages in the infiltrated area of the tumor tissue. Subsequently, the uptake of tumor cells by antigen-presenting cells (APC) is reduced, indirectly leading to a decrease in T cell activation; in addition, CD47 activation on naive T cells promotes the formation of immunosuppressive regulatory T-cell Tregs and inhibits Th1 effector cells. Formation (Avice MN et al, CD 47ligation selective inhibits the development of human naive T cells into Th1 effectors, J. Immunol., October 2000, 165(8): 4624-4631).

A number of anti-CD47 antibodies have been reported so far, for example, US Patent No. 2015/0183874 A1 reports a human IgGl type chimeric monoclonal antibody derived from B6H12 and a humanized B6H12 antibody produced by CDR grafting. Chinese patent application number CN201710759828.9 reports a monoclonal antibody "ADI-29341" which can specifically bind to CD47.

The triple-stranded anti-CD47/PD-L1 bispecific antibody or multispecific antibody of the present invention targets at least the antibodies of CD47 and PD-L1 at the same time, and the three antigen-binding sites bind to the CD47 and/or PD-L1 molecules, respectively. As described above, although CD47 is overexpressed on cancer cells, expression of CD47 in many normal tissues results in non-specific binding of antibodies targeting only CD47 to normal blood system cells, causing antigen sinking. phenomenon. The anti-CD47/PD-L1 bispecific antibody of the present invention simultaneously targets PD47 and PD-L1 on tumor cells, and promotes the anti-CD47/PD-L1 double of the present invention by specifically binding to PD-L1 on tumor cells. The selective binding of specific antibodies to tumor cells avoids binding to CD47 expressed in many normal tissues, whereby the triple-stranded anti-CD47/PD-L1 bispecific antibody of the present invention reduces side effects while enhancing phagocytosis.

In one embodiment, a triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds PD-L1 or CD47 a first antigen binding site and a single domain second antigen binding site on the third polypeptide chain that specifically binds to PD-L1 or CD47 and a single domain third antigen binding site that specifically binds PD-L1 or CD47 point. In one embodiment, the triplex antibody comprises a first antigen binding site comprising a VH1/VL1 pair that specifically binds to CD47 on a first polypeptide chain and a second polypeptide chain, and a third polypeptide chain The specificity binds to the first VHH and the second VHH of PD-L1. In another embodiment, the tri-chain antibody comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 on the first polypeptide chain and the second polypeptide chain, and a third The first VHH and the second VHH that specifically bind to CD47 on the peptide chain.

For the first antigen binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 or CD47, the "VH1/VL1 pair" comprises an anti-PD-L1 antibody derived from any of the prior art reports (eg, The anti-PD-L1 antibody exemplified herein and the 6 CDRs of the anti-PD-L1 antibody VH1/VL1 pair developed in the future or one, two or three of the CDRs of the 6 CDRs a sequence of four, or five amino acid changes (eg, amino acid substitutions or deletions); or comprising an anti-CD47 antibody (eg, an anti-CD47 antibody exemplified above) derived from any of the prior art and developed in the future The six CDRs of the VH1/VL1 pair of anti-CD47 antibodies or one, two, three, four, or five amino acid changes with one or more of the six CDRs (eg, amino acid substitutions or deletions) )the sequence of.

For the single domain antigen binding site that specifically binds to PD-L1 or CD47, which comprises a heavy chain variable domain (VH), a light chain variable domain (VL) that specifically binds to PD-L1 or CD47 a heavy chain variable domain in camelid antibodies consisting of only two heavy chains, which are naturally free of light chains from camelid serum, a VH-like single domain of IgNAR from sharks, and a camelized human VH structure. Domain, humanized camelid antibody heavy chain variable domain.

In one embodiment, a triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a first antigen chain comprising a VH1/VL1 pair that specifically binds to CD47 on a first polypeptide chain and a second polypeptide chain a single domain and a second antigen binding site on the third polypeptide chain that specifically binds to PD-L1, wherein the single domain and the third antigen binding specifically bind to PD-L1 The site binds to the same epitope on the PD-L1 or to a different epitope.

In one embodiment, the first antigen binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises GSIIEYYWS derived from the anti-CD47 antibody ADI-29341 ( VH CDR2, YHYYTGSTNYNPSLKS (SEQ ID NO: 4) shown in SEQ ID NO: 3), VH CDR3, RASQGISRWLA (SEQ ID NO: 10) shown by ARGKTGSAA (SEQ ID NO: 5) VL CDR2 represented by VL CDR1, AASSLQS (SEQ ID NO: 11) and VL CDR3 represented by QQTVSFPIT (SEQ ID NO: 12), or one or two with one or more of the 6 CDRs Sequence of three, four, or five amino acid changes (eg, amino acid substitutions or deletions).

In one embodiment, the first antigen binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises the SEQ ID derived from the anti-CD47 antibody ADI-29341 NO: 2/9 of the paired heavy chain variable region sequence/light chain variable region sequence, or at least 90%, 91%, 92 with the paired heavy chain variable region sequence/light chain variable region sequence %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequences of sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

The type of the heavy chain constant region of the immunoglobulin in the first polypeptide chain and the third polypeptide chain in the tri-chain antibody of the present invention is not particularly limited, and is preferably a heavy chain constant region of an IgG1, IgG2 or IgG4 immunoglobulin, Or a sequence that is substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical). More preferably, the heavy chain constant region is, or substantially identical to, the heavy chain constant region of a human IgGl immunoglobulin (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or more of the same).

In one embodiment, a triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for use in IgG4 (eg, human IgG4). In yet another embodiment, a triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for IgG1 (eg, human IgG1). For example, a hinge region having a "CPPC" amino acid residue is contained in the Fc domain of the first polypeptide chain and the third polypeptide chain of the triple-chain antibody, respectively, and/or Y349C and S354C are respectively contained ("EU according to Kabat" The number "", whereby the first polypeptide chain and the third polypeptide chain form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain and/or the third polypeptide chain of a triplex antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function. In a specific embodiment, the amino acid substitution is a LALA mutation.

In still another embodiment, the second polypeptide chain of a triplex anti-CD47/PD-L1 bispecific antibody of the invention comprises a kappa light chain constant region or a lambda light chain constant region, eg, a human kappa light chain constant region or a human λ light chain constant region. In one embodiment, the light chain constant region comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 13 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) , 98%, 99% or more of the same sequence.

In one embodiment, the Fc domain of each of the first polypeptide chain and the third polypeptide chain of the triplex anti-CD47/PD-L1 bispecific antibody of the invention comprises a "association" stable association, respectively. In one embodiment, an amino acid substitution T366W is included in one of the first polypeptide chain and the third polypeptide chain, and is included in the other of the first polypeptide chain and the third polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering). Thus the protrusions in one strand can be placed in the cavities in the other strand, facilitating the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain of the triplex anti-CD47/PD-L1 bispecific antibody of the invention and the immunoglobulin CH1 domain and CL domain of the second polypeptide chain respectively comprise a bulge or an empty a hole, and the protrusion or cavity in the CH1 domain can be placed in the hole or protrusion in the CL domain, respectively, such that the first polypeptide chain and the second polypeptide chain also form each other A stable association of “knot-in”.

In one embodiment, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises a first polypeptide chain set forth in SEQ ID NO: 1, a second polypeptide chain set forth in SEQ ID NO: 8, And the third polypeptide chain of SEQ ID NO: 14 or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, Sequence of 99% or higher identical).

In yet another embodiment, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain of SEQ ID NO: 1 and the second polypeptide chain of SEQ ID NO: And the third polypeptide chain of SEQ ID NO: 22, or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%) , 99% or higher of the same sequence.

The triple-stranded anti-CD47/PD-L1 bispecific antibody of the present invention is capable of binding to both PD-L1 and CD47 proteins, and maintains the affinity constant of the parent antibody, thereby blocking the SIRPα/CD47 signaling pathway and blocking PD1/PD-L1 signaling pathway.

In some embodiments, the conditions associated with the SIRPα/CD47 signaling pathway and the PD1/PD-L1 signaling pathway are various hematological and solid tumors including, but not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia , acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), lymphoma, breast cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, ovarian cancer, cervical cancer, kidney Cancer, pancreatic cancer, bladder cancer, glioma, melanoma and other solid tumors. In addition, human stem cell implantation in NOD mouse lines can be enhanced by blocking the SIRPα/CD47 signaling pathway (WO 2009/046541), therefore, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the present invention is also useful. Potential benefits in human stem cell transplantation.

Chinese Patent No. ZL201080064426.3 discloses that a tetravalent CD47-antibody constant region fusion protein that blocks the SIRPα/CD47 signaling pathway is capable of treating, preventing or diagnosing autoimmune diseases and inflammatory conditions mediated by SIRPα+ cells, for example, allergies Asthma or ulcerative colitis. These conditions include acute and chronic inflammatory conditions, allergic and allergic diseases, autoimmune diseases, ischemic conditions, severe infections, and cell or tissue or organ transplant rejection, including non-human tissue grafts (xenografts). Therefore, the triple-stranded anti-CD47/PD-L1 bispecific antibody of the present invention is expected to also function in the treatment, prevention or diagnosis of these diseases.

Ii) In one embodiment, the tri-chain antibody is an anti-4-1BB/PD-L1 bispecific antibody or a multispecific antibody.

PD-L1 is an immunological checkpoint inhibitor molecule. PD-L1 expressed on tumor cells activates PD-1/PD-L1 signaling pathway by binding to PD-1 on T lymphocytes, thereby inhibiting T lymphocyte activity, making T lymphocytes unable to effectively exert tumors. Killing effect, which is one of the reasons why tumor cells can escape the immune system in the body and lose control of proliferation (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12 ( 2): 130-146).

4-1BB (also known as CD137, TNFRSF9) is an activation-inducible co-stimulatory receptor expressed on activated T cells and natural killer (NK) cells and is a member of the tumor necrosis factor receptor superfamily. 4-1BB has an amino acid sequence as provided by GenBank Accession No. AAA62478.2, or an equivalent amino acid sequence from a non-human species such as mouse, rodent, monkey, donkey, and the like. The 4-1BB ligation on T cells triggers a signaling cascade that leads to upregulation of anti-apoptotic molecules, cytokine secretion and enhanced effector function. In dysfunctional T cells with reduced cytotoxic capacity, 4-1BB linkages show potent ability to restore effector function (Li SY, Liu Y. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137. Clin Pharmacol, 2013) , 5 (Suppl1): 47–53). On NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity.

An agonistic monoclonal antibody against 4-1BB has been developed to utilize 4-1BB signaling for cancer immunotherapy (Ye Q, Song DG, Poussin M, Yamamoto T, Best A, Li C et al, CD137 well identified And enriches for naturally occurring tumor-reactive T cells in tumor. Clin Cancer Res, 2014, 20(1): 44-55). Preclinical results of various induced tumor models and spontaneous tumor models indicate that targeting 4-1BB with agonistic antibodies can result in tumor clearance and sustained anti-tumor immunity. WO2004010947A2 discloses a humanized anti-4-1BB monoclonal antibody that binds to human 4-1BB and allows human 4-1BB to bind to a human 4-1BB ligand.

An anti-PD-L1 antibody molecule and a 4-1BB receptor targeting agent (for example, an antibody that stimulates signaling via 4-1BB (CD-137), for example, PF-2566, is generally mentioned in WO 2016/061142. ) Apply together.

Since tumor-immunization, therapeutic protocols targeting multiple targets can cooperate with each other, it is advantageous to prevent tumor immune escape. At present, co-administration of antibodies targeting different targets in tumor immunity has also entered clinical trials. . However, co-administration requires the injection of two separate antibody products or a single injection of a combination of two different antibodies. Although the two injections allowed flexibility in the amount and timing of the administration, it caused inconvenience and pain to the patient. In addition, although the combined preparation may provide some flexibility in the amount of administration, it is often difficult to find a formulation condition that allows the chemical and physical stability of the two antibodies in solution because the molecular characteristics of the two antibodies are different. Moreover, co-administration and combination therapy of two different antibodies may increase the additional cost to the patient and/or payer, and therefore, alternative immunotherapies for treating tumors are needed, and preferably such alternative immunotherapies involve bispecific antibodies.

The anti-4-1BB/PD-L1 bispecific antibody or multispecific antibody of the present invention is an antibody that targets at least 4-1BB and PD-L1 at the same time, and the three antigen binding sites respectively bind 4-1BB and/or The PD-L1 molecule is capable of activating the 1-1BB/4-1BB ligand signaling pathway in T cells and natural killer (NK) cells by blocking the PD-1/PD-L1 signaling pathway. In one embodiment, the triplex antibody comprises a first antigen binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 or 4-1BB on a first polypeptide chain and a second polypeptide chain, and A single domain second antigen binding site on the triple polypeptide chain that specifically binds to PD-L1 or 4-1BB and a single domain third antigen binding site that specifically binds to PD-L1 or 4-1BB. In one embodiment, the triplex antibody comprises a first antigen binding site comprising a VH1/VL1 pair that specifically binds 4-1BB on a first polypeptide chain and a second polypeptide chain, and a third polypeptide The first VHH and the second VHH of the PD-L1 are specifically bound on the chain. In another embodiment, the tri-chain antibody comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 on the first polypeptide chain and the second polypeptide chain, and a third Specific binding on the peptide chain binds to the first VHH and the second VHH of 4-1BB.

For the first antigen binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 or 4-1BB, the "VH1/VL1 pair" comprises an anti-PD-L1 antibody derived from any of the prior art reports (eg, , the anti-PD-L1 antibody exemplified above) and the 6 CDRs of the anti-PD-L1 antibody VH1/VL1 pair developed in the future or one or more of the CDRs of the 6 CDRs, a sequence of three, four, or five amino acid changes (eg, amino acid substitutions or deletions); or an anti-4-1BB antibody derived from any of the prior art reports (eg, the anti-4-1BB exemplified above) The antibody and the 6 CDRs of the anti-4-1BB antibody VH1/VL1 pair developed in the future or one, two, three, four or five amino acids with one or more of the 6 CDRs A sequence of changes (eg, amino acid substitutions or deletions).

For the single domain antigen binding site that specifically binds to PD-L1 or 4-1BB, it comprises a heavy chain variable domain (VH), a light chain variable structure that specifically binds to PD-L1 or 4-1BB Domain (VL), heavy chain variable domain in camelid antibodies consisting of only two heavy chains, naturally free of light chains from camelid serum, VH-like single domain of IgNAR from sharks, camelization Human VH domain, humanized camelid antibody heavy chain variable domain.

In one embodiment, a triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds 4-1BB a first antigen binding site and a single domain second and third antigen binding site on the third polypeptide chain that specifically binds to PD-L1, wherein the single domain that specifically binds to PD-L1 is second The third antigen binding site binds to the same epitope or different epitope on PD-L1.

In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a BMS-derived from an anti-4-1BB antibody. All 6 heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 26/28 of 663513, or One or more of the CDRs of the CDRs have one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions).

In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a BMS-derived from an anti-4-1BB antibody. The paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 26/28 of 663513, or at least 90% with the paired heavy chain variable region sequence/light chain variable region sequence, Sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

The type of the heavy chain constant region of the immunoglobulin in the first polypeptide chain and the third polypeptide chain of the triplex anti-4-1BB/PD-L1 bispecific antibody of the present invention is not particularly limited, and is preferably IgG1, IgG2 Or the heavy chain constant region of an IgG4 immunoglobulin, or substantially the same (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence. More preferably, the heavy chain constant region is, or substantially identical to, the heavy chain constant region of a human IgGl immunoglobulin (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or more of the same).

In one embodiment, a triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for use in IgG4 (eg, human IgG4). In yet another embodiment, a triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for IgG1 (eg, human IgG1). For example, a hinge region having a "CPPC" amino acid residue is contained in the Fc domain of the first polypeptide chain and the third polypeptide chain of the triple-chain antibody, respectively, and/or Y349C and S354C are respectively contained ("EU according to Kabat" The number "", whereby the first polypeptide chain and the third polypeptide chain form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain and/or the third polypeptide chain of a triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function . In a specific embodiment, the amino acid substitution is a LALA mutation.

In still another embodiment, the second polypeptide chain of a triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises a kappa light chain constant region or a lambda light chain constant region, eg, a human kappa light chain constant region Or human λ light chain constant region. In one embodiment, the light chain constant region comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 13 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) , 98%, 99% or more of the same sequence.

In one embodiment, the Fc domains of the first polypeptide chain and the third polypeptide chain of the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention each comprise a "stable" Hehe. In one embodiment, an amino acid substitution T366W is included in one of the first polypeptide chain and the third polypeptide chain, and is included in the other of the first polypeptide chain and the third polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering). Thus the protrusions in one strand can be placed in the cavities in the other strand, facilitating the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain of the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention and the immunoglobulin CH1 domain and CL domain of the second polypeptide chain respectively comprise a bulge Or a cavity, and the protrusions or holes in the CH1 domain may be respectively placed in the holes or protrusions in the CL domain such that the first polypeptide chain and the second polypeptide chain are in contact with each other It also forms a stable association of “knots”.

In one embodiment, the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO: 25, and the second polypeptide set forth in SEQ ID NO: a strand, and a third polypeptide chain set forth in SEQ ID NO: 14, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98) Sequence of %, 99% or higher identical).

In yet another embodiment, the triplex anti-4-1BB/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO: 25, and the second more represented by SEQ ID NO: a peptide chain, and a third polypeptide chain set forth in SEQ ID NO: 22, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or higher identical).

The triple-stranded anti-4-1BB/PD-L1 bispecific antibody of the present invention is capable of binding to both PD-L1 and 4-1BB proteins simultaneously, and maintains the affinity constant of the parent antibody, thereby being able to block PD-1/PD The -L1 signaling pathway and activates the 4-1BB/4-1BB ligand signaling pathway in T cells and natural killer (NK) cells. The triplex anti-4-1BB/PD-L1 bispecific antibodies of the invention can be used in the treatment, prevention or diagnosis of diseases associated with such signaling pathways.

Iii) In one embodiment, the tri-chain antibody is an anti-LAG-3/PD-L1 bispecific antibody or a multispecific antibody.

This embodiment relates to an anti-LAG-3/PD-L1 bispecific antibody or multispecific antibody that blocks the PD-1/PD-L1 signaling pathway and acts on the LAG-3 signaling pathway.

LAG-3 (lymphocyte activation gene-3, also known as CD223) is a cell surface molecule expressed on activated T cells and B cells, and is a cell depletion marker having immunosuppressive activity, which has been shown to be a cell surface molecule. Play a role in CD8+ T cell depletion.

Class II histocompatibility complex (MHC-II) is a ligand for LAG-3, and other ligands for LAG-3 (such as L-selectin and galectin-3) have also been identified (Anderson AC et al) Human, Lag-3, tim-3, and TIGIT: Co-inhibitory receptors with specialized functions in immune regulation, Immunity, 2016, 44(5): 989-1004; Kouo T. et al., Galectin-3shapes antitumor immune responses by Suppressing CD8 ⁺ T cells via LAG-3 and inhibiting expansion of plasmacytoid dendritic cells, Cancer Immunol Res. 2015 April; 3(4): 412–423).

In cancer, Regulatory T cells (Tregs) expressing LAG-3 have enhanced anti-tumor activity, while cytotoxic CD8+ T cells expressing LAG-3 have reduced proliferation rate and effector cytokine production. (Scurr M. et al., Highly prevalent colorectal cancer-infiltrating LAP ⁺ Foxp3 ^- T cells exhibit more potent immunosuppressive activity than Foxp3 ⁺ regulatory T cells, Mucosal Immunol. 2014, 7(2): 428-439). The splice variant of LAG-3, which is cleaved by metalloproteinases and secreted in the cellular microenvironment, has immunological activation when bound to MHC-II on antigen presenting cells (Casati C. et al., Soluble human LAG-3 molecule amplifies the in vitro Generation of type 1 tumor-specific immunity, Clinical Cancer Research. American Association for Cancer Research; 2006, 66(8): 4450-4460).

LAG-3+ tumor infiltrating lymphocytes (TIL) have been reported in patients with melanoma, colon cancer, pancreatic cancer, breast cancer, lung cancer, hematopoietic cancer, and head and neck cancer (Demeure CE et al., T Lymphocytes infiltrating various tumour types express) The MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts, Eur. J. Cancer, 2001, 37(13): 1709-1718). Antibody-based LAG-3 blockade restored CD8+ effector T cells and reduced Treg population in a variety of cancer mouse models.

The present inventors have developed a monoclonal antibody that inhibits the LAG-3 signaling pathway, which is the anti-LAG-3 antibody ADI-31853, having the paired heavy chain variable region sequence/light chain of SEQ ID NO: 30/35 Variable region sequence.

The triple-stranded anti-LAG-3/PD-L1 bispecific antibody or multispecific antibody of the present invention targets at least the antibodies of LAG-3 and PD-L1 at the same time, and the three antigen binding sites respectively bind LAG-3 and / Or PD-L1 molecule. In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody comprises a VH1/ comprising a first polypeptide chain and a second polypeptide chain that specifically binds PD-L1 or LAG-3 a first antigen binding site of the VL1 pair and a single domain second antigen binding site on the third polypeptide chain that specifically binds to PD-L1 or LAG-3 and a single specific binding to PD-L1 or LAG-3 Domain third antigen binding site. In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody comprises a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to LAG-3 An antigen binding site, and a first VHH and a second VHH on the third polypeptide chain that specifically bind to PD-L1. In another embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody comprises a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds PD-L1 A first antigen binding site, and a first VHH and a second VHH that specifically bind to LAG-3 on the third polypeptide chain.

For the first antigen binding site comprising a VH1/VL1 pair that specifically binds to PD-L1 or LAG-3, the "VH1/VL1 pair" comprises an anti-PD-L1 antibody derived from any of the prior art reports (eg, , the anti-PD-L1 antibody exemplified above) and the 6 CDRs of the anti-PD-L1 antibody VH1/VL1 pair developed in the future or one or more of the CDRs of the 6 CDRs, a sequence of three, four, or five amino acid changes (eg, amino acid substitutions or deletions); or an anti-LAG-3 antibody derived from any of the prior art reports (eg, anti-LAG-3 exemplified above) The antibody and the 6 CDRs of the anti-LAG-3 antibody VH1/VL1 pair developed in the future or one, two, three, four or five amino acids with one or more of the 6 CDRs A sequence of changes (eg, amino acid substitutions or deletions).

For the single domain antigen binding site that specifically binds to PD-L1 or LAG-3, it comprises a heavy chain variable domain (VH), a light chain variable structure that specifically binds to PD-L1 or LAG-3 Domain (VL), heavy chain variable domain in camelid antibodies consisting of only two heavy chains, naturally free of light chains from camelid serum, VH-like single domain of IgNAR from sharks, camelization Human VH domain, humanized camelid antibody heavy chain variable domain.

In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to LAG-3 a first antigen binding site and a single domain second and third antigen binding site on the third polypeptide chain that specifically binds to PD-L1, wherein the single domain that specifically binds to PD-L1 is second The third antigen binding site binds to the same epitope or different epitope on PD-L1.

In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from an anti-LAG-3 antibody ADI- VH CDR2, VIV CDR3, QVRQDISNYLN (SEQ ID NO: 33), VH CDR2, QVRQDANYLN (SEQ ID NO: 33), GSI CDR1, SIVYSGYTYYNPSLKS (SEQ ID NO: 32), SEQ ID NO: 31 36) VL CDR2 represented by VL CDR1, DASNLET (SEQ ID NO: 37) and VL CDR3 represented by QQVLELPPWT (SEQ ID NO: 38), or one or more CDRs of the 6 CDRs A sequence having one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions). In one embodiment, the first antigen-binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from an anti-LAG-3 antibody ADI- The paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 30/35 of 31853, or at least 90% with the paired heavy chain variable region sequence/light chain variable region sequence, Sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise CDR1, SEQ ID NO: CDR2 shown in 18 and CDR3 shown in SEQ ID NO: 19, or one, two, three, four, or five amino acid changes with one or more of the three CDRs (eg, Sequence of amino acid substitutions or deletions. In yet another embodiment, the single domain second and third antigen binding sites on the third polypeptide chain that specifically bind to PD-L1 comprise SEQ ID NO: 15 and/or SEQ ID NO: 16 The amino acid sequence shown, or a sequence substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).

The type of the heavy chain constant region of the immunoglobulin in the first polypeptide chain and the third polypeptide chain of the triplex anti-LAG-3/PD-L1 bispecific antibody of the present invention is not particularly limited, and is preferably IgG1, IgG2 Or the heavy chain constant region of an IgG4 immunoglobulin, or substantially the same (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) sequence. More preferably, the heavy chain constant region is, or substantially identical to, the heavy chain constant region of a human IgGl immunoglobulin (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or more of the same).

In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for use in IgG4 (eg, human IgG4). In yet another embodiment, a triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises a heavy chain constant region for IgG1 (eg, human IgG1). For example, a hinge region having a "CPPC" amino acid residue is contained in the Fc domain of the first polypeptide chain and the third polypeptide chain of the triple-chain antibody, respectively, and/or Y349C and S354C are respectively contained ("EU according to Kabat" The number "", whereby the first polypeptide chain and the third polypeptide chain form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain and/or the third polypeptide chain of a triplex anti-LAG-3/PD-L1 bispecific antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function . In a specific embodiment, the amino acid substitution is a LALA mutation.

In still another embodiment, the second polypeptide chain of a triplex anti-LAG-3/PD-L1 bispecific antibody of the invention comprises a kappa light chain constant region or a lambda light chain constant region, eg, a human kappa light chain constant region Or human λ light chain constant region. In one embodiment, the light chain constant region comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 13 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) , 98%, 99% or more of the same sequence.

In one embodiment, the Fc domains of the first polypeptide chain and the third polypeptide chain of the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention each comprise a "stable" Hehe. In one embodiment, an amino acid substitution T366W is included in one of the first polypeptide chain and the third polypeptide chain, and is included in the other of the first polypeptide chain and the third polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering). Thus the protrusions in one strand can be placed in the cavities in the other strand, facilitating the correct pairing of the first polypeptide chain and the third polypeptide chain.

In one embodiment, the first polypeptide chain of the triplex anti-LAG-3/PD-L1 bispecific antibody of the invention and the immunoglobulin CH1 domain and CL domain of the second polypeptide chain respectively comprise a bulge Or a cavity, and the protrusions or holes in the CH1 domain may be respectively placed in the holes or protrusions in the CL domain such that the first polypeptide chain and the second polypeptide chain are in contact with each other It also forms a stable association of “knots”.

In one embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO:29, and the second polypeptide set forth in SEQ ID NO:34 a strand, and a third polypeptide chain set forth in SEQ ID NO: 14, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98) Sequence of %, 99% or higher identical).

In yet another embodiment, the triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the invention comprises the first polypeptide chain set forth in SEQ ID NO:29, and the second more represented by SEQ ID NO:34 a peptide chain, and a third polypeptide chain set forth in SEQ ID NO: 22, or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, Sequence of 98%, 99% or higher identical).

The triple-stranded anti-LAG-3/PD-L1 bispecific antibody of the present invention is capable of binding to both PD-L1 and LAG-3 proteins simultaneously, and maintains the affinity constant of the parent antibody, thereby enabling blocking of PD-1/ The PD-L1 signaling pathway blocks the LAG-3 signaling pathway. The triple-stranded anti-LAG-3/PD-L1 bispecific antibodies of the invention can be used in the treatment, prevention or diagnosis of diseases associated with such signaling pathways.

III. Tri-chain antibody variants of the invention

In certain embodiments, amino acid sequence variants of the bispecific antibodies exemplified herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of bispecific antibodies. Amino acid sequence variants of bispecific antibodies can be made by introducing appropriate modifications to the nucleotide sequence encoding the bispecific antibody or by peptide synthesis. Such modifications include, for example, deletion of residues from within the amino acid sequence of an antibody and/or insertion of residues into and/or substitution of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses a desired characteristic, such as antigen binding.

Conservative substitutions are shown in Table 1 under the heading "Conservative substitutions." The more pronounced changes are further described below in Table 1 under the heading "Exemplary Displacement" and with reference to the amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for the desired activity, for example, retained/improved antigen binding or reduced immunogenicity.

Table 1

Primitive residue Exemplary replacement Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu, Val; Met; Ala; Phe; norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu;Phe;Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr;Phe Tyr Tyr(Y) Trp;Phe;Thr;Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu; Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

(3) Acidity: Asp, Glu;

(4) Basic: His, Lys, Arg;

((5) Residues affecting the chain direction: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will cause members of one of these categories to be exchanged for members of another classification.

IV. Immunoconjugate

The triplex antibodies of the invention are capable of recombinant fusion or chemical conjugation (including covalent and non-covalent conjugation) to a heterologous protein or polypeptide to produce a fusion protein. Methods of fusing or conjugating a protein, polypeptide or peptide to an antibody are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929 and EP 367,166.

In addition, the tri-chain antibodies of the invention can be fused to a labeling sequence (such as a peptide) to facilitate purification. In a preferred embodiment, the labeled amino acid sequence is a hexahistidine peptide, such as the one provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), etc., many of which are commercially available. . As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides convenient purification of the fusion protein. Other peptide tags for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37: 767) and "flag" label.

In other embodiments, a triple chain antibody of the invention is conjugated to a diagnostic or detectable agent. Such antibodies can be used as part of a clinical test (eg, to determine the efficacy of a particular therapy) for monitoring or predicting the onset, formation, progression, and/or severity of a disease or condition. Such diagnosis and detection can be accomplished by coupling an antibody to a detectable substance, including but not limited to a variety of enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactose Glycosidase or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, isothiocyanate , rhodamine, dichlorotriazinamide fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin and jellyfish Photoprotein; radioactive material such as, but not limited to, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), antimony ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In , ¹¹² In and ¹¹¹ In), 锝 ( ⁹⁹ Tc), 铊 ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd ), molybdenum ( ⁹⁹ Mo ), ytterbium ( ¹³³ Xe ), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, 47 Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Tin; and positron emission metal and non-radiomagnetic paramagnetic used in various positron emission tomography Metal ion.

The invention also encompasses the use of a tri-chain antibody conjugated to a therapeutic moiety. The tri-chain antibody can be conjugated to a therapeutic moiety, such as a cytotoxin (eg, a cytostatic or cytotoxic agent), a therapeutic agent, or a radioactive metal ion, such as an alpha emitter. The term "cytotoxin" or "cytotoxic agent" includes any substance that is detrimental to a cell.

Additionally, a tri-chain antibody can be conjugated to a therapeutic moiety or drug moiety that modulates a given biological response. The therapeutic or drug moiety should not be construed as being limited to classical chemotherapeutics. For example, the drug moiety can be a protein, peptide or polypeptide possessing the desired biological activity. Such proteins may, for example, include toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha interferon, beta interferon, nerve Growth factors, platelet-derived growth factors, tissue plasminogen activators, apoptotic agents, anti-angiogenic agents, or biological response modifiers, such as lymphokines.

In addition, the antibody can be conjugated to a therapeutic moiety such as a radioactive metal ion, such as an alpha-emitter such as ²¹³ Bi or can be used to catalyze the emission of metal ions (including but not limited to ¹³¹ In, ¹³¹ LU, ¹³¹ Y, ¹³¹ Ho, ¹³¹ Sm) A macrocyclic chelating agent conjugated to the polypeptide. In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be passed through a linker The molecule attaches to the antibody. Such linker molecules are well known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90, each of which is incorporated by reference in its entirety.

Techniques for conjugating a therapeutic moiety to an antibody are well known, see, for example, Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", cited from Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), Pp. 243-256 (Alan R. Liss, Inc. 1985).

The antibody may also be linked to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

V. Production and purification of the three-chain antibody of the present invention

The triple-chain antibodies of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, the polynucleotide encoding the first polypeptide chain of the tri-chain antibody, the polynucleotide of the second polypeptide chain, and/or the polynucleotide of the third polypeptide chain are separated and inserted into one or more The vector is for further cloning and/or expression in a host cell. The polynucleotide can be easily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.

Expression vectors can be constructed using methods well known to those of skill in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).

Once an expression vector comprising one or more polynucleotides of the invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, a host cell comprising an expression vector of the invention is provided. As used herein, the term "host cell" refers to any type of cellular system that can be engineered to produce a three-chain antibody of the invention. Host cells suitable for replicating and supporting the expression of a triplex antibody of the invention are well known in the art. Such cells can be transfected or transduced with a specific expression vector as needed, and a large number of cells containing the vector can be cultured for inoculating a large-scale fermenter to obtain a sufficient amount of the tri-chain antibody of the present invention for clinical use. Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0. For a review of mammalian host cell lines suitable for protein production see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

Standard techniques for expressing foreign genes in these host cell systems are known in the art. In one embodiment, a method of producing a three-chain antibody of the invention is provided, wherein the method comprises culturing a host cell as provided herein under conditions suitable for expression of the tri-chain antibody, the host cell comprising a coding The polynucleotide of the tri-chain antibody, and the tri-chain antibody is recovered from a host cell (or host cell culture medium).

The tri-chain antibody prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art.

The purity of the tri-chain antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the tri-chain antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.

VI. Pharmaceutical Compositions and Kits

In another aspect, the invention provides compositions, for example, pharmaceutical compositions comprising a tri-chain antibody as described herein formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

Also disclosed herein are compositions obtained by combining a three-chain antibody described herein with one or more therapeutic agents selected from one, two or all of the following categories (i)-(iii): (i) drugs that enhance antigen presentation (eg, tumor antigen presentation); (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); or (iii) reduce immunosuppression drug.

The compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. A common preferred composition is in the form of an injectable solution or an infusible solution. A preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the tri-chain antibody is administered by intravenous infusion or injection. In another preferred embodiment, the tri-chain antibody is administered by intramuscular, intraperitoneal or subcutaneous injection.

The phrases "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., a tri-chain antibody) in a suitable amount in a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients. A coating agent such as lecithin or the like can be used. In the case of dispersions, the proper fluidity of the solution can be maintained by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.

In certain embodiments, the tri-chain antibodies of the invention can be administered orally, e.g., orally, with an inert diluent or an edible carrier. The tri-chain antibodies of the invention may also be enclosed in hard or soft-shell gelatin capsules, compressed into tablets or incorporated directly into the subject's diet. For oral therapeutic administration, the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice papers It is used in the form of a wafer or the like. In order to administer a tri-chain antibody of the invention by parenteral administration methods, it may be desirable to coat or co-administer the tri-chain antibody with a material that prevents its inactivation. Therapeutic compositions can also be administered using medical devices known in the art.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a triplex antibody of the invention. "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required. The therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual. A therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%. The ability of the triplex antibodies of the invention to inhibit measurable parameters (e.g., tumor volume) can be assessed in an animal model system that predicts efficacy in human tumors.

By "prophylactically effective amount" is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the earlier stage of the disease or at an earlier stage of the disease.

Kits comprising a triple chain antibody as described herein are also within the scope of the invention. The kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

VII. Use of tri-chain antibodies

The triple-chain antibodies disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, eg, a human subject, to treat, prevent, and/or diagnose a variety of antigen-related diseases, such as cancer, autoimmune diseases. , acute and chronic inflammatory diseases, infectious diseases (for example, chronic infectious diseases or sepsis).

In one aspect, the invention provides a diagnostic method for detecting a biological sample, such as serum, semen, or a urine or tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in vitro or in vivo. The diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with a tri-chain antibody as described herein or administering the tri-chain antibody to a subject under conditions that allow interaction to occur and ( Ii) detecting the formation of a complex between the tri-chain antibody and the sample (and optionally, the control sample). Formation of the complex indicates the presence of a relevant antigen and may indicate the suitability or need for the treatment and/or prevention described herein.

In some embodiments, the relevant antigen is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (eg, RT-PCR), or in vivo imaging techniques. Generally, tri-chain antibodies used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates. Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.

In some embodiments, the level and/or distribution of the relevant antigen is determined in vivo, eg, in a non-invasive manner (eg, by detecting using a suitable imaging technique (eg, positron emission tomography (PET) scan)) The labeled tri-chain antibody of the present invention. In one embodiment, the correlation is determined in vivo, for example, by detecting a tri-chain antibody of the present invention which is detectably labeled with a PET reagent (for example, ¹⁸ F-fluorodeoxyglucose (FDG)). The level and/or distribution of antigen.

In one embodiment, the invention provides a diagnostic kit comprising a triple chain antibody as described herein and instructions for use.

In another aspect, the present invention relates to the use of a tri-chain antibody in vivo for the treatment or prevention of a disease in which an immune response is required to be modulated in a subject, thereby inhibiting or reducing related diseases such as cancerous tumors, autoimmune diseases, acute and chronic inflammatory diseases. The onset or recurrence of a disease, an infectious disease (eg, a chronic infectious disease or sepsis). The tri-chain antibody of the present invention can be used alone. Alternatively, the tri-chain antibody can be administered in combination with other cancer therapeutic/preventive agents. When a tri-chain antibody of the invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.

Accordingly, in one embodiment, the invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a tri-chain antibody described herein. In another embodiment, the invention provides a method of preventing the onset or recurrence of a disease in a subject, the method comprising administering to the subject a prophylactically effective amount of a tri-chain antibody described herein.

In some embodiments, cancers treated and/or prevented with a tri-chain antibody include, but are not limited to, solid tumors, hematological cancers (eg, leukemias, lymphomas, myeloma, eg, multiple myeloma), and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems, such as invasive lungs, breasts, ovaries, lymphoid, gastrointestinal (eg, colon), anal, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), nasopharynx (eg, differentiated or undifferentiated) Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Cancer can be in early, intermediate or advanced stages or metastatic cancer.

In some embodiments, the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC) ), ovarian cancer, pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, hematological malignancies (eg, lymphoma).

In some embodiments, infectious diseases treated and/or prevented with a tri-chain antibody include pathogens that are currently in the absence of an effective vaccine or pathogens to which conventional vaccines are not fully effective. These include, but are not limited to, HIV, (A, B, and C) hepatitis, flu, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. The blocking effect of the tri-chain antibody exemplified by the present invention on PD-L1 is particularly useful for combating infections established by pathogens (e.g., HIV) in which a variant antigen occurs as the infection progresses. These variant antigens can be regarded as foreign antigens upon administration of an anti-human PD-L1 antibody, whereby the tri-chain antibody exemplified by the present invention is capable of eliciting a strong T cell response which is not inhibited by a negative signal by PD-L1.

In some embodiments, the immune system is downregulated by treating and/or preventing inflammatory and autoimmune diseases and graft versus host disease (GvHD) with a tri-chain antibody of the invention. Examples of autoimmune diseases that can be treated and/or prevented by administration of the tri-chain antibodies of the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, Crohn's disease, lupus erythematosus, ulcerative colitis, uveal uv Inflammation and so on. Examples of inflammatory diseases that can be treated and/or prevented by administration of the tri-chain antibodies of the invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, allergic diseases, septic shock, pulmonary fibrosis, arthritis, and chronic diseases Chronic inflammation resulting from toxic or bacterial infections.

The following examples are described to aid in the understanding of the invention. The examples are not intended to be construed as limiting the scope of the invention in any way.

Example

Example 1. Construction, expression, purification, characterization and efficacy test of anti-CD47/PD-L1 bispecific antibody

Example 1.1. Construction of anti-CD47/PD-L1 bispecific antibody

In this example, two anti-CD47/PD-L1 bispecific antibodies were constructed and named as (1) bispecific antibody Kh2NF-PC, the structure of which is shown in Figure 1A; and (2) bispecific The structure of the antibody Kh2NF-PC-NL is shown in Figure 1B.

As can be seen from the structural diagram of Fig. 1A, the bispecific antibody Kh2NF-PC consists of three polypeptide chains, and the peptide chain #1 has the amino acid sequence of SEQ ID NO: 1, which comprises SEQ ID NO derived from the anti-CD47 antibody ADI29341. a VH amino acid sequence of 2, a CH1 amino acid sequence represented by SEQ ID NO: 6 derived from human IgG1 at the C-terminus of the VH amino acid sequence, and SEQ ID NO derived from human IgG1 at the C-terminus of the CH1 amino acid sequence : Fc region amino acid sequence of 7; peptide chain #2 has the amino acid sequence of SEQ ID NO: 8, which comprises the VL amino acid sequence of SEQ ID NO: 9 derived from the anti-CD47 antibody ADI29341, and the VL The human kappa light chain constant region (CL) amino acid sequence of SEQ ID NO: 13 at the C-terminus of the amino acid sequence; and the peptide chain #3 has the amino acid sequence set forth in SEQ ID NO: 14, which comprises the sequence shown in SEQ ID NO: First and second anti-PD-L1 VHH amino acid sequences, a linker peptide amino acid sequence of SEQ ID NO: 20 between said first and second anti-PD-L1 VHH amino acid sequences, and said second antibody The C-terminus of the PD-L1 VHH amino acid sequence is derived from the Fc region of SEQ ID NO: 21 of human IgG1. Base acid sequence.

As can be seen from the structural diagram of FIG. 1B, Kh2NF-PC-NL is also composed of three polypeptide chains, peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 1, and peptide chain #2 has the sequence shown in SEQ ID NO: An amino acid sequence, and peptide chain #3 has an amino acid sequence of SEQ ID NO: 22 from N-terminus to C-terminus, said peptide chain #3 comprising first and second anti-PD-L1 represented by SEQ ID NO: VHH amino acid sequence (no linker peptide between the first and second anti-PD-L1 VHH amino acid sequences): and SEQ ID NO: 21 derived from IgG1 at the C-terminus of the second anti-PD-L1 VHH amino acid sequence The Fc region amino acid sequence shown.

Three strands of the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC and Kh2NF-PC-NL were constructed as follows: The VH C-terminus of the CD47 antibody ADI-29341 was ligated to the N-terminus of the constant region of human IgG1 to obtain the peptide chain #1 , wherein the Fc region comprises a LALA mutation to attenuate the effector function of the antibody of the invention, and comprises a "binding" mutation for stable association with peptide chain #3; peptide chain #2 is derived from VL and human of ADI-29341 κ light chain constant region; peptide chain #3 comprises first and second anti-PD-L1 VHHs in tandem, no linker peptide between the two anti-PD-L1 VHHs (in the bispecific antibody Kh2NF-PC-NL Or a flexible peptide linked with 20 amino acid residues (G ₄ S) ₄ (in the case of the bispecific antibody Kh2NF-PC), the second anti-PD-L1 VHH C-terminus is linked to an Fc derived from IgG1 The N-terminus of the amino acid sequence of the region obtains peptide chain #3, wherein the Fc region comprises a LALA mutation to attenuate the effector function of the antibody of the present invention, and comprises a "binding" mutation to stably associate with peptide chain #1.

Example 1.2. Expression and purification of anti-CD47/PD-L1 bispecific antibody

In this example, the nucleotide sequences encoding the three strands of the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC and Kh2NF-PC-NL constructed in Example 1.1 were ligated into the market through a multiple cloning site. The eukaryotic expression vector pTT5 was sold and expressed in eukaryotic cells, and the bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL were obtained. The specific operation is as follows.

The nucleotide sequence encoding the above three strands of the bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL was synthesized by Genewiz. The coding nucleotide sequences of the three strands synthesized were digested with restriction endonucleases XbaI (New England Biolabs) and NotI (New England Biolabs), respectively, and then treated with T4 DNA ligase (New England Biolabs). Also ligated with the vector pTT5 digested with XbaI and NotI, three recombinant vectors containing the three coding nucleotide sequences, respectively, were obtained.

The three recombinant vectors were verified by sequencing and used for subsequent expression.

HEK293 cells (purchased from Invitrogen) were subcultured in Expi293 cell culture medium (purchased from Invitrogen). The cell culture was centrifuged one day before the transfection to obtain a cell pellet, and the cells were suspended with fresh Expi293 cell culture medium to adjust the cell density to 1 × 10 ⁶ cells/ml. The HEK293 cells were further cultured so that the cell density in the culture on the day of transfection was about 2 x 10 ⁶ cells/ml. A final volume of HEK293 cell suspension of 1/10 F17 medium (purchased from Gibco, Cat. No. A13835-01) was used as a transfection buffer. To each ml of transfection buffer, 10 μg of a 1:1:1 molar ratio of the recombinant plasmids prepared above containing the nucleotide sequence encoding peptide chain #1, peptide chain #2 or peptide chain #3, respectively, was mixed and mixed. Then, add polyethylenimine (PEI) (Polysciences, catalog number: 23966) 30ug, mix and incubate for 10 minutes at room temperature, then gently pour the PEI/DNA mixture into the HEK293 cell suspension. Mix gently, and incubate overnight at 8% CO ₂ at 36.5 °C.

After overnight culture, the culture flask was supplemented with FEED (Sigma, catalog number: H6784-100G) at a concentration of 1/50 of the culture volume after transfection and a concentration of 1/50 of the culture volume after transfection. 200 g / L of glucose solution, gently mixed, placed in 8% CO ₂ , 36.5 ° C continue to culture. After 20 hours, VPA (Gibco, catalog number: 11140-050) was added to a final concentration of 2 mM/L. After continuous culture until day 7 or cell viability ≤ 60%, the culture was collected, centrifuged at 7500 rpm for 30 minutes, and the cell supernatant was taken and filtered using SARTOPORE (Sartorius, catalog number: 5441307H4) in the AKTApure system (GE). Purification by affinity chromatography and ion exchange chromatography on Healthcare).

The specific affinity chromatography purification step is: using MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column, and placed in the AKTApure system. The AKTApure system equipped with a MabSelect SuRe affinity chromatography column was detoxified overnight with 0.1 M NaOH, and then the system was washed with 5 column volumes of binding buffer (Tris 20 mM, NaCl 150 mM, pH 7.2) and the column was equilibrated. The supernatant of the above filtered cells was passed through a column. The cells were re-equilibrated with 5 to 10 column volumes of binding buffer and monitored for UV-leveling using an ultraviolet detection device equipped with an AKTApure system. Then, the antibody was eluted with an elution buffer (citric acid + sodium citrate 100 mM, pH 3.5), and samples were collected according to the ultraviolet absorption value. Each 1 ml of the collection solution was neutralized with 80 ul of neutralizing buffer (Tris-HCl 2M) for further ion exchange chromatography.

The specific ion exchange chromatography purification operation step is: using Superdex 200 Increase 10/300GL (GE Healthcare, catalog number: 10245605) ion exchange chromatography column, and placed in the AKTApure system. The AKTApure system equipped with Superdex 200 Increase 10/300 GL ion exchange chromatography column was detoxified overnight with 0.1 M NaOH. Then, the system and the column were washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (citric acid + sodium citrate 100 mM, pH 5.0) until the conductance and pH were stable. The obtained neutralizing buffer (Tris-HCl 2M) containing the sample was subjected to buffer exchange, exchanged as a loading buffer, and then loaded; and rebalanced using 5 column volumes of the loading buffer. Linear elution with a gradient of 0-100% of elution buffer 2 (citric acid + sodium citrate 100 mM, NaCl 1M, pH 5.0), a total of 20 column volumes, and samples were collected according to the UV absorption value. .

The purity of the samples in the collected fractions was measured by size exclusion chromatography (SEC). Samples in fractions with a purity greater than 95% were combined according to SEC results. The SEC results are shown in Figures 2A and 2B. The purity of the bispecific antibody Kh2NF-PC was 97.76%, and the purity of Kh2NF-PC-NL was 97.86%.

The purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes using a 15 ml ultrafiltration centrifuge tube. The protein was diluted with PBS, centrifugation was continued, and centrifugation was performed at 4500 rpm for 30 minutes, and the operation was repeated twice to replace the buffer. The antibodies after buffer exchange were combined and the antibody concentration was measured.

Example 1.3. Determination of Dissociation Constants of Anti-CD47/PD-L1 Bispecific Antibodies

The equilibrium solution of the above two exemplary anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL binding to CD47 and PD-L1 of the present invention was determined by a kinetic binding assay using the Octet system (manufactured by ForteBio). Off constant (K _D ). The ForteBio affinity assay was performed according to the method reported in the literature (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): p. 270-278). Briefly, AHC sensor (Pall, Cat. No. 1506091) was immersed in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%) at room temperature for half an hour before the start of the experiment. 100 μl of SD buffer was added to the wells of a 96-well black polystyrene half-well microplate (Greiner) as a blank control (for background subtraction), 100 μl of 100 nM purified bispecific antibody Kh2NF-PC, Kh2NF-PC- NL and anti-PD-L1 humanized Nb-Fc antibody (PCT/CN2017/095884), anti-CD47 antibody ADI 29341, 100 μl of rh PD-L1 (100 nM) and h CD47 diluted in SD buffer as antigen (100 nM) (Acrobiosystems) solution. The anti-human IgG Fc biosensor AHC was immersed in wells containing the antibody solution, respectively, and immersed at room temperature for 600 seconds. The sensor was then washed in SD buffer until baseline was reached and then immersed in wells containing 100 ul of antigen solution to monitor antibody association with antigen. The sensor was then transferred to a well containing 100 ul of SD buffer to monitor antibody dissociation. The speed was 400 rpm and the temperature was 30 °C. The background corrected association curves and dissociation curves were fitted by Octet analysis software (ForteBio) to generate association (k _on ) and dissociation (k _dis ) rate constants which were subsequently used to calculate the equilibrium dissociation constant (K _D ). The _on , k _dis and K _D data for the antibodies are shown in Table 2, Table 3 and Figure 3.

Table 2. Dissociation constants (K _D ) of antibodies against rh PD-L1 as determined by the ForteBio Kinetic Binding Assay

Antibody name K _on (M ^-1 s ^-1 ) k _dis (s ^-1 ) K _D (M) Humanized Nb-Fc 2.33E+05 3.71E-03 1.60E-08 Kh2NF-PC-NL 2.28E+05 3.67E-03 1.61E-08 Kh2NF-PC 2.12E+05 4.09E-03 1.92E-08 ADI 29341 N.B. N.B. N.B.

Note: N.B: Undetectable.

Table 3. Solution h CD47 dissociation constant (K _D) by the kinetics of antibody determined ForteBio binding assays

Antibody name K _on (M ^-1 s ^-1 ) k _dis (s ^-1 ) K _D (M) Humanized Nb-Fc N.B. N.B. N.B. Kh2NF-PC-NL 1.19E+06 1.28E-03 1.07E-09 Kh2NF-PC 1.18E+06 1.55E-03 1.31E-09 ADI 29341 7.74E+05 1.80E-03 2.32E-09

As can be seen from the above data, the bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention are capable of simultaneously binding to PD-L1 and CD47 proteins in solution, and maintain the affinity constant of the parent antibody.

Example 1.4. Binding analysis of anti-CD47/PD-L1 bispecific antibodies of the invention with CHO cells overexpressing CD47 or PD-L1

Binding of the anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention to CHO cells overexpressing CD47 or PD-L1 was measured by FACS.

Briefly, ExpiCHO ^TM Expression System Kit (Invitrogen, catalog number: A29133), according to the manufacturer's instructions embodiment operates as follows: carrying pCHO1 cloned into the multiple cloning site MCS human PD-L1 cDNA (Sino Biological) a. The 0 vector (Invitrogen) was transfected into Chinese hamster ovarian cancer cells (CHO-S) (Invitrogen) to produce CHO cells (CHO-S-PD-L1 cells) overexpressing human PD-L1. CHO-S-PD-L1 cells were counted, diluted to 1 × 10 ⁶ cells/ml with cell culture medium, and added to a U-bottom 96-well plate at 100 μl/well. The cell culture medium was removed by centrifugation at 400 g for 5 minutes on a centrifuge. 100 μl of the serially diluted bispecific antibody Kh2NF-PC, Kh2NF-PC-NL of the present invention and humanized Nb-Fc as a control were separately added to the U-shaped plate and the cells were resuspended and allowed to stand on ice for 30 minutes. After centrifugation at 400 g for 5 minutes, the supernatant was removed, and unbound antibody was removed by washing the cells with PBS. Centrifuge at 400g for 5 minutes to remove PBS. 100 μl of 1:200 diluted PE-conjugated anti-human Fc antibody (Jackson Immuno Research) was added to each well and incubated on ice for 30 minutes in the dark. Centrifuge at 400g for 5 minutes and remove the supernatant. Unbound PE-conjugated anti-human Fc antibody was removed by washing the cells with PBS. The cells were resuspended in 100 μl of PBS, and binding of the antibody to the cells was detected by FACS. The result is shown in Figure 4A.

As seen from Fig. 4A, both the bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention are capable of binding to PD-L1 expressed on the cell surface and maintaining the binding EC50 of the parent antibody.

Similarly, overexpression of human CD47 was generated by transfecting pCHO1.0 vector (Invitrogen) carrying human CD47 cDNA (Sino Biological) cloned into the multiple cloning site MCS into Chinese hamster ovarian cancer cells (CHO-S) (Invitrogen). CHO-S cells (CHO-S-CD47 cells).

FACS detection was performed on CHO-CD47 except that the cells used were different and the ADI 29341 antibody was used as the positive control antibody and IgG1 was used as the negative control antibody, and the other experimental procedures were the same as the FACS detection of the above CHO-S-PD-L1 cells. The IgG1 negative control used in the present application has the heavy chain (HC) amino acid sequence shown in SEQ ID NO: 23 and the light chain (LC) amino acid sequence shown in SEQ ID NO: 24.

The result is shown in Figure 4B. As can be seen from Fig. 4B, the bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention are both capable of binding to CD47 expressed on the cell surface and maintaining the binding EC50 of the parent antibody.

Example 1.5. Soluble detection of anti-CD47/PD-L1 bispecific antibodies of the invention

Polyethylene glycol (PEG) is a polar nonionic precipitant. The anti-CD47/PD of the present invention was detected by a PEG precipitation method (Li li et al., Application of a PEG precipitation method for solubility screening: A tool for developing high protein concentration formulations. Protein Science, 2013. 22: p. 1118-23). -L1 bispecific antibody dissolution in different concentrations of PEG.

The anti-CD47/PD-L1 bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL solutions of the present invention were each concentrated to a concentration of 5 mg/ml. 40 μl/well of the bispecific antibody was added to a 96-well cell culture plate. Humira was used as a positive control. 30% PEG6000 (Sigma, Cat. No. 81255-250G), 26.7 μl, 40 μl, 46.7 μl, 53.3 μl, 60 μl, 66.7 μl, 73.3 were added to the first to eleven columns from left to right in a 96-well cell culture plate. Ll, 80 μl, 86.7 μl, 93.3 μl, 100 μl. Each well was made up to a total volume of 200 μl with PBS, whereby the final concentration of the antibody in each well was 1 mg/ml, and the PEG concentration gradients in the first to eleventh columns of the 96-well cell culture plate from left to right were respectively 4%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%. Leave at room temperature for 1 hour. The absorbance at OD 500 nm was measured. The experimental results are shown in Figure 5.

As can be seen from Figure 5, the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC of the present invention has similar solubility to Sumex.

Example 1.6 Thermal Stability Detection of Anti-CD47/PD-L1 Bispecific Antibody of the Present Invention

Differential scanning fluorimetry (DSF) provides information about the structural stability of a protein based on the fluorescence changes in the protein profile, detects changes in the conformation of the protein, and obtains the melting temperature (T _m ) of the protein. In this example, the T _m value of the anti-CD47/PD-L1 bispecific antibody of the present invention was determined by the DSF method.

The anti-CD47/PD-L1 bispecific antibody Kh2NF-PC and Kh2NF-PC-NL solution of the present invention were each diluted to 1 mg/ml with a PBS solution.

To 4 μl of SYPRO Orange Protein Gel Stain (Gibco, catalog number: S6650), 196 μl of PBS was added, and SYPRO Orange Protein Gel Stain was diluted 50-fold.

To a 96-well PCR plate (Nunc), 50 μl of the above-described bispecific antibody at a concentration of 1 mg/ml was added, and 10 μl of the above 50-fold diluted SYPRO Orange Protein Gel Stain was added, followed by the addition of 40 μl of water. The assay was performed on a 7500 Real Time PCR system (Themro Fisher). Set the system temperature to increase by 0.5 degrees per minute. When the absolute value of the fluorescence curve peaks, the corresponding temperature is the Tm of the protein.

The experimental results are shown in Table 4 below. The bispecific antibodies of the present invention exhibit three T _m values, both > 55 ° C, and therefore, have better thermal stability.

Table 4.

Further, after the bispecific antibody of the present invention was allowed to stand at 40 ° C for 10, 20, and 30 days, changes in purity and biological activity were examined, and long-term thermal stability evaluation of the antibody was carried out. Specifically, the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC and Kh2NF-PC-NL solution of the present invention were separately concentrated in PBS to a concentration of 5 mg/ml, and then dispensed in an EP tube at 200 μl/tube. , protected from light at 40 ° C.

One tube was taken on days 0, 1, 3, 7, 10, 20, and 30 for SEC-HPLC to determine the purity of the anti-CD47/PD-L1 bispecific antibody of the present invention after being placed at 40 °C. The experimental results are shown in Figure 6. The anti-CD47/PD-L1 bispecific antibody of the present invention has high antibody purity even after being left at 40 ° C for up to 30 days.

Further, the binding activity of the anti-CD47/PD-L1 bispecific antibody of the present invention to CHO-S cells expressing CD47 or PD-L1 was detected on Days 0 and 30 as described in Example 1.4 above. The experimental results are shown in Figures 7A and 7B.

As seen from Fig. 6, Fig. 7A and Fig. 7B, the bispecific antibody of the present invention has good long-term thermal stability and maintains biological activity.

Example 1.7. Detection of anti-PD-L1 activity of the bispecific antibody of the present invention based on MOA method

Considering that the exploration of antibodies should be based on the understanding and biological activity of its mechanisms of action (MOA), this example uses PD-1/PD-L1 Blockade Bioassay, Cell Propagation Model (Promega) The anti-PD-L1 biological activity of the bispecific antibody of the present invention was investigated.

Promega's PD-1/PD-L1 Blockade Bioassay is a biologically relevant MOA-based assay for determining the potency and stability of antibodies that block PD-1/PD-L1 interaction. The assay consists of two genetically engineered cell lines:

PD-1 effector cells: Stable expression of human PD-1 and Jurkat T cells expressing luciferase by a nuclear factor of activated T cells (NFAT).

PD-L1 aAPC/CHO-K1 cells: CHO-K1 cells stably expressing human PD-L1 and cell surface proteins that activate the corresponding TCR in an antigen-independent manner.

PD-1 binds to PD-L1 to block the transduction of NFAT downstream signals, thereby inhibiting the expression of luciferase. When PD-1 antibody or PD-L1 antibody is added, this blocking effect is reversed. The photozyme is expressed to thereby detect a fluorescent signal. The detection method has good sensitivity, specificity and accuracy, and the stability is very good.

Test according to the manufacturer's product specifications. PD-L1 aAPC/CHO-K1 cells were subcultured 1-2 days prior to the implementation of the MOA method. The culture supernatant was discarded, and the cells were washed with PBS (Gibco). Digestion with appropriate amount of trypsin (Gibco) was carried out at 37 ° C / 5% CO _{2 for} 3 to 5 minutes. Then, a medium of 4 times trypsin volume was added, and the cells were transferred to a 50 ml centrifuge tube and counted. Take the required volume of cells and centrifuge at 230g for 10 minutes. RPMI 1640 medium (Gibco) was added and the cells were resuspended to 4 x 10 ⁵ cells/ml. The cell suspension was added to a 96-well white cell culture plate (Nunclon) at 100 μl/well, and added to a solution of PBS to 200 μl/well. The cells were cultured overnight in a 37 ° C / 5% CO ₂ incubator.

Similarly, PD-1 effector cells were subcultured 1-2 days prior to the implementation of the MOA method. After counting, the required volume of cells was taken and centrifuged at 170 g for 5 minutes. The cells were resuspended in test buffer (RPMI 1640 medium + 1% FBS) to 1.25 x 10 ⁶ cells/ml.

95 μl/well of PD-L1 aAPC/CHO-K1 cell culture supernatant was discarded. Then, 40 μl of each test antibody, an anti-PD-L1 humanized Nb-Fc antibody as a control, and an IgG1 negative control were added, respectively. 40 μl of the PD-1 effector cells prepared above were added to the wells of the assay plate. Incubate for 6 hours in a 37 ° C / 5% CO ₂ incubator.

The Bio-Glo ^TM buffer (Promega Corporation) to melt, added Bio-Glo ^TM substrate mix. The obtained Bio-Glo ^TM Reagent at 80μl / well of the culture plate after detection hole 6 hours. Leave at room temperature for 5 to 10 minutes and read the fluorescence signal value.

As a result, as shown in Fig. 8, the bispecific antibody of the present invention can abolish the inhibition of the NFAT signaling pathway by the PD-1/PD-L1 interaction, and the activity is superior to the humanized Nb-Fc which is an anti-PD-L1 antibody. The antibody is used alone.

Example 1.8. Detection of the ability of the bispecific antibody of the present invention to promote macrophage phagocytosis of tumor cells

The antibodies Kh2NF-PC and Kh2NF-PC-NL of the invention are tested for their ability to phagocytose tumor cells by macrophages in a flow cytometry based assay.

Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation of fresh blood taken from donors. From the PBMC isolated according to EasySep ^TM Human CD14 Positive Selection Kit (Stemcell Corporation USA) protocol specification, to give the mononuclear CD14 positive cells. Briefly, 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D Systems) was added for adherent culture for 7 consecutive days; on day 5, 20 ng/mL interferon-γ (IFN-γ, AcroBiosystem) was stimulated for 1 hour, and then stimulated by adding 100 ng/mL lipopolysaccharide (LPS, Sigma) for 48 hours to induce differentiation of precursor cells in peripheral blood mononuclear cells into macrophages.

Human PD-L1 was transformed into tumor cell CCRF-CEM (purchased from ATCC) by electroporation to construct target tumor cell CCRF-CEM-PD-L1. The target tumor cells CCRF-CEM-PD-L1 according CellTrace ^TM CFSE Cell Proliferation Kit: instructions (Invitrogen, Cat. No. C34554), and with a fluorescent dye carboxyfluorescein diacetate succinimidyl ester (carboxyfluorescein diacetate, succinimidyl ester, CFSE ) Perform fluorescent labeling. The labeled tumor cells were co-cultured with the above-described macrophages which had undergone induction of differentiation at a cell ratio of 4:1, and simultaneously incubated with different concentrations of the test antibody for 3 hours at 37 °C. The cells were then washed at least twice, allylphycocyanin (APC)-labeled CD14 antibody (purchased from BD) was added, and incubated on ice (in the dark) for 30 minutes in PBS containing 0.1% BSA. The cells were washed at least twice with PBS and analyzed by flow cytometry. The population of cells that are engulfed is a population of cells that are positive for CD14 in living cells and that are also positive for the fluorescent dye CFSE. The experimental results are shown in Figure 9.

As can be seen from Figure 9, the anti-CD47/PD-L1 bispecific antibody can effectively induce macrophage to exert phagocytosis on target cells co-expressed by CD47 and PD-L1, and its induction activity is similar to that of anti-CD47 monoclonal antibody.

Example 1.9 Detection of erythrocyte agglutination activity by anti-CD47/PD-L1 bispecific antibody of the present invention

It is known in the art that most anti-CD47 antibodies have side effects that promote red blood cell agglutination, and thus the therapeutic applications of these anti-CD47 antibodies are limited. To this end, the inventors further studied the red blood cell agglutination of the anti-CD47/PD-L1 bispecific antibody of the present invention. The specific detection methods are as follows:

Fresh human blood was collected, washed with PBS three times to prepare a 10% human red blood cell suspension, human erythrocytes and test antibodies (maximum concentration of 60 ug / ml, three-fold serial dilution, a total of 11 diluted concentrations) at 37 ° C Incubation was carried out for 2-6 hours and the reaction was carried out in a 96-well round bottom plate. After the reaction is finished, take a picture and judge the result. The result was judged to be that if the red blood cells settled at the bottom of the well and were tiled in a net shape, a red blood cell agglutination reaction occurred (see the results of Hu5F9 ("5F9" antibody in US2015/0183874 A1 in Fig. 10), if the red blood cells did not agglutinate. Upon reaction, red blood cells will sink to the bottom of the well (see IgG1 control in Figure 10).

In the experiments conducted as described in the above assay, the results of the hemagglutination reaction are shown in FIG. The erythrocyte agglutination activity of the antibody Kh2NF-PC of the present invention is very weak, and its activity for promoting erythrocyte agglutination is significantly lower than that of the control group Hu5F9. It can be seen that the anti-CD47/PD-L1 bispecific antibody of the present invention has a markedly reduced red blood cell agglutination, and thus has a markedly reduced side effect in clinical treatment, and can be widely applied to the treatment of various cancers.

Example 1.10 Selective binding activity of anti-CD47/PD-L1 bispecific antibody of the present invention to tumor cells

CD47 protein is expressed on the surface of normal red blood cells in the human body, and most anti-CD47 monoclonal antibodies bind to normal red blood cells, which is one of the main causes of side effects of anti-CD47 monoclonal antibodies. In this example, tumor cells and human erythrocytes were co-incubated, and the selective binding properties of the bispecific antibody of the present invention to tumor cells were examined. The specific experimental process is as follows:

50 ml of fresh blood of the donor was taken, 2.5 times volume of PBS solution was added, and then gently added to 12.5 ml of FiColl (Thermo), divided into 4 tubes, centrifuged at 400 g for 30 minutes, and the centrifugation was stopped at zero deceleration. The upper layer of serum and PBMC were removed to obtain the lowest layer of red blood cells.

Tumor cells H292 (ATCC) according CellTrace ^TM CFSE Cell Proliferation Kit: instructions (Invitrogen, Cat. No. C34554), and fluorescently labeled with a fluorescent dye CFSE. The labeled tumor cells were co-cultured with the above-dissociated human erythrocytes at a cell ratio of 1:20, and simultaneously incubated with different concentrations of the test antibody for 30 minutes at 4 °C. The cells were then washed at least twice with PBS, and an allophycocyanin (APC)-labeled human Fc antibody (purchased from Biolegend, catalog number: 409306) was added and incubated on ice (in the dark) in PBS containing 0.1% BSA. minute. The cells were washed at least twice with PBS and analyzed by flow cytometry.

The experimental results are shown in Table 5 and Figure 11. The bispecific antibodies Kh2NF-PC and Kh2NF-PC-NL of the present invention are more inclined than the anti-CD47 monoclonal antibody ADI-29341 at an antibody concentration of 1.111 nM to 0.041 nM. In combination with tumor cells (at 0. 2 nM, Kh2NF-PC and Kh2NF-PC-NL had 88.51% and 83.24% binding to H292 cell surface, respectively, while ADI-29341 only bound 5.74% to H292 cell surface).

Table 5. Proportion of anti-CD47/PD-L1 bispecific antibodies binding to H292 cells

Example 1.11 Antitumor effect of anti-CD47/PD-L1 bispecific antibody of the present invention in vivo

In the present example, tumor-bearing mice were produced by inoculating NOD-SCID mice with Raji-PD-L1 cells, and the antitumor effect of the CD47/PD-L1 antibody of the present invention was measured.

NOD-SCID mice:

Female NOD-SCID mice (42-62 days old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The grade is SPF, the number is 100, and the quality inspection unit is Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the certificate number is NO.11400700284978. The mice were domesticated for 7 days after arrival and then the study was started.

Culture cells and inoculate mice:

The pJHO1.0 vector (Invitrogen) carrying human PD-L1 cDNA (Sino Biological) cloned into the multiple cloning site MCS was transfected into Raji host cells purchased from ATCC, and Raji cells stably expressing human PD-L1 were obtained by pressure screening. (Raji-PD-L1 cells). Raji-PD-L1 cells were routinely subcultured for subsequent in vivo experiments in mice.

The cultured Raji-PD-L1 cells were collected by centrifugation, and the cells were dispersed in 1×PBS, and Raji-PD-L1 cells having a cell density of 10×10 ⁶ /ml were 1:1 with Matrigel gel (Corning, catalog number: 356231). The cells were mixed to prepare a cell suspension having a cell density of 5 × 10 ⁶ /ml. A Raji-PD-L1 tumor-bearing mouse model was established by inoculating 0.2 ml of the cell suspension on day 0 into the right abdomen region of NOD-SCID mice.

Grouping and administration of tumor-bearing mice:

Eight days after tumor cell inoculation, the tumor-bearing volume of each mouse was examined, and mice with a tumor-bearing volume ranging from 50.56 mm ³ to 115.39 mm ³ were selected and grouped into tumor-sized volume (6 mice per group). The dosage and administration method are shown in Table 6. H-IgG (purchased from Equitech-Bio, lot number: 160308-02, specification 1 g/vial, formulated with PBS 10 mg/ml) was used as a negative control. Specifically, the mice were administered on the 8th, 10th, 12th, 14th, 16th, 18th, and 20th day after the Raji-PD-L1 cell inoculation, and the mouse tumor volume and the mouse body weight were monitored 2-3 times a week. The monitoring ended after 33 days. The relative tumor inhibition rate (TGI%) was calculated on the 33rd day after inoculation, and the formula was as follows: TGI%=100%* (h-IgG control tumor volume – treatment group tumor volume)/(h-IgG control tumor volume –h - IgG control group before tumor volume). The average tumor volume before administration of the h-IgG control group was 80.86 mm ³ . Tumor volume determination: The maximum long axis (L) and the largest broad axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated as follows: V = L x W ² /2. The weight was measured using an electronic balance.

Table 6. Experimental design table

*Dosage every 2 days for 7 times

The tumor inhibition rate results are shown in Figure 17 and Table 7. On the 33rd day after inoculation, compared with h-IgG, ADI-29341 0.1 mg/kg single drug tumor inhibition rate was 88%; ADI-29341 0.1 mg/kg and The tumor inhibition rate of humanized Nb-Fc 0.1mg/kg combination was 107%; the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC 0.17mg/kg had a tumor inhibition rate of 111%, and the tumor inhibition effect was obvious. It is superior to the 10% tumor inhibition rate of humanized Nb-Fc 0.1 mg/kg, and the anti-CD47/PD-L1 bispecific antibody Kh2NF-PC has the best tumor suppressing effect. The results of body weight testing in mice showed no significant difference in body weight between groups of mice.

Table 7. Tumor inhibition rate on day 33

Example 2. Construction, expression, purification and characterization of anti-4-1BB/PD-L1 bispecific antibody

Example 2.1. Construction of anti-4-1BB/PD-L1 bispecific antibody

In this example, an anti-4-1BB/PD-L1 bispecific antibody was constructed and designated as the bispecific antibody Kh2NF-P4, and its structural schematic is shown in Figure 1A.

As can be seen from the structural diagram of FIG. 1A, the bispecific antibody Kh2NF-P4 is composed of three polypeptide chains, and the peptide chain #1 has the amino acid sequence of SEQ ID NO: 25, which comprises the anti-4-1BB antibody BMS-663513. a VH amino acid sequence of SEQ ID NO: 26, a CH1 amino acid sequence represented by SEQ ID NO: 6 derived from human IgG1 at the C-terminus of the VH amino acid sequence; and a human IgG1 derived from the C-terminus of the CH1 amino acid sequence The Fc region amino acid sequence set forth in SEQ ID NO: 7; peptide chain #2 has the amino acid sequence set forth in SEQ ID NO: 27, which comprises SEQ ID NO: 28 derived from the anti-4-1BB antibody BMS-663513 a VL amino acid sequence, and a human kappa light chain constant region (CL) amino acid sequence set forth in SEQ ID NO: 13 at the C-terminus of the VL amino acid sequence; and peptide chain #3 having the amino acid sequence set forth in SEQ ID NO: It comprises the first and second anti-PD-L1 VHH amino acid sequences set forth in SEQ ID NO: 16; the linkage shown in SEQ ID NO: 20 between the first and second anti-PD-L1 VHH amino acid sequences Peptide amino acid sequence; and SEQ ID derived from human IgG1 at the C-terminus of the second anti-PD-L1 VHH amino acid sequence NO: The Fc region amino acid sequence shown by 21.

Three strands of the bispecific antibody Kh2NF-P4 against 4-1BB/PD-L1 were constructed as follows: The VH C-terminus of the 4-1BB antibody BMS-663513 was ligated to the N-terminus of the constant region of human IgG1 to obtain peptide chain #1, wherein The Fc region contains a LALA mutation to attenuate the effector function of the antibody of the invention, and includes a "binding" mutation to stably associate with peptide chain #3; peptide chain #2 is derived from VL of VL and gamma of human κ Chain constant region; peptide chain #3 comprises first and second anti-PD-L1 VHHs in tandem, linked by a flexible peptide of 20 amino acid residues (G ₄ S) ₄ between the two anti-PD-L1 VHHs Peptide chain #3 was obtained by ligating the second anti-PD-L1 VHH C-terminus to the N-terminus of the Fc region derived from IgG1, wherein the Fc region comprises a LALA mutation to attenuate the effector function of the antibody of the present invention, and The mutation is mutated to form a stable association with peptide chain #1.

Example 2.2. Expression and purification of anti-4-1BB/PD-L1 bispecific antibody

In this example, the nucleotide sequences encoding the three strands of the anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 constructed in Example 2.1 were ligated into the eukaryotic expression vector through the multiple cloning site. pTT5 (Biotechnology Research Institute; Montreal, Canada), expression and purification in eukaryotic cells, the bispecific antibody Kh2NF-P4 was obtained.

Plasmid transfection, expression and purification of the anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 were identical to those described in Example 1.2 above. The SEC results of the bispecific antibody Kh2NF-P4 are shown in Figure 12, and the purity was 98.42%.

Example 2.3. Binding analysis of anti-4-1BB/PD-L1 bispecific antibody of the present invention to CHO cells expressing 4-1BB or PD-L1

Binding of the anti-4-1BB/PD-L1 bispecific antibody Kh2NF-P4 of the present invention to CHO cells expressing 4-1BB or PD-L1 was measured by FACS essentially as described in Example 1.4 above. Briefly, CHO-S-PD-L1 cells were counted, diluted to 1 × 10 ⁶ cells/ml with cell culture medium, and added to a U-bottom 96-well plate at 100 μl/well. The cell culture medium was removed by centrifugation at 400 g for 5 minutes on a centrifuge. 100 μl of the serially diluted bispecific antibody Kh2NF-P4 of the present invention and the humanized Nb-Fc as a control were separately added to the U-shaped plate and the cells were resuspended and allowed to stand on ice for 30 minutes. After centrifugation at 400 g for 5 minutes, the supernatant was removed, and unbound antibody was removed by washing the cells with PBS. Centrifuge at 400g for 5 minutes to remove PBS. 100 μl of 1:200 diluted PE-conjugated anti-human Fc antibody (Jackson Immuno Research) was added to each well and incubated on ice for 30 minutes in the dark. Centrifuge at 400g for 5 minutes and remove the supernatant. Unbound PE-conjugated anti-human Fc antibody was removed by washing the cells with PBS. The cells were resuspended in 100 μl of PBS, and binding of the antibody to the cells was detected by FACS. The result is shown in Figure 13B.

As seen from Fig. 13B, the bispecific antibody Kh2NF-P4 of the present invention is capable of binding to PD-L1 expressed on the cell surface, and has a binding ability similar to that of the parent antibody.

Similarly, overexpression was generated by transfecting the human 4-1BB cDNA (Sino Biological) pCHO1.0 vector (Invitrogen) carrying the clone into the multiple cloning site MCS into Chinese hamster ovarian cancer cells (CHO-S) (Invitrogen). Human 4-1BB CHO-S cells (CHO-S-4-1BB cells). FACS detection was performed on CHO-S-4-1BB, except that the cells used were different and the antibody control used was BMS-663513 antibody, and the other experimental procedures were the same as those of the CHO-S-PD-L1 cells described above. The result is shown in Figure 13A.

As seen from Fig. 13A, the bispecific antibody Kh2NF-P4 of the present invention is capable of binding to 4-1BB expressed on the cell surface, and its binding ability is similar to that of the parent antibody.

Example 3. Construction, expression, purification and characterization of anti-LAG-3/PD-L1 bispecific antibody

Example 3.1. Construction of anti-LAG-3/PD-L1 bispecific antibody

In this example, an anti-LAG-3/PD-L1 bispecific antibody was constructed and designated as the bispecific antibody Kh2NF-PL, and its structural schematic is shown in FIG. 1B.

As can be seen from the structural diagram of Fig. 1B, the bispecific antibody Kh2NF-PL is composed of three polypeptide chains, and the peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 29, which comprises an anti-LAG-3 antibody derived from ADI-31853. The VH amino acid sequence of SEQ ID NO: 30, the CH1 amino acid sequence of SEQ ID NO: 6 derived from human IgG1 at the C-terminus of the VH amino acid sequence, and the human IgG1 derived from the C-terminus of the CH1 amino acid sequence. The Fc region amino acid sequence set forth in SEQ ID NO: 7; peptide chain #2 has the amino acid sequence set forth in SEQ ID NO: 34, which comprises SEQ ID NO: 35 derived from the anti-LAG-3 antibody ADI-31853 a VL amino acid sequence, and a human kappa light chain constant region (CL) amino acid sequence set forth in SEQ ID NO: 13 at the C-terminus of the VL amino acid sequence; and peptide chain #3 having the amino acid sequence set forth in SEQ ID NO: It comprises the first and second anti-PD-L1 VHH amino acid sequences set forth in SEQ ID NO: 16 with no linker peptide amino acid sequence between the first and second anti-PD-L1 VHH amino acid sequences; The C-terminus of the second anti-PD-L1 VHH amino acid sequence is derived from the Fc region of SEQ ID NO: 21 of human IgG1. Acid sequence.

The three strands of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL of the present invention were constructed as follows: the VH C-terminus of the LAG-3 antibody ADI-31853 was ligated to the N-terminus of the constant region of human IgG1 to obtain a peptide chain #1 , wherein the Fc region comprises a LALA mutation to attenuate the effector function of the antibody of the invention, and comprises a "binding" mutation for stable association with peptide chain #3; peptide chain #2 is derived from VL and human of ADI-31853 κ light chain constant region; peptide chain #3 comprises first and second anti-PD-L1 VHHs in tandem, no linker peptide between the two anti-PD-L1 VHHs, and a second anti-PD-L1 VHH C-terminus Linking to the N-terminus of the amino acid sequence of the Fc region derived from IgG1, the peptide chain #3 is obtained, wherein the Fc region comprises a LALA mutation to attenuate the effector function of the antibody of the present invention, and comprises a "binding" mutation to interact with the peptide chain #1 Stable association.

Example 3.2. Expression and purification of anti-LAG-3/PD-L1 bispecific antibody

In this example, the nucleotide sequences encoding the three strands of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL constructed in Example 3.1 were ligated into the eukaryotic expression vector through the multiple cloning site. pTT5 (Biotechnology Research Institute; Montreal, Canada), expression and purification in eukaryotic cells, the bispecific antibody Kh2NF-PL was obtained.

Plasmid transfection, expression and purification of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL were identical to those described in Example 1.2 above. The SEC results of the bispecific antibody Kh2NF-PL are shown in Figure 14, with a purity of 97.84%.

Example 3.3. Binding analysis of anti-LAG-3/PD-L1 bispecific antibody of the present invention to cells expressing LAG-3 or PD-L1

Binding of the anti-LAG-3/PD-L1 bispecific antibody Kh2NF-PL of the present invention to cells expressing LAG-3 or PD-L1 was measured by FACS essentially as described in Example 1.4 above. Briefly, CHO-S-PD-L1 cells were counted, diluted to 1 × 10 ⁶ cells/ml with cell culture medium, and added to a U-bottom 96-well plate at 100 μl/well. The cell culture medium was removed by centrifugation at 400 g for 5 minutes on a centrifuge. 100 μl of the serially diluted bispecific antibody Kh2NF-PL of the present invention and the anti-PD-L1 humanized Nb-Fc antibody as a control were separately added to the U-shaped plate and the cells were resuspended and allowed to stand on ice for 30 minutes. After centrifugation at 400 g for 5 minutes, the supernatant was removed, and unbound antibody was removed by washing the cells with PBS. Centrifuge at 400g for 5 minutes to remove PBS. 100 μl of 1:200 diluted PE-conjugated anti-human Fc antibody (Jackson Immuno Research) was added to each well and incubated on ice for 30 minutes in the dark. Centrifuge at 400g for 5 minutes and remove the supernatant. Unbound PE-conjugated anti-human Fc antibody was removed by washing the cells with PBS. The cells were resuspended in 100 μl of PBS, and binding of the antibody to the cells was detected by FACS. The result is shown in Figure 15A.

As seen from Fig. 15A, the bispecific antibody Kh2NF-PL of the present invention is capable of binding to PD-L1 expressed on the cell surface, and the binding ability is similar to that of the parent antibody.

HEK293 cells overexpressing human LAG-3 (293-LAG) were transfected into HEK293 cells (Invitrogen) by carrying the pCHO1.0 vector (Invitrogen) carrying human LAG-3 cDNA (Sino Biological) cloned into the multiple cloning site MCS. -3 cells).

Similarly, FACS detection was performed on 293-LAG-3 cells, except that the cells used were different and the antibody control used was the anti-LAG-3 antibody ADI-31853, and the other experimental procedures were performed with the above CHO-S-PD-L1 cells. The same is true for FACS testing. The result is shown in Figure 15B.

As seen from Fig. 15B, the bispecific antibody Kh2NF-PL of the present invention is capable of binding to LAG-3 expressed on the cell surface, and has a binding ability similar to that of the parent antibody.

Example 3.4 Activation of human T cells by anti-LAG-3/PD-L1 bispecific antibody of the present invention

In this example, the activation effect of the anti-LAG-3/PD-L1 bispecific antibody of the present invention on human T cells was examined using a mixed lymphocyte assay, and the specific experimental procedure is as follows.

50 ml of fresh blood of the donor was taken, 2.5 times volume of PBS solution was added, and then gently added to 12.5 ml of FiColl (Thermo), divided into 4 tubes, centrifuged at 400 g for 30 minutes, and the centrifugation was stopped at zero deceleration. The middle white strip was pipetted into PBS and washed twice with PBS to obtain isolated PBMC.

5 ml of T cell culture medium X-VIVO 15 (LONZA) was added to 50 ml of isolated PBMC cells, and cultured in an incubator at 37 ° C, 6% CO _{2 for} 2 hours, and the suspension cell suspension was aspirated for CD4+ cell separation. In addition, for the cells remaining after the suspension of the cell suspension, 3 ml of dendritic cell (DC) medium (X-VIVO 15 (Lonza) 99%, human AB serum (Access) 1%, HEPES 10 mM, β-) was added thereto. Me 50μM, IL-4 (R&D Systems) 1000U/ml, GM-CSF (R&D Systems) 1000U/ml), add 3ml DC medium after 2 days of culture, and add rTNFα (R&D Systems) (1000U) on the 5th day of culture. /ml), IL-1β (R&D Systems) (5 ng/ml), IL-6 (R&D Systems) (10 ng/ml) and 1 μM PGE2 (R&D Systems) were cultured for 2 days to obtain isolated DC cells for use as mixed lymphocytes DC cells used in cellular responses (MLR).

CD4+ cell separation was performed according to the instructions of Untouched CD4+T cell Isolation Kit (Invitrogen, Cat. No. 11346D). Briefly, the suspension cell suspension obtained by static incubation of PBMC for 2 hours was placed in a 20 ml centrifuge tube, centrifuged at 200 g for 10 minutes, and 500 μl of the separation solution, 100 μl of AB-type serum, and 100 μl of the kit were added to the cell pellet. The purified antibody was incubated at 4 ° C for 20 minutes, washed once with the separation solution, and then incubated with 500 μl of bead buffer for 15 minutes. The beads were removed by magnetic field, washed once with T cell medium, and resuspended in 8 ml medium at 37 ° C. The obtained CD4+ cells were cultured in 6% CO ₂ .

1×10 ⁷ CD4+ cells were resuspended in 4 ml of X-VIVO 15 medium (LONZA), Dynabeads Human T-Activator CD3/CD28 (Invitrogen) was added 1:1, cultured for 3 days, and beads stimulation was performed on CD4+ cells. .

The mixed lymphocyte reaction (MLR) was carried out as follows. The above isolated DC cells were mixed with bead-stimulated CD4+ cells in a 96-well cell culture plate (Nunc) at a volume of 200 μl per well, 10,000 DC cells, and 100,000 CD4+ cells, supplemented with serially diluted antibodies and 1 nM SEE ( Toxin technology), using the self-made IgG4 antibody as a negative control, mixed culture for 3 days, and measuring IL2 concentration using an IL2 kit (Cisbio). The IgG4 control antibody has the heavy chain (HC) amino acid sequence set forth in SEQ ID NO: 39 and the light chain (LC) amino acid sequence set forth in SEQ ID NO:40.

Experimental Results As shown in Fig. 16, the bispecific antibody of the present invention can activate T cells in vitro, and its activation effect is stronger than that of the anti-PD-L1 or anti-LAG-3 antibody alone. The activation of T cells by the bispecific antibodies of the invention in vitro is similar to the combination of anti-PD-L1 and anti-LAG-3 antibodies.

While certain representative embodiments and details have been shown for the purposes of the present invention, it will be apparent to those skilled in the art In this regard, the scope of the invention is limited only by the following claims.

Claims (26)

a three-chain antibody comprising three polypeptide chains, wherein the first polypeptide chain comprises a first heavy chain variable domain and the second polypeptide chain comprises a first light chain variable domain, the first heavy chain variable domain Pairing with the first light chain variable domain forms a first antigen binding site; and the third polypeptide chain comprises a single domain second antigen binding site and a single domain third antigen binding site. The triplex antibody of claim 1, wherein the first polypeptide chain comprises a first heavy chain variable domain and an immunoglobulin CH1 domain, and the second polypeptide chain comprises a first light chain variable domain And immunoglobulin CL domain. The triplex antibody according to claims 1 and 2, wherein a single domain second antigen binding site and a single domain third antigen binding site of the third polypeptide chain have or have no linking peptide, Preferably, the third polypeptide chain does not comprise an immunoglobulin CH1 domain. The triplex antibody according to any one of claims 1 to 3, wherein the single domain second antigen binding site and the single domain third antigen binding site are respectively selected from a heavy chain variable domain (VH) a light chain variable domain (VL), a heavy chain variable domain of an antibody that naturally lacks a light chain (eg, a heavy chain variable domain of a naturally occurring heavy chain antibody in camelid species), a VH-like single domain in a novel antigen receptor (NAR) immunoglobulin (such as a naturally occurring IgNAR in shark serum), and a recombinant single domain antigen binding site (eg, a camelized human VH domain) a humanized camelid antibody heavy chain variable domain), for example, the single domain second antigen binding site and the single domain third antigen binding site are respectively selected from the naturally occurring weight of camelid species a heavy chain variable domain of a chain antibody, a camelized human VH domain, and a humanized camelid antibody heavy chain variable domain (which are simply referred to as "VHH"), whereby the single domain The second antigen binding site and the single domain third antigen binding site are the first VHH and The second VHH, the sequences of the first VHH and the second VHH are the same or different, and bind the same or different antigenic epitopes. The triplex antibody according to any one of claims 1 to 4, wherein the first polypeptide chain comprises a first heavy chain variable domain, an immunoglobulin CH1 domain and an Fc domain from the N-terminus to the C-terminus; The second polypeptide chain comprises a first light chain variable domain and an immunoglobulin CL domain from the N-terminus to the C-terminus; and the third polypeptide chain comprises a single domain second antigen-binding site from the N-terminus to the C-terminus A single domain third antigen binding site and an Fc domain, preferably, the immunoglobulin is an IgGl, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgGl immunoglobulin. The triplex antibody according to claim 5, wherein the Fc domain of the first polypeptide chain and the third polypeptide chain comprises a hinge region of an immunoglobulin constant portion, and the first polypeptide chain and the third polypeptide The polypeptide chains are stably associated with each other by a disulfide bond at the hinge region, for example, comprising a "CPPC" amino acid residue in the Fc domain of the first polypeptide chain and the third polypeptide chain of the tri-chain antibody a hinge region of the base such that the first polypeptide chain and the third polypeptide chain are stably associated with each other by a disulfide bond formed between amino acid residues at the hinge region; Preferably, the first polypeptide chain and the third polypeptide chain further comprise Y349C and S354C or S354C and Y349C (according to the "EU numbering" of Kabat) in the respective Fc domains, such that the first polypeptide The chain and the third polypeptide chain further form an interchain disulfide bond in the Fc region. The triplex antibody of claim 5 or 6, wherein the first polypeptide chain and/or the third polypeptide chain comprise a mutation in the Fc domain that affects antibody effector function, for example, a LALA mutation. The tri-chain antibody according to any one of claims 5 to 7, wherein the first polypeptide chain and the third polypeptide chain respectively comprise protrusions or holes in respective Fc domains, and the first plurality The protrusions or holes in the Fc domain of the peptide chain can be placed in the holes or bulges of the Fc domain of the third polypeptide chain, respectively, whereby the first polypeptide chain and the third plurality The peptide chains form a stable association of "binding" with each other. The tri-chain antibody according to any one of claims 2 to 8, wherein the immunoglobulin CH1 domain and the CL domain respectively comprise a protrusion or a hole, and the protrusion or the CH1 domain The holes may be placed in the holes or protrusions in the CL domain, respectively, whereby the first polypeptide chain and the second polypeptide chain form a stable association of "binding" with each other. The triplex antibody according to any one of claims 1 to 9, wherein the first antigen binding site, the second antigen binding site and the third antigen binding site bind to the same antigen or an epitope on a different antigen , For example, the first antigen binding site binds to an epitope of a first antigen, and the second antigen binding site and a third antigen binding site bind to the same or different epitopes on the second antigen, thereby A tri-chain antibody is a bispecific antibody directed against a first antigen and a second antigen; The first antigen binding site binds to an epitope of the first antigen, and the second antigen binding site and the third antigen binding site respectively bind to an epitope of the second antigen and an epitope of the third antigen, thereby The tri-chain antibody is a trispecific antibody. The tri-chain antibody according to any one of claims 3 to 10, wherein the linker peptide comprises a glycine (G) and a serine (S) residue, for example, comprising a GGGGS repeat, preferably, comprising 4 GGGGS repeats. The tri-chain antibody according to any one of claims 1 to 11, wherein the antigen is a cytokine, a growth factor, a hormone, a signaling protein, an inflammatory mediator, a ligand, a cell surface receptor or a fragment thereof. The tri-chain antibody according to claim 12, wherein the antigen is selected from the group consisting of a tumor-associated antigen, an immunological checkpoint molecule, and a costimulatory molecule in the immune system, and ligands and/or receptors of these molecules. The tri-chain antibody according to claim 13, wherein the antigen is selected from the group consisting of CD47, PD1, PD-L1, PD-L2, LAG-3, and 4-1BB (CD137). The tri-chain antibody according to any one of claims 1 to 14, wherein the tri-chain antibody is an anti-CD47/PD-L1 bispecific antibody, and three antigen-binding sites bind to CD47 and/or PD-, respectively. L1 molecule, For example, the tri-chain antibody comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to CD47 on a first polypeptide chain and a second polypeptide chain, and a specific binding on a third polypeptide chain a first VHH and a second VHH of PD-L1; or a first antigen-binding site comprising a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to PD-L1, and a third a first VHH and a second VHH on the polypeptide chain that specifically bind to CD47; Preferably, the first antigen-binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises GESIHYYWS (SEQ ID NO) derived from the anti-CD47 antibody ADI-29341 :3) VH CDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4), VH CDR3, ORFQGISRWLA (SEQ ID NO: 10), VL CDR1 , VL CDR2 represented by AASSLQS (SEQ ID NO: 11) and VL CDR3 represented by QQTVSFPIT (SEQ ID NO: 12), or one, two, three with one or more of the six CDRs Sequence of four, four, or five amino acid changes (eg, amino acid substitutions or deletions); the second domain of the third polypeptide chain that specifically binds to PD-L1, both the second and third antigen binding sites CDR3 represented by SEQ ID NO: 17, CDR2 represented by SEQ ID NO: 18, and CDR3 represented by SEQ ID NO: 19, or one or two CDRs with one or more of the three CDRs a sequence of three, four, or five amino acid changes (eg, amino acid substitutions or deletions), More preferably, the first antigen binding site of the VH1/VL1 pair that specifically binds to CD47 on the first polypeptide chain and the second polypeptide chain comprises SEQ ID NO: derived from the anti-CD47 antibody ADI-29341: a paired heavy chain variable region sequence/light chain variable region sequence of 2/9, or at least 90%, 91%, 92% with the paired heavy chain variable region sequence/light chain variable region sequence, a sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, a single domain second on the third polypeptide chain that specifically binds to PD-L1 And the third antigen binding site comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 15 and/or SEQ ID NO: 16 (eg, at least 90%, 92%, 95%, 97%, a sequence of 98%, 99% or more of the same), Most preferably, the tri-chain antibody comprises a first polypeptide chain set forth in SEQ ID NO: 1, a second polypeptide chain set forth in SEQ ID NO: 8, and SEQ ID NO: 14 or SEQ ID NO: 22 The third polypeptide chain shown, or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher) )the sequence of. The tri-chain antibody according to any one of claims 1 to 14, wherein the tri-chain antibody is an anti-4-1BB/PD-L1 bispecific antibody, and three antigen-binding sites respectively bind 4-1BB and / or PD-L1 molecule, For example, the tri-chain antibody comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain, and a specificity on the third polypeptide chain First binding to the first VHH and second VHH of PD-L1; or a first antigen binding site comprising a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to PD-L1, and a first VHH and a second VHH that specifically bind to 4-1BB on the third polypeptide chain; Preferably, the first antigen binding site of the VH1/VL1 pair that specifically binds to 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a pair derived from SEQ ID NO: 26/28 All six heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the heavy chain variable region sequence/light chain variable region sequence, or one or more of the six CDRs, a sequence of two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); a single domain second and third antigen on the third polypeptide chain that specifically binds to PD-L1 The binding site comprises CDR1 represented by SEQ ID NO: 17, CDR2 represented by SEQ ID NO: 18, and CDR3 represented by SEQ ID NO: 19, or has one or more CDRs of said three CDRs a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions), More preferably, the first antigen binding site comprising the VH1/VL1 pair of the specific 4-1BB on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from SEQ ID NO: 26/28. a heavy chain variable region sequence/light chain variable region sequence, or at least 90%, 91%, 92%, 93%, 94% with the paired heavy chain variable region sequence/light chain variable region sequence a sequence of 95%, 96%, 97%, 98%, 99% or more sequence identity, a single domain on the third polypeptide chain that specifically binds to PD-L1, a second and a third antigen binding The sites all comprise, or are substantially identical to, the amino acid sequence set forth in SEQ ID NO: 15 and/or SEQ ID NO: 16 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) a sequence of 98%, 99% or more of the same), Most preferably, the tri-chain antibody comprises a first polypeptide chain set forth in SEQ ID NO: 25, a second polypeptide chain set forth in SEQ ID NO: 27, and SEQ ID NO: 14 or SEQ ID NO: 22 The third polypeptide chain shown, or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher) )the sequence of. The tri-chain antibody according to any one of claims 1 to 14, wherein the tri-chain antibody is an anti-LAG-3/PD-L1 bispecific antibody, and three antigen-binding sites bind to LAG-3 and / or PD-L1 molecule, For example, the tri-chain antibody comprises a first antigen-binding site comprising a VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain, and a specificity on the third polypeptide chain First binding to the first VHH and second VHH of PD-L1; or a first antigen binding site comprising a VH1/VL1 pair comprising a first polypeptide chain and a second polypeptide chain that specifically binds to PD-L1, and a first VHH and a second VHH that specifically bind to LAG-3 on the third polypeptide chain; Preferably, the first antigen binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises GSIYSESYYWG derived from the anti-LAG-3 antibody ADI-31853 VH CDR2, VIV CDR3, QASQDISNYLN (SEQ ID NO: 36) represented by VH CDR1, SIVYSGYTYYNPSLKS (SEQ ID NO: 32), and VH CDR3 (SEQ ID NO: 33) shown by (SEQ ID NO: 31) VL CDR2 represented by VL CDR1, DASNLET (SEQ ID NO: 37) and VL CDR3 represented by QQVLELPPWT (SEQ ID NO: 38), or one or more CDRs of the six CDRs, a sequence of two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions); a single domain second and third antigen on the third polypeptide chain that specifically binds to PD-L1 The binding site comprises CDR1 represented by SEQ ID NO: 17, CDR2 represented by SEQ ID NO: 18, and CDR3 represented by SEQ ID NO: 19, or has one or more CDRs of said three CDRs a sequence of one, two, three, four, or five amino acid changes (eg, amino acid substitutions or deletions), More preferably, the first antigen binding site of the VH1/VL1 pair that specifically binds to LAG-3 on the first polypeptide chain and the second polypeptide chain comprises a derivative derived from SEQ ID NO: 30/35. a heavy chain variable region sequence/light chain variable region sequence, or at least 90%, 91%, 92%, 93%, 94% with the paired heavy chain variable region sequence/light chain variable region sequence a sequence of 95%, 96%, 97%, 98%, 99% or more sequence identity, a single domain on the third polypeptide chain that specifically binds to PD-L1, a second and a third antigen binding The sites all comprise, or are substantially identical to, the amino acid sequence set forth in SEQ ID NO: 15 and/or SEQ ID NO: 16 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) a sequence of 98%, 99% or more of the same), Most preferably, the tri-chain antibody comprises a first polypeptide chain set forth in SEQ ID NO: 29, a second polypeptide chain set forth in SEQ ID NO: 34, and SEQ ID NO: 14 or SEQ ID NO: 22 The third polypeptide chain shown, or substantially identical to any of the sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher) )the sequence of. A polynucleotide encoding the first polypeptide chain, the second polypeptide chain and/or the third polypeptide chain of the tri-chain antibody of any one of claims 1-17. A vector, preferably an expression vector comprising a polynucleotide encoding a first polypeptide chain, a second polypeptide chain and/or a third polypeptide chain of the three-chain antibody of any one of claims 1-17 . A host cell comprising the polynucleotide of claim 18 or the vector of claim 19, for example, the host cell is a mammalian cell, preferably a CHO cell, a HEK293 cell; the host cell is a prokaryotic cell Preferably, it is an E. coli cell. A method for producing the tri-chain antibody according to any one of claims 1 to 17, which comprises the step of (i) cultivating the host cell of claim 20 under conditions suitable for expression of the tri-chain antibody And (ii) recovering the tri-chain antibody from the host cell or the culture medium. A pharmaceutical composition comprising the tri-chain antibody of any one of claims 1-17 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 22, which further comprises at least one other active ingredient. Use of a tri-chain antibody according to any one of claims 1 to 17 and a pharmaceutical composition according to claims 22 to 23 for the treatment or prevention of a disease in an individual or for the diagnosis of a disease Tool, preferably, the individual is a mammal, more preferably a human. Use according to claim 24 for autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases (for example, chronic infectious diseases or sepsis), treatment and/or prevention or diagnosis of tumors, for example, said tumors It is selected from solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma, eg, multiple myeloma) and metastatic lesions. Use of a triple-chain antibody according to claim 24 for increasing hematopoietic stem cell implantation in a subject in need thereof.

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