TW202140546A - Bispecific anti-PD-L1/VEGF antibody and uses thereof useful for the treatment of cancers - Google Patents

Abstract

The present invention provides a bispecific anti-VEGF x PD-L1 antibody or its antigen-binding portion, wherein the bispecific antibody or its antigen-binding portion includes a PD-L1 antigen-binding module that is combined with a VEGF antigen-binding module, the PD-L1 antigen-binding module including complementarity-determining region 1 (CDR1) including SEQ ID NO: 1, CDR2 including SEQ ID NO: 2, and CDR3 including SEQ ID No: 3; and the VEGF antigen-binding module includes heavy chain complementarity-determining region 1 (HCDR1) including SEQ ID NO: 4, HCDR2 including SEQ ID NO: 5, and HCDR3 including SEQ ID No: 6, light chain complementarity-determining region 1 (LCDR1) including SEQ ID NO: 7, LCDR2 including SEQ ID NO: 8, and LCDR3 including SEQ ID No: 9; a method for preparing the bispecific antibody or its antigen-binding portion; and uses of the bispecific antibody or its antigen-binding portion in the treatment of diseases such as cancers.

Description

A bispecific anti-PD-L1/VEGF antibody and its use

The present disclosure generally relates to a bispecific anti-PD-L1 x VEGF antibody, a method for preparing the antibody and its use.

Angiogenesis is essential for tumor growth and metastasis progression. Controlling tumor-related angiogenesis is a promising cancer treatment strategy. Vascular endothelial growth factor (VEGF) is a key mediator of blood vessel production and has been validated in various types of human cancers. Tumor cells release growth factors (such as VEGF) that bind to nearby endothelial cells, which initiates a signal transduction cascade that stimulates endothelial cells to divide and form new blood vessels. The signal transmission of VEGF through its receptor VEGFR plays a vital role in the angiogenesis and growth of many solid tumors. Anti-angiogenic drugs that target the VEGF pathway, such as Avastin (Bevacizumab), have been clinically successful.

On the other hand, targeted immune checkpoint molecules such as programmed death ligand 1 (PD-L1) or its receptor programmed death 1 (PD-1) have shown promising clinical success. PD-L1 expression is closely related to the poor prognosis of various types of cancer. Anti-PD-L1 antibodies can target PD-L1 expressed on tumor cells and tumor infiltrating immune cells, prevent binding to PD-1 and B7.1 on the surface of T cells, and can also activate T cells and recruit other T cells To attack tumors, and then make the immune system able to fight many types of cancer.

In addition to the established anti-angiogenesis effect, anti-VEGF therapy can further enhance the ability of anti-PD-1/PD-L1 therapy to restore anti-cancer immunity by inhibiting VEGF-related immunosuppression, promoting T cell tumor infiltration and Initiate and initiate T cell responses to tumor antigens. Therefore, the development of VEGF and PD-L1 bispecific antibodies that combine anti-angiogenesis therapy and immune checkpoint suppression can achieve promising results in cancer treatment.

Although targeting VEGF and targeting PD-1/PD-L1 therapy have obvious benefits, there are still a large number of unmet needs. 15%-20% of patients do not respond to anti-VEGF therapy, and more and more evidence shows that long-term use of anti-VEGF agents in cancer treatment can enhance tumor resistance. 3%-9% of patients develop the immunogenicity of the treatment. In addition, the overall survival time is limited and safety issues are limited, including changes in bone morphology, glomerular lesions with inflammation of the kidneys, and reduction in the formation of vacuoles in the adrenal glands with inflammation. Immune checkpoint inhibitors that block the PD-1/PD-L1 pathway (such as nivolumab, pembrolizumab, and atezolizumab) represent standard treatment options for patients with multiple cancers. However, the response rate of these drugs in the unselected population is 14-23%, and the response rate in patients with PD-L1-expressing tumors is 16-48%. These drugs can be found in some but not all patients. Provide better treatment effect.

Therefore, there is a great need to develop new anti-PD-L1/anti-VEGF bispecific antibodies. In this disclosure, a dual specificity that can simultaneously bind human PD-L1 and VEGF with high affinity, block the signal transmission of both PD-1/PD-L1 and VEGF/VEGFR, and show superior anti-tumor efficacy Sex antibody.

Broadly speaking, the present disclosure relates to compounds, methods, compositions, and products that provide antibodies with improved efficacy. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that can react with various targets.

In one aspect, the present disclosure provides a bispecific antibody or antigen-binding portion thereof, which comprises a PD-L1 antigen-binding module combined with a VEGF antigen-binding module, wherein: The PD-L1 antigen binding module comprises: a complementarity determining region (CDR) comprising SEQ ID NO:1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; and The VEGF antigen binding module comprises: a heavy chain complementarity determining region (HCDR) comprising SEQ ID NO: 4, an HCDR comprising SEQ ID NO: 5, an HCDR 3 comprising SEQ ID NO: 6, and a HCDR comprising SEQ ID NO: 7 Light chain complementarity determining region (LCDR) 1, LCDR2 comprising SEQ ID NO: 8 and LCDR3 comprising SEQ ID NO: 9.

In certain embodiments, the PD-L1 antigen binding module as disclosed herein comprises a variable domain comprising the amino acid sequence of SEQ ID NO: 10 or at least 85%, 90%, or 95% of SEQ ID NO: 10 The same amino acid sequence.

In certain embodiments, the VEGF antigen binding module as disclosed herein comprises: a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or at least 85%, 90%, or 95% of SEQ ID NO: 11 % Identical amino acid sequence; and A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 12.

In certain embodiments, the PD-L1 antigen binding module is fused to the N-terminus of the VEGF antigen binding module. In some other embodiments, the PD-L1 antigen binding module is fused to the C-terminus of the VEGF antigen binding module.

In certain embodiments, the PD-L1 antigen binding module is derived from a single domain antibody (sdAb), such as a VHH antibody. The VHH can be derived from camelid animals, including alpaca and llama. Preferably, the VHH is a humanized VHH.

In certain embodiments, the PD-L1 antigen binding module is operably linked to the N-terminus of the light chain or heavy chain of the VEGF antigen binding module, optionally via a linker. The linker may comprise or consist of 2 to 4 copies of GGGGS (G4S), for example, the linker may be (G4S) ₂ .

In certain embodiments, the bispecific antibody or antigen binding portion thereof as disclosed herein comprises a heavy chain and a light chain, wherein: The heavy chain comprises a domain operably linked as in VH-CH1-hinge-Fc, wherein VH-CH1 is derived from the VEGF antigen binding module; and The light chain comprises a domain operably linked as in VHH-VL-CL, where VHH comes from the PD-L1 antigen binding module and VL-CL comes from the VEGF antigen binding module.

In certain embodiments, the Fc region is a human IgG Fc region, preferably a human IgG1 Fc region.

In certain embodiments, the present disclosure provides a bispecific antibody or antigen binding portion thereof, wherein the heavy chain comprises SEQ ID NO: 13, and the light chain comprises SEQ ID NO: 14.

In certain embodiments, the bispecific antibody or antigen binding portion thereof as disclosed herein is a humanized antibody.

In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a bispecific antibody or antigen binding portion thereof as disclosed herein.

In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule as disclosed herein. In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or vector as disclosed herein.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the bispecific antibody or antigen binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method for producing the bispecific antibody or antigen-binding portion thereof as disclosed herein, including the following steps: -Express the antibody or its antigen-binding portion in host cells; and -Isolate the antibody or its antigen-binding portion from the host cell.

In one aspect, the present disclosure provides a method for modulating an immune response in a subject, comprising administering to the subject the bispecific antibody or antigen binding portion or pharmaceutical composition thereof as disclosed herein.

In one aspect, the present disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding portion or pharmaceutical composition thereof as disclosed herein.

In one aspect, the present disclosure provides a method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding portion or pharmaceutical composition thereof as disclosed herein. The cancer can be selected from colon cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastric cancer, lymphoma, melanoma, liver cancer And head and neck cancer. In certain embodiments, the cancer is colon cancer or colorectal cancer.

In some embodiments, the bispecific antibody or antigen-binding portion thereof can be administered in combination with chemotherapeutic agents, radiotherapy, and/or other agents used in cancer immunotherapy.

In one aspect, the present disclosure provides a bispecific antibody or antigen-binding portion thereof as disclosed herein, for use in: i) Regulate the immune response related to PD-L1/VEGF; ii) Enhance T cell proliferation and cytokine production; and/or iii) Stimulate immune response or function, for example, stimulate immune response against cancer cells.

In one aspect, the present disclosure provides the bispecific antibody or antigen binding portion thereof as disclosed herein for use in the diagnosis, prevention or treatment of cancer.

In one aspect, the present disclosure provides the use of the bispecific antibody or antigen binding portion thereof as disclosed herein in the preparation of a medicament for regulating the immune response in a subject or inhibiting the growth of tumor cells.

In one aspect, the present disclosure provides the use of the bispecific antibody or antigen binding portion thereof as disclosed herein in the preparation of a medicament for the treatment or prevention of cancer.

In one aspect, the present disclosure provides a kit comprising the bispecific antibody or antigen binding portion thereof as disclosed herein. The kit can be used for the detection, diagnosis, prognosis or treatment of diseases or conditions (such as cancer).

The above content is an overview, and therefore contains simplifications, generalizations and omissions of details when necessary; therefore, those skilled in the art will recognize that the overview is only illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and/or devices and/or other subjects described herein will become apparent from the teachings shown herein. An overview is provided to briefly introduce some selected concepts, which will be further described in the detailed description below. This summary is not intended to determine the key features or basic features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In addition, the contents of all references, patents, and disclosed patent applications cited throughout this application are incorporated herein by reference in their entirety.

Although the present invention can be implemented in many different forms, what is disclosed herein are specific illustrative embodiments that verify the principles of the present invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. In addition, any chapter headings used in this article are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular form shall include the plural form, and terms in the plural form shall include the singular form. More specifically, as used in this specification and the scope of the appended application, unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" include plural referents. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells and the like. In this application, the use of "or" means "and/or" unless stated otherwise. In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not limiting. In addition, the range provided in the specification and the appended application patent scope includes all values between the endpoint and the interruption point.

Generally, the terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein, and nucleic acid chemistry and hybridization described herein and their techniques are well-known and commonly used terms in the art. Unless otherwise specified, the methods and techniques of the present invention are generally performed according to conventional methods known in the art, and performed as described in various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th edition, WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as laboratory procedures and techniques are well-known and commonly used terms in the art. definition

In order to better understand the present invention, definitions and explanations of related terms are provided as follows.

The term "antibody" or "Ab" is used in the broadest sense herein and encompasses a variety of antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (eg, bispecific antibodies). A natural intact antibody generally refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chain of an antibody can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light chain and the heavy chain, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDR)) separated by relatively conserved regions (called framework regions (FR)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal. The variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site. The distribution of amino acids in various regions or domains follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901 -917; Chothia et al. (1989) Nature 342: 878-883 as defined. Antibodies can have different antibody isotypes, such as IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody can be used interchangeably in the context of this application, and refers to a polypeptide comprising a fragment of a full-length antibody, which retains the antigen-specific binding of the full-length antibody. Ability, and/or its ability to compete with the full-length antibody for binding to the same antigen. In general, see Fundamental Immunology, Ch. 7 (Paul, W. Ed., Second Edition, Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can use any suitable Standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains are derived from, for example, whole antibody molecules. Such DNA is known And/or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biotechnology, for example, to combine one or The plural variable and/or constant domains are arranged into a suitable configuration, or codons are introduced, cysteine residues are generated, amino acids are modified, added or deleted, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragment; and (vii) the smallest recognition unit (such as isolated complementarity determining region (CDR), such as CDR3 peptide) or restricted FR3-CDR3-FR4 peptide consisting of amino acid residues in the hypervariable region of an antibody mimicking . Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanoantibodies (e.g. Monovalent nanoantibodies, bivalent nanoantibodies, etc.), small modular immunological drugs (SMIP) and shark variable IgNAR domains are also included in the expression "antigen-binding fragments" used in the article. In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. The variable domain and the constant domain may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region can be composed of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids, which cause flexibility between adjacent variable and/or constant domains in a single polypeptide molecule Sexual or semi-flexible connection.

As used herein, the term "variable domain/variable domain" of an antibody refers to an antibody variable region or fragments thereof comprising one or more CDRs. Although the variable domain can contain a complete variable region (such as HCVR or LCVR), it can also contain less than the complete variable region but still retain the ability to bind antigen or form an antigen binding site.

As used herein, the term "antigen-binding module" refers to an antibody fragment formed from a part of an antibody that contains one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain a complete natural antibody structure. Unlike the term "antigen-binding site" which usually refers to a variable domain, an antigen-binding module may include a constant domain in addition to the variable domain. Examples of antigen binding modules include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab') ₂ , Fv fragments, disulfide bond stabilized Fv fragments (dsFv), ( dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), multispecific antibody, camelized single domain antibody, nanobody (nanobody), domain antibody, and two Valence domain antibody. The antigen binding module can bind to the same antigen as the parent antibody. In certain embodiments, the antigen binding module may be a Fab fragment or a VHH antibody. In some embodiments, the antigen binding module may comprise one or more CDRs from a specific human antibody grafted onto the framework regions from one or more different human antibodies. For more descriptions of the detailed format of the antigen binding module, see Spiess et al., Molecular Immunology, 67(2), pp. 95-106 (2015), and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which is fully incorporated into this article by reference.

The "Fab" of an antibody refers to the following part of an antibody, which is composed of a single light chain (both variable and constant regions) combined with the variable and first constant regions of a single heavy chain through disulfide bonds. In certain embodiments, both the constant regions of the light chain and the heavy chain are replaced by the TCR constant region.

The "Fc" of an antibody refers to the following part of the antibody, which contains the second (CH2) and third (CH3) constant regions of the first heavy chain in conjunction with the second and third constant regions of the second heavy chain via disulfide bonds. When referring to the Fc region, depending on the context, it can refer to one chain or two chains of the Fc region. The Fc part of an antibody is responsible for multiple effector functions such as ADCC and CDC, but does not play a role in antigen binding. The ability of an antibody to initiate and regulate effector functions via its Fc domain is a key part of its protective activity in vivo. Although it was previously believed that the neutralizing activity of antibodies was only the result of the Fab-antigen interaction, more and more evidences show that its in vivo activity is highly dependent on the interaction between the IgG Fc domain and its related receptor Fcγ receptor (FcγR). In effect, these receptors are expressed on the surface of effector lymphocytes.

The term "PD-L1", also known as programmed death ligand 1, is a 40 kDa type 1 transmembrane protein that is presumed to play a major role in suppressing the adaptive arm of the immune system. PD-L1 is the main ligand of programmed death 1 (PD-1). PD-1 is a co-inhibitory receptor that can be constitutively expressed or induced in myeloid, lymphoid, normal epithelial cells and cancer. As used herein, the term "PD-L1" when referring to the amino acid sequence of the PD-L1 protein includes the full-length PD-L1 protein, or the extracellular domain of PD-L1 (PD-L1 ECD) or a fragment containing PD-L1 ECD; It also includes PD-L1 ECD fusion proteins, such as fragments fused with mouse or human (mFc or hFc) IgG Fc. In addition, as understood by those skilled in the art, the PD-L1 protein will also include those naturally or artificially introduced mutations in the amino acid sequence (including but not limited to substitutions, deletions and/or additions) that do not affect biological functions.

As used herein, the term "antibody that binds PD-L1" or "anti-PD-L1 antibody" includes antibodies that specifically recognize PD-L1 and antigen-binding fragments thereof. The antibodies and antigen-binding fragments of the present disclosure can bind to soluble PD-L1 protein and/or PD-L1 expressed on the cell surface. Soluble PD-L1 includes native PD-L1 protein and recombinant PD-L1 protein variants that lack a transmembrane domain or are not associated with cell membranes. As used herein, the expression "anti-PD-L1 antibody" includes monovalent antibodies with a single specificity, as well as those comprising a first antigen-binding site that binds to PD-L1 and a second antigen-binding site that binds to a second (target) antigen. A bispecific antibody, wherein the anti-PD-L1 antigen binding site comprises any one or more of the heavy chain variable region/light chain variable region or CDR sequences as described in Table A herein. Examples of anti-PD-L1 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

The term "VEGF" (vascular endothelial growth factor, also known as VEGF-A) is a signal protein produced by cells that stimulates blood vessel formation. VEGF is a subfamily of the platelet-derived growth factor family of cystine-knot growth factor. They are important signal transduction proteins involved in angiogenesis (de novo formation of the embryonic circulatory system) and blood vessel production (blood vessel growth from existing blood vessels). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF (placental growth factor), VEGF-E (Orf-VEGF) and Trimeresurus flavoviridis svVEGF.

As used herein, "VEGF receptor" or "VEGFR" refers to the receptor for vascular endothelial growth factor (VEGF). There are three subtypes of VEGFR, numbered 1, 2 and 3. Depending on its alternative splicing, the VEGF receptor can be membrane-bound or soluble. In the VEGF receptor, VEGFR-1 binds VEGF-A, PlGF and VEGF-B.

As used herein, a "bispecific" antibody refers to an artificial antibody having fragments derived from two different monoclonal antibodies and capable of binding two different epitope elements. Two epitopes can be present on the same antigen, or they can be present on two different antigens.

The term "bispecific antigen binding molecule" refers to at least a first antigen binding domain (also referred to herein as the first antigen binding site) and a second antigen binding domain (also referred to herein as the second antigen binding site). Binding site) protein, polypeptide or molecular complex. In some embodiments, the "bispecific antigen binding molecule" is a "bispecific antibody." Each antigen-binding domain in a bispecific antibody contains at least one CDR, which specifically binds to a specific antigen alone or in combination with one or more additional CDRs and/or FRs. In the context of the present invention, the first antigen binding site specifically binds to a first antigen (eg PD-L1), and the second antigen binding site specifically binds to a second different antigen (eg VEGF).

The terms "anti-PD-L1/anti-VEGF antibody", "anti-PD-L1/anti-VEGF bispecific antibody", "antibody against PD-L1 and VEGF", "anti-PD-L1×VEGF bispecific antibody", "PD-L1×VEGF antibody" is used interchangeably herein, and refers to a bispecific antibody that specifically binds PD-L1 and VEGF.

As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies show a single binding specificity and affinity for a specific epitope.

As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, where the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from Human antibodies.

The term "humanized antibody" means an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence. Other framework region modifications can be made within the human framework sequence.

The term "operably linked" refers to the juxtaposition (with or without spacers or linkers) of two or more biological sequences of interest so that they are in a relationship that allows them to function in the intended manner. In terms of polypeptides, it refers to the connection of polypeptide sequences in a manner that allows the product to be connected to have the expected biological function. For example, the antibody variable region can be operably linked to the constant region so as to provide a stable product with antigen binding activity. The term can also be used in relation to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc.), it means a polynucleotide sequence to allow regulation of the polypeptide from the polynucleotide The way of expression is connected.

As used herein, the term "Ka" is intended to indicate the association rate of a specific antibody-antigen interaction, and the term "Kd" as used herein is intended to indicate the dissociation rate of a specific antibody-antigen interaction. The Kd value of an antibody can be determined using a method well established in the art. As used herein, the term "KD" is intended to denote the dissociation constant of a specific antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (ie, Kd/Ka) and expressed as a molar concentration (M). The preferred method of determining the antibody Kd is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore® system.

As used herein, the term "high affinity" of an IgG antibody is an indicator of the target antigen having 1×10 ^-7 M or lower, more preferably 5×10 ^-8 M or lower, even more preferably 1×10 ^-8 M or Lower, even better, 5×10 ^-9 M or lower, and even better 1×10 ^-9 M or lower K _D antibody.

As used herein, the term "EC _50", also referred to as "EC50" means the concentration inducing 50% of the drug response between the baseline and maximum, an antibody or agent after a certain exposure time. In the context of this application, _{the unit of EC 50} is "nM".

As used herein, the term "IC _50", also called "half maximal inhibitory concentration" of a substance is a measure of inhibitory potency of a particular biological or biochemical functions of. In the context of the present application, the unit IC ₅₀ is "nM".

As used herein, the ability to "inhibit binding" means that an antibody or antigen-binding fragment thereof inhibits the binding of two molecules (for example, human PD-L1/VEGF and human PD-1/VEGFR) to any detectable level. In certain embodiments, the binding of two molecules can be achieved by the antibody or antigen-binding fragment thereof at a rate of no more than 50 nM, no more than 30 nM, no more than 10 nM, no more than 5 nM, no more than 1 nM, or even lower IC ₅₀ inhibition.

As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also called "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and usually have a specific three-dimensional structure and specific charge characteristics. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, eds. (1996).

As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component occurs naturally, it may be because it has changed in nature, or the substance has been separated from nature, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a certain moving object, and the same high-purity polynucleotide or polypeptide isolated from the natural state is called an isolated polynucleotide or polypeptide. The term "isolated" neither excludes mixed man-made or synthetic materials, nor does it exclude other impure materials that do not affect the activity of the separated materials.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the PD-L1/VEGF protein is substantially free of specific binding Antibodies to antigens other than PD-L1/VEGF protein). However, isolated antibodies that specifically bind to human PD-L1/VEGF protein may have cross-reactivity with other antigens, such as PD-L1/VEGF protein from other species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemical substances.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector allows the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can be transformed, transduced or transfected into the host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC); bacteriophages such as lambda phage Or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papilloma virus, and milk Long-short virus (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication.

As used herein, the term "host cell" refers to a cellular system that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs or hamsters), such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or Hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only the specific test cell, but also the progeny of this cell. Certain modifications may occur in the offspring due to mutations or environmental influences. Such offspring may be different from the parent cell, but are still included in the scope of the term "host cell".

As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by alignment and comparison of sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps (if any) in the comparison are preferably addressed by specific mathematical models or computer programs (ie "algorithms"). Methods that can be used to calculate the identity of nucleic acids or polypeptides for comparison are included in Computational Molecular Biology, (Lesk, AM Ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW Ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (edited by Griffin, AM and Griffin, HG), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J. eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48: 1073 Of those.

As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the properties of antigens that stimulate specific immune cells to activate, proliferate and differentiate to ultimately produce immune effect substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes to stimulate organisms with antigens. It is then formed in the organism's immune system. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunity.

As used herein, the term "transfection" refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. The protocols and techniques used for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13 : 197. In a specific embodiment of the present invention, the human PD-L1/VEGF gene is transfected into 293F cells.

As used herein, the term "SPR" or "Surface Plasma Resonance" refers to and includes an optical phenomenon that allows the analysis of instant biospecific interactions by detecting changes in protein concentration within the matrix of the biosensor, for example, using the BIAcore system ( Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For detailed description, see Examples and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jönsson, U., et al. (1991) Biotechniques 11: 620-627; Johnson, B ., Et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time according to the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting" . 2017-11-09). The instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, CA), Epics C from Coulter Epics Division (Hialeah, FL), and MoFlo from Cytomation (Colorado Springs, Colorado).

The term "subject" includes any human or non-human animal, preferably a human.

As used herein, the term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis mediated solid tumor and non-solid tumor such as leukemia that causes a medical condition.

The terms "treatment", "treatment" and "treatment" used herein in the context of the treatment of a condition generally refer to the treatment and therapy of humans or animals, in which some desired therapeutic effects are achieved, for example, inhibition of the progression of the condition, including a decrease in the rate of progression , The progress rate is stagnant, the condition subsides, the condition improves and the condition is cured. It also includes treatment as a preventive measure (ie prevention). For cancer, "treatment" may mean inhibiting or slowing the growth, proliferation or metastasis of tumor or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the development of the tumor, or some combination thereof.

As used herein, the term "effective amount" refers to the amount of the active compound or the amount of the material, composition, or dose containing the active compound, which is effective to produce a reasonable benefit/risk ratio when administered in accordance with the desired treatment regimen. Some of the desired therapeutic effects. For example, when used in combination with the treatment of CD3/CD20 related diseases or disorders, "effective amount" refers to the amount or concentration of the antibody or antigen binding portion thereof effective to treat the disease or disorder.

As used herein, the terms "prevent", "prevent" or "prevent" in relation to a certain disease condition in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, forming agent, and/or its salt is chemically and/or physically compatible with other ingredients in the formulation, and is physiologically compatible with the recipient. Allow.

As used herein, the term "pharmaceutically acceptable carrier and/or forming agent" refers to a carrier and/or forming agent that is pharmacologically and/or physiologically compatible with the subject and the active agent. Is well-known (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th edition, Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancement Agent. For example, pH adjusting agents include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a non-specific immune enhancer, which can enhance the immune response to the antigen or change the immune response in the organism when it is delivered to the organism together with the antigen or is delivered to the organism in advance. type. There are many adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Coryne bacterium parvum, lipid Polysaccharides, cell factor, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials. Bispecific antibodies and their antigen binding parts

In certain embodiments, the antibodies and antigen binding portions thereof provided herein are bispecific. In some embodiments, the antibodies and antigen-binding portions thereof provided herein have a first specificity for PD-L1 and a second specificity for VEGF.

According to certain exemplary embodiments, the present disclosure includes a bispecific antibody or an antigen-binding portion thereof, which includes a first antigen-binding module that specifically binds to PD-L1 and a second antigen-binding module that specifically binds to VEGF . Such antibodies may be referred to herein as, for example, "anti-VEGF/anti-PD-L1" or "anti-PD-L1/VEGF" or "anti-PD-L1xVEGF" or "PD-L1xVEGF" bispecific antibodies, or other similar Naming.

The bispecific antibody of the present disclosure can bind human PD-L1 and human VEGF with high affinity. The binding of the antibodies of the present disclosure to PD-L1 or VEGF can be assessed using one or more techniques established in the art (such as ELISA). The binding specificity of the antibody of the present disclosure can also be determined by monitoring the binding of the antibody to cells expressing PD-L1 protein or VEGF protein (for example, flow cytometry). For example, the antibody can be tested by a serial flow cytometry assay in which the antibody is reacted with a cell line expressing human PD-L1, such as a CHO cell transfected to express PD-L1 on its cell surface. Additionally or alternatively, binding assays may be tested in an antibody BIAcore, including binding kinetics (e.g., K _D value). Other suitable binding assays include ELISA or FACS assays, for example recombinant PD-L1 protein can be used.

For example, the present disclosure antibody 1 × 10 ^-7 M K _D or less, 5 × 10 ^-8 M or less K _D, 2 × 10 ^-8 M or less K _D, 1 × 10 ^{- 8} M or lower K _D , 5×10 ^-9 M or lower K _D , 4×10 ^-9 M or lower K _D , 3×10 ^-9 M or lower K _D , 2× ^{Combination of} 10 -9 M or lower K _D , 1×10 ^-9 M or lower K _D , 5×10 ^-10 M or lower K _D , 1×10 ^-10 M or lower K _D Human PD-L1 protein or human VEGF protein, as measured by surface plasmon resonance.

As verified in the examples, the bispecific antibody of the present disclosure can bind human PD-L1 and human VEGF (same as cynomolgus monkey VEGF) with higher affinity; bind cynomolgus monkey and mouse PD-L1; effectively inhibit off the PD-1 / PD-L1 and VEGFR / VEGF pathway two different signals, for example, nM level IC _50; block VEGF induced HUVEC proliferation; and produce a strong agonist effect on the factor-secreting cells. PD-L1 antigen binding module

The PD-L1 antigen binding module as defined herein can have various forms (for example, VHH, scFv, Fab), as long as it can specifically bind to the antigen. Generally, the PD-L1 binding module contained in the bispecific antibody is derived from a monospecific anti-PD-L1 antibody, which may be an antibody known in the art or a newly developed antibody. In some embodiments according to the present application, the PD-L1 antigen binding module may be derived from a parent single domain antibody (sdAb), such as a VHH antibody, which generally refers to an antibody composed of a single antibody variable domain. Similar to intact antibodies, single domain antibodies can also selectively bind to specific antigens. In some other embodiments, the PD-L1 antigen binding module can be derived from heavy chain antibodies without light chains.

The term "single variable domain" or "heavy chain variable region of a heavy chain antibody" and the terms "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "V _HH " or "nanometre "Antibody" can be used interchangeably. _{The V HH} molecule derived from camelid antibodies is the smallest known complete antigen binding domain (about 15 kDa, or 10 times smaller than conventional IgG), so it is very suitable for delivery to dense tissues and into the limited space between large molecules .

The parental VHH antibodies disclosed herein can be prepared by a skilled person according to methods known in the art or any future method. For example, VHH can be obtained using methods known in the art, for example, by immunizing camels and obtaining hybridomas therefrom, or by using molecular biology techniques known in the art to select a library of VHH of the present invention, and then by using phage display. choose.

For example, VHH antibodies can be obtained by immunizing alpaca or llama with the desired antigen, and then isolating the mRNA encoding the heavy chain antibody. Through reverse transcription and polymerase chain reaction, a gene library containing millions of cloned single domain antibodies was generated. Screening techniques such as phage display and ribosome display, for example, can be used to help identify colonies that bind antigen. In phage display, a library of (for example, human) antibodies is synthesized on phage, the library is screened with the target antigen or its antibody binding portion, and the phage that binds to the antigen is isolated to obtain immune response fragments. Methods of preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP ^™ Phage display kit, catalog number 240612). There are other methods and reagents that can be used to generate and screen antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991)).

The humanization of VHH antibodies can be achieved by many established methods in the art. For example, the amino acid sequence of the VHH framework region can be blasted against the human germline V gene database, and according to the Kabat CDR definition, the high-hit sequence can be defined by The human CDR sequence is replaced with a VHH CDR sequence to generate a humanized VHH sequence. In addition, certain residues in the framework region can be backmutated to maintain affinity.

In some embodiments, the PD-L1 antigen binding module includes one or more CDRs selected from the following group: (i) CDR1 containing SEQ ID NO: 1 or an amino acid sequence of amino acid additions, deletions, or substitutions that differ from SEQ ID NO: 1 by no more than 2 amino acids; (ii) CDR2 comprising SEQ ID NO: 2 or an amino acid sequence of amino acid additions, deletions, or substitutions that differ from SEQ ID NO: 2 by no more than 2 amino acids; and (iii) CDR3 containing SEQ ID NO: 3 or an amino acid sequence of amino acid addition, deletion, or substitution that differs from SEQ ID NO: 3 by no more than 2 amino acids.

In some embodiments, the PD-L1 antigen binding module is a VHH antibody, which comprises: (i) CDR1 comprising or consisting of SEQ ID NO: 1; (ii) comprising or consisting of SEQ ID NO: 2 CDR2; and (iii) CDR3 comprising SEQ ID NO: 3 or consisting of it.

In some embodiments, the VHH of the PD-L1 antigen binding module comprises: (i) the amino acid sequence of SEQ ID NO: 10; (ii) at least 85%, 90%, or 95% identical to SEQ ID NO: 10 Amino acid sequence; or (iii) Compared with SEQ ID NO: 10, it has one or plural (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or The substituted amino acid sequence. VEGF antigen binding module

Similarly, the VEGF antigen binding modules provided herein can be derived from anti-VEGF monospecific antibodies. In some embodiments according to the present application, the VEGF antigen binding module is a Fab fragment of an anti-VEGF complete antibody, that is, the VH and CH1 regions containing the heavy chain, and the VL and CL regions containing the light chain.

The anti-VEGF complete antibody used as the parent antibody may be a monoclonal antibody known in the art (for example, bevacizumab) or a newly developed one. Preferably, the anti-VEGF antibody is a fully human antibody or a humanized antibody.

In some embodiments, the VH region of the VEGF antigen binding module comprises one or more heavy chain CDRs (HCDR) selected from the following group: (i) HCDR1 composed of SEQ ID NO: 4 or HCDR1 whose amino acid sequence differs from SEQ ID NO: 4 by no more than 2 amino acids with amino acid additions, deletions, or substitutions; (ii) HCDR2 composed of SEQ ID NO: 5 or HCDR2 whose amino acid sequence differs from SEQ ID NO: 5 by no more than 2 amino acids; and (iii) HCDR3 composed of SEQ ID NO: 6 or HCDR3 whose amino acid sequence differs from SEQ ID NO: 6 by no more than 2 amino acids with amino acid additions, deletions, or substitutions; and/or The VL region contains one or more light chain CDRs (LCDR) selected from the following group: (i) LCDR1 composed of SEQ ID NO: 7 or LCDR1 whose amino acid sequence differs from SEQ ID NO: 7 by no more than 2 amino acids; (ii) LCDR2 composed of SEQ ID NO: 8 or LCDR2 whose amino acid sequence differs from SEQ ID NO: 8 by no more than 2 amino acids with amino acid additions, deletions, or substitutions; and (iii) LCDR3 composed of SEQ ID NO: 9 or LCDR3 whose amino acid sequence differs from SEQ ID NO: 9 by no more than 2 amino acids added, deleted, or substituted.

In some embodiments, the VH comprises: (i) HCDR1 comprising or consisting of SEQ ID NO: 4; (ii) HCDR2 comprising or consisting of SEQ ID NO: 5; and (iii) comprising SEQ ID NO : 6 or HCDR3 consisting of it; and the VL comprises: (i) LCDR1 comprising or consisting of SEQ ID NO: 7; (ii) LCDR2 comprising or consisting of SEQ ID NO: 8; and (iii) Contains SEQ ID NO: 9 or LCDR3 consisting of it.

In some embodiments, the VH of the VEGF antigen binding module comprises: (i) the amino acid sequence of SEQ ID NO: 11; (ii) the amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11 Or (iii) Compared with SEQ ID NO: 11, there are one or plural (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or substitutions Amino acid sequence.

In some embodiments, the VL of the VEGF antigen binding module comprises: (i) the amino acid sequence of SEQ ID NO: 12; (ii) the amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 12 Or (iii) Compared with SEQ ID NO: 12, there are one or plural (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or substitutions Amino acid sequence.

Unless otherwise stated, the assignment of amino acids to each CDR can be based on one of the numbering schemes provided below: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th edition), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel edited (2007) Handbook of Therapeutic Antibodies, 3rd edition, Wily-VCH VerVEGF GmbH and Co..

The variable regions and CDRs in the antibody sequence can be identified according to general rules that have been developed in the art (as described above, such as the Kabat numbering system) or by aligning the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, Eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. An exemplary database of antibody sequences is described and available from the "Abysis" website on www.bioinf.org.uk/abs (maintained by AC Martin, Department of Biochemistry & Molecular Biology University College London, London, England) and VBASE2 Website www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). It is better to use the Abysis database for sequence analysis, which integrates sequence data from Kabat, IMGT and protein database (PDB) and structure data from PDB, see Protein Sequence and Structure Analysis in the book by Dr. Andrew CR Martin of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-VerVEGF, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk/abs) . The Abysis database website also includes general rules that have been developed to identify CDRs that can be used in accordance with the teachings of this article. Unless otherwise stated, all CDRs described in this article are obtained from Kabat's Abysis database website.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program ( Version 2.0), using the PAM120 weight residue table, the gap length penalty is 12, and the gap penalty is 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GCG software package (available from http: //www.gcg.com) In the GAP program, use Blossum 62 matrix or PAM250 matrix, the gap weight is 16, 14, 12, 10, 8, 6 or 4, and the length weight is 1, 2, 3, 4 , 5, or 6.

Additionally or alternatively, the protein sequence of the present invention can be further used as a "query sequence" to perform searches against public databases to, for example, identify related sequences. This search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. MoI. Biol. 215:403-10. XBLAST program can be used to perform BLAST protein search, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecule of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using BLAST and Gap BLAST programs, the default parameters of each program (for example, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

In other embodiments, the CDR amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% to the specific CDR amino acid sequence contained in the above-mentioned corresponding sequence. % Or 99% are the same. In other embodiments, the amino acid sequence of the variable region may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence described above.

Preferably, the CDR of the isolated antibody or antigen-binding portion thereof contains conservative substitutions of no more than 2 amino acids or no more than 1 amino acid. The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein/polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue with a similar side chain, such as a residue that is physically or functionally similar (for example, having similar size, shape, charge, chemical properties, including the formation of covalent bonds or The ability of hydrogen bonding, etc.) to the substitution of the corresponding amino acid residue. A family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), and uncharged polar sides Chain of amino acids (such as glycine, aspartamide, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) ) And amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999) ); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which is incorporated herein by reference). Production of bispecific antibodies

In order to construct an anti-PD-L1/VEGF bispecific antibody, the PD-L1 antigen binding module and the VEGF antigen binding module described above can be fused together in various forms. In certain embodiments, the PD-L1 antigen binding module is fused to the N-terminus of the VEGF antigen binding module. When the PD-L1 antigen-binding module is VHH, the single chain of the PD-L1 antigen-binding module can be operably connected to the heavy chain or the light chain of the VEGF antigen-binding module, optionally via a linker. Preferably, the PD-L1 antigen binding module is connected to the light chain of the VEGF antigen binding module. The linker may be a peptide linker, for example, it may contain 1-4 copies of GGGGS (G4S). In one embodiment, the linker is (G4S)2.

The bispecific antibodies and antigen-binding portions thereof provided herein can be prepared using any suitable method known in the art. A conventional method is to co-express two immunoglobulin heavy chain-light chain pairs in host cells to produce bispecific antibodies in a recombinant manner (see, for example, Milstein and Cuello, Nature, 305:537 (1983)), and then use Purification by affinity chromatography. The sequence encoding the heavy chain variable domain of the antibody against the two specificities can be fused with the immunoglobulin constant domain sequence, and then inserted into the expression vector, and the expression vector and the expression vector of the light chain sequence can be co-transfected into a suitable Host cells to recombinantly express bispecific antibodies (see, for example, WO 94/04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Fc region

In certain embodiments, the bispecific antibody comprises an Fc region operably linked to a VEGF antigen binding module. The Fc region of the bispecific antibody disclosed herein may be a human IgG Fc region. The IgG Fc region can have any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the Fc region is of the IgG1 isotype.

In the context of the bispecific antibody of the present disclosure, the Fc region may contain one or more amino acid changes (for example, insertions, deletions, or substitutions) compared with the specified chimeric form of the Fc region. For example, the present disclosure includes bispecific antigen binding molecules comprising one or more modifications in the Fc region, which results in a modified Fc region with modified binding interactions between Fc and FcRn or FcγR.

When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is usually used (for example, as reported in Kabat et al., see EU index above). "As in the EU number in Kabat" or "as in the EU index in Kabat" refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbering in the constant domain of an antibody refer to residue numbering through the EU numbering system.

In certain embodiments, the Fc region is operably linked to the VEGF antigen binding module via the hinge region. Optionally, the hinge region may be derived from human IgG1, IgG2, or IgG4. In certain embodiments, the hinge region is derived from human IgG1. Nucleic acid molecule encoding the disclosed antibody

In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a bispecific antibody or antigen binding portion thereof as disclosed herein. For example, the nucleic acid sequence may encode the heavy chain and/or light chain of a bispecific antibody, or the nucleic acid sequence may encode the variable region of the heavy chain or light chain of the PD-L1 antigen binding module, or the VEGF antigen binding module. The nucleic acid sequence can also encode the Fc region of a bispecific antibody.

In some aspects, the present disclosure relates to a vector comprising a nucleic acid sequence encoding as disclosed herein. In some embodiments, the expression vector further comprises a nucleotide sequence encoding the constant region of a bispecific antibody, such as a chimeric or humanized bispecific antibody.

In the case of the present invention, the download body can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences containing a set of suitable expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the CD20 or CD3 antibody-encoding nucleic acid is contained in a naked DNA or RNA vector, including, for example, linear expression elements (described in, for example, Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), compact nucleic acid vectors (Described in, for example, US 6,077, 835 and/or WO 00/70087), plasmid vectors such as pBR322, pUC 19/18 or pUC 118/119, "midge" minimum size nucleic acid vectors (described in, for example, Schakowski et al., Mol Ther 3, 793-800 (2001)) or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (described in, for example, WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978) and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

In one embodiment, the vector is suitable for expressing anti-PD-L1/VEGF bispecific antibodies in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison WI), etc.). The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al. Eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York ( 1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

The expression vector may also or alternatively be a vector suitable for expression in mammalian cells, for example, a vector containing glutamine synthetase as a selectable marker, as described in, for example, Bebbington (1992) Biotechnology (NY) 10:169- 175 in the carrier.

The nucleic acid and/or vector may also include a nucleic acid sequence encoding a secretion/localization sequence that can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or cell culture medium. Such sequences are known in the art and include secretion leader or signal peptides.

The expression vector may contain or be associated with any suitable promoters, enhancers and other expression promoting elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly(A) termination sequences, in E. coli The origin of replication of the plasmid product in the medium, the antibiotic resistance gene as a selectable marker, and/or a convenient selection site (for example, a polymer linker). The nucleic acid may also comprise an inducible promoter as opposed to a constitutive promoter, such as CMV IE.

In yet another aspect, the present disclosure relates to a host cell containing the vector described herein. Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the bispecific antibodies of the present invention.

PD-L1-specific antibodies can be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, which produce the antibodies of the invention as defined herein, or the bispecific antibodies of the invention as defined herein. VEGF-specific antibodies can likewise be expressed in recombinant eukaryotic or prokaryotic host cells, such as transfectomas, which produce the antibodies of the invention as defined herein or the bispecific antibodies of the invention as defined herein.

Examples of host cells include yeast, bacteria, plant and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells or lymphocytes. For example, in one embodiment, the host cell may comprise first and second nucleic acid constructs stably integrated into the genome of the cell. In another embodiment, the invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid or linear expression element, which comprises the first and second nucleic acid constructs described above.

In another aspect, the present invention relates to a transgenic non-human animal or plant comprising a nucleic acid encoding one or two sets of human heavy chains and human light chains, wherein the animal or plant produces the bispecific antibody of the present invention.

In yet another aspect, the present disclosure relates to hybridomas that produce antibodies for the bispecific antibodies of the present disclosure as defined herein.

In one aspect, the invention relates to an expression vector comprising (i) Nucleic acid sequence, which encodes the PD-L1 antigen binding module; (ii) Nucleic acid sequence, which encodes the heavy chain and/or light chain of the VEGF antigen binding module; (iii) Nucleic acid sequence, which encodes the Fc region; or (iv) Nucleic acid sequence, which encodes the heavy chain and/or light chain of a bispecific antibody.

A nucleic acid sequence that encodes the heavy chain and/or light chain of a bispecific antibody.

In one aspect, the present disclosure relates to a method for producing a bispecific antibody according to any of the embodiments disclosed herein, comprising culturing the host cell disclosed herein containing one or more expression vectors expressing the bispecific antibody, and purifying the bispecific antibody from the culture medium. antibody. In one aspect, the invention relates to a host cell comprising an expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell. Pharmaceutical composition

In some aspects, the present invention relates to a pharmaceutical composition comprising at least one bispecific antibody or antigen binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier. Components of the composition

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention can also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine, so that the anti-PD-L1/anti-VEGF bispecific antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, Chelating agents, diluents, adjuvants, forming agents or non-toxic auxiliary substances, combinations of various components known in the art or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins . Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methylbenzene Methyl ether, butylated hydroxytoluene and/or propyl arsenate. As disclosed in the present invention, one or more antioxidants such as methionine containing reduced antibodies or antigen-binding fragments thereof can be oxidized in a solvent containing antibodies or antigen-binding fragments of the composition disclosed in the present invention. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the invention provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides methods in which the antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine. Thus, the antibody or its antigen-binding fragment can be prevented from oxidation, so as to extend its shelf life and/or increase its activity.

For further explanation, the pharmaceutically acceptable carrier may include, for example, an aqueous carrier, such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's Injections, non-aqueous carriers such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents with antibacterial or fungicidal concentration, isotonic agents such as sodium chloride or glucose, buffers such as phosphate Or citrate buffer, antioxidant such as sodium bisulfate, local anesthetic such as procaine hydrochloride, suspending agent and dispersing agent such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose or polyvinylpyrrolidone, emulsifier Such as polysorbate 80 (TWEEN-80), insulating or chelating agents such as EDTA (ethylene diamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethylene glycol, polyethylene glycol, propylene glycol, sodium hydroxide, Hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to contain phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methyl paraben and propyl paraben, thimerosal, benzalkonium chloride and benzethon chloride Ammonium in a pharmaceutical composition in a multi-dose container. Suitable forming agents may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin . Administration, formulation and dosage

The pharmaceutical composition of the present invention can be administered in vivo to subjects in need through various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, and intracardiac , Intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or by implantation or inhalation. The composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants And aerosol. A suitable formulation and route of administration can be selected according to the intended application and treatment plan.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and their controlled release dosage forms.

Formulations suitable for parenteral administration (for example by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, active ingredients dissolved, suspended or otherwise provided (for example, in liposomes or other microparticles). Sterile liquid (eg solution, suspension). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and make the preparations compatible with the blood (or other related body fluids) of the intended recipient. ) Isotonic solute. Examples of the forming agent include, for example, water, alcohol, polyol, glycerin, vegetable oil, and the like. Examples of suitable isotonic vehicles for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosage regimen (including dose, time, and repetition) will depend on the individual and individual's medical history and empirical considerations such as pharmacokinetics (eg half-life, clearance rate, etc.).

The frequency of administration can be determined and adjusted during the course of treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing tumor cell proliferation or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

Those skilled in the art will understand that the appropriate dosage may vary from patient to patient. Determining the optimal dose usually involves balancing the level of therapeutic benefit with any risks or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the particular compound, administration, time of administration, compound clearance rate, duration of treatment, other drugs, compounds and/or materials used in combination, and severity of the disorder, As well as the species, the patient’s gender, age, weight, medical condition, general health and previous medical history. The amount of the compound and the route of administration are ultimately determined by the doctor, veterinarian, or clinician, but the dosage is usually selected to achieve the local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

Generally, the antibody or antigen-binding portion thereof of the present invention can be administered in various ranges. These include about 5 μg/kg body weight to about 100 mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kg body weight per dose; and about 100 μg/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 μg/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, each dose is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight.

In any case, the antibody or antigen-binding portion thereof of the present invention is preferably administered to a subject in need as needed. Those skilled in the art can determine the frequency of administration, for example, the attending physician considers based on the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health condition of the subject being treated, and the like.

In certain preferred embodiments, the course of treatment involving the antibody or antigen binding portion thereof of the present invention will involve multiple doses of the selected drug product administered over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present invention can be used every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two Administer every month, every ten weeks, or every three months. In this regard, it is understandable that the dose can be changed or the interval adjusted based on patient response and clinical practice.

The dosage and regimen of the disclosed therapeutic composition can also be determined empirically in individuals who are administered one or more administrations. For example, an individual can be administered incremental doses of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or decreased or reduced based on empirically determined or observed side effects or toxicity, respectively. In order to assess the efficacy of the selected composition, the markers of a particular disease, disorder or condition can be tracked as previously described. For cancer, these include direct measurement of tumor size through palpation or visual observation, indirect measurement of tumor size through X-ray or other imaging techniques; improvement of evaluation through direct tumor biopsy and microscopic examination of tumor samples; measurement according to the methods described herein Identified indirect tumor markers (such as PSA for prostate cancer) or tumorigenic antigens, reduction of pain or paralysis; improvement of speech, vision, breathing, or other disability related to the tumor; increase in appetite; or through acceptance The improvement of the quality of life or the extension of the life span as measured by the test. Those skilled in the art will understand that the dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and the treatments used in the past and concurrent treatments.

A compatible formulation for parenteral administration (eg, intravenous injection) will contain the antibodies or antigen binding portions thereof disclosed herein at a concentration of about 10 μg/ml to about 100 mg/ml. In certain selected embodiments, the concentration of the antibody or antigen-binding portion thereof will include 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg / μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml or 1 mg/ml. In other preferred embodiments, the concentration of the antibody or its antigen-binding portion will include 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg ml, 16 mg/ml, 18 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml. Application of the invention

In some aspects, the present invention provides a method of treating a condition in a subject, which comprises administering a therapeutically effective amount of an antibody or antigen-binding portion thereof as disclosed herein to a patient (eg, a human) in need of treatment. For example, this disease is a type of cancer.

The methods provided in the present disclosure can be used to treat or prevent a variety of cancers involving PD-L1 and/or VEGF, whether they are malignant or benign, and whether they are primary or secondary. These cancers can be solid cancers or hematological malignancies. Examples of these cancers include lung cancers such as bronchial carcinoma (such as squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, cartilage hamartoma (non-cancerous), and sarcoma (carcinoma Sex); Heart cancers such as myxoma, fibroids, and rhabdomyomas; bone cancers such as osteochondroma, chondroma, chondroblastoma, chondroid chondroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple bone marrow Tumors, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing’s tumor (Ewing’s sarcoma), and reticulocyte sarcoma; brain cancers such as gliomas (such as glioblastoma multiforme), anaplastic stars Form cell tumor, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, angioblastoma Cell tumors, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts and hemangiomas; cancers in the digestive system such as colon cancer, leiomyomas, epidermoid carcinomas, adenocarcinomas, leiomyosarcomas, gastric glands Carcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyps and colorectal cancer; liver cancer such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolaminoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma ; Kidney cancer such as renal adenocarcinoma, renal cell carcinoma, high adrenal adenoma and transitional cell carcinoma of the renal pelvis; bladder cancer; blood system cancers such as acute lymphocytic leukemia (acute lymphocytic leukemia), acute myeloid (myeloid, bone marrow) , Myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (such as Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) lymphoma, Huo Chikin’s lymphoma, non-Hodgkin’s lymphoma, B-cell lymphoma, mycosis fungoides and myeloproliferative diseases (including myeloproliferative diseases such as polycythemia vera, myelofibrosis, thrombocytosis, and chronic granulocytes) Leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi’s sarcoma and Paget’s disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma Cancer; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer and testicular cancer (eg seminoma, teratoma, embryonic carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer) ), cervical cancer (cervical tumor), ovarian cancer (ovarian tumor), vulvar cancer, vaginal cancer, fallopian tube cancer and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic or medullary cancer); chromaffin cells Tumors (adrenal glands); noncancerous growths of parathyroid glands; pancreatic cancer; and hematological cancers such as leukemia, myeloma, non-Hodgkin’s lymphoma, and Hodgkin’s lymphoma. In a specific embodiment, the cancer is colon cancer.

In some embodiments, examples of cancers include, but are not limited to, B-cell carcinomas, including B-cell lymphomas (including low-grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytes (SL) NHL; intermediate/ Follicular NHL; Intermediate-grade diffuse NHL; Immunoblast NHL; High-grade lymphoblastic NHL; High-grade small non-cutting cell NHL; Massive disease NHL; Mantle cell lymphoma; AIDS-related lymphoma; Waldenstedt Renal macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disease (PTLD), and related to keloid Abnormal vascular proliferation, edema (for example, related to brain tumors), B cell proliferative disorders and Meigs' syndrome. More specific examples include but are not limited to relapsed or refractory NHL, front-line low-grade NHL, III/IV grade NHL, Chemoresistant NHL, precursor Bly lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or young lymphocytic leukemia and/or small lymphocytic lymphoma , B-cell young lymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, Lymph node marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low-grade/follicular lymphoma, intermediate/follicular NHL, mantle cell lymphoma, follicular center lymphoma (including aggressive Frontline NHL and aggressive recurrent NHL), relapsed or recurred NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, high-grade Immunoblast NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, giant disease NHL, Burkitt’s lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or Sezari syndrome, skin (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

In some embodiments, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphoma (eg, B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma; b) small undivided cell lymphoma/Burkitt’s lymphoma (including endemic Burkitt’s lymphoma, sporadic Burkitt’s lymphoma) Tumor and non-Burkitt lymphoma); c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma ); d) Mantle cell lymphoma (MCL); e) Large cell lymphoma (including B cell diffuse large cell lymphoma (DLCL) cell lymphoma, immunoblastic lymphoma, and primary mediastinal B cell lymphoma , Angiocentric Lymphoma-Lung B-cell Lymphoma); f) Hairy cell leukemia; g) Lymphocytic lymphoma, Waldenstrom's macroglobulinemia; h) Acute lymphocytic leukemia (ALL), chronic lymphoma Cell leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell juvenile lymphocytic leukemia; i) plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytoma and/or j) Hodge King's disease.

In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with antibodies or antigen binding portions thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. Antibodies or antigen-binding portions thereof can also be used to treat or prevent infectious diseases, inflammatory diseases (such as allergic asthma) and chronic graft-versus-host disease. Used in combination with chemotherapy

The antibody or antigen binding portion thereof can be used in combination with an anticancer agent, a cytotoxic agent, or a chemotherapeutic agent.

The term "anti-cancer agent" or "anti-proliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapy Agents, targeted anti-cancer agents, BRM, therapeutic antibodies, cancer vaccines, cytokine, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutics. It should be understood that in selected embodiments as described above, such anti-cancer agents may comprise a conjugate and may bind to the disclosed site-specific antibody prior to administration. More specifically, in some embodiments, the selected anticancer agent is linked to the unpaired cysteine of the engineered antibody to provide the engineered conjugate as described herein. Therefore, such engineered conjugates are clearly considered within the scope of the present invention. In other embodiments, the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, bacteria (e.g., diphtheria toxin, pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A), fungi (e.g., sarcin) , Restricted toxin (restrictocin), plants (acacia soytoxin, ricin, capsula root toxin, quercetin, pokeweed antiviral protein, saponin, gelonin, momoridin, trichosanthin, hordein , Aleurites fordii protein, caryophyllin protein, Phytolacca mericana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, Jatropha toxin protein, croton toxin, Saponaria officinalis inhibitor Agent, gelonin, mitigellin, limited aspergillin, phenomycin, neomycin and trichothecenes) or animal (for example, cytotoxic RNAse, such as extracellular pancreatic RNAse; DNAse I, including its Fragments and/or variants) of small molecule toxins or enzymatically active toxins.

For the purpose of the present invention, "chemotherapeutic agents" include chemical compounds (such as cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemicals generally target intracellular processes required for cell growth or division, and are therefore particularly effective for cancer cells that usually grow and divide rapidly. For example, Changchunxin depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become sexual or produce tumorigenic offspring (eg, TIC). These agents are usually used in combination and are usually the most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anticancer agents (as a component of a site-specific conjugate or in an unconjugated state) that can be used in combination with the site-specific construct of the present invention include, but are not limited to, alkylating agents, alkyl sulfonates , Aziridine, ethyleneimine and methylmelamine, polyacetogenins, camptotheca, bryostatin, callistatin (callystatin), CC-1065, cryptophycins, do Lastatin, docarmycin, eleutherobin, sarcodictyin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphines Acid salt, espotomycin, chromophore of pigment protein enediyne antibiotic, aclacinomysins, actinomycin, antoxomycin, azoserine, bleomycin, actinomycin C, carla Carabicin, carmine, oncinomycin, chromomycinis, dactinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L- ^{Norleucine, ADRIAMYCIN ®} doxorubicin, epirubicin, esorubicin, idarubicin, methiromycin, mitomycin, mycophenolic acid, nogamycin, olivemycin, Pelomycin, potfiromycin (potfiromycin), puromycin, triiron doxorubicin, rhodoubicin, streptozotocin, streptozotocin, tuberculin, ubiquitin, jingsi Statin, Zorubicin; Anti-metabolite, Erlotinib, Verofenib, Crizotinib, Sorafenib, Ibrutinib, Enzalutamide, Folic Acid Analogs, Purine Analogs , Androgens, anti-adrenaline, folic acid supplements such as frolinic acid (frolinic acid), acetoglucurolactone, aldophosphamide glycosides, aminoacetyl propionic acid, enuracil, amsacrine, Besbusch (Bestrabucil), bisantrene, edatrexa, defofamine, colchicine, diacrquinone, elfornithine, ammonium acetonide, eboxilone, etoglu, gallium nitrate , Hydroxyurea, lentinan, clonidamine, maytansinoids, mitogen hydrazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, methionine Mustard, Pirubicin, Losoxantrone, Podophyllic Acid, 2-Ethylhydrazine, Procarbazine, PSK® Polysaccharide Complex (JHS Natural Products, Eugene, OR), Razosan; Rhizomycin; Sizo Pyran; germanium spiroamine; tinuzoric acid; triimine quinone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A ( verracurin A), bacillus sporine A, and stilbine); urethane; vindesine; dacarbazine; mannitol Stine; Dibromomannitol; Dibromodulcol; Papporphan; Gacytosine; Arabinoside ("Ara-C");Cyclophosphamide;Thiotepa; Taxanes Chlorambucil (chloranbucil); GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; Mitoxantrone; Vinorelbine, NAVELBINE® Vinorelbine; Nuoxiaolin; Teniposide; Edartrexa; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Iriti Kang (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine; retinoid; capecitabine; combstatin; methytetrahydrofolate; oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents used to modulate or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, which inhibit the aroma of aromatase that regulates estrogen production in the adrenal glands. Enzyme inhibitors, and anti-androgens; and troxatabine (1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 Expression inhibitor; vaccine, PROLEUKIN ^® rIL-2; LURTOTECAN ^® topoisomerase 1 inhibitor; ABARELIX ^® rmRH; vinorelbine and espermycin, and any of the above pharmaceutically acceptable salts, acids or derivative. Used in combination with radiation therapy

The present invention also provides antibodies or antigen-binding portions thereof and radiotherapy (ie, any mechanism used to locally induce DNA damage in tumor cells, such as γ-irradiation, X-ray, UV-irradiation, microwave, electron emission, etc.) The combination. Combination therapy using targeted delivery of radioisotopes to tumor cells is also considered, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted means. Generally, radiation therapy is administered in pulses over a period of about 1 week to about 2 weeks. Radiation therapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy can be administered as a single dose or as a plurality of sequential doses. Drug packaging and kits

Pharmaceutical packages and kits containing one or more containers containing one or more doses of antibodies or antigen binding portions thereof are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding portion thereof, with or without one or more other agents. For other embodiments, this unit dose is supplied in a single-use pre-filled injection syringe. In other embodiments, the composition contained in the unit dose may include saline, sucrose, or the like; buffers, such as phosphate, etc.; and/or formulated in a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid (eg, sterile water or saline solution). In certain preferred embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with one or more containers indicates that the encapsulated composition is used to treat the selected neoplastic disease condition.

The present invention also provides kits for the production of antibodies and optionally single-dose or multi-dose administration units of one or more anticancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container may be formed of a variety of materials, such as glass or plastic, and contain a pharmaceutically effective amount of the disclosed antibody in conjugated or unconjugated form. In other preferred embodiments, one or more of the containers include a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). Such kits generally contain a pharmaceutically acceptable formulation of the antibody in a suitable container, and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for diagnosis or combination therapy. For example, in addition to the antibody or antigen-binding portion thereof of the present invention, such a kit may contain any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapeutics; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents. Cancer agents; cytotoxic agents; and/or other anti-cancer agents.

More specifically, the kits may have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without additional components, or they may have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or one component more than the other. Alternatively, the antibody of the kit and any optional anti-cancer agent may be stored separately in different containers before administration to the patient. The kit may also contain a second/third for containing sterile pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and glucose solution Container device.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit can be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent can also be provided in another container.

As briefly mentioned above, the kit may also contain tools for administering the antibody or its antigen-binding portion and any optional components to the patient, such as one or more needles, IV bags or syringes, or even eye drops, pipettes or Other similar devices, through which the preparation can be injected or introduced into the animal body or applied to the diseased area of the body. The kit of the present invention usually also includes a device for containing vials or the like and other tightly closed components for commercial sales, such as injection or blow-molded plastic containers, in which the required vials and other devices are placed and held. Overview of Sequence Listing

A sequence listing containing many amino acid sequences is attached to this application. Table A below provides an overview of the sequences included.

An exemplary anti-VEGF/anti-PD-L1 bispecific antibody as disclosed herein is called W3256-U15T2.G6-1.uIgG1 (can be abbreviated as W3256 throughout the specification). Table A SEQ ID NO. describe 1 The amino acid sequence of CDR1 of PD-L1 binding module 2 The amino acid sequence of CDR2 of PD-L1 binding module 3 The amino acid sequence of CDR3 of PD-L1 binding module 4 The amino acid sequence of the heavy chain CDR1 of the VEGF binding module 5 The amino acid sequence of the heavy chain CDR2 of the VEGF binding module 6 The amino acid sequence of the heavy chain CDR3 of the VEGF binding module 7 Amino acid sequence of light chain CDR1 of VEGF binding module 8 Amino acid sequence of light chain CDR2 of VEGF binding module 9 Amino acid sequence of light chain CDR3 of VEGF binding module 10 Amino acid sequence of VHH of PD-L1 binding module 11 Amino acid sequence of VH of VEGF binding module 12 Amino acid sequence of VL of VEGF binding module 13 Amino acid sequence of heavy chain of W3256 antibody 14 Amino acid sequence of light chain of W3256 antibody Example

The present invention generally described herein will be more easily understood by referring to the following examples, which are provided by way of illustration and are not intended to limit the present invention. These examples are not intended to show that the following experiments are all or only experiments performed. Example 1 Preparation of materials, reference antibodies and cell lines 1.1 Preparation of materials

Table 1 provides information on commercially available materials used in the examples. Material Sellers Item No. Expi293 expression system Thermo Fisher Scientific A14635 Protein A column GE healthcare 17-5438-02 Ni column GE healthcare 17-5247-01 HPLC-SEC TOSOH 0008541 NuPAGE 4%-12% Bis-Tris gel Invitrogen NP0322BOX Human VEGF Sino Biological 11066-HNAB Mouse VEGF Sino Biological 50159-MNAB Human VEGF, biotin labeled ACRO Biosystems VE5-H8210 Human VEGFR1, hFc tag Sino Biological 10136-H02H Mouse VEGFR1, hFc and his tags R&D 471-F1 Human PD-L1, His tag Sino Biological 10084-H08H Cynomolgus PD-L1, His tag Sino Biological 90251-C08H Mouse PD-L1, His tag Sino Biological 50010-M08H Goat anti-human IgG Fc-HRP antibody Bethyl A80-304P RPMI 1640 Gibco 22400-089 Ficoll-Paque Stemcell 07851 Monocyte Enrichment Kit MiltenyiBiotec 130-050-201 CD4 ⁺ T cell enrichment kit Stemcell 19052 Goat x-human IgG Fc FITC conjugation Bethyl A80-304F Recombinant human GM-CSF R&D 215-GM-010 Recombinant human IL-4 R&D 204-IL-010 LPS Sigma L5418 Recombinant human IL-2 standard R&D 202-IL-050 Recombinant human IFN standard PeproTech 300-02 Anti-hIL-2 purified mouse single clone IgG2A clone 5355 R&D MAB602 Anti-hIL-2 biotinylated goat IgG R&D BAF202 Human IFNγ Mab selection 2G1 Thermo prod M700A Human IFNγ Mab biotin labeling Thermo prod M701B Streptavidin HRP Invitrogen SNN1004 H3-thymidine and MicroScint Perkin Elmer NET027001MC Human serum complement Quidel A113 Fetal Bovine Serum (FBS) ExCell Bio FND500 Penicillin-Streptavidin (PS) Invitrogen SV30010 CellTiter-Glo Luminescent Cell Survival Assay Kit Promega G7573 1.2 Antigen preparation

Encoding human VEGF (Uniport code: P15692), mouse VEGF (Uniport code: Q00731) and human PD-L1 extracellular domain sequence (Uniport code: Q9NZQ7), mouse PD-L1 (Uniport code: Q9EP73), human The DNA sequences of PD-1 (Uniport code: Q15116) and mouse PD-1 (Uniport code: Q02242) were synthesized by Sangon Biothech (Shanghai, China), and then sub-cloned into the modified pcDNA3.3 expression vector. Different tags (for example, 6xhis, human Fc, or mouse Fc) at the end.

The purified expression vector was used to transfect Expi293 cells (Thermo Fisher Scientific, A14527). The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Ni-NTA column (GE Healthcare, 175248), a protein A column (GE Healthcare, 175438) or a protein G column (GE Healthcare, 170618). Human VEGF obtained by SDS-PAGE and the SEC, human PD-L1, mouse PD-L1, PD-1 human and mouse PD-1 for quality control, and then stored at -80 ^o C. 1.3 Production of benchmark antibody (BMK Ab)

A fragment encoding the anti-VEGF antibody bevacizumab (named WBP325-BMK3 or WBP325-BMK3.uIgG1, sequence from Drug Bank, Drug Bank number: DB00112) was synthesized in Sangon Biothech (Shanghai, China) or Genewiz (Suzhou, China) The DNA sequence was then subcloned together with the Fc region of human IgG1 into the modified pcDNA3.3 expression vector.

The anti-PD-L1 VHH antibody (W3156-AP3R2-1A3-z12) was generated by alternately immunizing alpaca with the extracellular domain of human PD-L1 and the extracellular domain of mouse PD-L1, and PBMC was isolated for phage library construction. After panning, screening and sequencing, a unique positive VHH fragment was identified (SEQ ID No: 4 disclosed in Patent Application No. PCT/CN2020/117351).

The anti-PD-L1 antibody Atezolizumab (named W315-BMK8.uIgG1K (RKNA) or abbreviated as W315-BMK8) developed by Roche was used as a control antibody.

To produce WBP325-BMK3, a plasmid containing recombinant VH and VL genes was co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a protein A column (GE Healthcare, 175438) or a protein G column (GE Healthcare, 170618). The antibodies obtained were analyzed by SDS-PAGE and SEC, and then stored at -80°C. 1.4 Establishment of stable cell lines/cell pools Cell lines expressing PD-L1

Using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) or PlasFect (Bioline, 46026), CHO-K1 or 293F cells were used for expression of genes encoding full-length human PD-L1 or mouse PD-L1 or cynomolgus PD-L1 Vector transfection. The cells are cultured in a medium containing appropriate selection markers. The human PD-L1 high-expressing stable cell line (WBP315.CHO-K1.hPro1.C11), the mouse PD-L1 high-expressing stable cell line (WBP315.293F.mPro1.C1) and the cynomolgus PD-L1 were obtained by limiting dilution Highly expressing stable cell line (WBP315.293F.cPro1.2A).

Insert the gene of human PD-L1 or mouse PD-L1 or cynomolgus PD-L1 into the expression vector pcDNA 3.3, respectively. The plasmids were then transfected into CHO-K1 cells or 293 cells, respectively. In short, for CHO-K1 cells, 5× ^{10 5} CHO-K1 cells were seeded into one well of a 6-well tissue culture plate one day before transfection, and incubated at 5% CO2 and 37°C. Use 3ml of fresh non-selective medium (F12-K, 10% FBS) to culture the cells. Prepare the transfection reagent in a 1.5 ml tube, including 4 μg DNA mixed with 10 μg Lipofectamine 2000, so that the final volume is 200 μl in Opti-MEM medium. Add the solution in the pipette dropwise to the cells. 6-8 hours after transfection, wash the cells with PBS and add 3 ml of fresh non-selective medium. 24-48 hours after transfection, the expressing cells were harvested with trypsin and inoculated into selective medium (F12-K, 10% FBS, 10 μg/ml blasticidin) in a T75 flask. For 293F cells, 20 μg DNA was mixed with 50 μl PlasFect to make the final volume 200 μl in Opti-MEM medium. This mixture was added to 20 ml (1×10 ⁶ /ml) of suspended 293F cells and cultured in Freestyle293 medium in a 125 ml flask. 48 hours after transfection, blasticidin was added as a selection marker. After selecting two or three times, the cells were detected with anti-PD-L1 antibody. Stable single cell colonies were isolated by limiting dilution and screened by FACS using anti-PD-L1 antibody. Target-expressing cell line

Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from ScienceCell (Cat. No.: 8000) and prepared in an endothelial cell culture medium (ECM, ScienceCell, Item No.: 1001) cultivated in. Culture the cells in an incubator at 37°C and 5% CO2. For long-term storage, the cells were frozen in complete growth medium supplemented with 5% (v/v) DMSO and stored in a liquid nitrogen phase. Example 2 Production of anti-PD-L1/VEGF bispecific antibodies 2.1 Construction of expression vector

The DNA sequence encoding the VHH antibody W3156-AP3R2-1A3-z12 connected by a flexible G4S linker was placed at the N-terminus of the light chain of WBP325-BMK3 (bevacizumab). The heavy chain was constructed using the same sequence as that of WBP325-BMK3 (bevacizumab). The recombinant DNA sequences were cloned into the modified pcDNA3.3 expression vector. The constructed antibody was named W3256-U15T2.G6-1.uIgG1 (can be abbreviated as "W3256" throughout the disclosure).

As mentioned above, the heavy chain of W3256-U15T2.G6-1.uIgG1 contains the variable heavy chain region of WBP325-BMK3 (bevacizumab) and the constant heavy chain region of human IgG1 (CH1-CH3). The light chain of W3256-U15T2.G6-1.uIgG1 consists of the variable light chain region of WBP325-BMK3 (bevacizumab) and the constant light chain region (CL) of human IgG1 and the VHH antibody W3156-AP3R2- at the N-terminus. 1A3-z12 structure, as shown in Figure 1. The specific sequences of the W3256 antibody are listed in Table 2-4 below. Table 2 Anti-VEGF HCDR1 HCDR2 HCDR3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 GYTFTNYGMN WINTYTGEPTYAADFKR YPHYYGSSHWYFDV LCDR1 LCDR2 LCDR3 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SASQDISNYLN FTSSLHS QQYSTVPWT Anti-PD-L1 VHH CDR1 CDR2 CDR3 SEQ ID NO:1 SEQ ID NO: 2 SEQ ID NO: 3 GHFSNLAVN GILWSGGSTFYADSVKG GTN table 3 The amino acid sequence of the VH of the VEGF binding module: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS (SEQ ID NO: 11) The amino acid sequence of the VL of the VEGF binding module: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK (SEQ ID NO: 12) The amino acid sequence of the VHH of the PD-L1 binding module: EVQLVESGGGLVQPGGSLRLSCAASGHFSNLAVNWFRQAPGKERELVAGILWSGGSTFYADSVKGRFTISRGNAENMLYLQMNSLRAEDTAVYYCNTGTNWGQGTLVTVSS (SEQ ID NO: 10) Table 4 The heavy chain amino acid sequence of W3256-U15T2.G6-1.uIgG1: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13) Light chain amino acid sequence of W3256-U15T2.G6-1.uIgG1: EVQLVESGGGLVQPGGSLRLSCAASGHFSNLAVNWFRQAPGKERELVAGILWSGGSTFYADSVKGRFTISRGNAENMLYLQMNSLRAEDTAVYYCNTGTNWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14) 2.2 Transfection, expression and purification

According to the manufacturer's instructions, the heavy chain and light chain expression plasmids were co-transfected into Expi293 cells using the Expi293 expression system kit (ThermoFisher-A14635). Five days after transfection, the supernatant was collected and protein A column (GE Healthcare-17543802) was used for protein purification. The antibody concentration is measured by NanoDrop. The purity of the protein was evaluated by SDS-PAGE and HPLC-SEC. After expression and purification, bispecific antibodies were obtained, including W3256-U15T2.G6-1.uIgG1. 2.3 Production of bispecific antibodies for in vivo research (including endotoxin control and testing)

According to the manufacturer's instructions, use the Expi293 expression system kit (ThermoFisher-A14635) to transfect the W3256-U15T2.G6-1.uIgG1 expression plasmid into Expi293 cells. Five days after transfection, the supernatant was collected, and protein A column (GE Healthcare-17543802) was used for protein purification under endotoxin-controlled conditions. The endotoxin test kit (GenScript-L00350) was used to confirm low endotoxin levels (less than 10 EU/mg).

The antibody concentration was measured by NanoDrop, and the yield of W3256-U15T2.G6-1.uIgG1 was 44.8 mg/l. The purity of the protein was evaluated by SDS-PAGE (Figure 2) and HPLC-SEC (Figure 3). According to HPLC-SEC, after protein A is purified in one step, the purity of W3256-U15T2.G6-1.uIgG1 is 93.43%. Example 3 In vitro characterization of bispecific antibodies 3.1 Differential Scanning Fluorescence (DSF)

The 7500 fast real-time PCR system (Applied Biosystems) was used for DSF determination. In short, 19μl of bispecific antibody solution was mixed with 1μl of 62.5X SYPRO Orange solution (ThermoFisher-S6650), and then added to a 96-well plate. The plate was heated from 26°C to 95°C at a rate of 2°C/min, and the resulting fluorescence data was collected. The data is automatically analyzed through its operating software, and Th is calculated by obtaining the maximum value of the negative derivative of the obtained fluorescence data with respect to temperature. _Ton can be roughly determined as the temperature at which the negative derivative graph begins to decrease from the baseline before the transition.

4, W3256 antibody T _h 1 is 65.7 ° C. 3.2 Human/cynomolgus VEGF binding (ELISA)

The binding of the antibody to the human VEGF antigen (WBP325-hPro1, Sino Biological, 11066-HNAB) was tested by a binding ELISA assay. Since the amino acid sequence of cynomolgus monkey VEGF is the same as that of human VEGF, the binding result represents the binding of human VEGF and the binding of cynomolgus VEGF. In short, a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher, 442404) was used in carbonate-bicarbonate buffer (20 mM Na ₂ CO3, 180 mM NaHCO ₃ , pH 9.2) with 0.25 μg/ml human VEGF Coat overnight at 4°C. After a 1-hour blocking step with 2% (w/v) bovine serum albumin (Pierce) dissolved in PBS, the serial dilutions of W3256-U15T2.G6-1.UIgG1 in PBS were placed on the plate at room temperature Incubate for 2 hours. After incubation, the plate was washed 3 times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. Add 100 ng/ml goat anti-human IgG Fc-HRP (Bethyl, #A80-304P) and incubate on the plate at room temperature for 1 hour. After washing 6 times, add tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) for detection. After approximately 8 minutes, the reaction was terminated by adding 100 μl of 2 M HCl to each well. Use a multiwell plate reader (SpectraMax® M5 ^e ) to measure the absorbance of the wells at 450 nm.

W3256 showed a binding ability comparable to WBP325-BMK3.uIgG1 on human VEGF, with an EC ₅₀ of about 0.095 nM (Figure 5). 3.3 Human PD-L1 binding (FACS)

The engineered human PD-L1 expressing cells (WBP315.CHO-K1.hPro1.C11) were seeded in a U-shaped bottom 96-well plate ^{at 1×10 5 cells/well.} A serial dilution of W3256-U15T2.G6-1.UIgG1 was added to the cells. The plate was incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well, and the plate was incubated at 4°C for 1 hour. The binding of antibodies to cells was tested by a serial flow cytometer, and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

W3256 also showed a binding capacity comparable to W3156-AP3R2-1A3-z12-hIgG1 on human PD-L1, with an EC ₅₀ of about 0.128 nM (Figure 6). 3.4 Double binding of human VEGF/human PD-L1 (ELISA)

To test whether the bispecific antibody can bind to both VEGF and PD-L1, the following ELISA assay was developed. Use a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) at 4°C with 1 μg/ml antigen-1 (VEGF, WBP325-hPro1, Sino Biological) or 1 μg/ml antigen-2 (PD-L1.ECD.mFc, W3153-hPro1.ECD.mFc) was coated overnight in carbonate-bicarbonate buffer. After blocking for 1 hour, the serial dilutions of W3256-U15T2.G6-1.UIgG1 in casein buffer were incubated on the plate at room temperature for 1 hour. After incubation, the plate was washed 3 times with 300 μL per well of PBS containing 0.5% (v/v) Tween 20. Add 0.5 μg/ml antigen-2.Biotin (PD-L1-ECD, WBP315-hPro1.ECD.mFc) or 0.5 μg/ml antigen-1.Biotin (VEGF, WBP325-hPro1.his) into the plate and incubate 1 Hour. After washing the plate 3 times, add HRP-labeled detection secondary antibody and incubate the plate for 1 hour at room temperature. After washing the plate 6 times, add tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) for detection. After about 10 minutes, the reaction was stopped by adding 100 μL of 2 M HCl per well. Use a multiwell plate reader (SpectraMax® M5 ^e ) to measure the absorbance of the wells at 450 nm.

The results showed that the binding of W3256 to VEGF did not affect the subsequent PD-L1 binding (Figure 7), and vice versa (Figure 8). 3.5 Cross-species binding (FACS/ELISA) Cynomolgus PD-L1 binding (FACS)

The engineered cynomolgus monkey PD-L1 expressing cells (WBP315.293F.cPro1.2A) were seeded in a U-shaped bottom 96-well plate at a density of ^{1×10 5 cells/well.} A serial dilution of W3256-U15T2.G6-1.UIgG1 was added to the cells. The plate was incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well, and the plate was incubated at 4°C for 1 hour. The binding of antibodies to cells was tested by serial flow cytometry, and the mean fluorescence intensity (MFI) was analyzed by FlowJo. Mouse VEGF binding (ELISA)

The binding of the antibody to the mouse VEGF antigen (WBP325-mPro1, Sino Biological, 50159-MNAB) was performed by the same ELISA assay as the human VEGF binding described above, except that the coating protein was 100 μL 0.25 μg/mL mice VEGF. Mouse PD-L1 binding (FACS)

The engineered mouse PD-L1 expressing cells (WBP315.293F.mPro1.C1) were seeded into a U-shaped bottom 96-well plate at a density of ^{1×10 5 cells/well.} Add serial dilutions of different antibodies to the cells. The plate was incubated at 4°C for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-095-008) was added to each well, and the plate was incubated at 4°C for 1 hour. The binding of W3256-U15T2.G6-1.UIgG1 on the cells was tested by serial flow cytometry, and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

The amino acid sequence of cynomolgus VEGF is the same as that of human VEGF. Therefore, W3256 also has binding activity to cynomolgus VEGF. The cross-species binding activity of W3256 with cynomolgus PD-L1, mouse VEGF, and mouse PD-L1 was also evaluated. Figure 9, Figure 10 and Figure 11 show their binding activity to VEGF or PD-L1. W3256 showed a binding ability comparable to that of the parent antibody on cynomolgus monkey PD-L1, with an EC ₅₀ of about 2.618 nM (Figure 9). W3256 cannot bind to mouse VEGF (Figure 10). W3256 also showed a binding ability comparable to that of the parent antibody on mouse PD-L1, with an EC ₅₀ of about 1.687 nM (Figure 11). 3.6 Binding affinity to VEGF and PD-L1 (SPR)

SPR technology was used to measure the binding rate constant (ka) and dissociation rate constant (kd) of the antibody to the ECD of VEGF or PD-L1. Therefore, the affinity constant (KD) is determined.

Biacore T200, S series sensor chip CM5, amine coupling kit and 10x HBS-EP were purchased from GE Healthcare. The antibody was captured on the surface of anti-human Fc IgG (Jackson, 109-005-098) immobilized on a CM5-biosensor chip (GE Healthcare Inc.). The measurement was performed at 25°C with HBS-EP + buffer (GE Healthcare Inc.) as the running buffer and the dilution buffer. Inject serially diluted PD-L1 antigen or VEGF antigen (W315-hPro1.ECD.his or W325-hPro1.his) and running buffer for detection in the binding phase and subsequent dissociation phase. Regeneration of the wafer surface was achieved by injecting 10 mM glycine at pH 1.5.

The affinity constant (KD) of W3256 is measured according to SPR technology. At the same time, the association rate constant (ka) and dissociation rate constant (kd) were also measured. The final data of each interaction is subtracted from the reference channel and buffer channel data. Analyze the experimental data, as shown in Figure 12 and Figure 13. The kinetic affinity results of the antibodies are listed in Table 5. Table 5. Kinetic affinity results of antibodies Analyte Ligand ka1 (1/Ms) kd1 (1/s) KD1 (M) W315-hPro1.ECD.his (PD-L1) W3256 2.14E+06 3.33E-04 1.55E-10 W3156-AP3R2-1A3-z12-hIgG1 1.82E+06 2.91E-04 1.60E-10 W315-BMK8.uIgG1K(RKNA) 1.53E+06 9.29E-05 6.08E-11 W325-hPro1.ECD.his (VEGF) W3256 2.38E+06 3.64E-05 1.53E-11 W325-BMK3.uIgG1 3.94E+06 3.69E-05 9.36E-12 3.7 Ligand Competition Assay (ELISA) Human VEGFR1 Competition Assay

The inhibitory effect on the binding of VEGF to human VEGF receptor 1 (W325-hpro1R1.ECD.hFc, Sino Biological, 10136-H02H) was determined by competitive ELISA. Put a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher, 442404) at 4°C with 2 μg/mL W325-hpro1R1.ECD.His in carbonate-bicarbonate buffer (20 mM Na ₂ CO ₃ , 180 mM NaHCO ₃ , PH9.2) medium-coated overnight. All wells were washed 3 times with 300 μL of PBS/0.5‰ Tween-20 (v/v) per well, and all subsequent washing steps in the assay were the same. Then the wells were blocked with 200 μL/well of 50% casein (Thermo SCIENTIFIC, 37528) for 1 hour, and then after the washing step, serially diluted antibodies and 0.02 μg/ml biotin-labeled human VEGF (W325-hPro1. Biotin, ACRO Biosystems, VE5-H8210) mixture was added to the wells and incubated at 25°C for 2 hours. The plate was washed 3 times, and then peroxidase-linked streptavidin (Jackson, 016-030-084) diluted 1:10000 in PBS/50% casein was added. Incubate the plate at 25°C for 1 hour, then wash 6 times as before. Add 100 μL/well of tetramethylbenzidine (TMB) substrate (Sigma, 860336) to all wells for 8 minutes, and then terminate the reaction with 100 μL 2M HCl. The degree of human VEGF-biotin binding to W325-hpro1R1.ECD.hFc is determined by measuring OD ₄₅₀ ^{with a multi-well plate reader (SpectraMax® M5 e} ). Analysis of binding IC ₅₀ values obtained by a four-parameter nonlinear regression using GraphPad Prism 5 software.

W3256 parent antibody exhibited comparable binding ability and competition hVEGFR1, which binds VEGF IC ₅₀ of approximately 3.86 nM (FIG. 14). Mouse VEGFR1 competition assay

The effect of antibody blocking the binding of VEGF to mouse VEGF receptor 1 (W325-mpro1R1.ECD.hFc.his (R&D, 471-F1) was evaluated by competitive ELISA. The mouse VEGFR1 competition ELISA method is similar to the human VEGFR1 competition ELISA, only However, the pre-coated protein on the 96-well ELISA plate is 2μg/ml W325-mpro1R1.ECD.hFc.his, the blocking buffer is PBS/2%BSA, and the concentration of W325-hPro1.Biotin is 0.44 μg/ml .

W3256 exhibits mVEGFR1 competitive (FIG. 16) comparable to the parent antibody, which binds VEGF IC ₅₀ of approximately 31 nM. Human PD-1 Competition Assay

The competition of W3256 with human PD-1 was measured by a FACS-based competition assay. CHO-K1 cells expressing human PD-L1 (WBP315.CHO-K1.hPro1.C11) were seeded into a 96-well U-shaped bottom plate at a density of ^{1×10 5 cells/well.} The serial dilutions of the test antibody are premixed with 5 μg/ml human PD-1 with mouse Fc label, then added to the cells, and incubated at 4°C for 1 hour. After washing, PE-labeled anti-mouse IgG was added and incubated with the cells at 4°C for 45 minutes. The cells were washed twice, and the MFI of the cells was measured by a serial flow cytometer and analyzed by FlowJo.

W3256 exhibit competitive with comparable parent antibody, which blocks binding hPD-1 IC ₅₀ of PD-L1 of about 0.048 nM (FIG. 15). Mouse PD-1 Competition Assay

CHO-K1 cells (WBP315.293F.mPro1.C1) expressing mouse PD-L1 were seeded into a 96-well U-shaped bottom plate ^{at 1×10 5 cells/well.} The serial dilutions of the test antibody are pre-mixed with 5 μg/ml mouse Fc-labeled mouse PD-1, then added to the cells, and incubated at 4°C for 1 hour. After washing, PE-labeled anti-mouse IgG was added and incubated with the cells at 4°C for 1 hour. The cells were washed twice, and the MFI of the cells was measured by a serial flow cytometer and analyzed by FlowJo.

W3256 exhibit competitive with comparable parent antibody, which blocks binding to PD-1 IC mouse PD-L1 is approximately ₅₀ 1.687 nM (FIG. 17). 3.8 HUVEC cell proliferation assay

The biological activity of W3256 in VEGF-induced HUVEC proliferation was evaluated. HUVEC cells are routinely cultured in ECM + 5% FBS + 1% ECGS. Collect nearly confluent cells with trypsin, and dilute to 1×10 ⁵ cells/mL with ECM + 1% FBS + 0.05% ECGS. The cells were seeded in a 96-well transparent bottom black plate (Greiner, 655090) at a density of 5000 cells/well. Add serially diluted antibodies and 50 ng/mL human VEGF (WBP325-hPro1, Sino Biological, 11066-HNAB). The plate was returned to the incubator for 4 days, and then CellTiter Glo (Promega, G7573) was used to evaluate cell viability. Wells without addition of ligand were used as controls for ligand-stimulated cell growth. By subtracting the background (no ligand) luminescence, and comparing the luminescence value with or without the addition of the antibody (ligand only), the effect of the test antibody on inhibiting the cell growth stimulated by the ligand is calculated. Using GraphPad Prism 5 software using a four-parameter nonlinear regression analysis to obtain the ₅₀ values proliferation inhibition IC.

W3256 effectively blocked VEGF-induced HUVEC proliferation in a concentration-dependent manner, with an IC ₅₀ of about 12 nM and a maximum inhibition rate of 101% (Figure 18). 3.9 Reporter gene determination

The conventionally cultured CHOK1-OKT3-PD-L1 cells were harvested by trypsin and seeded in a 96-well transparent bottom black plate (Greiner, 655090) at a density of 20,000 cells/well. Added serially diluted antibodies, and integrated the reporting signal NFAT-RE-Luc2p and Jurkat cells expressing full-length PD-1. Return the plate to the incubator for 4 hours, then add 50 μl One-glo Luciferase Assay Buffer/Substrate Mix. The luminescence is measured in the multiwell plate reader M5e.

W3256 showed function in the reporter gene assay (Figure 19), indicating that the antibody induces the PD-1 signaling pathway. 3.10 Determination of mixed lymphocyte reaction (MLR)

MLR was used to test the agonistic effect of PD-L1 antibody on cytokine, human IFN-γ secretion and activated human CD4 ^{+ T cell proliferation.}

Human peripheral blood mononuclear cells (PBMC) were freshly separated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. Culture isolated PBMC in complete RPMI-1640 (containing 10% FBS and 1% PS) supplemented with 100 U of recombinant human IL-2 (SL PHARM). Use the human monocyte enrichment kit to isolate monocytes according to the manufacturer's instructions. In complete RPMI-1640 medium supplemented with 800 U/ml recombinant human GM-CSF and 50 μg/ml IL-4, the cell concentration was adjusted to 2×10 ⁶ cells/ml. The cells are cultured for 5 to 7 days to differentiate into dendritic cells (DC). Replenish the cytokine by replacing half of the medium with fresh medium supplemented with the cytokine every 2-3 days. 18 to 24 hours before MLR, 1 µg/ml LPS was added to the culture to induce DC maturation. ^{The human CD4 +} T cell enrichment kit was used to isolate human CD4 ⁺ T cells according to the manufacturer's protocol.

Complete RPMI-1640 medium was used to establish MLR in 96-well round bottom plates (Nunc, 163320). Add CD4 ⁺ T cells, various concentrations of antibodies, and mature DC to the plate. The plate was incubated at 37°C, 5% CO _2. The production of IFN-γ was measured on the 5th day.

Human IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs. Recombinant human IFN-γ (PeproTech, 300-02) was used as a standard. The plate was pre-coated with a capture antibody specific for human IFN-γ (Pierce, M700A). After sealing, pipette the standard or sample into each well and incubate at ambient temperature for 2 hours. After removing unbound substances, a biotin-conjugated detection antibody (Pierce, M701B) specific for IFN-γ was added to the wells and incubated for 1 hour. Then horseradish peroxidase (HRP) conjugated with streptavidin (Invitrogen, SNN1004) was added to the wells and incubated at ambient temperature for 30 minutes. The color was developed by adding TMB substrate and then stopped with 2M HCl. Use Microplate spectrophotometer to read absorbance at 450 and 540 nm.

Figure 20 shows the effect of antibodies on hCD4 ⁺ T cell IFN-γ secretion in the mixed lymphocyte reaction assay. The results show that W3256 can induce IFN-γ secretion in MLR in a concentration-dependent manner. Example 4 Study on anti-tumor effect in vivo 4.1 Study on pharmacokinetics in mice

Purchased male C57BL/6 mice (6-8 weeks, 18-22 g) from Shanghai SLAC or BK Laboratory Co., LTD. WuXi Biologics ( They are raised in animal facilities in Shanghai, China.

Blood samples of male C57BL/6 mice (n = 5) were collected at 0, 0.5, 6, 24, 48, 72, 120, and 168 hours after a single intravenous injection of equimolar W3256 antibody. Human IgG ELISA quantitative method and dual antigen binding ELISA method were used to measure the serum antibody concentration.

According to the results of total IgG binding, when injected at a dose of 11.5 mg/kg via the intravenous route, the half-life of W3256 is 129 hours (Figure 21). When injected at a kg dose, the half-life of W3256 is 104 hours (Figure 22). 4.2 Efficacy of PBMC-RKO mouse model

In a medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, RKO tumor cells are maintained in vitro in a monolayer culture _{at 37ºC and 5% CO 2.} Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. The cells grown in the exponential growth phase were harvested and counted for tumor inoculation.

Female NCG mice (6-10 weeks old) were purchased from Shanghai SLAC or Laboratory Co., LTD. and performed in the animal facility of WuXi Biologics (Shanghai, China) under specific pathogen-free conditions and free access to food and water Rearing.

Each mouse was subcutaneously inoculated with RKO tumor cells (2 x 10 ⁶ ) in 0.2 ml PBS containing 50% matrix gel to form tumors, and 4 x 10 ⁶ PBMCs (Hemacare) were injected intraperitoneally on day 0. Treatment starts on the 7th day.

The primary endpoint is to see whether it can delay tumor growth or cure mice. Use a caliper to measure the size of the tumor twice a week, and use the following formula to ^{express the volume in mm 3} : V = 0.5 axb ² , where a and b represent the long and short diameters of the tumor, respectively.

Provide a summary of the statistical data of each group of tumor volumes at each time point, including the mean and the standard error of the mean (SEM). The statistical analysis of the difference in tumor volume between the groups and the analysis of drug interactions were performed on the data obtained at the optimal treatment time point after the final dose (the 28th day after the start of dosing). A two-way ANOVA analysis was performed to compare the tumor volume between the groups, and then Dunnett's t test for multiple comparisons after the fact (all compared with the IgG group) was performed. Use SPSS 17.0 or Prism 5 to analyze all data. p <0.05 was considered statistically significant.

W3256 showed the anti-tumor effect in the PBMC-RKO colorectal cancer model of NCG mice, which was significantly better than W315-BMK8 and comparable to W325-BMK3 (Figure 23).

Those skilled in the art can recognize and understand the description of this patent, and the present invention can be implemented in other specific forms without departing from its essence or basic characteristics. Since the foregoing description of the present invention only discloses exemplary embodiments thereof, other changes should be understood as being within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the scope of the appended application to indicate the scope and content of the present invention.

MFI: mean fluorescence intensity PBS: phosphate buffered saline

Figure 1 shows a schematic diagram of the W3256 antibody. Figure 2 shows the SDS-PAGE of the W3256 antibody (lane 1). In NuPAGE (Novex 4-12% Bis-Tris) gel, NR: non-reduced state, R: reduced state, M: PageRuler™ unstained protein gradient. Figure 3 shows the HPLC-SEC results of the W3256 antibody. Figure 4 shows the DSF profile of the W3256 antibody. Figure 5 shows the results of ELISA binding of W3256 antibody to human VEGF (same as cynomolgus VEGF). WBP332-1.80.12×Ab.hIgG1 is the isotype control. Figure 6 shows the result of FACS binding of W3256 antibody to human PD-L1. Figure 7 shows the result of dual binding of W3256 antibody to VEGF and PD-L1 later. Figure 8 shows the results of dual binding of W3256 antibody to PD-L1 and later VEGF. Figure 9 shows the FACS binding result of W3256 antibody to cynomolgus monkey PD-L1. Figure 10 shows the results of ELISA binding of W3256 antibody to mouse VEGF. Figure 11 shows the results of FACS binding of W3256 antibody to mouse PD-L1. Figures 12 to 13 show the SPR sensing patterns of human PD-L1 (Figure 12) and VEGF (Figure 13) by W3256 antibody. Figures 14 to 15 show the competition of W3256 antibody with human VEGFR1 on human VEGF binding (Figure 14) and the competition with human PD-1 on human PD-L1 binding (Figure 15). Figures 16 to 17 show the competition of W3256 antibody with mouse VEGFR1 on human VEGF binding (Figure 16) and the competition with mouse PD-1 on mouse PD-L1 binding (Figure 17). Figure 18 shows the inhibition of HUVEC cell proliferation by antibodies. Figure 19 shows the effect of the antibody reporter gene assay (RGA). Figure 20 shows the effect of antibodies on IFN-γ secretion by hCD4+ T cells in MLR. "Combo" means the combination of WBP325-BMK3.uIgG1 and W315-BMK8.uIgG1K (RKNA). Figure 21 shows the mouse PK analysis of the W3256 antibody after a single intravenous injection of an equimolar dose of the antibody in the total IgG binding assay. "Mpk" is an abbreviation of "mg/kg". Figure 22 shows the mouse PK analysis of the W3256 antibody after a single intravenous injection of an equimolar dose of the antibody in the dual antigen binding assay. Figure 23 shows the efficacy of antibodies in the PBMC-RKO mouse cancer model.

MFI: mean fluorescence intensity

PBS: phosphate buffered saline

Claims (23)

A bispecific antibody or antigen-binding portion thereof, which comprises a PD-L1 antigen-binding module combined with a VEGF antigen-binding module, wherein: The PD-L1 antigen binding module includes: Comprising the complementarity determining region 1 (CDR1) of SEQ ID NO: 1, CDR2 comprising SEQ ID NO: 2, and CDR3 comprising SEQ ID NO: 3; and The VEGF antigen binding module includes: The heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 4, the HCDR2 comprising SEQ ID NO: 5, the HCDR3 comprising SEQ ID NO: 6, and the light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 7 ), LCDR2 comprising SEQ ID NO: 8 and LCDR3 comprising SEQ ID NO: 9. The bispecific antibody or antigen-binding portion thereof according to claim 1, wherein the anti-PD-L1 antigen-binding module comprises a variable domain, the variable domain comprising the amino acid sequence of SEQ ID NO: 10 or the same as SEQ ID NO: 10 has an amino acid sequence that is at least 85%, 90%, or 95% identical. The bispecific antibody or antigen-binding portion thereof according to claim 1 or claim 2, wherein the VEGF antigen-binding module comprises: A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 11; and A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 12. The bispecific antibody or antigen-binding portion thereof according to any one of the preceding claims, wherein the PD-L1 antigen-binding module is fused to the N-terminus of the VEGF antigen-binding module. The bispecific antibody or antigen-binding portion thereof according to any one of the preceding claims, wherein the PD-L1 antigen-binding module is derived from a single domain antibody (sdAb), such as a VHH antibody. The bispecific antibody or antigen-binding portion thereof according to claim 5, wherein the VHH is derived from camelid animals, including alpaca and llama. The bispecific antibody or the antigen-binding portion thereof according to claim 5 or claim 6, wherein the PD-L1 antigen-binding module is operably linked to the N-terminus of the light chain or the heavy chain of the VEGF antigen-binding module , Optionally via a linker. The bispecific antibody or antigen-binding portion thereof according to claim 7, wherein the linker comprises 1 to 4 copies of GGGGS (G4S) or consists of the same. The bispecific antibody or antigen-binding portion thereof according to any one of the preceding claims, which comprises a heavy chain and a light chain, wherein: The heavy chain comprises a domain operably linked as in VH-CH1-hinge-Fc, wherein VH-CH1 is derived from the VEGF antigen binding module; and The light chain comprises a domain operably linked as in VHH-VL-CL, where VHH comes from the PD-L1 antigen binding module and VL-CL comes from the VEGF antigen binding module. The bispecific antibody or antigen-binding portion thereof according to claim 9, wherein the Fc region is a human IgG Fc region, preferably a human IgG1 Fc region. The bispecific antibody or antigen-binding portion thereof according to any one of the preceding claims, wherein the heavy chain comprises SEQ ID NO: 13, and the light chain comprises SEQ ID NO: 14. The bispecific antibody or antigen-binding portion thereof according to any one of the preceding claims, wherein the bispecific antibody is a humanized antibody. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the bispecific antibody or antigen-binding portion thereof according to any one of Claims 1 to 12. A vector, which is the nucleic acid molecule according to claim 13. A host cell comprising the nucleic acid molecule according to claim 13 or the vector according to claim 14. A pharmaceutical composition comprising the bispecific antibody or antigen binding portion thereof according to any one of claim 1 to claim 12 and a pharmaceutically acceptable carrier. A method for producing the bispecific antibody or antigen-binding portion thereof according to any one of claim 1 to claim 12, comprising the following steps: -Express the antibody or antigen-binding portion thereof as described in any one of claim 1 to claim 12 in the host cell described in claim 15; and -Isolate the antibody or its antigen-binding portion from the host cell. The bispecific antibody or antigen-binding portion thereof according to any one of claim 1 to claim 12 is used for the following purposes: i) Regulate PD-L1/VEGF-related immune response; ii) Inhibit the growth of tumor cells; iii) Enhance T cell proliferation and cell factor production; and/or iv) Stimulate immune response or function, for example, stimulate immune response against cancer cells. Use of the bispecific antibody or antigen binding portion thereof according to any one of claim 1 to claim 12 for the diagnosis, treatment or prevention of cancer. The use according to claim 19, wherein the cancer is selected from colon cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastric cancer , Lymphoma, melanoma, liver cancer, and head and neck cancer. The use according to claim 19 or claim 20, wherein the cancer is colon cancer or colorectal cancer. The use according to any one of claim 18 to claim 21, wherein the bispecific antibody or antigen-binding portion thereof is administered in combination with a chemotherapeutic agent, radiotherapy and/or other agents for cancer immunotherapy. A kit comprising the bispecific antibody or antigen-binding portion thereof according to any one of claim 1 to claim 12.

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