CN113214400A - Bispecific anti-PD-L1/VEGF antibody and application thereof - Google Patents

Abstract

The present disclosure provides bispecific anti-VEGF x PD-L1 antibodies or antigen-binding portions thereof, methods of producing the bispecific antibodies or antigen-binding portions thereof, and uses of the bispecific antibodies or antigen-binding portions thereof for treating diseases, such as cancer.

Description

Bispecific anti-PD-L1/VEGF antibody and application thereof

Cross reference to related applications

The present application claims priority from chinese patent application 202010070440.X, filed on 21/1/2020, the entire contents of which are incorporated herein by reference.

Sequence listing

This application contains a sequence listing in electronic form, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates generally to bispecific anti-PD-L1 x VEGF antibodies, methods for making the antibodies, and uses thereof.

Background

Angiogenesis is essential for tumor growth and metastatic progression. Controlling tumor-associated angiogenesis is a promising cancer treatment strategy. Vascular Endothelial Growth Factor (VEGF), a key mediator of angiogenesis, has been demonstrated in various types of human cancers. Tumor cells release growth factors (e.g., VEGF) that bind to nearby endothelial cells, thereby initiating a signaling cascade that stimulates endothelial cells to divide and form new blood vessels. The signaling of VEGF through its receptor, VEGFR, plays a crucial role in 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, targeting 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 associated with a poor prognosis in 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, then enable the immune system to fight multiple types of cancer.

In addition to established anti-angiogenic effects, anti-VEGF therapy may 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 eliciting and activating T cell responses against tumor antigens, among other things. Therefore, the development of a bispecific antibody to VEGF and PD-L1 that combines anti-angiogenic therapy with immune checkpoint inhibition could lead to promising results in cancer therapy.

Despite the clear benefits of targeted VEGF and targeted PD-1/PD-L1 therapies, there remains a substantial unmet need. 15% -20% of patients do not respond to anti-VEGF therapy, and there is increasing evidence that long-term use of anti-VEGF agents for cancer therapy will enhance tumor resistance. Between 3% and 9% of patients develop therapeutic immunogenicity. Furthermore, there is limited overall survival time and safety issues, including changes in bone morphology, glomerulopathy with kidney inflammation, and a reduction in adrenal vacuole formation with inflammation. Immune checkpoint inhibitors that block the PD-1/PD-L1 pathway (e.g., nivolumab, pembrolizumab, and atezolizumab) represent standard treatment options for patients with a variety of cancers. However, these drugs have a response rate of 14-23% in the unselected population and 16-48% in patients with tumors expressing PD-L1, and provide better therapeutic results in some but not all patients.

Therefore, the development of a novel anti-PD-L1/anti-VEGF bispecific antibody is highly desirable. In the present disclosure, bispecific antibodies were generated that were capable of simultaneously binding to human PD-L1 and VEGF with high affinity, blocking signaling of both PD-1/PD-L1 and VEGF/VEGFR, and showed superior anti-tumor efficacy.

Summary of The Invention

Broadly, the present disclosure relates to compounds, methods, compositions, and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present disclosure are broadly applicable to the field of antibody therapy and diagnosis, and may be used in conjunction with antibodies capable of reacting with various targets.

In one aspect, the present disclosure provides a bispecific antibody, or antigen-binding portion thereof, comprising a PD-L1 antigen-binding moiety in association with a VEGF antigen-binding moiety, wherein:

the PD-L1 antigen binding moiety comprises: complementarity Determining Region (CDR)1 comprising SEQ ID NO. 1, CDR2 comprising SEQ ID NO. 2, and CDR3 comprising SEQ ID NO. 3; and

the VEGF antigen binding moiety comprises: heavy chain complementarity determining region (HCDR)1 comprising SEQ ID NO. 4, HCDR2 comprising SEQ ID NO. 5, HCDR3 comprising SEQ ID NO. 6, light chain complementarity determining region (LCDR)1 comprising SEQ ID NO. 7, LCDR2 comprising SEQ ID NO. 8, and LCDR3 comprising SEQ ID NO. 9.

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

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

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

In certain embodiments, the PD-L1 antigen binding moiety is from a single domain antibody (sdAb), e.g., a VHH antibody. The VHH may be derived from camelids including alpaca (alpaca) and llama (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 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, a bispecific antibody or antigen-binding portion thereof as disclosed herein comprises a heavy chain and a light chain, wherein:

the heavy chain comprises domains operably linked as in VH-CH 1-hinge-Fc, wherein VH-CH1 is from the VEGF antigen binding moiety; and

the light chain comprises domains operably linked as in VHH-VL-CL, wherein VHH is from the PD-L1 antigen binding module and VL-CL is from the VEGF antigen binding module.

In certain embodiments, the Fc region is a human IgG Fc region, preferably a human IgG1Fc 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, a 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 a nucleic acid molecule or vector as disclosed herein.

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

In one aspect, the present disclosure provides a method of producing a bispecific antibody or antigen-binding portion thereof as disclosed herein, comprising the steps of:

-expressing the antibody or antigen-binding portion thereof in a host cell; and

-isolating the antibody or antigen-binding portion thereof from the host cell.

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

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

In one aspect, the present disclosure provides a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding portion thereof or pharmaceutical composition as disclosed herein. The cancer may 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 a chemotherapeutic agent, radiation therapy, and/or other agent for cancer immunotherapy.

In one aspect, the present disclosure provides a bispecific antibody or antigen-binding portion thereof as disclosed herein for use in:

i) modulating a PD-L1/VEGF related immune response;

ii) enhance T cell proliferation and cytokine production; and/or

iii) stimulating an immune response or function, e.g., stimulating an immune response against cancer cells.

In one aspect, the present disclosure provides a 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 a bispecific antibody or antigen-binding portion thereof as disclosed herein in the manufacture of a medicament for modulating an immune response or inhibiting tumor cell growth in a subject.

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

In one aspect, the present disclosure provides a kit comprising a bispecific antibody or antigen-binding portion thereof as disclosed herein. The kit may be used for detection, diagnosis, prognosis or treatment of a disease or condition (e.g., cancer).

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings presented herein. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential 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 published patent applications cited throughout this application are incorporated by reference herein in their entirety.

Brief Description of Drawings

Fig. 1 shows a schematic of the W3256 antibody.

FIG. 2 shows SDS-PAGE (lane 1) of the W3256 antibody. NuPAGE (Novex 4-12% Bis-Tris) gel, NR: non-reducing state, R: reduced state, M: PageRuler^TMUnstained protein gradient.

FIG. 3 shows HPLC-SEC results for the W3256 antibody.

Figure 4 shows the DSF profile of W3256 antibody.

Figure 5 shows the results of ELISA binding of the W3256 antibody to human VEGF (same as cynomolgus monkey VEGF). WBP332-1.80.12 × ab. higg1 is an isotype control.

Figure 6 shows FACS binding results of the W3256 antibody to human PD-L1.

Figure 7 shows the results of dual binding of the W3256 antibody to VEGF followed by PD-L1.

Figure 8 shows the results of dual binding of the W3256 antibody to PD-L1 and then VEGF.

Figure 9 shows FACS binding results of W3256 antibody to cynomolgus monkey PD-L1.

Figure 10 shows the results of ELISA binding of the W3256 antibody to mouse VEGF.

Figure 11 shows FACS binding results of W3256 antibody to mouse PD-L1.

FIGS. 12-13 show SPR sensorgrams of the W3256 antibody to human PD-L1 (FIG. 12) and VEGF (FIG. 13).

Fig. 14-15 show competition of the W3256 antibody with human VEGFR1 on human VEGF binding (fig. 14) and with human PD-1 on human PD-L1 binding (fig. 15).

Fig. 16-17 show competition of the W3256 antibody with mouse VEGFR1 on human VEGF binding (fig. 16), and with mouse PD-1 on mouse PD-L1 binding (fig. 17).

FIG. 18 shows inhibition of HUVEC cell proliferation by antibodies.

FIG. 19 shows the effect of antibody Reporter Gene Assay (RGA).

FIG. 20 shows the effect of antibodies on IFN- γ secretion from hCD4+ T cells in MLR. "Combo" indicates the combination of WBP325-BMK3.uIgG1 and W315-BMK8.uIgG1K (RKNA).

Figure 21 shows a mouse PK analysis of W3256 antibody after a single intravenous injection of equimolar doses of antibody in a total IgG binding assay. "mpk" is an abbreviation for "mg/kg".

Figure 22 shows a mouse PK analysis of W3256 antibody after a single intravenous injection of equimolar doses of antibody in a dual antigen binding assay.

FIG. 23 shows the efficacy of the antibody in a PBMC-RKO mouse cancer model.

Detailed Description

While this invention is susceptible of embodiment in many different forms, there is disclosed herein specific illustrative embodiments thereof which are indicative of the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells and the like. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms (such as "includes" and "including") is not limiting. Moreover, the ranges provided in the specification and the appended claims include all values between the endpoints and breakpoints.

Generally, the terminology associated with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Abbas et al, Cellular and Molecular Immunology,6 th edition, w.b. saunders Company (2010); sambrook J. & Russell D. molecular Cloning A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); ausubel et al, Short Protocols in Molecular Biology A Complex of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); harlow and Lane use Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al, Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature associated with the analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmacochemistry and the laboratory procedures and techniques described herein are those well known and commonly used in the art.

Definition of

For a better understanding of the present invention, the definitions and explanations of the relevant terms are provided below.

The term "antibody" or "Ab" is used herein in the broadest sense and encompasses a variety of antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). Natural intact antibodies generally refer to Y-shaped tetrameric proteins comprising two heavy (H) and two light (L) chain polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α and ε, which define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH 3). Each light chain consists 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 (referred to as Complementarity Determining Regions (CDRs)) separated by relatively conserved regions (referred to as Framework Regions (FRs)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen binding sites, respectively. 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. The antibodies may be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refer to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and/or that competes for binding to the same antigen as the full-length antibody. In general, see Fundamental Immunology, Ch.7(Paul, W. eds., second edition, Raven Press, N.Y. (1989), which is incorporated herein by reference for all purposes the antigen-binding fragments of antibodies can be derived, for example, from whole antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains Addition or deletion of amino acids, and the like.

Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment; (ii) a F (ab')2 fragment; (iii) (ii) a fragment of Fd; (iv) (iv) an Fv fragment; (v) single chain fv (scFv) molecules; (vi) a dAb fragment; and (vii) the smallest recognition unit (e.g., an isolated Complementarity Determining Region (CDR), such as a CDR3 peptide, or the limiting FR3-CDR3-FR4 peptide) consisting of amino acid residues that mimic a hypervariable region of an antibody. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), Small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragments" used herein. In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. The variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5,10,15,20,40,60 or more) amino acids that result in flexible or semi-flexible connections between adjacent variable and/or constant domains in a single polypeptide molecule.

As used herein, the term "variable domain" of an antibody refers to the variable region of an antibody or a fragment thereof comprising one or more CDRs. Although the variable domain may comprise the entire variable region (e.g., HCVR or LCVR), it may comprise less than the entire variable region while still retaining the ability to bind to an antigen or form an antigen-binding site.

As used herein, the term "antigen binding moiety" refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs, or any other antibody fragment that binds an antigen but does not comprise the entire native antibody structure. In contrast to the term "antigen binding site" which generally refers to a variable domain, an antigen binding module may comprise 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 ', F (ab')₂Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)₂Bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabodies (ds diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies (nanobodies), domain antibodies, and bivalent domain antibodies. The antigen binding moiety is capable of binding the same antigen as the parent antibody. In certain embodiments, the antigen binding moiety may be a Fab fragment or a VHH antibody. In some embodiments, the antigen binding module can comprise one or more CDRs from a particular human antibody grafted onto a framework region from one or more different human antibodies. Further description of the detailed form of the antigen binding module is found in Spiess et al, Molecular Immunology,67(2), pages 95-106 (2015), and in Brinkman et al, mAbs,9(2), pages 182-212 (2017), which are incorporated herein by reference in their entirety.

"Fab" of an antibody refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) in association with the variable and first constant regions of a single heavy chain by a disulfide bond. In certain embodiments, both constant regions of the light and heavy chains are replaced by TCR constant regions.

The "Fc" of an antibody refers to the portion of an antibody comprising the second (CH2) and third (CH3) constant regions of a first heavy chain in combination with the second and third constant regions of a second heavy chain via a disulfide bond. Reference to an Fc region may refer to one or both chains of the Fc region, depending on the context. The Fc portion of an antibody is responsible for a variety of effector functions such as ADCC and CDC, but does not function in antigen binding. The ability of an antibody to initiate and modulate effector functions via its Fc domain is a critical part of its protective activity in vivo. Although the neutralizing activity of antibodies was previously thought to be the result of only Fab-antigen interactions, there is increasing evidence that their in vivo activity is also highly dependent on the interaction between the IgG Fc domain and its associated receptor Fc γ receptors (Fc γ R), which are expressed on the surface of effector lymphocytes.

The term "PD-L1", also known as programmed death ligand 1, is a 40kDa type 1 transmembrane protein, presumably playing a major role in suppressing the adaptive arm of the immune system. PD-L1 is the primary ligand for programmed death 1(PD-1), and PD-1 is a co-inhibitory receptor that can be constitutively expressed or induced in myeloid, lymphoid, normal epithelial cells and cancers. As used herein, the term "PD-L1" when referring to the amino acid sequence of 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; also included are fusion proteins of PD-L1 ECD, such as a fragment fused to mouse or human (mFc or hFc) IgG Fc. Furthermore, as will be appreciated by those skilled in the art, PD-L1 proteins will also include those proteins that have naturally or artificially introduced mutations (including but not limited to substitutions, deletions and/or additions) into the amino acid sequence that do not affect biological function.

As used herein, the term "antibody that binds to PD-L1" or "anti-PD-L1 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize PD-L1. The antibodies and antigen-binding fragments of the present disclosure may bind to soluble PD-L1 protein and/or cell surface-expressed PD-L1. Soluble PD-L1 includes native PD-L1 protein as well as recombinant PD-L1 protein variants that lack a transmembrane domain or are not associated with a cell membrane. As used herein, the expression "anti-PD-L1 antibody" includes monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first antigen-binding site that binds PD-L1 and a second antigen-binding site that binds a second (target) antigen, wherein the anti-PD-L1 antigen-binding site comprises any one or more of the heavy chain variable/light chain variable regions or CDR sequences as set forth 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 cell-produced signaling protein that stimulates angiogenesis. VEGF is a subfamily of the platelet-derived growth factor family of cystine-knot (cysteine-knob) growth factors. They are important signaling proteins involved in angiogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels 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 flavoviris svVEGF.

As used herein, "VEGF receptor" or "VEGFR" refers to a receptor for Vascular Endothelial Growth Factor (VEGF). There are three subtypes of VEGFR, numbered 1, 2 and 3. The VEGF receptor may be membrane-bound or soluble, depending on its alternative splicing. In the VEGF receptor, VEGFR-1 binds to 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 to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

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

The terms "anti-PD-L1/anti-VEGF antibody", "anti-PD-L1/anti-VEGF bispecific antibody", "antibody directed against PD-L1 and VEGF", "anti-PD-L1 x VEGF bispecific antibody", "PD-L1 x VEGF antibody" are used interchangeably herein and refer 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 a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

The term "chimeric antibody" as used herein refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, for example, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

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

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

As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using well established methods in the art. As used herein, the term "KD" is intended to mean the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of KD to Ka (i.e., KD/Ka) and expressed as molar concentration (M). Determination of antibodiesA preferred method of bulk Kd is by using surface plasmon resonance, preferably using a biosensor system such as

Provided is a system.

The term "high affinity" for an IgG antibody as used herein means having 1 × 10 affinity for the target antigen^-7M or less, more preferably 5X10^-8M or less, even more preferably 1X 10^-8M or less, even more preferably 5X10^-9M or less, and even more preferably 1X 10^-9K of M or less_DThe antibody of (1).

As used herein, the term "EC₅₀", also referred to as" half effective concentration ", refers to the concentration of drug, antibody or toxin agent that induces a response of 50% between the baseline and maximum values after a particular exposure time. In the context of the present application, EC₅₀In units of "nM".

As used herein, the term "IC₅₀", also referred to as" half maximal inhibitory concentration, "is a measure of the efficacy of a substance in inhibiting a particular biological or biochemical function. In the context of the present application, IC₅₀In units of "nM".

As used herein, the ability to "inhibit binding" refers to an antibody or antigen-binding fragment thereof that inhibits the binding of two molecules (e.g., human PD-L1/VEGF and human PD-1/VEGFR) to any detectable level. In certain embodiments, the binding of two molecules may be by an antibody or antigen-binding fragment thereof with an IC of no more than 50nM, no more than 30nM, no more than 10nM, no more than 5nM, no more than 1nM, or even lower₅₀And (4) inhibiting.

As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "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, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, Vol.66, G.E.Morris, eds (1996).

As used herein, the term "isolated" refers to a state that is obtained from a natural state by artificial means. An "isolated" substance or component may be one that, if it occurs in nature, is naturally occurring, is separated from the nature, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same high purity polynucleotide or polypeptide isolated from that natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" neither excludes mixed artificial or synthetic substances nor other impurities which do not affect the activity of the isolated substance.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds PD-L1/VEGF protein is substantially free of antibodies that specifically bind antigens other than PD-L1/VEGF protein). However, isolated antibodies that specifically bind to human PD-L1/VEGF protein may be cross-reactive to other antigens, such as PD-L1/VEGF protein from other species. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be used to express the carried genetic material element in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda bacteriophage or M13 bacteriophage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV 40). The vector may contain a number of 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 comprise 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, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2/0, YB 2/0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such a cell. Because certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

The term "identity," as used herein, refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "percent identity" refers to the percentage of identical residues between amino acids or nucleotides in a comparison molecule and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in comparative Molecular Biology, (Lesk, eds., A.M.), 1988, New York: Oxford University Press; biocomputing information and Genome Projects, (Smith, eds. D.W.), 1993, New York: Academic Press; computer Analysis of Sequence Data, Part I, (Griffin, A.M. and Griffin, eds. H.G.), 1994, New Jersey: human Press; von Heinje, G.,1987, Sequence Analysis in Molecular Biology, New York: Academic Press; sequence Analysis Primer, (Gribskov, M. and Devereux, eds., J.), 1991, New York: M.Stockton Press; and those described in Carillo et al, 1988, SIAMJ. applied Math.48: 1073.

As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or primed lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation and differentiation of specific immune cells to eventually produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be developed in the immune system of an organism after stimulating the organism with the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunization.

As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are known in the art and are disclosed herein. See, e.g., 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 invention, the human PD-L1/VEGF gene is transfected into 293F cells.

As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes optical phenomena that allow analysis of real-time biospecific interactions by detecting changes in protein concentration within a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a detailed description, see the examples and

U.S. et al (1993) Ann.biol.Clin.51: 19-26;

U.S. et al (1991) Biotechniques 11: 620-627; johnsson, B.et al (1995) J.mol.Recognit.8: 125-131; and Johnnson, B., et al (1991) anal. biochem.198: 268-.

As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method of Sorting a heterogeneous mixture of biological cells into two or more containers, one Cell at a time, based on the specific light scattering and Fluorescence characteristics of each Cell (flowmetric. Instruments for performing 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 FACScat 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 solid and non-solid tumors mediated by growth, proliferation or metastasis of any tumor or malignant cell that causes a medical condition, such as leukemia.

The terms "treatment", "treating" and "treatment" as used herein in the context of treating a condition generally relate to the treatment and therapy of humans or animals in which some desired therapeutic effect is achieved, for example, inhibition of the progression of the condition, including a decrease in the rate of progression, arrest in the rate of progression, regression of the condition, amelioration of the condition, and healing of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For cancer, "treating" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation or metastasis or some combination thereof. For a tumor, "treating" includes removing all or a portion of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

As used herein, the term "effective amount" refers to an amount of active compound or of a material, composition, or dose comprising the active compound that, when administered in accordance with a desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit/risk ratio. For example, an "effective amount," when used in combination with the treatment of a CD3/CD 20-associated disease or disorder, refers to an amount or concentration of an antibody, or antigen-binding portion thereof, that is effective to treat the disease or disorder.

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

The term "pharmaceutically acceptable" as used herein means that the carrier, diluent, excipient, and/or salt thereof is chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active agent, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR, 19 th edition, Pennsylvania: machine Publishing Company,1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen in the organism or alter the type of immune response. There are a variety of adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., freund's complete adjuvant and freund's incomplete adjuvant), corynebacterium parvum (corynebacterium parvum), lipopolysaccharides, cytokines, and the like. 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 antigen-binding portions thereof

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 antigen-binding portion thereof, comprising a first antigen-binding moiety that specifically binds to PD-L1 and a second antigen-binding moiety that specifically binds to VEGF. Such antibodies may be referred to herein, for example, as "anti-VEGF/anti-PD-L1" or "anti-PD-L1/VEGF" or "anti-PD-L1 xVEGF" or "PD-L1 xVEGF" bispecific antibodies, or other similar nomenclature.

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

For example, the antibodies of the disclosure are administered at 1 × 10^-7K of M or less_D、5×10^-8K of M or less_D、2×10^-8K of M or less_D、1×10^-8K of M or less_D、5×10^-9K of M or less_D、4×10^-9K of M or less_D、3×10^-9K of M or less_D、2×10^-9K of M or less_D、1×10^-9K of M or less_D、5×10^-10K of M or less_D、1×10^-10K of M or less_DBinds to human PD-L1 protein or human VEGF protein as measured by surface plasmon resonance.

As demonstrated in the examples section, bispecific antibodies of the disclosure are capable of binding to human PD-L1 and human VEGF (same as cynomolgus monkey VEGF) with higher affinity(ii) a Binding to cynomolgus monkey and mouse PD-L1; effectively block both PD-1/PD-L1 and VEGFR/VEGF signaling pathways, e.g., IC in nM₅₀(ii) a Block VEGF-induced HUVEC proliferation; and produce a strong agonist effect on cytokine secretion.

PD-L1 antigen binding module

The PD-L1 antigen binding moiety as defined herein may have various forms (e.g. VHH, scFv, Fab) as long as it is capable of specifically binding to an antigen. Generally, the PD-L1 binding moiety comprised in a 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 moiety may be derived from a parent single domain antibody (sdAb), e.g. a VHH antibody, which generally refers to an antibody consisting of a single antibody variable domain. Like whole antibodies, single domain antibodies also selectively bind to specific antigens. In some other embodiments, the PD-L1 antigen binding moiety may be derived from a heavy chain antibody without a light chain.

The term "single variable domain" or "heavy chain variable region of a heavy chain antibody" is in contrast to the terms "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "V_HH"or" nanobodies "and the like may be used interchangeably. V derived from camelidae antibodies_HHThe molecule is the smallest known intact antigen-binding domain (about 15kDa, or 10-fold smaller than conventional IgG) and is therefore well suited for delivery to dense tissues and into the confined space between macromolecules.

The parent VHH antibodies disclosed herein may be prepared by the skilled person according to methods known in the art or any future method. For example, VHHs may be obtained using methods known in the art, for example by immunising camels and obtaining hybridomas therefrom, or by cloning libraries of VHHs of the invention using molecular biology techniques known in the art, followed by selection using phage display.

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 the reverse transcription and the polymerase chain reaction,a gene library was generated containing single domain antibodies from millions of clones. Screening techniques such as phage display and ribosome display, for example, can be used to help identify clones that bind the antigen. In phage display, a library of (e.g., human) antibodies is synthesized on phage, the library is screened with an antigen of interest or an antibody-binding portion thereof, and the phage that binds to the antigen is isolated, such that immunoreactive fragments can be obtained. Methods for making 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, Cat. No. 27-9400-01; and Stratagene SurfZAP^TMPhage display kit, cat No. 240612). Other methods and reagents useful for generating and screening antibody display libraries also exist (see, e.g., Barbas et al, Proc. Natl. Acad. Sci. USA 88: 7978-.

Humanization of VHH antibodies can be achieved by a number of established methods in the art, for example, the amino acid sequence of the VHH framework region can be found in the human germline V gene database blast and the humanized VHH sequence can be generated by replacing the human CDR sequences in the high hit sequence with VHH CDR sequences according to the Kabat CDR definition. In addition, certain residues in the framework regions may be back-mutated to maintain affinity.

In some embodiments, the PD-L1 antigen binding module comprises one or more CDRs selected from the group consisting of:

(i) a CDR1 comprising SEQ ID NO. 1 or an amino acid sequence that differs from SEQ ID NO. 1 by NO more than 2 amino acid additions, deletions, or substitutions;

(ii) CDR2 comprising SEQ ID NO 2 or an amino acid sequence that differs from SEQ ID NO 2 by NO more than 2 amino acid additions, deletions, or substitutions; and

(iii) CDR3 comprising SEQ ID NO 3 or an amino acid sequence that differs from SEQ ID NO 3 by NO more than 2 amino acid additions, deletions, or substitutions.

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

In some embodiments, the VHH of the PD-L1 antigen binding module comprises: (i) 10, the amino acid sequence of SEQ ID NO; (ii) an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO 10; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7,6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or substitutions as compared to SEQ ID NO: 10.

VEGF antigen binding modules

Similarly, the VEGF antigen binding moieties provided herein can be derived from anti-VEGF monospecific antibodies. In some embodiments according to the present application, the VEGF antigen binding moiety is a Fab fragment of an anti-VEGF whole antibody, i.e., comprising the VH and CH1 regions of the heavy chain, and the VL and CL regions of the light chain.

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

In some embodiments, the VH region of the VEGF antigen-binding module comprises one or more heavy chain cdrs (hcdrs) selected from the group consisting of:

(i) HCDR1 consisting of SEQ ID NO. 4 or HCDR1 having an amino acid sequence that differs from SEQ ID NO. 4 by NO more than 2 amino acid additions, deletions, or substitutions;

(ii) HCDR2 consisting of SEQ ID NO. 5 or HCDR2 having an amino acid sequence that differs from SEQ ID NO. 5 by NO more than 2 amino acid additions, deletions, or substitutions; and

(iii) HCDR3 consisting of SEQ ID NO 6 or HCDR3 having an amino acid sequence that differs from SEQ ID NO 6 by NO more than 2 amino acid additions, deletions, or substitutions; and/or

The VL region comprises one or more light chain cdrs (lcdr) selected from the group consisting of:

(i) LCDR1 consisting of SEQ ID NO. 7 or LCDR1 with an amino acid addition, deletion, or substitution that differs by NO more than 2 amino acids from SEQ ID NO. 7;

(ii) LCDR2 consisting of SEQ ID NO. 8 or LCDR2 with an amino acid sequence that differs from SEQ ID NO. 8 by NO more than 2 amino acid additions, deletions, or substitutions; and

(iii) LCDR3 consisting of SEQ ID NO.9 or LCDR3 having an amino acid sequence that differs from SEQ ID NO.9 by NO more than 2 amino acid additions, deletions, or substitutions.

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) HCDR3 comprising or consisting of SEQ ID NO 6; 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) LCDR3 comprising or consisting of SEQ ID NO 9.

In some embodiments, the VH of the VEGF antigen-binding module comprises: (i) 11, the amino acid sequence of SEQ ID NO; (ii) an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO 11; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7,6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or substitutions as compared to SEQ ID NO: 11.

In some embodiments, the VL of the VEGF antigen-binding moiety comprises: (i) 12, SEQ ID NO; (ii) an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO 12; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7,6, 5, 4, 3, 2, 1) amino acid additions, deletions and/or substitutions as compared to SEQ ID NO: 12.

Unless otherwise indicated, the assignment of amino acids to each CDR can be according to one of the numbering schemes provided below: kabat et al (1991) Sequences of Proteins of Immunological Interest (5 th edition), US depth 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 eds (2007) Handbook of Therapeutic Antibodies, 3 rd edition, Wily-VCH VerVEGF GmbH and Co.

The variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as described above, e.g., the Kabat numbering system) or by aligning the sequence to 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. Exemplary databases of antibody sequences are described in and available from the "Absysis" website (maintained by Department of Biochemistry & Molecular Biology University College London, London, A.C. Martin of England) and the VBASE2 website www.vbase2.org on www.bioinf.org.uk/abs, as described in Retter et al, Nucl. acids Res.,33(Database issue): D671-D674 (2005). The sequences are preferably analyzed using the Abysis database, which integrates Sequence data from the Kabat, IMGT, and Protein Database (PDB) with structural data from the PDB, see Protein Sequence and Structure Analysis of Antibody Variable Domains in the book by Dr.Andrew C.R.Martin in: Antibody Engineering Lab Manual (ed: Duebel, S. and Kontermann, R., Springer-VerVEGF, Heidelberg, ISBN-13: 978-one 35413547, also available on the website Biooil for. uk/abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein. Unless otherwise indicated, all CDRs described herein were obtained from the Abysis database website of Kabat.

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 a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, 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 GAP program in the GCG software package (available from http:// www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, with a GAP weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to perform searches against public databases, for example to identify related sequences. Such a search may be performed using the XBLAST program (version 2.0) of Altschul, et al (1990) J.MoI.biol.215: 403-10. BLAST protein searches using the XBLAST program can be performed with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gap alignments for comparison purposes, gap BLAST can be used, as described in Altschul et al, (1997) Nucleic Acids Res.25(17): 3389-3402. When BLAST and gapped BLAST programs are used, the default parameters for each program (e.g., 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% or 99% identical to the specific CDR amino acid sequence contained in the corresponding sequence described above. 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 CDRs of the isolated antibody or antigen binding portion thereof comprise 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 alter the basic properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include those in which an amino acid residue is substituted with another amino acid residue having a similar side chain, e.g., a physically or functionally similar residue (e.g., of similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, 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, e.g., 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 are incorporated herein by reference).

Generation of bispecific antibodies

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

Bispecific antibodies and antigen-binding portions thereof provided herein can be prepared using any suitable method known in the art. One conventional approach is to recombinantly produce bispecific antibodies by co-expressing two pairs of immunoglobulin heavy chain-light chains in a host cell (see, e.g., Milstein and Cuello, Nature,305:537(1983)), followed by purification by affinity chromatography. Bispecific antibodies can also be recombinantly expressed by fusing sequences encoding the heavy chain variable domains of antibodies specific for both specificities separately to immunoglobulin constant domain sequences, followed by insertion into an expression vector, and co-transfecting the expression vector with an expression vector for the light chain sequence into a suitable host cell (see, e.g., 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 moiety. The Fc region of the bispecific antibodies disclosed herein can be a human IgG Fc region. The IgG Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG 4. In certain embodiments, the Fc region is of the IgG1 isotype.

In the context of bispecific antibodies of the present disclosure, the Fc region may comprise one or more amino acid alterations (e.g., insertions, deletions, or substitutions) as compared to a 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 that result in a modified Fc region with modified binding interactions between Fc and FcRn or fcyr.

When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is typically used (e.g., the EU index as reported in Kabat et al, supra). "EU numbering as in Kabat" or "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody. Unless otherwise indicated herein, reference to residue numbering in the constant domain of an antibody refers to residue numbering by the EU numbering system.

In certain embodiments, the Fc region is operably linked to the VEGF antigen binding moiety via a hinge region. Optionally, the hinge region may be derived from human IgG1, IgG2, or IgG 4. In certain embodiments, the hinge region is derived from human IgG 1.

Nucleic acid molecules encoding antibodies of the disclosure

In some aspects, the 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 and/or light chain of a bispecific antibody, or the nucleic acid sequence may encode the heavy or light chain variable region of a PD-L1 antigen-binding module, or a VEGF antigen-binding module. The nucleic acid sequence may also encode the Fc region of a bispecific antibody.

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

The vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, CD20 or CD3 antibody-encoding nucleic acids are contained in naked DNA or RNA vectors, including, for example, linear expression elements (described, for example, in Sykes and Johnston, Nat Biotech 17,355-59(1997)), compact nucleic acid vectors (described, for example, in US 6,077,835 and/or WO 00/70087), plasmid vectors, for example pBR322, pUC 19/18 or pUC 118/119, "midge" minimum size nucleic acid vectors (described, for example, in Schakowski et al, Mol Ther 3,793-800(2001)), or as precipitated nucleic acid vector constructs, for example, Cap 04-precipitated constructs (described, for example, in WO200046147, Benvennisty and Reshef, PNAS USA 83,9551-55(1986), Wigler et al, Cell14,725(1978) and Coraro and Pearson, Somatotics Genetics 7, 1981). Such nucleic acid vectors and uses thereof are well known in the art (see, e.g., US5,589,466 and US5,973,972).

In one embodiment, the vector is suitable for expressing an anti-PD-L1/VEGF bispecific antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-. 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 may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters, such as alpha factor, alcohol oxidase and PGH (reviewed in: 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 comprising glutamine synthetase as a selectable marker, for example as described in Bebbington (1992) Biotechnology (NY)10: 169-.

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

The expression vector may comprise or be associated with any suitable promoter, enhancer, and other expression promoting element. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), efficient poly (a) termination sequences, origins of replication of plasmid products in e.coli, antibiotic resistance genes as selectable markers, and/or convenient cloning sites (e.g., polymeric linkers). The nucleic acid may also comprise an inducible promoter as opposed to a constitutive promoter, such as CMV IE.

In yet another aspect, the disclosure relates to a host cell comprising a vector described herein. Thus, the invention also relates to recombinant eukaryotic or prokaryotic host cells, e.g. transfectomas, producing the bispecific antibodies of the invention.

The PD-L1-specific antibody may be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein, or a bispecific antibody of the invention as defined herein. The VEGF-specific antibody may likewise be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein or a bispecific antibody of the invention as defined herein.

Examples of host cells include yeast, bacterial, plant and mammalian cells, such as CHO, CHO-S, HEK293, HEK-293F, Expi293F, PER. C6 or NS0 cells or lymphocytes. For example, in one embodiment, a host cell can 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-integrating nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising the first and second nucleic acid constructs described above.

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

In yet another aspect, the present disclosure relates to a hybridoma that produces an antibody for a bispecific antibody of the present disclosure as defined herein.

In one aspect, the invention relates to an expression vector comprising

(i) A nucleic acid sequence encoding a PD-L1 antigen binding module;

(ii) a nucleic acid sequence encoding the heavy and/or light chain of a VEGF antigen-binding module;

(iii) a nucleic acid sequence encoding an Fc region; or

(iv) A nucleic acid sequence encoding the heavy and/or light chain of a bispecific antibody.

In one aspect, the disclosure relates to nucleic acid constructs encoding one or more of the amino acid sequences listed in the sequence listing.

In one aspect, the disclosure relates to a method of producing a bispecific antibody according to any one of the embodiments disclosed herein, comprising culturing a host cell comprising one or more expression vectors expressing a bispecific antibody disclosed herein, and purifying the antibody from the culture medium. In one aspect, the present 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 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 invention may also be administered in combination with, for example, another immunostimulant, an anti-cancer agent, an anti-viral agent, or a vaccine, such that the anti-PD-L1/anti-VEGF bispecific antibody enhances the immune response to the vaccine. The pharmaceutically acceptable carrier may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending/dispersing agent, chelating agent, diluent, adjuvant, excipient or nontoxic auxiliary substance, a combination of various components known in the art or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorants, thickeners, colorants, 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, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl arsenate. As disclosed herein, the antibody or antigen-binding fragment of the disclosed compositions may be oxidized in a solvent containing the antibody or antigen-binding fragment of the disclosed composition comprising one or more antioxidants, such as methionine, that reduce the antibody or antigen-binding fragment thereof. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the present invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibody or antigen-binding fragment thereof is mixed with one or more antioxidants, such as methionine. Thus, the antibody or antigen-binding fragment thereof may be prevented from oxidation, to extend its shelf-life and/or increase activity.

To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous carriers such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactate ringer's injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobials at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or glucose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethyl cellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80(TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic 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 may be added to the pharmaceutical composition in a multi-dose container containing phenol or cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancing agents or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

Administration, formulation and dosage

The pharmaceutical compositions of the present invention may be administered in vivo to a subject in need thereof by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention may be formulated in solid, semi-solid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and treatment regimen.

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

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, ringer's solution or lactated ringer's injection. Similarly, the particular dosage regimen (including dose, time and repetition) will depend on the particular individual and the individual's medical history and empirical considerations such as pharmacokinetics (e.g., half-life, clearance, 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 such a reduction in tumor cells, reducing proliferation of tumor cells or delaying the development of metastases. In some embodiments, the dose administered may be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the present invention may be suitable.

One skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risk or deleterious side effects. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the particular compound, the administration, the time of administration, the rate of clearance of the compound, the duration of the treatment, other drugs, compounds and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health and prior medical history of the patient. The amount of the compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect, without causing substantial deleterious or adverse side effects.

In general, the antibodies of the invention, or antigen binding portions thereof, can be administered in a variety of ranges. These include from about 5 μ g/kg body weight to about 100mg/kg body weight per dose; about 50 μ g/kg body weight to about 5mg/kg body weight per dose; about 100. mu.g/kg body weight to about 10mg/kg body weight per dose. Other ranges include from about 100 μ g/kg body weight to about 20mg/kg body weight per dose and from about 0.5mg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, each dose is at least about 100 μ g/kg body weight, at least about 250 μ g/kg body weight, at least about 750 μ g/kg body weight, at least about 3mg/kg body weight, at least about 5mg/kg body weight, at least about 10mg/kg body weight.

In any event, the antibodies of the invention or antigen-binding portions thereof are preferably administered to a subject in need thereof as needed. The frequency of administration can be determined by one skilled in the art, for example, by the attending physician based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like.

In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding portions thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the invention can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

The dosage and regimen of the disclosed therapeutic compositions can also be determined empirically in individuals given one or more administrations. For example, an individual may be administered a incremental dose of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or decreased or lessened based on empirically determined or observed side effects or toxicity, respectively. To assess the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopy of tumor samples; measuring the reduction in pain or paralysis of an indirect tumor marker (e.g., PSA for prostate cancer) or tumorigenic antigen identified according to the methods described herein; improvement in speech, vision, respiration or other disability associated with the tumor; appetite increase; or an increase in quality of life or an increase in survival as measured by accepted tests. Those skilled in the art will appreciate that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other locations in the individual, and the treatment used in the past and the treatment used concurrently.

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

Application of the invention

In some aspects, the invention provides a method of treating a disorder in a subject, comprising administering to a patient (e.g., a human) in need of treatment a therapeutically effective amount of an antibody or antigen-binding portion thereof as disclosed herein. For example, the disease is a cancer.

Various cancers involving PD-L1 and/or VEGF, whether malignant or benign, and whether primary or secondary, may be treated or prevented using the methods provided by the present disclosure. These cancers may be solid cancers or hematologic malignancies. Examples of such cancers include lung cancers such as bronchial carcinomas (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondrogenic hamartoma (noncancerous) and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondrosis, chondroma, chondroblastoma, chondroid chondroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, ewing's tumor (ewing's sarcoma) and reticulocytoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannoma, ependymomas, meningiomas, pituitary adenomas, pinealomas, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermatocysts, and hemangiomas; cancers in the digestive system such as colon cancer, leiomyomas, epidermoid cancers, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, large intestinal polyps and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma and angiosarcoma; renal cancers such as renal adenocarcinoma, renal cell carcinoma, anadroma and transitional cell carcinoma of the renal pelvis; bladder cancer; hematologic cancers such as acute lymphocytic leukemia (acute lymphocytic leukemia), acute myelogenous (myelogenous, myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myelogenous (myelogenous, granulocytic) lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia and chronic granulocytic leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, kaposi's sarcoma, and paget's disease; head and neck cancer; cancers associated with the eye, such as retinoblastoma and intraocular melanoma; cancers of the male reproductive system such as benign prostatic hyperplasia, prostate cancer and testicular cancer (e.g., seminoma, teratoma, embryonic carcinoma and choriocarcinoma); breast cancer; cancers of the female reproductive system 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 carcinoma); pheochromocytoma (adrenal gland); non-cancerous growth of parathyroid gland; 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 cancer include, but are not limited to, B cell cancers including B cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL); Small Lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulk disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; Chronic Lymphocytic Leukemia (CLL); Acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myelocytic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus, edema (e.g., associated with brain tumors), B cell proliferative disorders and Meigs ' syndrome, more specific examples including, but not limited to, relapsed or refractory NHL, a prodeline low grade NHL, a grade III/IV NHL, a chemotherapy-resistant NHL, a precursor Bly lymphoblastic leukemia and/or lymphoma, a small lymphocytic lymphoma, a B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, a B-cell prolymphocytic lymphoma, an immune cell tumor and/or lymphoplasmacytic lymphoma, a marginal zone B-cell lymphoma, a splenic marginal zone lymphoma, an extranodal marginal zone-MALT lymphoma, a lymph node marginal zone lymphoma, a hairy cell leukemia, a plasmacytoma and/or a plasma cell myeloma, a low grade/follicular lymphoma, a medium grade/follicular NHL, a mantle cell lymphoma, a follicular central lymphoma (including an aggressive prodeline NHL and an aggressive relapsing NHL), a relapsed or relapsed NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, bulky disease NHL, burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or sezary syndrome, cutaneous (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, lymphomas (e.g., B cell non-hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, B) small non-dividing/burkitt's lymphoma (including endemic burkitt's lymphoma, sporadic burkitt's lymphoma and non-burkitt's 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), immunoblastic lymphoma, primary mediastinal B cell lymphoma, angiocentric lymphoma-pulmonary B cell lymphoma, f) hairy cell leukemia, g) lymphocytic lymphoma, waldenstrom's macroglobulinemia, h) acute Lymphocytic Leukemia (ALL), chronic lymphocytic leukemia CLL)/Small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytoma and/or j) Hodgkin's disease.

In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that can be treated with the antibodies or antigen-binding portions thereof include autoimmune encephalomyelitis, lupus erythematosus and rheumatoid arthritis. The antibodies, or antigen-binding portions thereof, may also be used to treat or prevent infectious diseases, inflammatory diseases (e.g., 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 anti-cancer agent, cytotoxic agent, or chemotherapeutic agent.

The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful in the treatment of cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiation therapy and anti-metastatic agents, and immunotherapeutic agents. It is to be understood that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may be conjugated to the disclosed site-specific antibodies prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present invention. In other embodiments, the disclosed anti-cancer 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 agent 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 (staphyloccal enterotoxin) a), fungi (e.g., alpha-sarcin, restrictocin), plants (abrin, ricin, calycanthin, quercetin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, Phytolacca merica protein (PAPI, PAPII and PAP-S), Momordica charantia (momordia charrantia) inhibitor, leprosy curcin, crotin, lycopodium (Saponaria officinalis) inhibitor, gelonin, mitellin, restrictocin, neomycin and trichothecene compounds) or trichothecene compounds (e.g., RNA, including fragments and/or variants thereof) or enzymatically active toxins.

For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic or cytostatic agents) that nonspecifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemicals are generally directed against intracellular processes required for cell growth or division and are therefore particularly effective against cancer cells which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. In general, a chemotherapeutic agent may include any chemical agent that inhibits or is designed to inhibit a cancer cell or a cell that may become sexually or produce tumorigenic progeny (e.g., TIC). These agents are often used in combination and are often most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with the site-specific constructs of the invention (as components or in an unconjugated state of the site-specific conjugates) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimine and methyl melamine, polyacetyls (acetogenins), camptothecins, bryostatins, calicheastatins (callystatins), CC-1065, croutoxins (cryptophycins), dolastatins, duocarmycins, eleutherobin (eleutherobin), coprostanin, saxodidin (sarcodictyins), spongin (spongistatin), mechlorethamine, antibiotics, enediynes, dynemics, bisphosphonates, epothilones, chromogens of chromogenes, clarithromycins (acacins), actinomycins, atramycins, azatrinins, bleomycin, actinomycins (carbapenems), caracinomycins (mycins), carcinomycins (mycins), mycins (mycins), Dactinomycin, daunorubicin, ditetracycline, 6-diazo-5-oxo-L-norleucine,

Doxorubicin, epirubicin, esorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, nogomycin, olivomycin, pelomycin, bodhimycin (potfiromycin), puromycin, triiron doxorubicin, roxobicin, streptonigrin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-epinephrine, folic acid supplements such as furinic acid (frilinic acid), acetoglucide, aldphosphoramide glycosides, aminoacetylpropionic acid, eniluracil, amsacrine, besubulic acid (bestraucil), bisantrene, edatrexate, difumamine (deffamine), colchicine, diazaquinone, efonicin (elfornitine), ethacrinide, epothilones, etoglu, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (maytansinoids), mitoguazatine, mitoxantrone, motdan (mopinnol), nitrene (netrarinone), pentostatin, amcinolone, piridoxuridine, carbohydrazide, 2-ethyl-propylate, propoxyphylzine, fossa, etoposide, leucinonide, leucinolone, and related compounds,

Polysaccharide complexes (JHS Natural Products, Eugene, OR), Razoxan; rhizomycin; a texaphyrin; a germanium spiroamine; (ii) zonecanoic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, Verlucurin A (verracurin A), bacillocin A and snakeheaded; uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; cassitoxin (gacytosine); arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chlorambucil (chlorenbucil);

gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; a platinum analog; vinblastine; platinum; etoposide (VP-16) (ii) a Ifosfamide; mitoxantrone; the concentration of the vincristine is controlled by the concentration of the vincristine,

vinorelbine; noxiaoling; (ii) teniposide; edatrexae; daunorubicin; aminopterin; (ii) Hirodad; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine; a retinoid; capecitabine; combretastatin; leucovorin; oxaliplatin; an inhibitor of PKC-alpha, Raf, H-Ras, EGFR, and VEGF-A (which reduces cell proliferation), and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing. 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, aromatase inhibitors that inhibit aromatase that modulates estrogen production in the adrenal glands, and anti-androgens; and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues); antisense oligonucleotides, ribozymes such as inhibitors of VEGF expression and inhibitors of HER2 expression; a vaccine is provided which comprises a vaccine,

rIL-2；

a topoisomerase 1 inhibitor;

rmRH; vinorelbine and epsipromycin, and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

Used in combination with radiotherapy

The invention also provides the combination of an antibody or antigen-binding portion thereof with radiotherapy (i.e., any mechanism used to induce DNA damage locally within tumor cells, such as gamma-irradiation, X-ray, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates may be used in conjunction with targeted anti-cancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy may be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple sequential doses.

Pharmaceutical pack and kit

Pharmaceutical packages and kits comprising one or more containers containing one or more doses of an antibody, or antigen-binding portion 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 additional agents. For other embodiments, such unit doses are supplied in single use prefilled syringe injections. In other embodiments, the composition contained in a unit dose may comprise saline, sucrose, or the like; buffers such as phosphate and the like; and/or formulated at a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted after addition of a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the compositions comprise one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the one or more containers indicates that the packaged composition is used to treat a selected neoplastic disease condition.

The invention also provides kits for producing single-dose or multi-dose administration units of an antibody and optionally one or more anti-cancer 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 can 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 includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits typically comprise a pharmaceutically acceptable formulation of the antibody in a suitable container, and optionally one or more anti-cancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for use in diagnosis or combination therapy. For example, such kits may contain, in addition to an antibody or antigen-binding portion thereof of the invention, any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapeutic agents; an anti-angiogenic agent; an anti-transfer agent; targeted anti-cancer agents; a cytotoxic agent; and/or other anti-cancer agents.

More specifically, kits can have a single container containing the disclosed antibodies or antigen-binding portions thereof, with or without additional components, or they can have different containers for each desired reagent. Where a combination therapeutic agent is provided for conjugation, the single solutions may be premixed in molar equivalent combinations or in a manner such that one component is more than the other. Alternatively, the antibody and any optional anti-cancer agent of the kit may be stored separately in separate containers prior to administration to a patient. The kit may also comprise a second/third container means for holding sterile pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), Phosphate Buffered Saline (PBS), ringer's solution and dextrose solution.

When the components of the kit are provided as one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous or saline solution. However, the components of the kit may be provided as a dry powder. When the agent or component is provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

As briefly mentioned above, the kit may also contain means for administering the antibody or antigen-binding portion thereof and any optional components to the patient, such as one or more needles, i.v. bags or syringes, or even eye droppers, pipettes, or other similar devices, through which the formulation may be injected or introduced into the animal or administered to the affected area of the body. The kits of the invention will also typically include a means for holding vials or the like, as well as other tightly closed components for commercial sale, such as injection or blow molded plastic containers, in which the desired vials and other devices are placed and retained.

Summary of sequence listing

The present application is accompanied by a sequence listing comprising a number of amino acid sequences. Table a below provides an overview of the sequences involved.

An exemplary anti-VEGF/anti-PD-L1 bispecific antibody as disclosed herein is referred to as W3256-u15t2.g6-1.uIgG1 (which may be abbreviated as W3256 throughout the specification).

TABLE A

SEQ ID NO.	Description of the invention
		1	Amino acid sequence of CDR1 of PD-L1 binding Module
2	Amino acid sequence of CDR2 of PD-L1 binding Module
		3	Amino acid sequence of CDR3 of PD-L1 binding Module
4	Amino acid sequence of heavy chain CDR1 of VEGF binding Module
		5	Amino acid sequence of heavy chain CDR2 of VEGF binding Module
6	Of VEGF binding modulesAmino acid sequence of heavy chain CDR3
		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

Examples

The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. These examples are not intended to be representative of the experiments below being all or only experiments performed.

Example 1

Preparation of materials, reference antibodies and cell lines

1.1 preparation of the Material

Information on commercially available materials used in the examples is provided in table 1.

TABLE 1

1.2 antigen preparation

DNA sequences encoding human VEGF (Uniport No.: P15692), mouse VEGF (Uniport No.: Q00731) and the extracellular domain sequence of human PD-L1(Uniport No.: Q9NZQ7), mouse PD-L1(Uniport No.: Q9EP73), human PD-1(Uniport No.: Q15116) and mouse PD-1(Uniport No.: Q02242) were synthesized by Sangon Biothech (Shanghai, China) and then subcloned into the modified pcDNA3.3 expression vector with a different tag (e.g., 6xhis, human Fc or mouse Fc) at the C-terminus.

Expi293 cells (Thermo Fisher Scientific, A14527) were transfected with the purified expression vector. 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). The obtained human VEGF, human PD-L1, mouse PD-L1, human PD-1 and mouse PD-1 were quality-controlled by SDS-PAGE and SEC, and then stored at-80 ℃.

1.3 Generation of reference antibody (BMK Ab)

DNA sequences encoding fragments of the anti-VEGF antibody bevacizumab (designated WBP325-BMK3 or WBP325-BMK3.uIgG1, sequences from Drug Bank, Drug Bank accession number: DB00112) were synthesized in either Sangon Biothech (Shanghai, China) or Genewiz (Suzhou, China) and subcloned into the modified pcDNA3.3 expression vector along with the Fc region of human IgG 1.

anti-PD-L1 VHH antibodies (W3156-AP3R2-1A3-z12) were generated by alternating immunization of alpacas with the ectodomain of human PD-L1 and the ectodomain of mouse PD-L1, and PBMCs were isolated for phage library construction. After panning, screening and sequencing, unique positive VHH fragments were identified (SEQ ID No:4 as disclosed in patent application No. PCT/CN 2020/117351).

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

To produce WBP325-BMK3, plasmids containing recombinant VH and VL genes were 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 obtained antibodies were analyzed by SDS-PAGE and SEC and then stored at-80 ℃.

1.4 establishment of Stable cell lines/cell pools

Cell line expressing PD-L1

CHO-K1 or 293F cells were transfected with expression vectors containing genes encoding full-length human PD-L1 or mouse PD-L1 or cynomolgus monkey PD-L1 using Lipofectamine 2000(Thermo Fisher Scientific, 11668019) or PlasFect (Bioline, 46026). The cells are cultured in a medium containing an appropriate selection marker. A human PD-L1 high-expression stable cell line (WBP315.CHO-K1.hPro1.C11), a mouse PD-L1 high-expression stable cell line (WBP315.293F. mPro1.C1) and a cynomolgus monkey PD-L1 high-expression stable cell line (WBP315.293F. cPro1.2A) are obtained by limiting dilution.

The genes of human PD-L1 or mouse PD-L1 or cynomolgus monkey PD-L1 are respectively inserted into expression vector pcDNA 3.3. The plasmids were then transfected into CHO-K1 cells or 293 cells, respectively. Briefly, for CHO-K1 cells, 5X10 was administered the day before transfection⁵A single well of 6-well tissue culture plates was seeded with CHO-K1 cells and incubated at 5% CO2 and 37 ℃. Cells were cultured with 3ml of fresh non-selective medium (F12-K, 10% FBS). Transfection reagents were prepared in 1.5ml tubes, including 4. mu.g DNA mixed with 10. mu.g Lipofectamine 2000 to give a final volume of 200. mu.l in Opti-MEM medium. The solution in the pipette is added dropwise to the cells. 6-8 hours after transfection, cells were washed with PBS and 3ml of fresh non-selective medium was added. 24-48 hours after transfection, expression cells were harvested with trypsin and inoculated into selective medium (F12-K, 10% FBS, 10. mu.g/ml blasticidin) in T75 flasks. For 293F cells, 20. mu.g of DNA was mixed with 50. mu.l of PlasFect to give a final volume of 200. mu.l in Opti-MEM medium. The mixture was added to 20ml (1X 10)⁶/ml) in 293F cells and cultured in Freestyle293 medium in 125ml flasks. Blasticidin was added as a selection marker 48 hours after transfection. After two or three selection passes, cells were detected with anti-PD-L1 antibody. Stable single cell clones were isolated by limiting dilution and screened by FACS using anti-PD-L1 antibody.

Cell lines expressing targets

Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from ScienceCell (cat # 8000) and cultured in endothelial cell culture medium (ECM, ScienceCell, cat # 1001) containing alkaline medium, 5% FBS, 1% endothelial cell growth factor (ECGS, ScienceCell, 1052). Cells were cultured in an incubator at 37 ℃ and 5% CO 2. For long-term storage, cells were frozen in complete growth medium supplemented with 5% (v/v) DMSO and stored in liquid nitrogen phase.

Example 2

Generation of anti-PD-L1/VEGF bispecific antibodies

2.1 construction of expression vectors

The DNA sequence encoding VHH antibody W3156-AP3R2-1A3-z12 linked by a flexible G4S linker was placed N-terminal to 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 vectors, respectively. The constructed antibody was named W3256-u15t2.g6-1.uIgG1 (may be abbreviated as "W3256" throughout the disclosure).

As described above, the heavy chain of W3256-u15t2.g6-1.uIgG1 comprises the variable heavy chain region of WBP325-BMK3 (bevacizumab) and the constant heavy chain region of human IgG1 (CH1-CH 3). The light chain of W3256-u15t2.g6-1.uIgG1 was composed of the variable light chain region of WBP325-BMK3 (bevacizumab) and the constant light chain region (CL) of human IgG1, as well as the VHH antibody W3156-AP3R2-1A3-z12 at the N-terminus, as shown in fig. 1. Specific sequences of the W3256 antibody are listed in tables 2-4 below.

TABLE 2

TABLE 3

TABLE 4

2.2 transfection, expression and purification

The heavy and light chain expression plasmids were co-transfected into Expi293 cells using the Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer's instructions. 5 days after transfection, the supernatant was collected and Protein purification was performed using Protein A column (GE Healthcare-17543802). Antibody concentration was measured by NanoDrop. The purity of the protein was assessed by SDS-PAGE and HPLC-SEC. Bispecific antibodies were obtained after expression and purification, including W3256-u15t2.g6-1.uIgG 1.

2.3 Generation of bispecific antibodies for in vivo studies including endotoxin control and testing

The W3256-U15T2.G6-1.uIgG1 expression plasmid was transfected into Expi293 cells using the Expi293 expression System kit (ThermoFisher-A14635) according to the manufacturer's instructions. Five days after transfection, the supernatant was collected and Protein purified using a Protein A column (GE Healthcare-17543802) under endotoxin control. Low endotoxin levels (below 10EU/mg) were confirmed using an endotoxin detection kit (GenScript-L00350).

The yield of W3256-U15T2.G6-1.uIgG1 was 44.8mg/l as measured by NanoDrop. The purity of the protein was assessed by SDS-PAGE (FIG. 2) and HPLC-SEC (FIG. 3). After one-step purification of protein A according to HPLC-SEC, the purity of W3256-U15T2.G6-1.uIgG1 was 93.43%.

Example 3

In vitro characterization of bispecific antibodies

3.1 differential scanning fluorescence method (DSF)

The DSF assay was performed using a 7500 rapid real-time PCR system (Applied Biosystems). Briefly, 19. mu.l of bispecific antibody solution was mixed with 1. mu.l of 62.5X SYPRO Orange solution (ThermoFisher-S6650) and then added to a 96-well plate. The plate was heated from 26 ℃ to 95 ℃ at a rate of 2 ℃/min and the resulting fluorescence data was collected. The data was automatically analyzed by its operating software and Th was calculated by taking the maximum of the negative derivative of the resulting fluorescence data with respect to temperature. T is_onThe temperature at which the negative derivative map begins to decrease from the pre-transition baseline may be determined approximately.

As shown in FIG. 4, T of W3256 antibody_hThe value of 1 is 65.7 ℃.

3.2 human/cynomolgus monkey VEGF binding (ELISA)

The binding of the antibody to human VEGF antigen (WBP325-hPro1, Sino Biological, 11066-HNAB) was tested by a binding ELISA assay. Since the amino acid sequence of cynomolgus VEGF is identical to that of human VEGF, the binding results represent binding of human VEGF and binding of cynomolgus VEGF. Briefly, 96-well ELISA plates (Nunc MaxiSorp, ThermoFisher, 442404) were placed in carbonate-bicarbonate buffer (20mM Na₂CO3、180mM NaHCO₃pH9.2) with 0.25. mu.g/ml human VEGF overnight at 4 ℃. After a1 hour blocking step with 2% (W/v) bovine serum albumin (Pierce) dissolved in PBS, serial dilutions of W3256-U15T2.G6-1.UIgG1 in PBS were incubated on the plates for 2 hours at room temperature. After incubation, the plates were used in 300. mu.L/wellPBS containing 0.5% (v/v) Tween 20 was washed 3 times. Goat anti-human IgG Fc-HRP (Bethyyl, # A80-304P) was added at 100ng/ml and incubated on the plate for 1 hour at room temperature. After 6 washes, Tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) was added for detection. After about 8 minutes, the reaction was stopped by adding 100. mu.l of 2M HCl per well. Using a multi-well plate reader (

M5^e) The absorbance of the wells was measured at 450 nm.

W3256 showed comparable binding capacity to WBP325-BMK3.uIgG1 on human VEGF, EC₅₀About 0.095nM (fig. 5).

3.3 human PD-L1 binding (FACS)

Engineered human PD-L1 expressing cells (wbp315.cho-k1.hpro1.c11) at 1 × 10⁵Cells/well were seeded in U-bottom 96-well plates. Serial dilutions of W3256-u15t2.g6-1.UIgG1 were added to the cells. The plates were incubated at 4 ℃ for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-. Binding of the antibody to the cells was tested by flow cytometry and Mean Fluorescence Intensity (MFI) was analyzed by FlowJo.

W3256 also showed comparable binding capacity, EC, to W3156-AP3R2-1A3-z12-hIgG1 on human PD-L1₅₀About 0.128nM (FIG. 6).

3.4 human VEGF/human PD-L1 double binding (ELISA)

To test whether bispecific antibodies can bind to both VEGF and PD-L1, the following ELISA assay was developed. 96-well ELISA plates (Nunc MaxiSorp, ThermoFisher) were coated overnight at 4 ℃ with 1. mu.g/ml antigen-1 (VEGF, WBP325-hPro1, Sino Biological) or 1. mu.g/ml antigen-2 (PD-L1.ECD. mFc, W3153-hPro1.ECD. mFc) in carbonate-bicarbonate buffer. After 1 hour of blocking, serial dilutions of W3256-U15T2.G6-1.UIgG1 in casein buffer were incubated on the plates for 1 hour at room temperature. After incubation, the plates were washed 3 times with 300. mu.L per well of PBS containing 0.5% (v/v) Tween 20. Mixing 0.5 μ g/ml antigen-2. Biotin (PD-L1-ECD, WBP315-hPro1.ECD. mFc) or 0.5 μ g/ml antigen-1. Biotin (VEG)F, WBP325-hpro1.his) were added to the plates and incubated for 1 hour. After washing the plate 3 times, HRP-labeled secondary detection antibody was added and incubated on the plate for 1 hour at room temperature. After washing the plate 6 times, Tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) was added for detection. After about 10 minutes, the reaction was stopped by adding 100. mu.L of 2M HCl per well. Using a multi-well plate reader (

M5^e) The absorbance of the wells was measured at 450 nm.

The results show that binding of W3256 to VEGF did not affect subsequent PD-L1 binding (fig. 7) and vice versa (fig. 8).

3.5 Cross-species binding (FACS/ELISA)

Cynomolgus monkey PD-L1 binding (FACS)

Engineered cynomolgus monkey PD-L1 expressing cells (WBP315.293F. cPro1.2A) at 1X 10⁵Cell/well density was seeded in U-bottom 96-well plates. Serial dilutions of W3256-u15t2.g6-1.UIgG1 were added to the cells. The plates were incubated at 4 ℃ for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-. Binding of the antibody to the cells was tested by flow cytometry and Mean Fluorescence Intensity (MFI) was analyzed by FlowJo.

Mouse VEGF binding (ELISA)

Binding of antibody to mouse VEGF antigen (WBP325-mPro1, Nano Biological, 50159-MNAB) was performed by the same ELISA assay as described above for human VEGF binding, except that the coating protein was 100. mu.L of 0.25. mu.g/mL mouse VEGF.

Mouse PD-L1 binding (FACS)

Engineered mouse PD-L1 expressing cells (wbp315.293f.mpro1.c1) at 1 × 10⁵The density of cells/well was seeded into U-bottom 96-well plates. Serial dilutions of different antibodies were added to the cells. The plates were incubated at 4 ℃ for 1 hour. After washing, FITC-labeled goat anti-human IgG (Jackson ImmunoResearch, 109-. W3256-U15T2.G6-1.UIgG1 was tested in thin sections by flow cytometryBinding on the cells and Mean Fluorescence Intensity (MFI) was analyzed by FlowJo.

Since cynomolgus VEGF has the same amino acid sequence as human VEGF, W3256 also has binding activity to cynomolgus VEGF. Cross-species binding activity of W3256 to cynomolgus monkey PD-L1, mouse VEGF and mouse PD-L1 was also assessed. FIG. 9, FIG. 10 and FIG. 11 show their binding activity to VEGF or PD-L1. W3256 showed comparable binding capacity, EC, to the parent antibody on cynomolgus monkey PD-L1₅₀Was about 2.618nM (FIG. 9). W3256 was unable to bind to mouse VEGF (fig. 10). W3256 also showed comparable binding capacity, EC, to the parent antibody on mouse PD-L1₅₀Was about 1.687nM (FIG. 11).

3.6 binding affinity (SPR) to VEGF and PD-L1

The SPR technique was used to measure the binding rate constant (ka) and dissociation rate constant (kd) of antibodies to either VEGF or the ECD of PD-L1. The affinity constant (KD) was thus determined.

Biacore T200, S series sensor chip CM5, amine coupling kit and 10x HBS-EP were purchased from GE Healthcare. The antibody was captured onto the surface of anti-human Fc IgG (Jackson, 109-005-098) immobilized on a CM 5-biosensor chip (GE Healthcare Inc.). The assay was performed with HBS-EP + buffer (GE Healthcare Inc.) as running buffer and dilution buffer at 25 ℃. Serial dilutions of PD-L1 antigen or VEGF antigen (W315-hpro1.ecd. his or W325-hpro1.his) and running buffer were injected for the binding phase and subsequent dissociation phase assays. Regeneration of the chip surface was achieved by injection of 10mM glycine pH 1.5.

The affinity constant (KD) of W3256 was measured according to SPR technique. The association rate constant (ka) and dissociation rate constant (kd) were also measured. The final data for each interaction is subtracted from the reference channel and buffer channel data. The experimental data were analyzed as shown in fig. 12 and 13. The kinetic affinity results for the antibodies are listed in table 5.

TABLE 5 kinetic affinity results for antibodies

3.7 ligand competition assay (ELISA)

Human VEGFR1 competition assay

Inhibition of VEGF binding to human VEGF receptor 1(W325-hpro1R1.ECD. hFc, Sino Biological, 10136-H02H) was determined by competitive ELISA. A96-well ELISA plate (Nunc Maxisorp, ThermoFisher, 442404) was incubated with 2. mu.g/mL W325-hpro1R1.ECD. His in carbonate-bicarbonate buffer (20mM Na. RTM.) at 4 ℃₂CO₃，180mM NaHCO₃pH9.2) was included overnight. All wells were washed 3 times with 300. mu.L of PBS/0.5% Tween-20(v/v) per well, and all subsequent washing steps in the assay were identical. The wells were then blocked with 200. mu.L/well of 50% casein (Thermo SCIENTIFIC, 37528) for 1 hour, and after a washing step, a mixture of serially diluted antibodies and 0.02. mu.g/ml biotin-labeled human VEGF (W325-hPro1.Biotin, ACRO Biosystems, VE5-H8210) was added to the wells and incubated at 25 ℃ for 2 hours. Plates were washed 3 times and then peroxidase-linked streptavidin diluted 1:10000 in PBS/50% casein (Jackson, 016. sup. 030. sup. 084) was added. The plates were incubated at 25 ℃ for 1 hour and then washed 6 times as before. 100 μ L/well of Tetramethylbenzidine (TMB) substrate (Sigma, 860336) was added to all wells for 8 min, and the reaction was then stopped with 100 μ L of 2M HCl. The extent of binding of human VEGF-biotin to W325-hpro1R1.ECD. hFc was determined by using a multi-well plate reader (

M5^e) Measuring OD₄₅₀To be determined. Bound IC was obtained by four parameter non-linear regression analysis using GraphPad Prism 5 software₅₀The value is obtained.

W3256 showed competitive binding to hVEGFR1 comparable to the parent antibody, its IC binding to VEGF₅₀Was about 3.86nM (FIG. 14).

Mouse VEGFR1 competition assay

The effect of the antibody to block VEGF binding to mouse VEGF receptor 1(W325-mpro1r1.ecd. hfc. his (R & D, 471-F1)) was assessed by competition ELISA the mouse VEGFR1 competition ELISA method was similar to the human VEGFR1 competition ELISA except that the pre-coated protein on the 96-well ELISA plate was 2 μ g/ml of W325-mpro1r1.ecd. hfc. his, the blocking buffer was PBS/2% BSA and the concentration of W325-hpro1.biotin was 0.44 μ g/ml.

W3256 showed comparable competition to mVEGFR1 with the parent antibody (fig. 16), its IC binding to VEGF₅₀About 31 nM.

Human PD-1 competition assay

Competition of W3256 with human PD-1 was measured by FACS-based competition assay. CHO-K1 cells (WBP315.CHO-K1.hPro1.C11) expressing human PD-L1 at 1X 10⁵The density of cells/well was seeded into a 96-well U-shaped bottom plate. Serial dilutions of the test antibody were premixed with 5 μ g/ml of mouse Fc-tagged human PD-1, then added to the cells and incubated at 4 ℃ for 1 hour. After washing, PE-labeled anti-mouse IgG was added and incubated with the cells for 45 min at 4 ℃. Cells were washed twice and MFI of cells was measured by flow cytometry and analyzed by FlowJo.

W3256 showed comparable competition ability to the parent antibody, blocking the IC of hPD-1 binding to PD-L1₅₀Was about 0.048nM (FIG. 15).

Mouse PD-1 competition assay

CHO-K1 cells (WBP315.293F.mPro1.C1) expressing mouse PD-L1 at 1X 10⁵Cells/well were seeded into 96-well U-shaped bottom plates. Serial dilutions of the test antibody were premixed with 5 μ g/ml mouse PD-1 with mouse Fc tag, then added to the cells and incubated at 4 ℃ for 1 hour. After washing, PE-labeled anti-mouse IgG was added and incubated with the cells for 1 hour at 4 ℃. Cells were washed twice and MFI of cells was measured by flow cytometry and analyzed by FlowJo.

W3256 showed comparable competitive capacity to the parent antibody, blocking the IC of mouse PD-1 binding to PD-L1₅₀Was about 1.687nM (FIG. 17).

3.8HUVEC cell proliferation assay

The biological activity of W3256 in VEGF-induced HUVEC proliferation was evaluated. HUVEC cells routinely in ECM + 5% FBS + 1% ECGSAnd (5) culturing. The near confluent cells were collected with trypsin and diluted to 1X 10 with ECM + 1% FBS + 0.05% ECGS⁵cells/mL. Cells were seeded at a density of 5000 cells/well in 96-well clear bottom black plates (Greiner, 655090). Serial dilutions of antibody were added, along with 50ng/mL human VEGF (WBP325-hPro1, Sino Biological, 11066-HNAB). The plates were returned to the incubator for 4 days, and cell viability was then assessed using CellTiter Glo (Promega, G7573). Wells without added ligand were used as controls for ligand stimulated cell growth. The effect of the test antibody on inhibiting ligand-stimulated cell growth was calculated by comparing the luminescence values with or without the addition of antibody (ligand only) after subtraction of background (no ligand) luminescence. Proliferation inhibition IC was obtained using GraphPad Prism 5 software using four parameter non-linear regression analysis₅₀The value is obtained.

W3256 effectively blocks VEGF-induced HUVEC proliferation, IC, in a concentration-dependent manner₅₀At about 12nM, the maximum inhibition was 101% (fig. 18).

3.9 reporter Gene assays

Routinely cultured CHOK1-OKT3-PD-L1 cells were harvested by trypsin and seeded at a density of 20000 cells/well in 96-well clear bottom black plates (Greiner, 655090). Serially diluted antibodies were added, as well as Jurkat cells that integrated the reporter signal NFAT-RE-Luc2p and expressed full-length PD-1. The plate was returned to the incubator for 4 hours, then 50. mu.l of One-glo luciferase assay buffer/substrate mixture was added. Luminescence was measured in a multiwell plate reader M5 e.

W3256 showed function in the reporter gene assay (fig. 19), indicating that the antibody induces the PD-1 signaling pathway.

3.10 Mixed Lymphocyte Reaction (MLR) assay

Use of MLR to test PD-L1 antibodies against cytokine, human IFN-gamma secretion and activated human CD4⁺Agonism of T cell proliferation.

Human Peripheral Blood Mononuclear Cells (PBMC) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. Isolated PBMCs were cultured in complete RPMI-1640 (containing 10% FBS and 1% PS) supplemented with 100U recombinant human IL-2(SL PHARM). Make itMonocytes were isolated using a human monocyte enrichment kit according to the manufacturer's instructions. Cell concentration was adjusted to 2X 10 in complete RPMI-1640 medium supplemented with 800U/ml recombinant human GM-CSF and 50. mu.g/ml IL-4⁶Cells/ml. The cells were cultured for 5 to 7 days to differentiate into Dendritic Cells (DCs). Cytokines were supplemented every 2-3 days by replacing half of the medium with fresh medium supplemented with cytokines. 1 μ g/ml LPS was added to the culture 18 to 24 hours before MLR to induce maturation of DCs. Using human CD4 according to the manufacturer's protocol⁺T cell enrichment kit for separating human CD4⁺T cells.

MLRs were established in 96-well round bottom plates (Nunc, 163320) using complete RPMI-1640 medium. Will CD4⁺T cells, various concentrations of antibodies and mature DCs were added to the plates. The plates were incubated at 37 ℃ with 5% CO₂And (4) incubating. IFN- γ production was measured on day 5.

Human IFN- γ was measured by enzyme linked immunosorbent assay (ELISA) using matched antibody pairs. Recombinant human IFN-. gamma. (PeproTech, 300-02) was used as a standard. The plates were pre-coated with a capture antibody specific for human IFN- γ (Pierce, M700A). After blocking, standards or samples were pipetted into each well and incubated at ambient temperature for 2 hours. After removal of unbound material, biotin-conjugated detection antibody (Pierce, M701B) specific for IFN- γ was added to the wells and incubated for 1 hour each. Streptavidin-conjugated horseradish peroxidase (HRP) (Invitrogen, SNN1004) was then added to the wells and incubated at ambient temperature for 30 minutes. Color was developed by addition of TMB substrate and then stopped with 2M HCl. The absorbance was read at 450 and 540nm using a Microplate spectrophotometer.

FIG. 20 shows antibody versus hCD4 in a mixed lymphocyte reaction assay⁺Influence of IFN-. gamma.secretion by T cells. The results show that W3256 induces IFN-. gamma.secretion in MLR in a concentration-dependent manner.

Example 4

Study of in vivo antitumor Effect

4.1 pharmacokinetic Studies in mice

Male C57BL/6 mice (6-8 weeks, 18-22g) were purchased from swac or BK Laboratory co, ltd, shanks in a specific pathogen-free condition and in the animal facility of WuXi biology (shanghai, china) where food and water are freely available.

Blood samples were collected from male C57BL/6 mice (n-5) at 0, 0.5, 6, 24, 48, 72, 120, 168 hours after a single intravenous injection of equimolar concentrations of the W3256 antibody. Serum antibody concentrations were measured using human IgG ELISA quantification and dual antigen binding ELISA.

Based on the total IgG binding results, the half-life of W3256 was 129 hours when injected by the intravenous route at a dose of 11.5mg/kg (fig. 21), and 104 hours when injected by the intravenous route at a dose of 11.5mg/kg in the dual antigen-binding PK assay (fig. 22).

4.2 efficacy of PBMC-RKO mouse model

In medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, at 37 ℃ and 5% CO₂RKO tumor cells were maintained in vitro in monolayer culture under conditions. Tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were harvested and counted for tumor inoculation.

Female NCG mice (6-10 weeks old) were purchased from the shanghai SLAC or Laboratory co, ltd, and were bred in the animal facilities of WuXi biology (shanghai, china) under specific pathogen-free conditions and with free access to food and water.

Each mouse was inoculated subcutaneously with RKO tumor cells (2X 10) in 0.2ml PBS containing 50% matrix gel⁶) To form tumors and injected intraperitoneally 4x 10 on day 0⁶Pbmc (hemacare). Treatment started on day 7.

The primary endpoint was to see if tumor growth could be delayed or mice cured. Tumor size was measured twice weekly using calipers and in mm using the following formula³Represents the volume: v ═ 0.5a x b²Wherein a and b represent the long and short diameters of the tumor, respectively.

Summary of statistical data including mean and Standard Error of Mean (SEM) for each group of tumor volumes at each time point is provided. Statistical analysis of tumor volume differences between groups and analysis of drug interactions were performed on data obtained at the optimal treatment time point after the final dose (day 28 after the start of dosing). Two-way ANOVA analysis was performed to compare tumor volumes between groups, followed by a t-test with post hoc multiple comparisons Dunnett (both compared to IgG group). All data were analyzed using SPSS 17.0 or Prism 5. p <0.05 is considered statistically significant.

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

Those skilled in the art will recognize and appreciate that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Since the foregoing description of the invention discloses only exemplary embodiments thereof, other variations should be understood to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the following claims as indicating the scope and content of the invention.

Sequence listing

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Val Asn Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala

35 40 45

Gly Ile Leu Trp Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala Glu Asn Met Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Gly Thr Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 11

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> VH VEGF

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL VEGF

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> HC

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 14

<211> 335

<212> PRT

<213> LC

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Phe Ser Asn Leu Ala

20 25 30

Val Asn Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala

35 40 45

Gly Ile Leu Trp Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala Glu Asn Met Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Thr Gly Thr Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

115 120 125

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

130 135 140

Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

145 150 155 160

Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His

165 170 175

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

180 185 190

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

195 200 205

Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys

210 215 220

Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

245 250 255

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

260 265 270

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

275 280 285

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

290 295 300

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

305 310 315 320

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

325 330 335

Claims (24)

1. A bispecific antibody, or antigen-binding portion thereof, comprising a PD-L1 antigen-binding moiety in association with a VEGF antigen-binding moiety, wherein:

the PD-L1 antigen binding moiety comprises:

comprises 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 moiety comprises:

heavy chain complementarity determining region 1 comprising SEQ ID NO. 4 (HCDR1), HCDR2 comprising SEQ ID NO. 5, HCDR3 comprising SEQ ID NO. 6, light chain complementarity determining region 1 comprising SEQ ID NO. 7 (LCDR1), LCDR2 comprising SEQ ID NO. 8, and LCDR3 comprising SEQ ID NO. 9.

2. The bispecific antibody or antigen-binding portion thereof of claim 1, wherein the anti-PD-L1 antigen-binding moiety comprises a variable domain comprising the amino acid sequence of SEQ ID NO 10 or an amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO 10.

3. The bispecific antibody, or antigen-binding portion thereof, of claim 1 or 2, wherein the VEGF antigen-binding moiety comprises:

a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence 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 at least 85%, 90%, or 95% identical to SEQ ID NO. 12.

4. The bispecific antibody, or antigen-binding portion thereof, of any one of the preceding claims, wherein the PD-L1 antigen-binding moiety is fused to the N-terminus of the VEGF antigen-binding moiety.

5. The bispecific antibody or antigen-binding portion thereof of any one of the preceding claims, wherein the PD-L1 antigen-binding moiety is from a single domain antibody (sdAb), such as a VHH antibody.

6. The bispecific antibody or antigen-binding portion thereof of claim 5, wherein the VHH is derived from a camelid, including alpaca (alpaca) and llama (llama).

7. The bispecific antibody or antigen-binding portion thereof of claim 5 or 6, wherein the PD-L1 antigen-binding moiety is operably linked to the N-terminus of the light or heavy chain of the VEGF antigen-binding moiety, optionally via a linker.

8. The bispecific antibody or antigen-binding portion thereof of claim 7, wherein the linker comprises or consists of 1 to 4 copies of GGGGS (G4S).

9. The bispecific antibody, or antigen-binding portion thereof, of any one of the preceding claims, comprising a heavy chain and a light chain, wherein:

the heavy chain comprises domains operably linked as in VH-CH 1-hinge-Fc, wherein VH-CH1 is from the VEGF antigen binding moiety; and

the light chain comprises domains operably linked as in VHH-VL-CL, wherein VHH is from the PD-L1 antigen binding module and VL-CL is from the VEGF antigen binding module.

10. The bispecific antibody or antigen-binding portion thereof of claim 9, wherein the Fc region is a human IgG Fc region, preferably a human IgG1Fc region.

11. The bispecific antibody, or antigen-binding portion thereof, of any one of the preceding claims, wherein the heavy chain comprises SEQ ID No. 13 and the light chain comprises SEQ ID No. 14.

12. The bispecific antibody, or antigen-binding portion thereof, of any one of the preceding claims, wherein the bispecific antibody is a humanized antibody.

13. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14.

16. A pharmaceutical composition comprising the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12 and a pharmaceutically acceptable carrier.

17. A method of producing the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12, comprising the steps of:

-expressing the antibody or antigen-binding portion thereof of any one of claims 1-12 in the host cell of claim 15; and

-isolating said antibody or antigen-binding portion thereof from the host cell.

18. Use of the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12 in the manufacture of a medicament for modulating an immune response in a subject.

19. Use of the bispecific antibody, or antigen-binding portion thereof, of any one of claims 1-12 in the manufacture of a medicament for inhibiting tumor cell growth in a subject.

20. Use of the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12 in the manufacture of a medicament for the diagnosis, prevention or treatment of cancer in a subject.

21. The use of claim 20, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, breast cancer, lung cancer, cervical cancer, kidney cancer, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, stomach cancer, lymphoma, melanoma, liver cancer, and head and neck cancer.

22. The use of claim 20 or 21, wherein the cancer is colon cancer or colorectal cancer.

23. The use of any one of claims 19-22, wherein the bispecific antibody or antigen-binding portion thereof is administered in combination with a chemotherapeutic agent, radiation therapy, and/or other agent for cancer immunotherapy.

24. A kit comprising the bispecific antibody or antigen-binding portion thereof of any one of claims 1-12.

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