WO2023241480A1

WO2023241480A1 - Anti-pd-l1, vegf and egfr trispecific antibody and use thereof

Info

Publication number: WO2023241480A1
Application number: PCT/CN2023/099420
Authority: WO
Inventors: 张震; 郎国竣; 朱益灵; 司远青; 邓强强
Original assignee: 三优生物医药(上海)有限公司
Priority date: 2022-06-13
Filing date: 2023-06-09
Publication date: 2023-12-21

Abstract

Provided are a trispecific antibody targeting PD-L1, VEGF and EGFR and a preparation method therefor. Further provided are a pharmaceutical composition containing the trispecific antibody, and the use thereof in treatment and diagnosis.

Description

Anti-PD-L1, VEGF and EGFR trispecific antibodies and their applications

Technical field

The invention belongs to the field of biomedicine, and specifically relates to a trispecific antibody targeting PD-L1, VEGF and EGFR and its preparation method and application.

Background technique

PD-L1 is a type I transmembrane protein on the cell membrane and is expressed on immune cells such as T cells and B cells as well as tumor cells. Tasuku Honjo et al. discovered and proved that when PD-L1 on the tumor cell membrane combines with PD-1 on immune cells such as T cells, the tumor cells send out inhibitory signals, and the T cells are unable to recognize tumor cells and kill them. , the body's immune function is suppressed (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). Current topics in microbiology and immunology, 410, 75-97). For the first time, Chen Pingping cured about 60% of mice with head and neck cancer through PD-L1 blocking antibodies combined with T cells (Chen, L et al. (2003). Cancer research, 63(19), 6501-6505.). Currently, 10 anti-PD-1/PD-L1 monoclonal antibodies have been approved for marketing in China, with indications targeting various solid tumors such as PD-L1-positive NSCLC, head and neck squamous cell carcinoma, and melanoma (Radvanyi, et al, P. (2013).letter.Clinical cancer research,19(19),5541). However, pathological detection indicators such as PD-L1 expression level and tumor mutation burden (TMB) predict a low response rate to anti-PD-1/PD-L1 antibodies. Currently, they are only effective in less than 40% of solid tumors, and 30% of patients have A certain degree of drug resistance appears after treatment. Moreover, current monospecific anti-PD-L1 antibodies are unable to differentiate between PD-L1 expressed on normal cells and tumor cells, so they have the potential to cause side effects, further limiting their application.

The most critical cytokine that promotes angiogenesis is VEGF-A (also known as VEGF). VEGF activates VEGFR2 (the main receptor tyrosine kinase receptor that mediates angiogenesis) to promote vascular endothelial cell mitosis and increase vascular permeability, thereby promoting cardiovascular sprouting. At the same time, the high expression of VEGF in the tumor microenvironment can amplify the immunosuppressive effect of PD-(L)1. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21(5):687-690.). The anti-VEGF monoclonal antibody bevacizumab has good efficacy and target safety in tumor treatment and eye diseases related to vascular dysplasia (Pfisterer, J., et al. (2020). Lancet Oncol 21( 5):699-709. Bhandari, S., et al. (2020). Ophthalmology 127(5):608-615.).

Human epidermal growth factor receptor (EGFR, ErbB-1 or HER1 for short) is a member of the epidermal growth factor receptor (HER) family. In related ligands such as EGF and transforming growth factor-α (transforming Under the action of growth factor α (TGFα), EGFR is converted from a monomer into a dimer and is activated, thereby further activating downstream signaling pathways and regulating cell proliferation. Abnormal EGFR function is related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis. Its functional abnormalities are mainly manifested in two aspects: one is excessive abnormal expression in tumor tissues, and the other is the continuous activation of EGFR mutants in tumor cells (without ligand stimulation or the formation of a self-circulating stimulation pathway). Relevant clinical data show that the amount of EGFR expression is closely related to the malignancy of tumors and the prognosis of tumor patients. However, due to the widespread expression of EGFR in normal tissues, clinical EGFR inhibitors have a high proportion of side effects such as skin reactions and diarrhea, which seriously affect the quality of life of patients.

In clinical patients who received EGFR treatment and were resistant to EGFR-TKI, the PD-L1 positive rate was as high as 58.8%, indicating that EGFR inhibitors can upregulate the expression of PD-L1 in tumors (Gainor, Justin F et al. Clin Cancer Res. 22 (18):4585-93). Activation of EGFR not only causes tumor proliferation, metastasis and blood vessel formation through signaling pathways, it further inhibits tumor-infiltrating lymphocytes and increases Treg cells, reducing MHC-I and MHC-II are expressed on the surface of tumor cells, leading to immune suppression (Li, Xue et al. Cancer Let; 418:1-9.). At the same time, the EGFR and VEGF signaling pathways are synergistic and complementary, and VEGF inhibitors can alleviate single-drug resistance caused by EGFR mutations (Le, Xiuning et al. J Thorac Oncol; 16(2):205-215). Therefore, there is an urgent need to develop a trispecific antibody targeting PD-L1, VEGF and EGFR to specifically kill tumor cells with good safety and provide more possibilities for cancer treatment.

Contents of the invention

Through in-depth research, the inventors combined the anti-PD-L1 nanobodies and anti-VEGF nanobodies developed with anti-EGFR antibodies to develop a trispecific antibody that simultaneously targets EGFR, VEGF and PD-L1. The anti-VEGF Nanobody part, the anti-PD-L1 Nanobody part, and the anti-EGFR antibody part in the trispecific antibody of the present invention show similar or better in vitro response than the already marketed Bevacizumab, Atezolizumab and Panitumumab. Antigen binding activity. At the same time, the trispecific antibody of the present invention is significantly better than the parent PD-L1 nanobody in cell-based PD-L1/PD-1 blocking activity in vitro. Furthermore, the affinity of the trispecific antibody of the present invention for binding to the PD-L1 end is higher than that of the EGFR end, which potentially improves the enrichment of the antibody in EGFR-positive tumor sites, reduces the exposure of normal tissue, and further reduces the binding of the antibody to EGFR-positive tumors. PD-L1 dosing. Furthermore, in a tumor-bearing mouse model, the trispecific antibody of the present invention also showed a complete alleviation trend for tumor growth, and compared with the combination of three parent monoclonal antibodies in equal molar amounts, it showed a comparable Or even better tumor growth inhibition effect, so it has very broad application prospects.

Therefore, in a first aspect, the invention provides a trispecific antibody, wherein the antibody includes a first antigen-binding domain that specifically binds a first antigen, a second antigen-binding domain that specifically binds a second antigen. , and a third antigen-binding domain that specifically binds a third antigen, wherein the first antigen, the second antigen, and the third antigen are different from each other and independently selected from the group consisting of PD-L1, VEGF, and EGFR;

Among them, the antigen-binding domain that specifically binds VEGF includes the CDR1-3 sequence contained in SEQ ID NO:2;

Among them, the antigen-binding domain that specifically binds PD-L1 includes the CDR1-3 sequence contained in SEQ ID NO:1; and,

Wherein, the antigen-binding domain that specifically binds to EGFR includes a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain includes the HCDR1-contained in SEQ ID NO:31 3. The VL domain includes LCDR1-3 contained in SEQ ID NO:32.

In some embodiments, the antigen-binding domain that specifically binds VEGF comprises the amino acid sequence set forth in SEQ ID NO:2, or is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:2 An identical amino acid sequence, or an amino acid sequence with one or more (preferably 1-10, more preferably 1-5) additions, deletions and/or substitutions of amino acids compared to SEQ ID NO:2. Most preferably, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:2, or consists of the amino acid sequence shown in SEQ ID NO:2.

In some embodiments, the antigen-binding domain that specifically binds PD-L1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or is at least 80%, 85%, 90%, 95%, or identical to SEQ ID NO: 1 An amino acid sequence that is 99% identical, or has one or more (preferably 1-10, more preferably 1-5) additions, deletions and/or substitutions of amino acids compared to SEQ ID NO: 1 . Most preferably, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.

In some embodiments, the antigen-binding domain that specifically binds EGFR comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain comprises SEQ ID NO: 31 The amino acid sequence shown, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 31, or having one or more (preferably 1-10 lands, More preferably 1-5) amino acid addition, deletion and/or substituted amino acid sequence; and the VL domain comprises the amino acid sequence shown in SEQ ID NO:32, or has at least 80% with SEQ ID NO:32 , an amino acid sequence that is 85%, 90%, 95% or 99% identical, or has one or more (preferably 1-10, more preferably 1-5) amino acids compared to SEQ ID NO:32 Added, deleted and/or substituted amino acid sequences. Most preferably, the antigen-binding domain of EGFR comprises the VH amino acid sequence of SEQ ID NO:31 and the VL amino acid sequence of SEQ ID NO:32.

In some preferred embodiments of the trispecific antibodies according to the present invention, the antigen-binding domain that binds EGFR comprises a Fab domain formed by the pairing of a VH-CH1 polypeptide chain and a VL-CL polypeptide chain. Accordingly, in some embodiments, the invention provides trispecific antibodies comprising (e.g., in a 1:1:1 ratio):

(i) The antigen-binding domain that binds EGFR is a Fab domain (Fab ^EGFR );

(ii) the antigen-binding domain that binds PD-L1 is a VHH domain (VHH ^PD-L1 ); and

(iii) The antigen-binding domain that binds VEGF is the VHH domain (VHH ^VEGF ). Preferably, the Fab ^EGFR domain, VHH ^PD- ^L1 domain and VHH ^VEGF domain are connected through a linker. Preferably, the linker contains 10-20 amino acids in length, and more preferably, contains the amino acid sequence ( G ₄ S) ₃ . In some embodiments, the VHH ^PD-L1 domain and the VHH ^VEGF domain are each linked to the Fab ^EGFR domain in separate forms. In yet another embodiment, the VHH ^PD-L1 domain and the VHH ^VEGF domain are linked in tandem to the Fab ^EGFR domain.

In further preferred embodiments of the trispecific antibody according to the invention, the trispecific antibody according to the invention further comprises an antigen-binding domain that binds EGFR, such as the C-terminus of a Fab ^EGFR domain (preferably, the The C-terminus of the Fab's VH-CH1 polypeptide chain) is connected to the immunoglobulin Fc region.

In any embodiment in which the trispecific antibody according to the invention comprises a Fab ^EGFR domain, preferably, the CH1 domain of the Fab ^EGFR domain is an IgG, especially a human IgG, such as a CH1 domain of a human IgG1 , and preferably comprises the amino acid sequence of SEQ ID NO:34. In yet another embodiment, the CL domain of the Fab ^EGFR domain is an immunoglobulin kappa or lambda light chain constant region, and preferably comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 6.

In any embodiment in which the above trispecific antibody according to the invention comprises an Fc region, preferably the Fc region is an IgG Fc region. More preferably, the Fc region consists of a hinge region, a CH2 domain and a CH3 domain. For example, the Fc region may be selected from an Fc region of a human IgG1, IgG2, IgG3 or IgG4 isotype, preferably an Fc region of human IgG1, such as a native sequence IgG1 Fc region or a variant sequence IgG1 Fc region. In one embodiment, the Fc region contains mutations that reduce effector function, for example, the mutations L234A and L235A. In a preferred embodiment, the Fc region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence with 99% identity, or consisting of.

In the above embodiment in which the trispecific antibody according to the present invention comprises a CH1 domain and an Fc region, preferably, the antibody comprises a human IgG1 heavy chain constant region, preferably comprising the amino acid sequence of SEQ ID NO: 4 or having the same amino acid sequence as SEQ ID NO: 4. or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In yet another aspect, the invention also provides nucleic acids encoding polypeptide chains of the antibody molecules of the invention, vectors and host cells containing the same.

In a further aspect, the invention also provides pharmaceutical compositions comprising the antibody molecules of the invention and their use in vitro and in vivo, especially in the treatment of diseases.

Description of the drawings

Figures 1A-1G show the schematic structures of candidate trispecific antibodies.

Figures 2A-2G show SEC-HPLC monomer detection patterns of candidate trispecific antibodies.

Figures 3A-3B show the binding activity of candidate trispecific antibodies to the recombinant protein VEGF-His.

Figures 4A-4B show the binding activity of candidate trispecific antibodies to recombinant protein PD-L1-His.

Figures 5A-5B show the binding activity of candidate trispecific antibodies to recombinant protein EGFR-His.

Figure 6 shows the binding activity of candidate trispecific antibodies to huPD-L1-CHO-K cells.

Figure 7 shows the binding activity of candidate trispecific antibodies to A431 cells.

Figures 8A-8C show the activity of candidate trispecific antibodies binding to multiple antigens simultaneously. Figure 8A shows the activity of the candidate trispecific antibody that first binds to the recombinant protein VEGF-Fc and then binds to the recombinant protein PD-L1-mFc; Figure 8B shows that the candidate trispecific antibody first binds to the recombinant protein VEGF-Fc and then binds to the recombinant protein Activity of protein EGFR-His binding; Figure 8C shows the activity of the candidate trispecific antibody first binding to recombinant protein PD-L1-mFc and then binding to recombinant protein EGFR-His.

Figures 9A-9C show the detection of blocking activity of candidate trispecific antibodies against PD-1/PD-L1 by luciferase reporter gene method.

Figures 10A-10B show the detection of blocking activity of candidate trispecific antibodies on VEGF/VEGFR2 by luciferase reporter gene method.

Figures 11A-11B show the inhibitory effect of candidate trispecific antibodies on tumor growth in mouse transplantation models.

Detailed description of the invention

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

I.Definition

The term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "comprises" or "includes" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

As used herein, the term "antibody" is used in its broadest sense to refer to a protein that contains an antigen-binding site.

The terms "antigen binding site" and "antigen binding domain" are used interchangeably herein to refer to the region of an antibody molecule that actually binds to the antigen. The antigen binding site for the antibody molecules of the invention is provided by the variable domain from the heavy chain antibody (i.e., "VHH") and the heavy/light chain variable domain pair from the conventional IgG antibody (i.e., VH and VL) .

As used herein, the term "trispecific" antibody refers to an antibody having at least three antigen-binding sites, each of the at least three antigen-binding sites binding to a different antigenic epitope, e.g., in The trispecific antibody according to the present invention binds to the antigens EGFR, VEGF and PD-L1 respectively.

As used herein, the expression "valency" in relation to an antibody refers to the total number of antigen-binding sites in the antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a 6-valent antibody means that the antibody molecule contains a total of 6 antigen-binding sites; the antibody molecule can be a "2+2+2-valent" trispecific antibody, that is, the antibody has three different antigens Binding specificity, where there are two identical antigen-binding sites for each antigen-binding specificity.

In this article, the term "VEGF" refers to vascular endothelial growth factor A (Vascular endothelial growth factor A, VEGF-A) protein. VEGF-A produces multiple isoforms through alternative splicing of exons during the transcription process, including isoforms VEGF121, VEGF165, VEGF189, and VEGF206. In this context, VEGF refers in particular to isoform VEGF165, such as the human VEGF165 protein described under UniProtKB-P15692 (in particular, the amino acid sequence of amino acids 27-191 without signal peptide). Enhanced expression of VEGF-A has been observed in human cancer cell lines and in cancer patients with different malignancies, including colorectal, breast, non-small cell lung and ovarian cancer, and correlates with intratumoral microvessel count (MVC). ) is directly related to increased neovascularization measured. As used herein, "antigen-binding specificity for VEGF", that is, "an antigen-binding domain that specifically binds VEGF", is provided by the VHH domain.

As used herein, the term "PD-L1" refers to programmed cell death 1 ligand 1 protein (eg, human PD-L1 protein under UniProtKB accession number Q9NZQ7). As an immune checkpoint molecule, PD-L1 is involved in mediating the activation threshold of T cells and limiting T effector cell responses, and is often up-regulated on tumor cells as one of the mechanisms of tumor immune evasion. In the antibody of the present invention, the "antigen-binding specificity for PD-L1", that is, the "antigen-binding domain that specifically binds to PD-L1" is provided by the VHH domain.

As used herein, the term "EGFR" refers to epidermal growth factor receptor (eg, human EGFR protein under UniProtKB accession number P00533). A large number of studies have shown that EGFR is highly expressed or abnormally expressed in most tumors, such as glial cell carcinoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and other tissues. In the antibody of the present invention, the "antigen-binding specificity for EGFR", that is, the "antigen-binding domain that specifically binds to EGFR" is provided by the pair of VH and VL domains.

As used herein, the term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are approximately 150,000 dalton heterotetrameric glycoproteins composed of two disulfide-bonded light chains and two heavy chains. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). In IgG molecules, the VH-CH1 of the heavy chain is usually paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules and two dimerized Fc regions connected by the immunoglobulin hinge region. The heavy chain of an immunoglobulin can be assigned to one of five categories based on the type of its constant region, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), where one These classes can be further divided into subclasses such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be classified into one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.

As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. In the case of heavy chain antibodies, such as those from the family Camelidae, a single VH domain (herein also referred to as a VHH domain) may be sufficient to confer antigen binding specificity. The VHH domain, like the heavy and light chain variable regions of conventional IgG antibodies, contains four conserved framework regions (FRs) and three complementarity determining regions (CDRs), with FR1-CDR1-FR2-CDR2-FR3 -Sequencing of CD3-FR4.

As used herein, a "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable region"). loops") and/or regions containing antigen contact residues ("antigen contact points"). CDRs are mainly responsible for binding to antigenic epitopes. In the VHH domain and VH/VL domain of the antibody of the present invention, the CDRs are numbered sequentially starting from the N-terminus and are generally referred to as CDR1, CDR2 and CDR3. The CDR sequences in a defined VHH domain and a defined VH/VL domain can be determined using methods known in the art. Those skilled in the art can easily determine the CDR sequence range of any given antibody variable region amino acid sequence at http://www.abysis.org/abysis/, including the Kabat, AbM, Chothia, Contact and IMGT schemes The defined regional scope of the CDR and its combination scope. Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways and combinations thereof.

As used herein, the term "VHH" is used to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain (sometimes referred to herein as a Nanobody), also referred to as a single variable domain fragment ( sVD). Therefore, VHH differs from conventional VH of four-chain immunoglobulins in that it does not need to be paired with a light chain variable domain to form an antigen-binding site. Such VHH molecules can be derived from antibodies produced in Camelidae species such as camels, alpacas, dromedaries, llamas and guanacos. Species other than camelids may also produce heavy chain antibodies that naturally lack light chains, and such VHHs are also within the scope of the invention. In some cases, for therapeutic applications of VHH, it is desirable to reduce its immunogenicity. Therefore, preferably, in one embodiment, the antibodies of the invention comprise a humanized VHH domain.

As used herein, the term "Fab domain" is used to refer to a structure consisting of a heavy chain variable region VH and a heavy chain constant region CH1 (VH-CH1) and a complementary light chain variable region VL and light chain similar to that in a conventional four-chain IgG antibody. The structure formed by the pairing of chain constant region CL (VL-CL). The term also encompasses structures in which CH1 and CL are exchanged, ie, structures formed by the pairing of VH-CL and VL-CH1. As shown in Figures 1A-1G, the Fab domain can be fused to the N-terminus of the immunoglobulin Fc region.

As used herein, "immunoglobulin heavy chain constant region" (CH) refers to the constant region domain from, obtained from, or derived from an immunoglobulin heavy chain (eg, human IgG1 heavy chain), including from the N-terminus to the C-terminus. The heavy chain constant region domains CH1, CH2, CH3, and optionally CH4 are covalently linked in sequence. In most cases, the heavy chain constant region domains CH1 and CH2 are connected through the heavy chain hinge region, but when appropriate, they can also be connected through a flexible connecting peptide. In some preferred embodiments of the invention, the antibody molecules of the invention comprise an immunoglobulin heavy chain constant region consisting of the heavy chain constant region domains CH1-hinge region-CH2-CH3. In this context, the domains of the immunoglobulin heavy chain constant region can be selected based on the expected function of the antibody molecule. For example, the constant region domain may be a constant region domain of IgA, IgD, IgE, IgG or IgM, especially a constant region domain of human IgG, for example, a constant region structure of human IgG1, IgG2, IgG3 or IgG4. domain, preferably the constant region domain of human IgG1. As an example, both the CH1 domain and the Fc region comprising the CH2 and CH3 domains of the antibody may be derived from IgG1, especially human IgG1.

As used herein, "immunoglobulin light chain constant region" refers to the constant region domain CL from, obtained from, or derived from an immunoglobulin light chain. The light chain CL constant region of an immunoglobulin, based on its amino acid sequence, can be a kappa light chain CL domain and a lambda light chain CL domain.

The terms "Fc region" and "Fc domain" are used interchangeably herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc region" contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain. Fc regions that can be used in the antibodies of the invention include, but are not limited to, Fc regions of IgGl, IgG2, IgG3, or IgG4 having native or variant sequences. Unless otherwise stated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to, e.g., Kabat et al., Sequences of Proteins of Immunological Interes, 5 They are numbered according to the EU numbering system (also called the EU index) described in Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the term "Fc region" or "Fc domain" does not include the heavy chain variable region VH and the light chain variable region VL as well as the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin.

In this article, the term "native sequence Fc region" encompasses the various naturally occurring immunoglobulin Fc region sequences, such as the various Ig subtypes and their allotype Fc region sequences (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions,20October 2014,doi:10.3389/fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal terminal lysine (Lys447) of the Fc region may or may not be present. In still some embodiments, the human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a native immunoglobulin at the N-terminus, such as the sequence E216 to T225 or the sequence D221 to T225 according to EU numbering.

As used herein, the term "variant sequence Fc region" refers to an Fc region polypeptide that contains modifications relative to a native sequence Fc region polypeptide. The modification may be the addition, deletion or substitution of amino acid residues. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Modifications may be aimed at altering the binding of the Fc region to its receptor and the resulting effector function.

As used herein, the term "effector function" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Depending on the intended use of the antibody molecule, the antibody molecule of the invention may have altered effector functions, such as reduced or eliminated ADCC activity, relative to an antibody molecule with a wild-type Fc region.

As used herein, some embodiments of the antibodies according to the invention comprise an Fc region. Herein, an antibody polypeptide chain that contains an Fc region is also called a heavy chain, and an antibody polypeptide chain that does not contain an Fc region is also called a light chain. In these embodiments, preferably the antibody does not have an antigen-binding domain covalently linked to the C-terminus of the heavy chain Fc region.

As used herein, the term "flexible linker peptide" or "linker" or "linker peptide" is used interchangeably and refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine ( S) and/or threonine residues (T), or from the hinge region of an immunoglobulin.

As used herein, "percent (%) identity" of an amino acid sequence refers to a candidate sequence after aligning it with the specific amino acid sequence shown in this specification and introducing gaps if necessary to achieve maximum percent sequence identity. The percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence shown in this specification, without considering any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein. , 85%, 90%, 95%, 97%, 98% or 99% or higher. The variants may contain conservative modifications.

With respect to a polypeptide sequence, "conservative modifications" include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. Such conservatively modified variants are in addition to and not exclusive of the polymorphic variants, interspecies homologs and alleles of the present invention. The following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valerine Amino acid (V); 6) Phenylalanine (F), tyrosine (Y), color amino acid (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, eg, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used inter alia to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by an enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by the ELISA assay described in Example 4.1, 4.2, 4.3 or 4.6 Detect the binding ability of the antibody to VEGF, PD-L1 or EGFR protein or both, or detect the binding ability of the antibody to cells expressing PD-L1 and/or EGFR on their surface by the FACS assay described in Example 4.4 or 4.5, or The affinity constant KD is detected by the biofilm interference technique described in Example 8 (for example, the Octet Fortebio detection system can be used).

In this article, the "blocking activity" of an antibody against PD-L1 means that the antibody blocks the binding of PD-L1 to the receptor PD-1 and/or reduces the signal transduction of PD-L1/PD-1. The assay used to measure this blocking activity can be, for example, a reporter gene-based signaling pathway blocking assay, such as the reporter gene-based assay described in Example 5. The blocking activity of the antibody to be tested on PD-L1 can be determined with reference to the PD-L1/PD-1 binding or PD-L1/PD-1 signaling levels in the absence of antibodies and/or in the presence of positive antibodies.

In this article, the "neutralizing activity" of an antibody against VEGF refers to the function of the antibody blocking the signaling pathway between VEGF and its receptor VEGFR2. The assay used to measure this activity may be, for example, a reporter gene-based signaling pathway blocking assay, such as the reporter gene-based assay described in Example 6. The neutralizing activity of the antibody to be tested on VEGF can be determined with reference to the signaling level of VEGF/VEGFR2 in the absence of the antibody and/or in the presence of a positive antibody.

As used herein, an antibody having "blocking activity" on EGFR means that the antibody blocks the binding of EGFR to its ligand, and/or reduces the function of signaling mediated through EGFR. An assay used to determine this blocking activity may be, for example, an in vitro proliferation inhibition assay based on EGFR expressing cells (eg, the A431 cell line). The blocking activity of the antibody to be tested on EGFR can be determined with reference to the level of proliferation inhibition in the absence of the antibody and/or in the presence of a positive antibody.

As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.

As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, viruses and adeno-associated viruses).

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

As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease progression or to slow the progression of disease. In the context of tumors or cancer treatments, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (e.g., administration of drugs such as antibodies of the invention), including, but not limited to, e.g., reduction in tumor volume, tumor Reduced cell number, reduced tumor cell proliferation, or reduced tumor cell survival.

As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, solid tumors, and liquid tumors. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include PD-L1 positive and/or EGFR positive tumors/cancers, including metastatic forms thereof.

II. Trispecific antibodies of the invention

In one aspect, the invention provides a trispecific antibody that specifically binds PD-L1, VEGF and EGFR, the antibody comprising an antigen-binding site (preferably a VHH domain, herein) that specifically binds PD-L1. (also abbreviated as VHH ^PD-L1 in ) and an antigen-binding site that specifically binds to VEGF (preferably a VHH domain, also abbreviated as VHH ^VEGF herein), and an antigen-binding site that specifically binds to EGFR (preferably composed of VH Paired with the VL domain). Preferably, in the antibody of the present invention, the PD-L1, VEGF and EGFR antigen binding sites are connected through a linker. In some embodiments, the antibodies of the invention further comprise an immunoglobulin constant region domain. For example, in some embodiments, a VH and VL domain pair that specifically binds EGFR is present in a trispecific antibody of the invention as contained in a Fab domain (also abbreviated herein as Fab ^EGFR ). In further embodiments, a trispecific antibody according to the invention further comprises an Fc region linked to the C-terminus of said Fab ^EGFR domain.

The components of the trispecific antibody of the present invention are described in detail below. It will be understood by those skilled in the art that any combination of any technical features of these components is within the scope of the present invention unless the context clearly indicates otherwise. Furthermore, one skilled in the art will understand that the antibodies of the invention (including antibodies in any form) may comprise any such combination of features unless the context clearly indicates otherwise.

antigen binding site

In trispecific antibodies according to the invention, in some embodiments, the antigen-binding site that specifically binds PD-L1 is provided by the VHH ^PD- ^L1 domain; and the antigen-binding site that specifically binds VEGF is provided by VHH ^VEGF The antigen binding site that specifically binds EGFR is provided by the complementary paired VH and VL domains.

VHH ^PD-L1 domains according to the invention comprise VHH domains from anti-PD-L1. Preferably the VHH comprises CDR1, CDR2 and CDR3 sequences having the variable region shown in SEQ ID NO:1. The CDR sequence range of the variable region SEQ ID NO: 1 amino acid sequence may be defined according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or may be defined according to any two, more, or all of these definition schemes. Those skilled in the art can easily access the CDR sequences defined by these definitions via http://www.abysis.org/abysis/ . In a preferred embodiment, the VHH ^PD-L1 domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region with SEQ ID NO:1.

In yet another preferred embodiment, the VHH ^PD-L1 domain according to the invention comprises

(i) CDR1 comprising or consisting of SEQ ID NO: 19;

(ii) CDR2 containing or consisting of SEQ ID NO:20; and

(iii) CDR3 containing or consisting of SEQ ID NO:21.

In one embodiment, the VHH ^PD-L1 domain comprises the amino acid sequence set forth in SEQ ID NO:1. In yet another embodiment, the VHH ^PD-L1 domain comprises at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1 and retains the ability to specifically bind PD-L1 amino acid sequence. In yet another preferred embodiment, the VHH ^PD-L1 domain comprises the addition of one or more (preferably 1-10, more preferably 1-5) amino acids compared to SEQ ID NO: 1, Amino acid sequences that are deleted and/or substituted (eg, conservative substitutions) and retain the ability to specifically bind PD-L1. Preferably, the addition, deletion and/or substitution of amino acids does not occur in the CDR region. Most preferably, in the trispecific antibody according to the invention, the VHH ^PD-L1 domain according to the invention comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.

VHH ^VEGF domains according to the invention comprise VHH domains from anti-VEGF. Preferably, the VHH comprises CDR1, CDR2 and CDR3 sequences having the variable region shown in SEQ ID NO:2. The CDR sequence range of the variable region SEQ ID NO:2 amino acid sequence may be defined according to the Kabat, AbM, Chothia, Contact, or IMGT schemes, or may be defined according to any two, more, or all of these definition schemes. Those skilled in the art can easily access the CDR sequences defined by these definitions via http://www.abysis.org/abysis/ . In a preferred embodiment, the VHH ^VEGF domain according to the invention comprises the CDR1 sequence, the CDR2 sequence and the CDR3 sequence defined according to AbM in the variable region with SEQ ID NO:2.

In yet another preferred embodiment, the VHH ^VEGF domain according to the invention comprises

(i) CDR1 containing or consisting of SEQ ID NO:22;

(ii) CDR2 containing or consisting of SEQ ID NO:23; and

(iii) CDR3 containing or consisting of SEQ ID NO:24.

In one embodiment, the VHH ^VEGF domain comprises the amino acid sequence set forth in SEQ ID NO:2. In yet another embodiment, the VHH ^VEGF domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:2 and retains the ability to specifically bind VEGF. In yet another preferred embodiment, the VHH ^VEGF domain comprises additions, deletions and additions of one or more (preferably 1-10, more preferably 1-5) amino acids compared to SEQ ID NO:2. or an amino acid sequence that is substituted (eg, conservatively substituted) and retains the ability to specifically bind VEGF. Preferably, the addition, deletion and/or substitution of amino acids does not occur in the CDR region. Most preferably, in the trispecific antibody according to the invention, the VHH ^VEGF domain according to the invention comprises the amino acid sequence of SEQ ID NO:2, or consists of the amino acid sequence shown in SEQ ID NO:2.

In one embodiment, an antigen binding site according to the invention that specifically binds EGFR comprises a complementary pair of VH domains and VL domains. Preferably, the VH domain includes the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO:31; and the VL domain includes the light chain variable region shown in SEQ ID NO:32. LCDR1, LCDR2 and LCDR3 sequences of variable regions. The CDR sequence ranges of the variable region SEQ ID NOs: 31 and 32 amino acid sequences may be defined according to the Kabat, AbM, Chothia, Contact or IMGT schemes, or may be defined according to any two, more or all of these definition schemes. Those skilled in the art can easily access the CDR sequences defined by these definitions via http://www.abysis.org/abysis/ . In a preferred embodiment, the VH domain and the VL domain are respectively comprised in the heavy chain variable region having SEQ ID NO: 31 or the light chain variable region having SEQ ID NO: 32 as defined by AbM CDR1 sequence, CDR2 sequence and CDR3 sequence.

In yet another preferred embodiment, the antigen binding site specifically binding to EGFR according to the present invention comprises

(i) HCDR1 containing SEQ ID NO:25 or consisting of SEQ ID NO:25;

(ii) HCDR2 containing or consisting of SEQ ID NO:26;

(iii) HCDR3 containing or consisting of SEQ ID NO:27;

(iv) LCDR1 containing or consisting of SEQ ID NO:28;

(v) LCDR2 containing or consisting of SEQ ID NO:29; and

(vi) LCDR3 containing or consisting of SEQ ID NO:30.

In one embodiment, the antigen-binding site that specifically binds EGFR according to the present invention comprises the amino acid sequence shown in SEQ ID NOs: 31 and/or 32, or has at least 80% similarity with SEQ ID NOs: 31 and/or 32. , 85%, 90%, 95% or 99% identity and retain the ability to specifically bind to EGFR. In yet another preferred embodiment, the antigen-binding site specifically binding EGFR according to the present invention comprises one or more (preferably 1-10, more preferably 1-10, more preferably SEQ ID NOs: 31 and/or 32). 1-5) amino acid additions, deletions and/or substitutions (eg, conservative substitutions) of amino acid sequences that retain the ability to specifically bind to EGFR. Preferably, the addition, deletion and/or substitution of amino acids does not occur in the CDR region. Most preferably, in the trispecific antibody according to the invention, the EGFR antigen binding site according to the invention comprises the VH amino acid sequence of SEQ ID NO: 31 and the VL amino acid sequence of SEQ ID NO: 32, or consists of SEQ ID NOs: The amino acid sequence composition shown in 31 and 32.

Immunoglobulin constant region domain

The trispecific antibody according to the present invention, in addition to the aforementioned antigen-binding site, in some embodiments, may also comprise an immunoglobulin constant region domain, such as one connected to the VH and VL domains respectively to form a Fab domain. Immunoglobulin CH1 and CL constant region domains, and/or an Fc region comprising immunoglobulin constant region domains CH2, CH3 and optionally CH4.

In the trispecific antibodies of the invention, suitable CH1 and CL domains may be CH1 and CL domains from any natural immunoglobulin molecule or derivatives thereof. In some embodiments, the CH1 domain comprises an amino acid sequence from a CH1 domain of an immunoglobulin IgG, especially IgG1. Preferably, the CH1 domain comprises or is at least 90%, 95% identical to SEQ ID NO: 34 , 96%, 97%, 98%, 99% or higher identity of the amino acid sequence. In further embodiments, the light chain CL domain of the antibody molecule comprises the amino acid sequence of the CL domain from an immunoglobulin kappa light chain or lambda light chain, preferably the CL domain comprises SEQ ID NO: 5 or 6 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto. In the antibody molecule of the present invention, the heavy chain CH1 domain connected to the VH domain is paired with the light chain CL domain connected to the VL domain to form a Fab domain.

In the trispecific antibodies of the invention, a suitable Fc region (or Fc domain) may be an Fc region from any natural immunoglobulin molecule or a derivative thereof. For example, the Fc region of an antibody of the invention may comprise two or three constant region domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain. Preferably, the Fc region of the antibody of the present invention includes: CH2-CH3 from N-terminus to C-terminus, and more preferably includes: hinge region-CH2-CH3 from N-terminus to C-terminus. In some embodiments, the Fc region of the antibody molecule is an Fc region from an IgG, eg, an Fc region from IgG1, IgG2 or IgG4, preferably an Fc region from human IgG1. Preferably, the Fc region comprises SEQ ID NO: 33 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto. As will be appreciated by those skilled in the art, antibody molecules of the invention may contain modifications in the Fc region that alter effector function, depending on the intended use of the antibody molecule. In one embodiment , one or more effector functions of the Fc region of the invention have been altered relative to a wild-type Fc region of the same isotype. The effector function of the Fc region can be altered by any method selected from altering glycosylation of the Fc region, using an Fc isoform that naturally possesses altered effector function, and modifying the amino acid sequence of the Fc region.

Accordingly, in some embodiments, the invention provides trispecific antibodies comprising an immunoglobulin CH1 domain and a CL domain. In other embodiments, the invention provides trispecific antibodies comprising an Fc region consisting of the immunoglobulin heavy chain constant region domains CH2-CH3. In yet another embodiment, the invention provides a trispecific antibody comprising an immunoglobulin CH1 domain and a CL domain and further comprising an Fc region, preferably wherein the CH1 domains are connected by an immunoglobulin hinge region. The Fc region polypeptide, and more preferably, the antibody comprises an immunoglobulin constant region polypeptide consisting of CH1-hinge region-CH2-CH3. In some embodiments, the immunoglobulin constant region domains contained in the trispecific antibodies according to the invention include CH1 and CL constant domains in the Fab structure and CH2 and CH3 constant domains in the Fc region (if present) may be, independently of each other, e.g., an immunoglobulin constant domain from human IgG, e.g., a constant domain from human IgG1, IgG2, IgG3 or IgG4, and preferably a constant domain from human IgG1. Furthermore, in further embodiments, the immunoglobulin constant region domains comprised in the trispecific antibodies according to the invention, including CH1 and CL and the constant regions CH2 and CH3 (if present), may independently of each other be Native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof, preferably native sequence constant domains.

In a preferred embodiment, the invention provides a trispecific antibody comprising a human IgG1 immunoglobulin heavy chain constant region, preferably the immunoglobulin heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 4 or a combination thereof. An amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity; and preferably, the trispecific antibody further comprises a kappa or lambda light chain constant region, for example, The amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

Connector

In the trispecific antibody according to the present invention, linkers can be used to connect antibody components. For example, the VHH ^PD- ^L1 and VHH ^VEGF domains according to the present invention can be connected to the N-terminal or C-terminal of the Fab ^EGFR domain respectively through a linker, or the VHH ^PD- ^L1 and VHH ^VEGF domains according to the present invention can be connected via The subunits are connected in series with each other and optionally further connected to the N-terminus of the Fab ^EGFR domain through a linker.

The linkers that can be used in the antibodies of the present invention are not particularly limited. Those skilled in the art can easily determine the available linker sequences based on the components to be connected and the location of the connection. In one embodiment, the linker is a flexible linker peptide of 5-50 amino acids, preferably a linker peptide comprising glycine (G) and/or serine (S) and/or threonine residues (T). In one embodiment, the linker is 5-50 amino acids in length, for example, 8, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In one embodiment, the linker comprises the amino acid sequence (G ₄ S) _n , wherein n is an integer equal to or greater than 1, for example, n is an integer 2, 3, 4, 5, 6, or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G ₄ S) ₃ . In another embodiment, the linker comprises the amino acid sequence TS( _G4S )n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In yet another embodiment, the linker is a hinge region from an immunoglobulin. Linkers that can be used for the antibody molecules of the present invention can also be, for example, but not limited to, the following amino acid sequences: (Gly ₃ Ser) ₂ , (Gly ₄ Ser) ₂ , (Gly ₃ Ser) ₃ , (Gly ₄ Ser) ₃ , (Gly ₃ Ser) ₄ , (Gly ₄ Ser) ₄ , (Gly ₃ Ser) ₅ , (Gly ₄ Ser) ₅ , (Gly ₃ Ser) ₆ , (Gly ₄ Ser), GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTGSSGKPGSGEGSTKG. Alternatively, suitable flexible linker peptides can be rationally designed using computer programs to simulate the three-dimensional structures of proteins and peptides, or through phage display methods.

Exemplary trispecific antibodies

In some embodiments, the invention provides trispecific antibodies wherein the VHH ^PD-L1 and VHH ^VEGF domains are linked to an antigen binding site that specifically binds EGFR, either in separate form or in tandem form. When the antigen-binding domain that binds EGFR includes a Fab domain formed by pairing a VH-CH1 polypeptide chain and a VL-CL polypeptide chain (ie, a Fab ^EGFR domain), the connection can occur, for example, but not limited to, The Fab domain occurs at the N-terminus of one or both of the two polypeptide chains, or occurs at the N-terminus of one of the two polypeptide chains (e.g., the VH-CH1 polypeptide chain) and the other polypeptide chain (e.g., the VL-CL polypeptide chain). ) of the C-terminal. Therefore, in some embodiments, the present invention provides trispecific antibodies, wherein the VHH ^PD-L1 domain and the VHH ^VEGF domain are linked to the N of the two variable regions (VH and VL) of the Fab ^EGFR domain, respectively. end; or wherein the VHH ^PD-L1 domain and the VHH ^VEGF domain are connected in series, and further connected at the N-terminus of one of the two variable regions (VH or VL) of the Fab ^EGFR domain. In other embodiments, the invention also provides trispecific antibodies, wherein the VHH ^PD-L1 domain is linked to the N-terminus of the heavy chain variable region (VH) of the Fab ^EGFR domain, and the VHH VEGF domain is linked to the N-terminus of the heavy chain variable region (VH) of the Fab ^EGFR domain. Fab The C-terminal linkage of the light chain constant region (CL) of the ^EGFR domain. In any of the embodiments described, the connection is preferably via a linker according to the invention.

Accordingly, in some preferred embodiments, the invention provides trispecific antibodies, wherein said trispecific antibodies comprise a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes a first VHH domain, a linker, a VH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus,

The second polypeptide chain includes a second VHH domain, a linker, a VL domain and a CL domain from the N-terminus to the C-terminus,

wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and wherein the VH domain is paired with the VL domain and specifically binds to the third antigen,

wherein the first antigen and the second antigen are different from each other and independently selected from PD-L1 and VEGF (preferably, the first antigen is PD-L1 and the second antigen is VEGF), and wherein the third antigen is EGFR .

In other preferred embodiments, the invention provides trispecific antibodies, wherein said trispecific antibodies comprise a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes a first VHH domain, a linker, a second VHH domain, a linker, a VH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus,

The second polypeptide chain includes a VL domain and a CL domain from the N-terminus to the C-terminus,

The first polypeptide chain includes VH domain, CH1 domain and Fc region from N-terminus to C-terminus,

The second polypeptide chain includes from N-terminus to C-terminus a first VHH domain, a linker, a second VHH domain, a linker, a VL domain, and CL domain,

The second polypeptide chain includes a VL domain, a CL domain, a linker and a second VHH domain from the N-terminus to the C-terminus,

The first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and the VH domain is paired with the VL domain and specifically binds to the third antigen, wherein the first antigen is PD-L1 , the second antigen is VEGF, and the third antigen is EGFR.

In some more preferred embodiments, the invention provides trispecific antibodies comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of: combination:

(a) Comprising the amino acid sequence shown in SEQ ID NO:7 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO: 13 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the second polypeptide chain of the amino acid sequence;

(b) Comprising the amino acid sequence shown in SEQ ID NO:8 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO:14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the second polypeptide chain of the amino acid sequence;

(c) Comprising the amino acid sequence shown in SEQ ID NO:9 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 15 or 16 , a second polypeptide chain with an amino acid sequence that is 98% or 99% identical;

(d) Contains the amino acid sequence shown in SEQ ID NO: 10 or 11 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto The first polypeptide chain has a unique amino acid sequence, and contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 17 , a second polypeptide chain with an amino acid sequence that is 98% or 99% identical; and

(e) Comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO: 18 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the amino acid sequence of the second polypeptide chain.

In one embodiment, the antibody according to the present invention comprises: a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:7; and a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:13 composed of the second polypeptide chain. In one embodiment, the antibody according to the present invention comprises: a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8; and a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14 composed of the second polypeptide chain. In one embodiment, an antibody according to the invention comprises: comprising SEQ The amino acid sequence shown in ID NO: 9 or a first polypeptide chain consisting thereof; and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 15 or 16 or consisting thereof. In one embodiment, the antibody according to the present invention comprises: a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10 or 11; and comprising the amino acid sequence shown in SEQ ID NO: 17 or The second polypeptide chain it consists of. In one embodiment, the antibody according to the present invention comprises: a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12; and a first polypeptide chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 18 composed of the second polypeptide chain.

In any of the above embodiments of the antibodies according to the invention, the invention also contemplates trispecific antibodies comprising two first polypeptide chains and two second polypeptide chains. Preferably, the two first polypeptide chains and the two second polypeptide chains associate to form a hexavalent monomeric antibody, wherein the Fc regions of the two first polypeptide chains pair with each other and dimerize, And preferably, one or more disulfide bonds are formed through the immunoglobulin hinge region included in the Fc region to stabilize the monomeric antibody structure. In one embodiment, the antibody is a "2+2+2-valent" type trispecific antibody, which contains 2 identical Fab ^EGFR domains, 2 identical VHH ^PD-L1 domains and 2 identical VHH ^VEGF domain, thereby conferring binding specificity against three different antigens. As will be understood by those skilled in the art, the two first polypeptide chains contained in the trispecific antibody need not be identical; and similarly, the two second polypeptide chains need not be identical. For example, asymmetric modifications can be introduced in one or both first polypeptide chains and/or the second polypeptide chain without affecting the desired target binding activity. In some aspects, however, preferably the two first polypeptide chains are identical and the two second polypeptide chains are also identical.

In any of the above embodiments of the antibodies according to the invention, the invention also contemplates embodiments in which the CH1 and CL domains in the first and second polypeptide chains are exchanged. In this embodiment with a CH1/CL domain exchange, preferably the Fc region is connected to the CL domain of the Fab domain by an immunoglobulin hinge region.

III. Production and Purification of Antibodies of the Invention

In yet another aspect, the invention provides methods for producing the antibodies of the invention. To produce the antibodies of the invention, the polypeptide chains of the antibodies of the invention can be obtained, for example, by solid-state peptide synthesis (eg Merrifield solid phase synthesis) or recombinant production, and assembled under appropriate conditions.

For recombinant production, the polynucleotide encoding any polypeptide chain and/or polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. The polynucleotide can be readily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of an antibody of the invention are provided. In yet another embodiment, the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Accordingly, in one embodiment, the invention provides a method for producing an antibody of the invention, said method comprising: culturing a host cell comprising a polypeptide chain encoding said antibody under conditions suitable for expression of said polypeptide chain; and The antibody is produced by assembling the polypeptide chain under conditions suitable for assembly of the polypeptide chain into the antibody.

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

In one embodiment, the invention also provides host cells comprising one or more polynucleotides of the invention. In some embodiments, host cells comprising expression vectors of the invention are provided. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffa Rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2/0, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

Antibodies prepared by the methods described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. After purification, the purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the antibodies provided herein can be identified, screened or characterized by a variety of assays known in the art.

In a preferred embodiment, the trispecific antibody of the invention exhibits good production properties in recombinant production in mammalian host cells such as CHO cells, in particular, good expression yield and good by-product profile. In one embodiment, the transient expression level of the antibody of the invention reaches at least about 20 μg/mL, preferably at least about 50 μg/mL, more preferably at least about 80 μg/mL, at least about 90 μg/mL, or at least about 100 μg/mL. In yet another embodiment, after one-step protein A affinity chromatography purification of the trispecific antibody of the present invention, SEC-HPLC is used to determine the purity of the product, which shows a purity of more than 95%, preferably 96%, 97%, 98% or 99%. purity above. Preferably, the transient expression level is measured according to the method described in Example 3; the product purity after one-step protein A purification is measured according to the SEC-HPLC monomer purity identification method described in Example 3.4.

IV. Pharmaceutical compositions, pharmaceutical combinations and kits

In one aspect, the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion). In some embodiments, the antibodies of the invention are the only active ingredient in the pharmaceutical composition. In other embodiments, a pharmaceutical composition may comprise an antibody described herein together with more than one therapeutic agent.

In another aspect, the invention also provides pharmaceutical combinations comprising an antibody described herein and more than one therapeutic agent.

Therapeutic agents suitable for use in pharmaceutical compositions and drug combinations of the present invention may be therapeutic agents selected from any one of the following categories (i)-(iv): (i) Drugs that enhance antigen presentation (e.g., tumor antigen presentation) ; (ii) drugs that enhance effector cell responses (e.g., B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects.

The compositions of the present invention may be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.

The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody of the present invention. A "therapeutically effective amount" means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time. The therapeutically effective amount may vary depending on a variety of factors such as disease state, age, gender and weight of the individual. A therapeutically effective amount is any amount in which the toxic or harmful effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%. The present invention can be evaluated in animal model systems predictive of efficacy in human tumors. A demonstrated antibody's ability to inhibit a measurable parameter (eg, tumor volume). A "prophylactically effective amount" means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Typically, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.

Kits containing the antibodies described herein are also within the scope of the invention. A kit may contain one or more other elements, including, for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject.

V. Uses and Methods of the Molecules of the Invention

In one aspect, the invention provides in vivo, in vitro uses and methods of using the antibodies of the invention.

In some embodiments, the uses and methods of the invention involve applying the antibodies of the invention in vivo and/or in vitro:

- Binds human PD-L1, human VEGF and human EGFR; or

-Block human PD-L1/PD-1 signaling; or

- Neutralize VEGF activity; or

-Block signaling mediated via EGFR; or

-Inhibit tumor cell growth,

or for the preparation of a medicament for any of the above uses.

In some embodiments, the antibody of the invention or a pharmaceutical composition comprising an antibody of the invention is used as a medicament for the treatment and/or prevention of disease in an individual or as a diagnostic tool for disease, preferably the individual is a mammal, more Preferably humans.

The antibody according to the invention is particularly suitable for use as an anti-tumor drug since it confers binding specificity to three different antigens, namely PD-L1, VEGF and EGFR. In some embodiments, antibodies according to the invention are used in the treatment of PD-L1 positive and EGFR positive cancers. Preferably, the PD-L1 positive and EGFR positive cancer is a solid tumor or a blood cancer, for example selected from the group consisting of cutaneous squamous cell carcinoma, head and neck cancer, melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial cancer Cancer, stomach cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma cancer, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma, liver cancer, microsatellite unstable solid tumors. Preferably, the cancer is cutaneous squamous cell carcinoma, colorectal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer.

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

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

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

Example

Example 1 Raw material preparation

1.1 Antigen preparation

The extracellular region of human PD-L1 (UniProtKB-Q9NZQ7), human VEGF (UniProtKB-P15692) and human EGFR (UniProtKB-P00533) were synthesized by Universal Biosystems (Anhui) Co., Ltd. PCR amplifies each target fragment, and introduces His tag or hIgG1 Fc (Uniprot No. P0DOX5, 218-449AA) or mouse Fc (Uniprot No. P01868) at the C-terminus through primers, and then constructs them respectively through homologous recombination. Eukaryotic expression vector pcDNA3.4 (Invitrogen). Transform the constructed expression vectors of each recombinant protein VEGF-His, VEGF-Fc, PD-L1-His, PD-L1-mFc, EGFR-Fc, and EGFR-His into E. coli DH5α respectively, culture them overnight at 37°C, and then Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression.

Each of the above recombinant proteins was expressed through the Expi293 transient expression system (ThermoFisher, A14635). For the transient transduction method, please refer to the instructions of the Expi293 ^TM expression system kit. 5-7 days after transfection, the cell expression supernatant was centrifuged at 15,000 g for 10 min. The obtained His-tagged recombinant protein expression supernatant was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentration of imidazole, and each eluted protein was passed through ultrasonic purification. The filter concentration tube (Millipore, UFC901096) was replaced into PBS buffer; the obtained Fc-tagged recombinant protein expression supernatant was filtered through a 0.22 μm filter membrane and purified using the Protein A/G affinity chromatography column affinity method. The target protein was eluted with 100mM glycinate (pH 3.0), then concentrated and replaced. After passing the SDS-PAGE identification and activity identification, it was frozen at -80°C.

1.2 Preparation of control antibodies

In this example, anti-human VEGF antibody P30-10-26 (VHH ^VEGF -hlgG1 Fc, VHH ^VEGF amino acid sequence shown in SEQ ID NO: 2) is derived from patent application CN202110995278.7; anti-human PD-L1 antibody D21- 4 (VHH ^PD-L1 -hlgG1 Fc, VHH ^PD-L1 amino acid sequence shown in SEQ ID NO: 1) derived from patent application PCT/CN2020/125301; anti-VEGF control antibody bevacizumab (Bevacizumab, abbreviated as Beva) sequence Sourced from Drug Bank (Drug Bank No: DB00112); anti-PD-L1 control antibody Atezolizumab (Ate) sequence comes from Drug Bank (Drug Bank No: DB11595); anti-EGFR control antibody panitumumab (Panitumumab, abbreviated as Pani) sequence is derived from IMGT (IMGT/mAb-DB ID: 196). The above amino acid sequences are converted into gene sequences respectively, and then the target fragment gene is synthesized by General Biotechnology Co., Ltd. Each target fragment was amplified by PCR and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) through homologous recombination.

The antibodies were all expressed using the transient transfer system (ExpiCHO). For the transient transfer method, please refer to the ExpiCHO ^TM Expression System Kit User Guide. The expressed cell suspension was centrifuged at high speed and the supernatant was taken. The supernatant was filtered through a 0.22 μm filter membrane and purified using the Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100mM glycinate (pH 3.0), then concentrated and replaced. After passing the SDS-PAGE identification and activity identification, it was frozen at -80°C.

1.3 Cell line construction

1.3.1 Construction of huPD-L1-CHO-K cell line

CHO-K cells (Thermo, A1461801) were passaged to 5 × 10 ⁶ cells/mL. The next day, the constructed cells containing full-length human PD-L1 were transferred using an electroporation kit (Invitrogen, MPK10096) and an electroporation instrument (Invitrogen, MP922947). (UniProtKB-Q9NZQ7) gene sequence plasmid was introduced into CHO-K cells. After electroporation, the cells were transferred to CD-CHO medium (Gibco, 10743029) and placed in a 37°C cell culture incubator. Raise for 48h. Then, 2000 cells/well were plated into a 96-well plate, and a final concentration of 30 μM MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C) were added for pressure screening. The single cell clones grown in the 96-well plate were picked, expanded and cultured, and then identified by FACS to obtain the huPD-L1-CHO-K cell line.

1.3.2 Construction of HEK293-VEGFR2-NFAT cell line

The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into HEK293 cells ( CRL-1573 ^TM ) to obtain the HEK293-NFAT cell line. HEK293-NFAT cells were passaged to 2 × 10 ⁵ cells/mL. The next day, the constructed cells containing the full-length human VEGFR2 gene sequence (NCBI Gene ID: 3791) plasmid was introduced into HEK293-NFAT cells. After electroporation, the cells were moved to DMEM medium (Gibco, 12634010) and placed in a 37°C cell culture incubator for 48 hours. Then 1,000 cells/well were plated into a 96-well plate, and puromycin at a final concentration of 2 μg/mL was added for pressure screening. Single cell clones grown in the 96-well plate were picked, and after expansion and culture, the HEK293-VEGFR2-NFAT cell line was identified by adding a luciferase catalytic substrate to detect the fluorescence signal.

1.3.3 Construction of Jurkat-PD-1-NFAT cell line

The pGL4.30 plasmid (promega, #E8481) containing the NF-AT-re nucleic acid sequence was electroporated into Jurkat cells ( TIB-152) to obtain Jurkat-NFAT cell line. On this basis, stably transfer the full-length expression gene sequence of PD-1 (NCBI Gene ID: 5133). The method is consistent with 1.3.2. Add puromycin and 0.5 μg/mL hygromycin B solution at a final concentration of 2 μg/mL. Monoclonal cell lines were screened, and the Jurkat-PD-1-NFAT cell line was obtained through FACS identification after expansion and culture.

1.3.4 Construction of CHO-PD-L1-CD3L cell line

The scFv sequence of OKT-3 (Drug Bank No: DB00075) was electroporated into CHO cells to obtain a CHO-CD3L stably transfected cell line stably expressing membrane-bound anti-CD3 scFv. On this basis, the full-length expression gene of PD-L1 was stably transfected. Sequence (NCBI Gene ID: 29126), the method is consistent with 1.3.2, add a final concentration of 30mM methionine iminosulfone and 8μg/mL puromycin to screen monoclonal cell lines, expand the culture and obtain CHO-PD-L1 through FACS identification -CD3L cell line.

Example 2 Construction of anti-PD-L1, VEGF and EGFR trispecific antibodies

This example describes the structure and construction of expression vectors for exemplary anti-PD-L1, VEGF, and EGFR trispecific antibodies (TriAbs). Eleven constructs were designed and constructed: VHH ^PD-L1 amino acid sequence comes from D21-4, and its variable region amino acid sequence is shown in SEQ ID NO:1; VHH ^VEGF amino acid sequence comes from P30-10-26, and its variable region The amino acid sequence is shown in SEQ ID NO:2; the VH ^EGFR and VL ^EGFR amino acid sequences are from Panitumumab; the linker amino acid sequence is shown in SEQ ID NO:3; the human IgG1 heavy chain constant region CH is shown in SEQ ID NO:4; The human Kappa light chain constant region CL is shown in SEQ ID NO:5. Exemplary TriAbs contain two identical first polypeptide chains and two identical second polypeptide chains. The constructs are shown in Table 1 and the corresponding amino acid sequences are provided in Table 2. Figures 1A-1G show structural schematics of candidate trispecific antibodies.

According to the structure of the construct, fragments of each antibody variable region and constant region were amplified by PCR, connected by overlap extension PCR, and then constructed into modified eukaryotic expression vectors by homologous recombination. Plasmid pcDNA3.4 (Invitrogen) constitutes the complete full-length gene of the polypeptide chain of the construct. The constructed vectors containing the full-length gene of the polypeptide chain of the construct were transformed into E. coli DH5α and cultured at 37°C overnight. Use endotoxin-free plasmid extraction kit (OMEGA, D6950-01) for plasmid extraction to obtain endotoxin-free The protein construct polypeptide chain plasmid is used for eukaryotic expression.

Table 1 Constructs of anti-PD-L1, VEGF and EGFR trispecific antibodies

Table 2 Amino acid sequences of anti-PD-L1/VEGF/EGFR trispecific antibodies

Example 3 Expression, purification, and physical and chemical property analysis of anti-PD-L1/VEGF/EGFR trispecific antibodies

3.1 Expression and purification of anti-PD-L1/VEGF/EGFR trispecific antibodies

The construct of Example 2 is expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133). The specific method is as follows: On the day of transfection, confirm that the cell density is about 7×10 ⁶ to 1×10 ⁷ cells/mL and the cells are viable. The rate is >98%. At this time, use fresh ExpiCHO expression medium pre-warmed at 37°C to adjust the cells to a final concentration of 6×10 ⁶ cells/mL. Dilute the target plasmid with OptiPRO ^TM SFM pre-cooled at 4°C (add 1 μg of plasmid to 1 mL of the medium). At the same time, dilute ExpiFectamine ^TM CHO with OptiPRO ^TM SFM. Mix the two in equal volumes and mix by gently pipetting. Prepare ExpiFectamine ^TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5 minutes, slowly add to the prepared cell suspension while shaking gently, and finally place in a cell culture shaker at 37°C, 8% _CO2 Cultivation. 18-22h after transfection, ExpiCHO ^TM Enhancer and ExpiCHO ^TM Feed were added to the culture medium, and the flask was placed on a 32°C shaker and 5% CO ₂ to continue culturing. On day 5 after transfection, add the same volume of ExpiCHO ^TM Feed and mix the cell suspension gently while adding slowly. 7-15 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes. The resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then purified with 100mM sodium acetate (pH 3.0). ) to elute the target protein, then neutralize with 1M Tris-HCl, and finally replace the obtained protein into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

3.2 Determination of the concentration of anti-PD-L1/VEGF/EGFR trispecific antibodies

The concentration of the purified trispecific antibody in Example 3.1 was measured using a verified ultra-trace spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd., Nano-300), and the measured A280 value was divided by the theoretical extinction coefficient of the antibody. The value is used as the antibody concentration value for subsequent studies, After passing the quality inspection, it is packaged and stored at -80℃.

3.3 SDS-PAGE identification of anti-PD-L1/VEGF/EGFR trispecific antibodies

Preparation of non-reducing solution: 1 μg of candidate trispecific antibody and reference product IPI (the IPI is the abbreviation of Ipilimumab, prepared by the method of Example 3.1) were added to 5×SDS loading buffer and 40mM Iodoacetamide, heated in a dry bath at 75°C for 10 minutes, cooled to room temperature, centrifuged at 12,000 rpm for 5 minutes, and took the supernatant. Preparation of reducing solution: 2 μg of candidate trispecific antibody and reference IPI were added to 5×SDS loading buffer and 5mM DTT, heated in a dry bath at 100°C for 10 minutes, cooled to room temperature, and centrifuged at 12,000 rpm for 5 minutes to take the supernatant. Add the supernatant to a Bis-tris 4-15% gradient gel (purchased from GenScript) and perform electrophoresis at a constant voltage of 110V. When Coomassie Brilliant Blue migrates to the bottom of the gel, stop running, take out the gel piece and place it in Coomassie Brilliant Blue staining solution. incubate for 1-2 hours, discard the staining solution, add destaining solution, replace the destaining solution 2-3 times as needed, destain until the gel background is transparent and then store it in deionized water. After decolorization, scan with an EPSON V550 color scanner, and calculate the purity of the reduced and non-reduced bands through ImageJ according to the peak area normalization method.

The results showed that the bands of the candidate trispecific antibody and the reference IPI reducing gel and non-reducing gel were all in line with the expected sizes, and the purity of the reducing gel was greater than 95%.

3.4 SEC-HPLC monomer purity identification of anti-PD-L1/VEGF/EGFR trispecific antibodies

Material preparation: 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Candidate trispecific antibodies are diluted to 0.5mg/mL with mobile phase solution. The Agilent HPLC 1100 column (XBridge BEH SEC 3.5μm, 7.8mm I.D.×30cm, Waters) flow rate was set to 0.8mL/min, the injection volume was 20μL, and the VWD detector wavelengths were 280nm and 214nm.

The SEC-HPLC results of the candidate trispecific antibodies are as follows: The percentages of high molecular polymers, antibody monomers and low molecular substances in the sample were calculated according to the area normalization method. The results are shown in Figures 2A-2G and Table 3.

Table 3 Physicochemical data of anti-PD-L1/VEGF/EGFR trispecific antibodies

Example 4 Affinity activity analysis of anti-PD-L1/VEGF/EGFR trispecific antibodies

4.1 Detection of the binding ability of candidate trispecific antibodies to recombinant protein VEGF-His based on ELISA method

Coat the recombinant protein VEGF-His on a 96-well ELISA plate and incubate at 4°C overnight. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, candidate trispecific antibodies and control antibody Bevacizumab at different concentrations were added and incubated for 1 h. Afterwards, the cells were washed three times with PBST and the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Using PRISM ^TM (GraphPad Software, San Diego, CA) analyzed the data and calculated _EC50 values.

The ELISA binding assay results are shown in Figures 3A-3B and Tables 4A-4B, and the candidate trispecific antibody exhibited an ability to bind to VEGF that was better than or equivalent to the control antibody Bevacizumab.

4.2 Detection of the binding ability of candidate trispecific antibodies to recombinant protein PD-L1-His based on ELISA method

Coat the recombinant protein PD-L1-His on a 96-well ELISA plate and incubate at 4°C overnight. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, different concentrations of candidate trispecific antibody and control antibody Atezolizumab were added and incubated for 1 h. Afterwards, the cells were washed three times with PBST and the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The ELISA binding assay results are shown in Figure 4A-4B and Table 4A-4B. The candidate trispecific antibodies TriAb3, TriAb4, TriAb6, TriAb7, TriAb8 and TriAb10 all showed comparable ability to bind to PD-L1 as the control antibody Atezolizumab.

4.3 Detection of the binding ability of candidate trispecific antibodies to recombinant protein EGFR-Fc based on ELISA method

Recombinant protein EGFR-Fc was coated on a 96-well ELISA plate and kept overnight at 4°C. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, different concentrations of candidate trispecific antibody and control antibody Panitumumab were added and incubated for 1 h. Afterwards, the cells were washed three times with PBST and the secondary antibody Anti-human-κ (Millipore, AP502P) was added and incubated for 1 h. After the incubation was completed, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The ELISA binding assay results are shown in Figures 5A-5B and Table 4A-4B. The candidate trispecific antibodies all showed comparable ability to bind to EGFR as the control antibody Panitumumab.

Table 4A Binding ability of anti-PD-L1/VEGF/EGFR trispecific antibodies based on ELISA method

Table 4B Binding ability of anti-PD-L1/VEGF/EGFR trispecific antibodies based on ELISA method

4.4 Test the binding ability of candidate trispecific antibodies to huPD-L1-CHO-K based on FACS method

Collect huPD-L1-CHO-K cells in the exponential growth phase, centrifuge at 300g to discard the supernatant, resuspend the cells in FACS buffer (PBS containing 1% BSA), count and adjust the cell suspension density to 2×10 ⁶ pieces/mL. Subsequently, huPD-L1-CHO-K cells were added into a 96-well round-bottom plate at 100 μL per well, and the supernatant was discarded by centrifugation. Add different concentrations of candidate trispecific antibody and control antibody D21-4 dilutions to the corresponding wells, resuspend the cells and incubate at 4°C for 30 minutes. Wash the incubated cell mixture three times, add PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596), resuspend and incubate at 4°C for 30 minutes. The incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The FACS binding assay results are shown in Figure 6 and Table 5. The candidate trispecific antibody showed comparable binding ability to PD-L1 expressed on cells as the control antibody D21-4.

Table 5 FACS-based PD-L1 binding ability of anti-PD-L1/VEGF/EGFR trispecific antibodies

4.5 Test the binding ability of candidate trispecific antibodies to A431 cells based on FACS method

Collect human epidermal cancer cell A431 cells with high expression of EGFR on the cell surface in the exponential growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu188), centrifuge and discard the supernatant, and reuse the cells with FACS buffer (PBS containing 1% BSA). Suspend, count and adjust the cell suspension density to 2×10 ⁶ cells/mL. Subsequently, A431 cells were added into a 96-well round-bottom plate at 100 μL per well, and the supernatant was discarded by centrifugation. Add different concentrations of candidate trispecific antibody and control antibody Panitumumab dilutions to the corresponding wells, resuspend the cells and incubate at 4°C for 30 minutes. Wash the incubated cell mixture three times, add PE-labeled anti-human-IgG-Fc flow cytometry antibody (Abcam, 98596), resuspend and incubate at 4°C for 30 minutes. The incubated cell mixture was washed three times, 200 μL of FACS buffer was added to resuspend the cells, and the cells were detected and analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The FACS binding assay results are shown in Figure 7 and Table 6. The candidate trispecific antibody showed comparable binding to the control antibody Panitumumab. Binding ability of A431 cells.

Table 6 PD-L1 binding capacity data of anti-PD-L1/VEGF/EGFR trispecific antibodies

4.6 Detection of the ability of candidate trispecific antibodies to simultaneously bind to antigens based on ELISA method

4.6.1 Detection of the ability of candidate trispecific antibodies to simultaneously bind to VEGF/PD-L1 based on ELISA method

Coat the recombinant protein VEGF-Fc on a 96-well ELISA plate and incubate at 4°C overnight. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, candidate trispecific antibodies of different concentrations were added and incubated for 1 h. Afterwards, wash the plate three times with PBST and add recombinant protein PD-L1-mFc. After washing the plate three times with PBST, add Anti-mouse Fc-HRP (Abcam, ab97265) and incubate at room temperature for 60 minutes. After completion, wash the plate 12 times with PBST. Add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The ELISA binding assay results are shown in Figure 8A. The trispecific antibodies TriAb3, TriAb4, and TriAb10 showed the ability to simultaneously bind to VEGF/PD-L1.

4.6.2 Detection of the ability of candidate trispecific antibodies to simultaneously bind to EGFR/VEGF based on ELISA method

Coat the recombinant protein VEGF-Fc on a 96-well ELISA plate and incubate at 4°C overnight. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, candidate trispecific antibodies of different concentrations were added and incubated for 1 h. Afterwards, wash the plate three times with PBST and add the recombinant protein EGFR-His. After washing the plate three times with PBST, add Anti-6×His-HRP (Proteintech, HRP-66005) and incubate at room temperature for 60 minutes. After completion, wash the plate with PBST for 12 seconds. times, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The ELISA binding assay results are shown in Figure 8B. The trispecific antibodies TriAb3, TriAb4, and TriAb10 showed the ability to bind to EGFR/VEGF simultaneously.

4.6.2 Detection of the ability of candidate trispecific antibodies to simultaneously bind to PD-L1/EGFR based on ELISA method

Recombinant protein PD-L1-mFc was coated on a 96-well ELISA plate and kept overnight at 4°C. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 h. After the plate was washed three times with PBST, candidate trispecific antibodies of different concentrations were added and incubated for 1 h. Afterwards, wash with PBST 3 After several times, add the recombinant protein EGFR-His, wash the plate 3 times with PBST, add Anti-6×His-HRP (Proteintech, HRP-66005), and incubate at room temperature for 60 minutes. After completion, wash the plate 12 times with PBST, add TMB (SurModics , TMBS-1000-01) color development. According to the color development results, 2M HCl was added to stop the reaction, and the plate was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). Data were analyzed using PRISM ^™ (GraphPad Software, San Diego, CA), and _EC50 values were calculated.

The ELISA binding assay results are shown in Figure 8C. The trispecific antibodies TriAb3, TriAb4, and TriAb10 showed the ability to simultaneously bind to PD-L1/EGFR.

Example 5 Detection of PD-1/PD-L1 blocking activity of anti-PD-L1/VEGF/EGFR trispecific antibody based on reporter gene method

This example uses a luciferase reporter gene system composed of CHO-PD-L1-CD3L cells and Jurkat-PD-1-NFAT cells to detect the activity of candidate trispecific antibodies in blocking the PD-1/PD-L1 signaling pathway. . In this system, the combination of PD-1 and PD-L1 can block CD3 downstream signal transduction and thereby inhibit luciferase expression. When a trispecific antibody containing anti-PD-L1 is added, the blocking effect is reversed, and luciferase Enzyme expression, stimulated with antibodies of different concentration gradients, will produce a fluorescence reading curve with antibody concentration dependence, so that the blocking activity of the antibody can be evaluated. The specific method is as follows:

Add a volume of 50 μL of 2 × 10 ⁴ cells/well of CHO-PD-L1-CD3L cells and 1 × 10 ⁵ cells/well of Jurkat-PD-1-NFAT cells into a 96-well white-sided bottom permeable cell culture plate. Add 50 μL of gradient diluted candidate trispecific antibody and control antibody D21-4, and incubate in a 37°C incubator for 6 hours. Add 50 μL Bright-Lite (vazyme, product number: DD1204-03) to each well, incubate in the dark for 10 minutes, and detect the fluorescence signal.

The blocking activity test results are shown in Figures 9A-9C. The candidate trispecific antibodies all showed better PD-1/PD-L1 blocking activity than the control antibody D21-4.

Example 6 Detection of VEGF neutralizing activity of anti-PD-L1/VEGF/EGFR trispecific antibodies based on reporter gene method

In this example, in order to determine the function of the candidate trispecific antibody in neutralizing the VEGF-VEGFR2 signaling pathway, the HEK293-VEGFR2-NFAT cell line was used (VEGF recombinant protein was added to this cell line culture system to activate the cell line through the VEGF-VEGFR2 signaling axis. The NFAT luciferase reporter gene is transcribed and expressed within the NFAT luciferase reporter gene, and a luciferase catalytic substrate is added to generate a fluorescent signal) as a material to detect the candidate trispecific antibody that neutralizes VEGF-VEGFR2 binding and thus blocks the expression of the downstream NFAT luciferase reporter gene. ability. The specific implementation is as follows:

Adjust the HEK293-VEGFR2-NFAT cell line to 4×10 ⁵ cells/mL, add 100 μL per well to a new 96-well cell culture plate, and place it in a 37°C cell culture incubator. At the same time, use DMEM culture medium to gradiently dilute the candidate trispecific antibody and control antibody P30-10-26, add 60ng/mL VEGF-Fc, mix and incubate at room temperature for 30 minutes. Subsequently, the co-incubated gradient diluted antibody and VEGF-Fc mixture was added to a 96-well cell culture plate and cultured in a 37°C incubator for 18 hours. After the culture, add 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) to each well, shake for 2 minutes, and then detect the fluorescence value of the 96-well plate.

The results are shown in Figure 10A-10B and Table 7A-7B. The trispecific antibody TriAb10 is better than the control antibody P30-10-26 in neutralizing VEGF. The other trispecific antibodies are as effective as the control antibody P30-10 in neutralizing VEGF. -26 is equivalent.

Table 7A VEGF neutralizing activity of anti-PD-L1/VEGF/EGFR trispecific antibodies

Table 7B VEGF neutralizing activity of anti-PD-L1/VEGF/EGFR trispecific antibodies

Example 7 In vivo tumor inhibition experiment of anti-PD-L1/VEGF/EGFR trispecific antibodies

Female NSG mice of 6-8 weeks old (purchased from Shanghai Nanmo Biology, strain: NOD-Prkdc ^scid Il2rg ^em1 /Smoc) were used. The experimental mice were kept in independent ventilation boxes with constant temperature and humidity, and the breeding room temperature was 21-24°C. Humidity 30-53%. 5×10 ⁶ A431 cells/mouse were injected subcutaneously on the right back (day 0), and then randomly divided into groups (6 mice per group): PBS-treated group, TriAb3 administration group, TriAb4 administration group, D21-4+Panitumumab+P30-10-26 administration group, D21-4+P30-10-26 administration group, each administration group has a low-dose group and a high-dose group. Dosage group. On the second day after tumor bearing, PBMC (C2106025) was injected through the tail vein. Each mouse was injected with 5×10 ⁶ PBMC cells. The first dose was given 2 hours later. The dose was administered twice a week, intraperitoneally ( i.p.) for a total of 3 weeks.

Observe and record the tumor length (mm) and width (mm) at any time, and calculate the tumor volume (V). The calculation method is V = (length × width ² )/2, and the tumor inhibition rate TGI (%) = (1-administration) The average tumor volume of the group/the average tumor volume of the PBS-treated group) × 100%. The tumor inhibition results are shown in Figure 11 and Table 8. It can be seen from the results that all administration groups showed tumor growth inhibition relative to the PBS-treated group; at high doses (equimolar), TriAb3 administration group, TriAb4 administration group and D21-4+Panitumumab+P30-10 All mice in the -26 combination group showed a trend of complete tumor remission; at the same time, the low-dose (equimolar) TriAb4 administration group had a better tumor inhibitory effect than the D21-4+Panitumumab+P30-10-26 combination group. , the tumor inhibition rate of trispecific antibodies is better than that of combination therapy.

Table 8 Tumor inhibition rate TGI (%) of different administration groups

Example 8 Affinity detection of anti-PD-L1/VEGF/EGFR trispecific antibodies

In this example, the Fortebio Octet RED96 instrument was used to detect the affinity of the candidate trispecific antibody and the control antibody to EGFR, PD-L1 and VEGF respectively.

Candidate antibodies were diluted to 10 μg/mL in 10×KB buffer (10×PBS containing 1% BSA, 0.5% Tween 20), and recombinant proteins (EGFR-His, PD-L1-His, and VEGF-His) were diluted to 10×KB The buffer is diluted into a concentration gradient series. Protect from light and pre-wet the sensor (Protein A Sensor, Gator, 20-5006) for at least 10 minutes before starting to test the sample plate (GreinierBio, PN655209). If the test is correct, proceed according to the preset procedure. First, the candidate antibody and the sensor are combined for 120 seconds. After the combination is completed, the sensor is continued to equilibrate in 10×KB buffer for 30 seconds. The sensor combined with the antibody is transferred to different concentrations of antigen diluent and combined for 120 seconds. After the signal is stable, it is transferred to 10 In ×KB buffer, the dissociation time is 180s. Finally, KD (affinity kinetic constant), Kon (binding constant) and Koff (dissociation constant) are obtained by fitting the binding and dissociation data of different concentrations of antigens. Kon can be written as Ka , Koff can be written as Kd.

The test results are shown in Table 9. The results show that the trispecific antibodies TriAb4 and TriAb10 have KD and Kd comparable to the control antibodies Panitumumab, D21-4 and P30-10-26.

Table 9 Affinity of anti-PD-L1/VEGF/EGFR trispecific antibodies

Sequence Listing Overview

This application is accompanied by a sequence listing containing a number of nucleic acid and amino acid sequences. The table below provides an overview of the included sequences.

Claims

A trispecific antibody, wherein the antibody includes a first antigen-binding domain that specifically binds a first antigen, a second antigen-binding domain that specifically binds a second antigen, and a third domain that specifically binds a third antigen. An antigen-binding domain, wherein the first antigen, the second antigen and the third antigen are different from each other and independently selected from the group consisting of PD-L1, VEGF and EGFR;

Among them, the antigen-binding domain that specifically binds VEGF includes the CDR1-3 sequence contained in SEQ ID NO:2;

Among them, the antigen-binding domain that specifically binds PD-L1 includes the CDR1-3 sequence contained in SEQ ID NO:1; and,

Wherein, the antigen-binding domain that specifically binds to EGFR includes a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain includes the HCDR1-contained in SEQ ID No: 31 3. The VL domain includes LCDR1-3 contained in SEQ ID No: 32.
The trispecific antibody of claim 1, wherein the antigen-binding domain that specifically binds VEGF comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 80%, 85%, 90%, or 80% of the amino acid sequence with SEQ ID NO: 2. An amino acid sequence that is 95% or 99% identical, or has one or more (preferably 1-10, more preferably 1-5) additions, deletions and/or substitutions of amino acids compared to SEQ ID NO:2 The amino acid sequence of

Most preferably, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:2, or consists of the amino acid sequence shown in SEQ ID NO:2.
The trispecific antibody of any one of claims 1-2, wherein the antigen-binding domain that specifically binds to PD-L1 includes the amino acid sequence shown in SEQ ID NO: 1, or has at least 80% similarity with SEQ ID NO: 1 , an amino acid sequence that is 85%, 90%, 95% or 99% identical, or has one or more (preferably 1-10, more preferably 1-5) amino acids compared to SEQ ID NO: 1 added, deleted and/or substituted amino acid sequences,

Most preferably, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1.
The trispecific antibody of any one of claims 1 to 3, wherein the antigen-binding domain that specifically binds to EGFR includes a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein,

The VH domain comprises the amino acid sequence shown in SEQ ID NO:31, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:31, or with SEQ ID NO:31. NO: 31 compared to an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) additions, deletions and/or substitutions of amino acids; and

The VL domain comprises the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO:32, or an amino acid sequence with SEQ ID NO:32. NO: 32 compared to an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) additions, deletions and/or substitutions of amino acids;

Most preferably, the antigen-binding domain comprises the VH amino acid sequence of SEQ ID NO: 31 and the VL amino acid sequence of SEQ ID NO: 32.
The trispecific antibody of any one of claims 1-4, wherein the antibody comprises:

(i) The antigen-binding domain that binds EGFR is a Fab domain;

(ii) the antigen-binding domain that binds PD-L1 is a VHH domain; and

(iii) the antigen-binding domain that binds VEGF is a VHH domain,

Wherein, the domains of (i), (ii) and (iii) are connected through a linker, and the linker contains 10-20 amino acids in length, preferably, contains the amino acid sequence (G 4 S) 3 .
The trispecific antibody of claim 5, wherein the antibody further comprises an immunoglobulin Fc region connected to the C-terminus of the Fab domain of (i), preferably, the Fc region is an IgG1 Fc region.
The trispecific antibody according to any one of claims 1 to 6, wherein the trispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes a first VHH domain, a linker, a VH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus,

The second polypeptide chain includes a second VHH domain, a linker, a VL domain and a CL domain from the N-terminus to the C-terminus,

wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and wherein the VH domain is paired with the VL domain and specifically binds to the third antigen,

wherein the first antigen and the second antigen are different from each other and independently selected from PD-L1 and VEGF (preferably, the first antigen is PD-L1 and the second antigen is VEGF), and wherein the third antigen is EGFR .
The trispecific antibody according to any one of claims 1 to 6, wherein the trispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes a first VHH domain, a linker, a second VHH domain, a linker, a VH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus,

The second polypeptide chain includes a VL domain and a CL domain from the N-terminus to the C-terminus,

wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and wherein the VH domain is paired with the VL domain and specifically binds to the third antigen,

wherein the first antigen and the second antigen are different from each other and independently selected from PD-L1 and VEGF (preferably, the first antigen is PD-L1 and the second antigen is VEGF), and wherein the third antigen is EGFR .
The trispecific antibody according to any one of claims 1 to 6, wherein the trispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes VH domain, CH1 domain and Fc region from N-terminus to C-terminus,

The second polypeptide chain includes a first VHH domain, a linker, a second VHH domain, a linker, a VL domain and a CL domain from the N-terminus to the C-terminus,

wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and wherein the VH domain is paired with the VL domain and specifically binds to the third antigen,

wherein the first antigen and the second antigen are different from each other and independently selected from PD-L1 and VEGF (preferably, the first antigen is PD-L1 and the second antigen is VEGF), and wherein the third antigen is EGFR .
The trispecific antibody according to any one of claims 1 to 6, wherein the trispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain includes a first VHH domain, a linker, a VH domain, a CH1 domain and an Fc region from the N-terminus to the C-terminus,

The second polypeptide chain includes a VL domain, a CL domain, a linker and a second VHH domain from the N-terminus to the C-terminus,

wherein the first VHH domain and the second VHH domain specifically bind to the first antigen and the second antigen respectively, and wherein the VH domain is paired with the VL domain and specifically binds to the third antigen,

The first antigen is PD-L1, the second antigen is VEGF, and the third antigen is EGFR.
The trispecific antibody of any one of claims 1-6, wherein the trispecific antibody comprises a first polypeptide chain and a second polypeptide chain selected from the group consisting of:

(a) Comprising the amino acid sequence shown in SEQ ID NO:7 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO: 13 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the second polypeptide chain of the amino acid sequence;

(b) Comprising the amino acid sequence shown in SEQ ID NO:8 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO:14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the second polypeptide chain of the amino acid sequence;

(c) Comprises the amino acid sequence shown in SEQ ID NO:9 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it The first polypeptide chain of the amino acid sequence, and contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 15 or 16 , a second polypeptide chain with an amino acid sequence that is 98% or 99% identical;

(d) Contains the amino acid sequence shown in SEQ ID NO: 10 or 11 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto The first polypeptide chain has a unique amino acid sequence, and contains or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 17 , a second polypeptide chain with an amino acid sequence that is 98% or 99% identical; and

(e) Comprising the amino acid sequence shown in SEQ ID NO: 12 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The first polypeptide chain of the amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO: 18 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity to the amino acid sequence of the second polypeptide chain.
A polynucleotide encoding the trispecific antibody of any one of claims 1-11.
A vector, preferably an expression vector, comprising the polynucleotide of claim 12.
A host cell comprising the polynucleotide of claim 12 or the vector of claim 13, for example, the host cell is a mammalian cell.
A method for producing the trispecific antibody of any one of claims 1-11, comprising:

Culturing a host cell comprising a polypeptide chain encoding the antibody under conditions suitable for expression of the polypeptide chain; and assembling the polypeptide chain into the antibody under conditions suitable for assembly of the polypeptide chain to produce the antibody.
A pharmaceutical composition comprising the trispecific antibody of any one of claims 1-11 and a pharmaceutically acceptable carrier.
Use of the trispecific antibody according to any one of claims 1-11 or the pharmaceutical composition according to claim 16, for use in vivo or in vitro

- Binds human PD-L1, human VEGF and human EGFR; or

-Block human PD-L1/PD-1 signaling; or

- Neutralize VEGF activity; or

-Block signaling mediated via EGFR; or

-Inhibit tumor cell growth,

or for the preparation of a medicament for any of the above uses.
The use of claim 17, wherein said trispecific antibody or pharmaceutical composition is used as a medicament for the treatment and/or prevention of disease in an individual, preferably said individual is a mammal, more preferably a human.
The use of claim 18, wherein the disease is PD-L1 positive and/or EGFR positive cancer, preferably solid tumors, such as cutaneous squamous cell carcinoma, head and neck cancer, melanoma, renal cell carcinoma, non-small cell lung cancer, bladder cancer , urothelial cancer, kidney cancer, gastric cancer, colon cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, cervical cancer, glioblastoma cancer, pancreatic cancer, prostate cancer, esophageal cancer, lymphoma, liver cancer, microbial cancer Satellite unstable solid tumors.