WO2022042715A1 - BIFUNCTIONAL FUSION PROTEIN TARGETING PD-L1 AND TGFβ, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF - Google Patents

Abstract

The present invention provides a bifunctional fusion protein targeting programmed cell death ligand-1 (PD-L1) and transforming growth factor-β (TGFβ), and also provides a nucleic acid molecule encoding the fusion protein of the present invention, an expression vector for expressing the fusion protein of the present invention, a host cell, a method, and a pharmaceutical composition containing the fusion protein of the present invention. The present invention also provides a medical application of the bifunctional fusion protein targeting PD-L1 and TGFβ in the treatment of tumor diseases.

Description

Bifunctional fusion protein targeting PD-L1 and TGFβ and preparation method and application thereof technical field

The invention relates to the field of biomedicine, in particular to a bifunctional fusion protein targeting PD-L1 and TGFβ and a preparation method and application thereof.

Background technique

Immunomodulation targeting PD-1 (Programmed Death 1) is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplantation survival. Two ligands of PD-1 have been found so far: PD-L1 and PD-L2. PD-L1, also known as B7-H1, is about 40 kDa in size and is a type I transmembrane protein. Under normal circumstances, the immune system will react to foreign antigens and promote the proliferation and activation of antigen-specific T cells. The activated T cells upregulate the expression level of PD-1, and combine with PD-L1 to form a negative feedback to avoid excessive T cells.

The up-regulation of PD-L1 expression in tumor cells is an important way to achieve immune escape. Studies have found that the PD-L1 protein is highly expressed in human tumor tissues such as breast cancer, lung cancer, gastric cancer, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer. And the expression level of PD-L1 is closely related to the clinical and prognosis of patients. In the tumor microenvironment, the up-regulation of PD-L1 expression can directly inhibit the anti-tumor response of T cells and mediate the immune escape of tumor cells through the PD-1 signaling pathway. At present, a number of companies are developing monoclonal antibodies against PD-1 or PD-L1, which can improve the patient's own immune response to tumors by blocking the combination between PD-L1/PD-1, thereby killing tumor cells. the goal of. Approved PD-L1 mAbs include Roche's Tecentriq (atezolizumab), AstraZeneca's Imfinzi (durvalumab), and Merck's Bavencio (avelumab).

In tumors, cancer cells may only occupy a small part, and most of them are stromal cells and infiltrating cells, including fibroblasts, myeloid cells, lymphocytes, and endothelial cells. Among them, stromal fibroblasts and fibroblast-like cells are known as cancer-associated fibroblasts (CAFs), which are interspersed among tumor cell populations, support tumor growth, expansion and metastasis, and pass through a variety of The pathway creates an immunosuppressive tumor microenvironment. TGFβ is an important immunosuppressive mediator in the tumor microenvironment. TGFβ belongs to the TGF family, there are three ligand isoforms, TGFβ1, 2 and 3, all of which are homodimers, and there are three TGFβ receptors, TGFβRI, II and III. TGFβ plays a dual role in tumors as a regulatory factor. TGFβ acts as a tumor suppressor before the tumor becomes malignant, and becomes a tumor promoter after the tumor becomes malignant.

According to previous reports, TGFβ1 is highly expressed in bladder cancer, triple-negative breast cancer, esophageal cancer, lung cancer, skin cancer and gastric cancer and is associated with subsequent activation of the TGFβ pathway. TGFβ secreted by tumor cells or stromal cells can promote angiogenesis, for example, can induce the expression of angiogenic factors VEGF and CTGF. Moreover, TGFβ secreted by tumor cells turns different cells into tumor-associated fibroblasts (CAFs). CAFs are one of the most important fibroblasts in the tumor microenvironment. TGFβ allows CAFs to form functional filopodia, thereby Invading the tumor microenvironment, approaching cancer cells, and exerting tumor-promoting functions, CAFs can also promote epithelial mesenchymalization (EMT) and induce tumor progression.

Since TGFβ plays a role in maintaining immune tolerance, it is helpful in promoting tumor immune escape and can promote tumor progression. Studies have found that TGFβ can regulate the proliferation and differentiation of T cells by interfering with TCR-related signals, and inhibit the autoimmune response. TGFβ secreted by tumors can induce DC apoptosis and inhibit DC migration; it can also prevent NK from recognizing tumor cells, thereby preventing NK cell-mediated killing. Meanwhile, tumor-secreted TGFβ affects T cell activation by directly targeting CTL-related factors perforin, granzymes A and B, and IFNγ, mediating tumor escape from immune surveillance. The most important role of TGFβ in promoting tumor immune escape is to stimulate Treg cells, induce autoimmune tolerance, and promote immunosuppression. Treg cells with surface-bound TGFβ inhibited CD8+T-mediated killing, decreased granzyme B expression, and increased the amount of PD-1 on the cell surface.

TGFβ also plays a role in tumor cell migration and local metastasis. For example, TGFβ-induced EMT not only induces "mesenchymal" in cancer cells, but also supports tumorigenesis, host immune surveillance evasion and chemoresistance. TGFβ signaling can promote the targeted migration of tumor lymphatic vessels and support the extravasation of tumor cells to other sites to form secondary tumors.

Existing studies have shown that in some solid tumors, PD-(L)1 antibody has no obvious benefit, which may be related to TGFβ, tumor cells or Treg cells autocrine or paracrine TGFβ, and TGFβ1 can lead to a decrease in markers of NK activity. , NK lytic activity or mediated ADCC activity is reduced, causing immunosuppression in the tumor microenvironment. At the same time, TGFβ is also associated with tumor mesenchymalization (mesenchymalization occurs in tumor metastasis or drug resistance and immune tolerance) and resistance to chemotherapy, and can also up-regulate the expression of PD-L1.

At present, there have been several different research stages in the treatment of bifunctional fusion protein drugs targeting both PD-L1 and TGFβ. US20150225483A1 describes a bifunctional fusion protein that combines anti-programmed death ligand 1 (PD-L1) antibodies with the soluble type II tumor growth factor beta receptor (TGFβRII) as a TGFβ neutralizing "Trap" The extracellular domain is composed of two anti-PD-L1 immunoglobulin light chains and two extracellular domains of human TGFβRII fused to the anti-PD-L1 heavy chain via a glycine-serine flexible linker The drug (M7824) has been clinically studied in gastric cancer, lung cancer, esophageal cancer, NSCLC, biliary tract cancer and other tumor diseases.

WO2018205985A1 discloses a fusion protein containing a TGF-beta receptor, comprising a targeting PD-L1 part and a TGF-beta receptor part, wherein the TGF-beta receptor part is the N of the extracellular region of TGF-betaRII A terminally truncated form of the drug (SHR-1701) has been clinically studied in nasopharyngeal carcinoma.

WO2020094122A1 discloses a pharmaceutical composition comprising a PD-L1/TGFβRII fusion protein that is more convenient for production and administration and has more stable performance, comprising: a TGF-β receptor fusion protein, and a buffer, the buffer is selected from the group consisting of Histidine buffer, succinate buffer, phosphate buffer and citrate buffer.

However, the currently disclosed bifunctional fusion protein against PD-L1 and TGFβ is a heterotetramer, and the antibody part is a full-length antibody, and the fusion protein formed by fusing the TGF-β receptor part at the C-terminus of the heavy chain The molecular weight is large and the protein space structure is complex, which leads to problems such as easy degradation, polymerization or low expression during the production process of fusion protein. Therefore, in actual production and clinical application, it is still necessary to develop a bifunctional fusion protein targeting PD-L1 and TGFβ through new research and development ideas, and obtain better affinity, drug structure or protein stability through protein engineering. performance products.

SUMMARY OF THE INVENTION

The present invention provides a bifunctional fusion protein that targets and binds PD-L1 and TGFβ, and also provides a nucleic acid molecule encoding the antibody of the present invention, an expression vector, host cell and method for expressing the antibody of the present invention, as well as a nucleic acid molecule encoding the antibody of the present invention. Pharmaceutical compositions of antibodies. The present invention also provides the medical use of the bifunctional fusion protein that targets and binds PD-L1 and TGFβ in the treatment of tumor diseases.

In a first aspect, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, comprising:

(a) human TGFβ receptor II (TGFβRII) that binds TGFβ, or a fragment thereof in an N-terminal truncated form; and

(b) a Nanobody that binds PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein:

(i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G;

(ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and

(iii) the sequence of CDR3 is shown as SEQ ID NO: 3;

In another embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, the fusion protein is shown in general formula (I):

VHH-L1-Fc-L2-TGFβRII (I)

Wherein, VHH is a Nanobody that binds to PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein:

(i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G;

(ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and

(iii) the sequence of CDR3 is shown as SEQ ID NO: 3;

Wherein, L1 and L2 are connecting peptides, and each is present or absent;

wherein, Fc is the Fc domain of a human IgG antibody;

Wherein, TGFβRII is human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ.

In another embodiment, in the bifunctional fusion protein targeting and binding to PD-L1 and TGFβ according to the present invention, the sequence of CDR1 is consistent with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4.

In another embodiment, in the bifunctional fusion protein targeting and binding to PD-L1 and TGFβ according to the present invention, the sequence of CDR2 is consistent with the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5.

In another embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ according to the present invention, wherein the heavy chain variable region of the Nanobody VHH contains as SEQ ID NO: 10, SEQ ID NO : 11, SEQ ID NO: 12 or SEQ ID NO: 13 any one of the identical amino acid sequences shown. In another preferred embodiment, wherein the heavy chain variable region sequence of the Nanobody VHH is the same as the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: SEQ ID NO: 13 Have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology.

In another embodiment, in the bifunctional fusion protein targeting and binding to PD-L1 and TGFβ according to the present invention, the general sequence formulas of connecting peptides L1 and L2 are respectively (G ₄ S) _n , (SG ₄ ) _n , G ₄ (SG ₄ ) _n or (G ₄ S) _n G, wherein n is an integer of 0-6, preferably 3-5. In another preferred embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ according to the present invention, the connecting peptide L1 does not exist, and the connecting peptide L2 is (G ₄ S) ₄ G (SEQ ID NOSEQ ID NO: ID NO: 27).

In another embodiment, in the bifunctional fusion protein targeting and binding PD-L1 and TGFβ according to the present invention, Fc is the Fc domain of human IgG antibody, selected from the Fc domain of human IgG1, IgG2, IgG3 or IgG4 . In yet another specific aspect, the human constant region Fc segment is an IgGl Fc, the sequence of which is set forth in SEQ ID NO:28. In yet another specific aspect, the human constant region is an IgGl Fc mutant whose sequence is set forth in SEQ ID NO:29. In yet another specific aspect, the human constant region Fc segment is an IgG4 Fc, the sequence of which is set forth in SEQ ID NO:30.

In another embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, wherein human TGFβRII capable of binding TGFβ refers to its extracellular domain sequence (ECD1-136), such as SEQ ID NOSEQ ID NO: 31. In another preferred embodiment, the N-terminal truncated form fragment of human TGFβRII is selected from the group consisting of a deletion of less than 26 consecutive amino acids at the N-terminus of SEQ ID NO: 31, more preferably a deletion of 14-21 consecutive amino acids at the N-terminus , such as ECD (15-136) (SEQ ID NO: 32) with a deletion of 14 N-terminal amino acids, ECD (20-136) with a deletion of 19 N-terminal amino acids (SEQ ID NO: 33), and a deletion with 21 N-terminal amino acids ECD of amino acids (22-136) (SEQ ID NO: 34) and the like.

In a particularly preferred embodiment of the present invention, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, the amino acid sequence of which is such as SEQ ID NO: SEQ ID NO: 35, SEQ ID NO: SEQ ID NO: 36, SEQ ID NOSEQ ID NO:37, SEQ ID NOSEQ ID NO:38, SEQ ID NOSEQ ID NO:39, SEQ ID NOSEQ ID NO:43 or SEQ ID NOSEQ ID NO:44.

In another embodiment, the present invention provides an isolated polynucleotide encoding the bifunctional fusion protein that targets binding PD-L1 and TGFβ.

The present invention provides an expression vector comprising an isolated polynucleotide encoding the bifunctional fusion protein targeted to bind PD-L1 and TGFβ, and a host cell comprising the expression vector. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

In another embodiment, the present invention provides a pharmaceutical composition comprising the bifunctional fusion protein targeted to bind PD-L1 and TGFβ and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a method for making a bifunctional fusion protein that targets PD-L1 and TGFβ, comprising expressing a bifunctional fusion protein that targets PD-L1 and TGFβ in a host cell and The fusion protein is isolated from the host cell.

In another embodiment, the present invention provides a method of treating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a bifunctional fusion protein targeted to bind PD-L1 and TGF[beta]. In yet another aspect, the present invention provides a method of modulating an immune response in a subject, comprising administering to the subject a bifunctional fusion protein of the present invention that targets binding PD-L1 and TGFβ, such that the immune response in the subject is Responses are regulated. Preferably, the antibodies of the invention enhance, stimulate or increase an immune response in a subject.

In another embodiment, the present invention provides the use of a bifunctional fusion protein that targets binding PD-L1 and TGFβ for the treatment of cancer or for the inhibition of tumor growth. The cancer or tumor may be selected from the group or site: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine , head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

In another embodiment, the subject or subject of administration of the bifunctional fusion protein targeting binding PD-L1 and TGFβ is a mammal, such as a mouse, monkey, dog, cow, horse or human, preferably a human.

Description of drawings

Figure 1 shows the results of binding affinity screening of VHH antibody lysate samples that bind to PD-L1.

Figure 2 shows the results of blocking assay screening of anti-PD-L1 candidate antibody molecules.

Figure 3 shows the results of cell binding experiments for candidate antibody molecules: (A) isotype control (human IgG1), (B) NB22D-21, (C) NB22gb-10, (D) positive control KN035.

Figure 4 shows the experimental verification results of the specific binding reaction of the NB22D-21 molecule.

Figure 5 shows the results of the mixed lymphocyte reaction experiment validation of the NB22D-21 molecule.

Figure 6 shows the results of the binding experiment validation of NB22D-21 molecule and its humanized derivative molecules, wherein the isotype control (isotype) is human IgG1.

Figure 7 shows the results of a human-mouse cross-reactivity experiment of the NB22D-21 molecule and its humanized derivatives, wherein the isotype is human IgG1.

Figure 8 shows the flow cytometry results of the human-mouse cross-reactivity experiment of the humanized derivative molecule NB22D-21-huVH1: (A) control molecule KN035, (B) NB22D-21-huVH1.

Figure 9 shows the results of a binding blocking experiment of NB22D-21 molecule and its humanized modified molecule, wherein the isotype control (isotype) is human IgG1.

Figure 10 shows the blocking activity of the druggable engineered molecule on PD-L1-CHO, where the isotype control is human IgG1.

Figure 11 shows the results of IFN-γ (A) and IL-2 (B) secretion in mixed lymphocyte reactions by druggable engineered molecules.

Figure 12 shows a sequence alignment of candidate antibody molecules with CDR sequences boxed.

Figure 13 shows the binding affinity of D21-4-T and D21-huVH2-T to PD-L1 detected by Octet.

Figure 14 ELISA shows that D21-4-T, D21-huVH2-T bind to PD-L1.

Figure 15 ELISA shows that D21-4-T and D21-huVH2-T can simultaneously bind to dual targets of PD-L1 and TGFβ.

Figure 16 ELISA shows that D21-4-T, D21-huVH2-T block PD-L1/PD-1 binding.

Figure 17 FACS shows that D21-4-T, D21-huVH2-T block PD-L1/PD-1 binding.

Figure 18 FACS shows D21-4-T, D21-huVH2-T endocytosis mediated by PD-L1 targets.

Figure 19 MLR shows that D21-4-T, D21-huVH2-T enhance the proliferative activation of T cells.

Figure 20 Antitumor efficacy in mice of D21-4-T and D21-huVH2-T.

Detailed description of the invention

definition

In order to make the present invention easier to understand, certain terms are first defined. Additional definitions will be set forth throughout the detailed description.

Unless otherwise stated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art and are Technical literature and general textbooks in the field are fully explained, such as Molecular Cloning:

A Laboratory Manual (Molecular Cloning: Laboratory Manual) et al.

The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-1" are interchangeable Used interchangeably, variants, isoforms, species homologs, and analogs that share at least one epitope with PD-1 are included in human PD-1. PD-1 is a member of the CD28/CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. The complete PD-1 sequence can be found under GenBank number U64863.

As used herein, the term "PD-L1" refers to programmed cell death ligand 1 (PD-L1, see eg Freeman et al. (2000) J. Exp. Med. 192:1027). Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1, B7H1, CD274, and B7-H, among others. A representative amino acid sequence of human PD-L1 is disclosed in NCBI Accession No. NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is shown under NCBI Accession No.: NM_014143.4. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found in many tumors or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells, and myeloid cells. Binding of PD-L1 and its receptor induces signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system during specific events (eg, pregnancy, autoimmune disease, tissue allografts) and is thought to allow tumors or cancer cells to bypass immune checkpoints and evade immune responses.

"Antibody" refers to any form of antibody that exhibits the desired biological activity (eg, inhibition of binding of a ligand to its receptor or by inhibition of ligand-induced receptor signaling). Thus, "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, nanobodies, and multispecific antibodies (eg, bispecific antibodies) . An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camelids may contain only heavy chains.

"Nanobodies", also known as single domain antibodies (sdAbs), are modified from heavy chain antibodies found in camelid animals, and are antibodies containing a single antibody heavy chain variable region domain. The heavy chain antibody found in camelid only contains one variable domain of heavy chain of HCAb (VHH) and two conventional CH2 and CH3 regions. The VHH region cloned and expressed alone has good Structural stability and antigen-binding activity. VHH is the smallest known unit that can bind target antigen, and its molecular weight is much smaller than that of IgG antibody, so VHH is also called Nanobody (nanobody).

As used herein, the terms "bind" and "specifically bind" refer to the binding of an antibody or antigen-binding portion to an epitope in an in vitro assay, preferably in bioluminescence interferometry (ForteBio) using purified wild-type antigen. In certain embodiments, an antibody or antigen-binding portion is said to specifically bind an antigen when it preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

"TGFβRII" or "TGFβ receptor II" refers to the human TGFβRII extracellular domain sequence (ECD1-136) capable of binding TGFβ, as set forth in SEQ ID NO: SEQ ID NO:31. The N-terminal truncated form fragment of human TGFβRII is selected from the deletion of 26 or less consecutive amino acids on the N-terminal of SEQ ID NO: 31, more preferably the deletion of 14-21 consecutive amino acids at the N-terminal, preferably the N-terminal of human TGFβRII Truncated form fragments comprise the amino acid sequence set forth in SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. TGFβRII can retain at least 50%, 75%, 90%, 95%, 99% or 100% of the TGFβ binding activity of the wild-type sequence.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The Fc can be selected from the Fc domains of human IgG1, IgG2, IgG3 or IgG4 or Fc domains mutated at one or several of these sites.

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. The majority of humanized antibodies are human immunoglobulins (acceptor antibodies) in which the hypervariable region residues of the acceptor antibody are replaced by hypervariable regions of a non-human species (donor antibody) with the desired specificity, affinity and capacity. Residue substitution, non-human species such as mouse, rat, rabbit or non-human primate. In certain instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies comprise substantially all of at least one and usually both variable domains, wherein all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, all or nearly all of the FR regions FR regions of human immunoglobulin sequences. The humanized antibody also optionally comprises at least a portion of an immunoglobulin (usually human immunoglobulin) constant region (Fc).

An "isolated" antibody is one that has been identified and separated from components of its natural environment, the contaminating components of which are substances that would interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other Protein solutes or non-protein solutes. In some embodiments, the antibody is purified to greater than 95% purity, more preferably greater than 99% purity, as determined by the Lowry method. Isolated antibodies are generally prepared by at least one purification step.

An "isolated" nucleic acid molecule is one that has been identified and separated from at least one contaminating nucleic acid molecule. An isolated nucleic acid molecule differs from its naturally occurring form or environment.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "immune cells" as used herein includes cells of hematopoietic origin and that play a role in immune responses. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, a "variant" of a sequence refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

As used herein, the term "about" refers to an index value that is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, "about" or "consisting essentially of" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean at most one order of magnitude or at most five times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for the specific value.

When using "administer" and "treat" in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to combining an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, or biological fluid. Person, cell, tissue, organ or biological fluid contact. "Administering" and "treatment" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of cells, eg, by agents, diagnostic agents, binding compositions, or by other cells.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular subject may vary depending on factors such as the disease being treated, the general health of the patient, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Patient" means a human or non-human animal (eg, a mammal).

"Cancer" or "tumor" refers to a collection of cells that proliferate in an abnormal manner.

Various aspects of the invention are described in further detail in the following subsections.

A bifunctional fusion protein targeting PD-L1 and TGFβ

In a first aspect, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, comprising:

(a) human TGFβ receptor II (TGFβRII) that binds TGFβ, or a fragment thereof in an N-terminal truncated form; and

(b) a Nanobody that binds PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein:

(i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G;

(ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and

(iii) the sequence of CDR3 is shown as SEQ ID NO: 3;

The bifunctional fusion protein that targets and binds to PD-L1 and TGFβ prepared by the present invention has at least three functions: first, it relieves the immunosuppressive effect related to TGFβ, reduces the inhibitory effect of Treg on the proliferation of CD4+T cells, and promotes CD8+T cells. and activation of NK cells; second, reverse TGFβ-mediated tumor cell mesenchymalization; third, block the binding of PD-L1 to PD-1. The advantage of dual-targeting protein over the combination of PD-L1 antibody and TGFβ antibody is that the PD-L1 antibody can be used to guide the TGFβRII cytokine trap to the tumor microenvironment, specifically bind TGFβ in the tumor microenvironment, and use PD-L1 antibody to guide the TGFβRII cytokine trap to the tumor microenvironment. L1-mediated targeted internalization brings TGFβ into cells for destruction. The above effects cannot be achieved by the combined treatment of the two. For more information on the mechanism of action of the anti-PD-L1/TGFβRII trap, please refer to the description in Chinese patent CN201580007865.3, which is incorporated herein by reference in its entirety.

In the past, the bifunctional fusion protein targeting PD-L1 and TGFβ was a heterotetramer, and the antibody part was a full-length antibody. The fusion protein formed by merging the TGFβ receptor part at the C-terminus of the heavy chain had a larger molecular weight and a protein spatial structure. It is complex, which leads to problems such as easy degradation, aggregation or low expression level in the production process of fusion protein. Compared with the reported anti-PD-L1/TGFβRII trap, the improvement of the present invention lies in that the antibody binding to PD-L1 in the bifunctional fusion protein of the present invention adopts nanobody (VHH).

Nanobodies were first reported by Belgian scientists in the journal Nature in 1993 (Hamers-Casterman C, et al; (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448.), in alpaca There is a naturally occurring light chain-deficient antibody in peripheral blood, which contains only a variable heavy chain (VHH) and two conventional CH2 and CH3 regions, but does not resemble an engineered single-chain antibody fragment (scFv) So easy to stick to each other, and even clumped together. More importantly, the individually cloned and expressed VHH structure has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind to the target antigen. VHH is extremely soluble, not easy to aggregate, can withstand high temperature, strong acid, strong alkali and other denaturing conditions, and its affinity is comparable to that of full-length human antibodies.

The bifunctional fusion protein targeting PD-L1 and TGFβ of the present invention, wherein the PD-L1-binding nanobody is derived from Chinese patent application CN202010765530.0, which is incorporated herein by reference in its entirety. The PD-L1 antibody in the form of nanobodies and its derivative molecules after humanization transformation or druggability transformation according to the present invention, while maintaining the advantages of high affinity and low molecular weight, carry out immunogenic modification and post-translational modification Point transformation has improved the druggability and has great therapeutic advantages. The molecular weight of the anti-PD-L1/TGF-β bifunctional fusion protein finally prepared by the present invention is only about 63% of that of the similar drugs M7824 and SHR-1701 under research, and the dosage can be reduced or can be increased at the same dosage. Drug molar concentration ratio to improve drug efficacy.

In another embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, the fusion protein is shown in general formula (I):

VHH-L1-Fc-L2-TGFβRII (I)

Wherein, VHH is a Nanobody that binds to PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein:

(i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G;

(ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and

(iii) the sequence of CDR3 is shown as SEQ ID NO: 3;

Wherein, L1 and L2 are connecting peptides, and each is present or absent;

wherein, Fc is the Fc domain of a human IgG antibody;

Wherein, TGFβRII is human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ.

In another embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ according to the present invention, the sequence of the heavy chain variable region CDR1 of the Nanobody is the same as SEQ ID NO: 1 or SEQ ID NO : The sequence shown in 4 is identical.

In another embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ of the present invention, the sequence of the heavy chain variable region CDR2 of the Nanobody is the same as SEQ ID NO: 2 or SEQ ID NO : The sequence shown in 5 is identical.

In a preferred embodiment of the present invention, in the bifunctional fusion protein targeting and binding to PD-L1 and TGFβ according to the present invention, wherein the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region of the Nanobody are respectively associated with SEQ ID NO, SEQ ID NO: 1, SEQ ID NO, SEQ ID NO: 2, SEQ ID NO, SEQ ID NO: 3, or SEQ ID NO, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 3 are identical in sequence.

In another embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ of the present invention, the heavy chain variable region of the Nanobody comprises a framework region (hereinafter referred to as FR region), so Said FR region comprises FR1, FR2, FR3 and FR4, and is spaced with CDR1, CDR2 and CDR3 on said heavy chain variable region to form from N-terminus to C-terminus FR1-CDR1-FR2-CDR2-FR3-CDR3- Structure of FR4. Wherein, FR1 is selected from the sequence shown in SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22; FR2 is selected from the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 19; FR3 is selected from the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 19; from the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20; FR4 is selected from the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 21;

In another embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ according to the present invention, wherein the heavy chain variable region of the Nanobody VHH contains as SEQ ID NO: 10, SEQ ID NO : 11, SEQ ID NO: 12 or SEQ ID NO: 13 any one of the identical amino acid sequences shown.

In another preferred embodiment, wherein the heavy chain variable region sequence of the Nanobody VHH has a sequence with the sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 An amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of amino acid additions, deletions and/or substitutions that retains the ability to specifically bind to PD-L1 , 97%, 98%, 99% or 100% homology and amino acid sequences that retain the ability to specifically bind PD-L1. In some preferred embodiments, antibodies with high (ie, 90% or higher) homology to Nanobody CDR regions or variable regions are obtained by conservative sequence modifications, including amino acid substitutions, additions and deletions, and the like. The one or more amino acid additions, deletions and/or substitutions (eg, conservative substitutions) are no more than five, preferably no more than three. The term "conservative sequence modification" is intended to mean that the amino acid modification does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Modification of nucleic acid molecules encoding variable region sequences can be accomplished by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are well described in the art. These families include those with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine) , tryptophan, histidine) amino acids. Thus, one or more amino acid residues outside the CDR regions of the antibodies of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibodies tested for retained function using the functional assays described herein. Preferred sites for site-directed mutagenesis or PCR-mediated mutagenesis are located outside of the variable regions CDR1-CDR3.

Exemplary substitution

original residue Exemplary substitution conservative substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp;Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg

original residue Exemplary substitution conservative substitution Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids can be grouped by the properties of common side chains:

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

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidity: Asp, Glu;

(4) Alkaline: His, Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions require exchanging members of one of these classes for another.

In another embodiment, in the bifunctional fusion protein targeting and binding PD-L1 and TGFβ according to the present invention, each of the connecting peptides L1 and L2 is present or absent, and the general sequence formula is (G ₄ S) _n , (SG ₄ ) _n , G ₄ (SG ₄ ) _n or (G ₄ S) _n G, wherein n is an integer of 0-6, preferably 3-5. In another preferred embodiment, in the bifunctional fusion protein targeting PD-L1 and TGFβ according to the present invention, the connecting peptide L1 is absent, and the connecting peptide L2 is (G ₄ S) ₄ G (SEQ ID NO. : 27).

In another embodiment, in the bifunctional fusion protein targeting and binding PD-L1 and TGFβ according to the present invention, Fc is the Fc domain of human IgG antibody, selected from the Fc domain of human IgG1, IgG2, IgG3 or IgG4 . In yet another specific aspect, the human constant region Fc segment is an IgGl Fc, the sequence of which is set forth in SEQ ID NO:28. In yet another specific aspect, the human constant region is an IgG1 Fc mutant that can remove the ADCC, ADCP or CDC effects of IgG1 Fc by site-directed mutagenesis, the preferred sequence is shown in SEQ ID NO: 29. In yet another specific aspect, the human constant region Fc segment is an IgG4 Fc, the sequence of which is set forth in SEQ ID NO:30.

In another embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, wherein the human TGFβRII extracellular domain sequence (ECD1-136) capable of binding TGFβ is shown in SEQ ID NO: 31 . In another preferred embodiment, the N-terminal truncated form fragment of human TGFβRII is selected from the group consisting of a deletion of less than 26 consecutive amino acids at the N-terminus of SEQ ID NO: 31, more preferably a deletion of 14-21 consecutive amino acids at the N-terminus , such as ECD (15-136) (SEQ ID NO: 32) with a deletion of 14 N-terminal amino acids, ECD (20-136) with a deletion of 19 N-terminal amino acids (SEQ ID NO: 33), and a deletion with 21 N-terminal amino acids ECD of amino acids (22-136) (SEQ ID NO: 34) and the like. In another embodiment, the human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ has at least 90%, 91%, 92 sequence homology with SEQ ID NO:31-SEQ ID NO:34 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, the fusion protein is shown in general formula (I):

VHH-L1-Fc-L2-TGFβRII (I)

Wherein, VHH is a Nanobody that binds PD-L1, wherein the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region of the Nanobody are respectively the same as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or The sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 3 are identical;

Wherein, the L1 linking peptide does not exist, and the general sequence formula of the L2 linking peptide is (G ₄ S) _n , (SG ₄ ) _n , G ₄ (SG ₄ ) _n or (G ₄ S) _n G, and n is 3- An integer of 5.

wherein, Fc is the Fc domain of a human IgG1 or IgG4 antibody;

Wherein, TGFβRII is human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ.

In another preferred embodiment of the present invention, the present invention provides a bifunctional fusion protein that targets and binds PD-L1 and TGFβ, and the fusion protein is shown in the general formula (I):

VHH-L1-Fc-L2-TGFβRII (I)

Wherein, VHH is a Nanobody that binds to PD-L1, wherein the heavy chain variable region of the Nanobody VHH contains any of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 A consensus amino acid sequence shown;

Wherein, the L1 linking peptide does not exist, the sequence of the L2 linking peptide is of the general formula (G ₄ S) _n G, and n is an integer of 3-5;

wherein, Fc is the Fc domain of a human IgG1 or IgG4 antibody;

Wherein, TGFβRII is human TGFβRII capable of binding TGFβ or its N-terminal truncated form fragment, the sequence of which is shown in any one of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 .

In a particularly preferred embodiment of the present invention, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, the amino acid sequence of which is such as SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37. SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 44. In another preferred embodiment, the present invention provides a bifunctional fusion protein targeting PD-L1 and TGFβ, wherein the amino acid sequence of the bifunctional fusion protein is the same as that of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO : 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 44 with at least 90%, 91%, 92%, 93%, 94%, 95% homology , 96%, 97%, 98%, 99% or 100%.

Nucleic acid molecules encoding fusion proteins of the present invention

Another aspect of the present invention pertains to nucleic acid molecules encoding the bifunctional fusion proteins of the present invention that target binding to PD-L1 and TGF[beta]. Nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids of the invention can be obtained using standard molecular biology techniques. Preferred nucleic acid molecules of the present invention are those encoding Nanobody CDR regions, variable regions, Nanobodies containing a human Fc segment, and full-length fusion proteins targeting bifunctional fusion proteins that bind PD-L1 and TGFβ shown in the present invention. The amino acid sequence of a nucleic acid molecule.

The DNA encoding the Nanobody VHH can be operably linked to another DNA molecule encoding the human heavy chain constant regions (CH1, CH2 and CH3). Sequences of human heavy chain constant region genes are known in the art (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), including DNA fragments of these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgGl, IgG2, IgG3 or IgG4 constant region, but is most preferably an IgGl constant region. An exemplary DNA sequence encoding Nanobody D21-4 is shown in SEQ ID NO:41.

In order to create a bifunctional fusion protein gene targeting PD-L1 and TGFβ, a DNA fragment encoding a nanobody that binds PD-L1, a DNA fragment encoding the Fc domain of a human IgG antibody, and a human TGFβRII or its A DNA fragment encoding an N-terminal truncated form of the fragment is operably linked to a DNA fragment encoding a flexible linker (L1 or L2), eg, a DNA fragment encoding the amino acid sequence ( _G4S )4G, to form a DNA fragment encoding VHH-L1-Fc-L2 _- TGFβRII DNA molecules of fusion proteins. Preferably, in the absence of L1, a DNA molecule encoding a VHH-Fc-L2-TGFβRII fusion protein is formed

The present invention provides polynucleotides encoding bifunctional fusion protein molecules that target binding PD-L1 and TGFβ. In some embodiments, an exemplary nucleotide sequence encoding a PD-L1/TGFβ bifunctional fusion protein (also referred to as PD-L1/TGFβ trap) D21-4-T (SEQ ID NO: 35) is as SEQ ID NO :42.

Expression vectors and host cells

The present invention provides expression vectors comprising the isolated polynucleotides, as well as host cells comprising the expression vectors.

The selection of a suitable vector will depend primarily on the size of the nucleic acid into which the vector is to be inserted and the particular host cell in which the vector is to be transformed. To express the protein of interest, DNA can be inserted into an expression vector by standard molecular biology techniques, thereby operably linking the gene to transcriptional and translational regulatory sequences.

The expression vector and expression control sequences are selected to suit the expression host cell used. The DNA molecule encoding the PD-L1 Nanobody-linking peptide-human TGFβRII fusion protein was inserted into the expression vector by standard methods. Alternatively, the recombinant expression vector may encode a signal peptide, which facilitates secretion of the fusion protein in the host cell. The fusion protein gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the fusion protein gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the fusion protein gene, the recombinant expression vectors of the present invention carry regulatory sequences such as those derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg, adenovirus major late promoter (AdMLP)) and polyoma A viral promoter and/or enhancer that regulates the expression of fusion protein genes in host cells. The term "regulatory sequences" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that regulate transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

To express the fusion protein, an expression vector encoding the fusion protein is transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the fusion proteins of the invention in either prokaryotic or eukaryotic host cells (eg bacterial, yeast or mammalian cells), expression in eukaryotic cells (most preferably mammalian host cells) is most preferred fusion protein. Preferred mammalian host cells for expressing the fusion protein of the present invention include Chinese hamster ovary cells (CHO cells), NSO myeloma cells, COS cells and SP2 cells, etc., preferably CHO cells.

When a recombinant expression vector encoding a fusion protein is introduced into a mammalian host cell, the fusion protein is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, the fusion protein is secreted into the culture of the cultured host cell. base. The fusion protein can be recovered from the broth of the culture using standard protein purification methods.

In another embodiment, the present invention provides a method for making a bifunctional fusion protein that targets PD-L1 and TGFβ, comprising:

(a) introducing a recombinant expression vector carrying a gene encoding a bifunctional fusion protein targeting binding to PD-L1 and TGFβ into a mammalian host cell, and culturing the recombinant host cell under conditions conducive to production of the fusion protein; and

(b) recovering the fusion protein.

In the production methods of the present invention, cells are cultured in a nutrient medium suitable for production of the fusion protein using methods known in the art. For example, small-scale or large-scale fermentation in laboratory or industrial fermentors (including continuous, batch , fed-batch or solid-state fermentation) to culture cells. Cultivation is carried out in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions.

If the fusion protein is secreted into the nutrient medium, the fusion protein can be recovered from the medium using methods known in the art. For example, it can be recovered from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Purification can be accomplished by a variety of methods known in the art including, but not limited to, chromatography (eg, Protein A affinity columns, ion exchange, hydrophobicity, chromatographic focusing and size exclusion) or any combination thereof.

pharmaceutical composition

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the bifunctional fusion protein targeted to bind PD-L1 and TGFβ and a pharmaceutically acceptable carrier.

In one aspect, the present invention provides a pharmaceutical composition comprising a bifunctional fusion protein targeting binding to PD-L1 and TGFβ formulated together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

Pharmaceutical compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated into a solution, a microemulsion, a liposome or a lyophilized powder for injection. Preferred routes of administration for the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal/spinal or other parenteral routes of administration, eg, by injection or infusion. For parenteral administration, administer 0.1 mg/kg-100 mg/kg, or 0.5 mg/kg-50 mg/kg, or 1 mg/kg-25 mg/kg, or 2 mg/kg-10 mg/kg, or 5 mg/kg-10 mg dose of fusion protein per kg. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. A "therapeutically effective amount" of a fusion protein of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of damage or disability resulting from the disease. For example, for the treatment of tumors, a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, more preferably by at least about 60%, relative to untreated subjects. Even more preferably up to at least about 80%. The ability of compounds to inhibit tumor growth can be assessed in animal model systems that can predict efficacy in human tumors. One of ordinary skill in the art would be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

Uses and Methods of the Invention

In another embodiment, the present invention provides a method of treating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a bifunctional fusion protein targeted to bind PD-L1 and TGF[beta].

In yet another aspect, the present invention provides a method of modulating an immune response in a subject, comprising administering to the subject a bifunctional fusion protein of the present invention that targets binding PD-L1 and TGFβ, such that the immune response in the subject is Responses are regulated. Preferably, the antibodies of the invention enhance, stimulate or increase an immune response in a subject.

The invention provides a method of treating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a fusion protein of the invention. The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, mice, rats, cats, cattle, horses, chickens, amphibians, and reptiles. In another embodiment, the subject or subject of administration of the bifunctional fusion protein targeting binding PD-L1 and TGFβ is a mammal, such as a mouse, monkey, dog, cow, horse or human, preferably a human.

In another embodiment, the present invention provides the use of a bifunctional fusion protein that targets binding PD-L1 and TGFβ for the treatment of cancer or for the inhibition of tumor growth. The cancer or tumor may be selected from the group or site: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine , head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. The present invention can also be used to treat metastatic cancer, especially metastatic cancer expressing PD-L1.

The optimal dose of the bifunctional fusion protein of the invention that targets binding to PD-L1 and TGF[beta] will depend on the disease being treated, the severity of the disease, and the presence or absence of side effects. Optimal dosages can be determined by routine experimentation. For parenteral administration, administer 0.1 mg/kg-100 mg/kg, or 0.5 mg/kg-50 mg/kg, or 1 mg/kg-25 mg/kg, or 2 mg/kg-10 mg/kg, or 5 mg/kg-10 mg /kg dose. Exemplary treatment regimens can be administered weekly, every two weeks, every three weeks, every four weeks, monthly, every 3 months, or every 3-6 months.

detailed description

The invention generally described herein will be more readily understood by reference to the following examples, which should not be construed as further limiting. The contents of all drawings and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Those skilled in the art should understand that, unless otherwise noted, the reagents, plasmids, cells, etc. used in the following examples are all commercially available products.

The CDR and variable region amino acid sequences of the PD-L1 Nanobodies and the ability and functional activity to bind target proteins are referred to in Examples 1-22, and Tables A, B and C below provide a summary of the sequences included (SEQ ID NO: 1-SEQ ID NO: 26), the sequence, data and accompanying drawings can refer to Chinese patent application CN202010765530.0, which is incorporated herein by reference in its entirety.

Examples 23-29 relate to the protein sequence and DNA sequence of a bifunctional fusion protein that binds PD-L1 and TGFβ, as well as the ability and functional activity to bind the two related target proteins, as shown in SEQ ID NO:27-SEQ ID NO:44 .

Example 1 Construction of cell lines stably expressing PD-L1

In this example, CHO-s cells expressing human PD-L1, monkey PD-L1 and mouse PD-L1 were constructed respectively, and Roche's control antibody was prepared.

1.1 Control antibody preparation

Whole gene synthesis control antibody-Atezolizumab (Roche) light chain and heavy chain gene sequences (gene synthesis supplier: Universal Bio), the control antibody was expressed using the ExpiCHO transient expression system (purchased from Thermo Fisher), cultured The base is ExpiCHO ^™ Expression Medium (Gibco, A29100-01), and the transfection kit is ExpiFectamine ^™ CHO Transfection Kit (Gibco, A29129).

The specific method is as follows: construct the ExpiCHO expression plasmids of the light chain and heavy chain genes of the atezolizumab antibody by molecular cloning, passage ExpiCHO cells (purchased from GibcoA29127) one day before transfection, and in a 25ml cell culture system, 25μg of the constructed plasmids (The plasmid mixture containing the light chain encoding gene and the heavy chain encoding gene with a mass ratio of 2:1) was mixed with the transfection reagent and added dropwise to 25ml of ExpiCHO cell culture, mixed well, and expressed at 37°C for 18-22 hours. , add the feeding medium according to the instructions in the kit, after feeding, the cells are cultured at 32°C, on the 5th day after transfection, add the second feed, and culture the cells at 32°C for 10-12 days After that, the expressed cell suspension was centrifuged at high speed to take the supernatant, and the obtained supernatant was filtered through a 0.22 μm filter membrane and purified by the Protein A/G affinity chromatography column affinity purification method, using 100 mM glycinate (pH3. 0) The target protein was eluted, followed by neutralization to pH 7.0 with 1M Tris-HCl. After a small amount of sampling, it was identified by SDS-PAGE, then packed and stored frozen.

The preparation method of control antibody KN035 (amino acid sequence is shown in Sequence Listing SEQ ID NO: 9) is as follows:

The complete gene sequence of the control antibody KN035 was fully synthesized, and the ExpiCHO expression plasmid of the KN035 antibody gene was constructed by molecular cloning. The ExpiCHO cells (purchased from Gibco A29127) were passaged one day before transfection, and in a 25ml cell culture system, the constructed After 25 μg of plasmid was mixed with transfection reagent, transfection was carried out according to the method for preparing control antibody-Atezolizumab, and the antibody was expressed.

1.2 Construction of stable cell lines

Recombinant vector plasmids expressing the full-length proteins of human PD-L1 (gi number: NP_054862.1), mouse PD-L1 (gi number: NP_068693) and rhesus monkey PD-L1 (gi number: ABO33163.1) were constructed respectively. The constructed plasmids were introduced into CHO-s cells (purchased from Thermo Fisher) and A375 melanoma cell line (ATCC, CRL-1619) by electroporation. The CHO-s cell line with high expression of PD-L1 protein of the above three species and the A375 cell line with high expression of human PD-L1 (PD-L1-A375) were obtained by screening.

1.2.1 Construction of plasmids expressing human PD-1 and PD-L1 ectodomain proteins

The expression vectors containing the full-length protein gene sequences of human PD-L1, mouse PD-L1 and rhesus monkey PD-L1 were synthesized by gene synthesis, and then introduced into E. coli after ligation. The E. coli monoclone was picked and sequenced to obtain the correct plasmid. Cloning, plasmid extraction and resequencing.

1.2.2 Construction of CHO-s cell lines expressing PD-1 and PD-L1 proteins

1.2.2.1 Electric transfer

CHO-s cells were cultured and maintained in CD-CHO serum-free medium (Gibco, 10743029), the cells were passaged to 5×10 ⁶ /mL the day before electroporation, and an electroporation kit (Invitrogen, Neon ^TM Kit, MPK10096) was used the next day. The constructed plasmids were respectively introduced into CHO-s cells using an electroporator (Invitrogen, NeonTM Transfection System, MP922947). The electroporated cells were added to 3 mL of CD-CHO medium and placed in a carbon dioxide incubator at 37°C for 48 hours.

1.2.2.2 Cell plating and culture

The electroporated CHO-s cells were plated at 2000 cells/well in a 96-well cell culture plate, and L-Methionine sulfoximine (MSX) (Millipore, GSS-1015-F) was added at a final concentration of 30 μM/mL. ), keep the cell culture volume at 100 μL/well and 1×GS supplement (Sigma, 58672C), place it in a 37°C carbon dioxide incubator, and add a medium containing 30 μM MSX and 1×GS supplement after 10 days 50 μL.

1.2.2.3 Clonal identification and cell expansion

The grown clones were picked and transferred to 24-well cell culture plates. The cell lines were identified by FACS, and clones with high expression were selected for expansion and cryopreservation. The relevant FACS identification methods are as follows:

1) Collect empty CHO-s cells and 2×10 ⁵ of each cloned CHO-s cells first, centrifuge at 300 g to remove the supernatant, and resuspend the cells in 200 μL of prepared FACS buffer (1×PBS+2% FBS). 96-hole round bottom plate;

2) Centrifuge the 96-well round bottom plate at 300g for 5min, and remove the supernatant;

3) Add anti-PD-L1 antibody diluent or negative control antibody diluent to the corresponding wells, blow the cells evenly with a drain gun and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS buffer to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and place them at 4°C to incubate for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

1.2.3 Preparation of A375 cell line expressing PD-L1

The PD-L1 high-expressing cell line (PD-L1-A375) of the A375 cell line was prepared by the same method as 1.2.2.1 for electroporation of CHO-s cells, which was used for the construction of the animal model of the A375 cell line.

Embodiment 2 Animal immunity and serum titer detection

2.1 Animal immunization

Purchase PD-L1 (NP_054862.1) extracellular domain protein (Yiqiao Shenzhou, 10084-H05H) to immunize 2 alpacas (Nanchang Dajia Technology). Each alpaca was immunized with 500 μg each time, once every 2 weeks, for a total of 4 times.

2.2 Serum titer detection

After the alpaca immunization was completed, the alpaca serum was taken for immune titer detection.

The immune titer assay is to measure the binding ability of immune serum to recombinant protein PD-L1 (Yiqiao Shenzhou, 10084-H05H) by ELISA, and determine the immune effect according to the titer of the binding antibody.

The specific method is as follows:

2.2.1 Antigen coating: One day before the immunotiter determination, the antigen recombinant protein PD-L1 was diluted with PBS to a final concentration of 2 μg/mL to obtain a dilution. 30 [mu]l of the obtained dilution was added to an ELISA plate and coated overnight at 4[deg.]C. On the day of immunotiter determination, the cells were rinsed three times with PBS, blocked with PBST containing 5% nonfat milk for two hours at room temperature, and rinsed three times with PBS.

2.2.2 Serum dilution: Dilute the unimmunized negative serum and the immunized serum with PBS on another dilution plate. The first well is diluted 200 times, and then the subsequent 7 wells are diluted by 3 times.

2.2.3 Antibody reaction: The diluted serum was added to the first ELISA plate, incubated at 37°C for 1 h, washed twice with PBS, and then added with secondary antibody goat anti-camelid IgG antibody (purchased from Nanjing GenScript) at 1:5000. .

2.2.4 Color reading: After washing the above secondary antibody 3 times with PBS, add color developing solution for 5 minutes, after adding stop solution, read the plate at OD450 by a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in the table. 1.

Table 1. Results of ELISA coloration experiments at different dilution ratios

dilution ratio negative serum NSY004 NSY005 1:2000 0.209 2.582 2.163 1:4000 0.133 2.286 2.216 1:8000 0.093 1.923 2.131

1:16000 0.052 1.817 1.868 1:32000 0.054 1.337 1.218 1:64000 0.048 1.048 0.792 1:128000 0.042 0.761 0.587 1:256000 0.048 0.404 0.473

Among them, the NSY004 and NSY005 columns are the ELISA color test results of the immunized sera of two alpacas after diluting different times, and the negative serum is the ELISA test results of the unimmunized alpacas serum. According to the results in Table 1, it can be seen that the IgG titers of the two alpacas have reached 256000, and the immunization effect is good, which can be used for the construction of the peripheral blood immune antibody library in the next step.

Example 3 Construction and preliminary screening of alpaca immune library

After animal immunization, 50 mL of fresh alpaca blood was taken, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation medium (GE, 17144003S), and anti-human PD-L1 antibody bacteriophage alpaca was tested. Immune library construction.

The specific method is as follows:

Dilute the collected alpaca blood with PBS at a ratio of 1:1 (v/v), take 15ml of Ficoll-Paque density gradient separation solution and slowly add it to a 50ml centrifuge tube, tilt the centrifuge tube and slowly add it along the tube wall to dilute it. 30mL of alpaca blood, so that the two liquids maintain a clear separation interface. Centrifuge at 4°C for 20 min, keeping the acceleration at 3 and 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is the plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is Ficoll-Paque PLUS, and there is a narrow white band dominated by PBMC at the junction of the upper and middle layers, that is, the PBMC cell layer. . Carefully use a pipette to transfer the PBMC cells in the middle to a new 50 mL centrifuge tube. Rinse twice with PBS, centrifuge horizontally at 1500 rpm for 10 min at 4°C, resuspend with 1.5 ml PBS, and count by microscope.

RNA was extracted from the isolated PBMC cells, and the extracted RNA was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A). Since the molecular form of alpaca antibody is different from that of ordinary antibodies, it does not contain light chain and heavy chain does not contain CH1. Therefore, firstly, common primers are designed on the front end of VH and CH2, and two fragments of different sizes are obtained by PCR. Small purpose fragments. Then, by aligning the amino acid sequences of all common VHH germlines (Germline), Germline-specific degenerate primers containing NcoI and NotI restriction sites at both ends were designed to amplify all VHH genes using the recovered product as a template Finally, the target antibody gene fragment is inserted into the phage display vector through double enzyme digestion and ligation. It should be pointed out that the C-terminus of the VHH gene on the expression vector is fused to the GIII gene in the phage expression vector. The ligation product was recovered by a recovery kit (Omega, D6492-02), and finally transformed into competent E. coli SS320 by electroporation (Bio-Rad, MicroPulser), and plated on ampicillin-resistant 2-YT solid plates. In order to calculate the library capacity, the total number of clones formed by all electrotransformation was calculated by taking 10 μl of the bacterial solution of the library for 10-fold gradient dilution, 2 μl of each dilution gradient was placed on the plate, and the clones formed on the plate were counted to calculate the total number of clones formed by all electrotransformation, that is, the library capacity. The capacity of this immune pool is 1×10 ⁹ .

Based on this storage capacity, take 10 times the bacterial volume (about 20 OD) of the storage capacity and add it to the fresh 2-YT liquid medium, and adjust the amount of medium added so that the initial OD value of the bacterial dilution is 0.05. Placed at 37°C, cultured at 220rpm to the logarithmic growth phase, at this time, VSCM13 helper phage was added in an amount 50 times the number of bacteria, mixed well, allowed to stand for 30min, then incubated at 220rpm for 1h, and then replaced by centrifugation at 10,000rpm for 5min into carbenicillin/kanamycin dual-resistant 2-YT medium, and cultured overnight at 30°C, 220 rpm. The next day, centrifuged at 13000g for 10min, and the supernatant was precipitated by adding 20% PEG/NaCl solution to obtain phage corresponding to the alpaca immune antibody library. After washing once with PBS, it was used for the target PD-L1 antibody screening.

Phage screening uses recombinant PD-L1 protein, using magnetic bead screening and immunotube screening. The specific methods are as follows.

3.1 Magnetic bead screening

Screening was performed based on the binding of biotin-labeled recombinant PD-L1 protein to avidin-coupled magnetic beads (purchased from Thermo Fisher, Cat. No. 11205D). For the labeling method, see the instructions of Roche's biotin labeling kit, catalog number: 11418165001), incubate the biotin-labeled PD-L1 protein with the magnetic beads, so that the PD-L1 protein is bound to the magnetic beads. The magnetic beads conjugated with PD-L1 antigen and the phage library with nanobody display were incubated at room temperature for 2 hours. After washing 6-8 times with PBST, the non-specifically adsorbed phages were removed, and trypsin (Gibco) was added and mixed gently for 20 min. , to elute the nanobody-displayed phage that specifically binds to the human PD-L1 protein. The eluted phages were then infected with log-phase SS320 cells (Lucigen, MC1061 F), and the phage-infected SS320 cells were spread on carbenicillin-resistant plates, cultured at 37°C overnight, and collected the next day. Bacteria. The phage was prepared by using SS320 bacteriophage, and the preparation method is described in the above-mentioned library phage preparation method. The resulting phage was continued to be used for the second round of screening, and eluted by trypsin at the end of the second round of screening. Phages obtained from the second round of screening were used for the third round of screening, and eluted with trypsin at the end of the third round of screening. Iteratively, sequence analysis was performed by randomly picking 10 clones per round. The results showed that the monoclonal phage obtained after 3 rounds of screening, and the sequenced monoclonal, the gene sequences of different clones were repeated, which proved that the sequence enrichment was obvious.

3.2 Immunotube screening

Immune tube screening is based on coating the surface of the immune tube with antigens and screening for antibody-displaying phages that bind to the target antigen. The day before screening, the immunotubes were coated with recombinant human PD-L1 protein in advance, and the immunotubes bound with PD-L1 antigen and the phage library with nanobody display were incubated at room temperature for 2 hours. After washing 6-8 times with PBST, the non-specific Heterotropically adsorbed phage was added with trypsin (Gibco) and mixed gently for 20 min to elute the nanobody-displayed phage that specifically binds to human PD-L1 protein. The eluted phages were then infected with log-phase SS320 cells (Lucigen, MC1061 F), and the phage-infected SS320 cells were spread on carbenicillin-resistant plates, cultured at 37°C overnight, and collected the next day. Bacteria. The phage was prepared by using SS320 bacteriophage, and the preparation method is described in the above-mentioned library phage preparation method. The resulting phage was continued for the second round of screening. At the end of the second round of screening, trypsin was used for elution. Phages obtained from the second round of screening were used for the third round of screening, and at the end of the third round of screening, trypsin was used for elution. Repeatedly, 10 clones were randomly selected in each round after sequencing and sequence analysis. The results showed that after 3 rounds of screening, the sequenced single clones had duplicated gene sequences of different clones, which proved that the sequence enrichment was obvious.

The phage libraries obtained by the two different screening methods were screened for single clones, and the positive clones in the third round of products of magnetic bead screening and immunotube screening were selected respectively. The specific method is as follows:

The day before the screening, the recombinant human PD-L1 protein was coated on a 96-well ELISA plate, and the induced phage supernatant was prepared in the 96-well plate the next day, and the positive clones against the recombinant human PD-L1 protein were screened by phage ELISA. All positive clones were sequenced and analyzed, and the clones with unique sequences were prepared as lysates. The preparation method was as follows: the bacterial solution of the clone was inoculated at 1:100 in 50 mL the day before, incubated at 37°C with constant shaking for 14 hours, centrifuged at 10,000 g at room temperature for 5 minutes, and then used The bacteria were resuspended in 1 mL of Tris-HCl buffer containing benzonase nuclease at pH 9.0, lysed on ice for 30 min, centrifuged at 10,000 g at 4°C for 10 min, and the supernatant was collected to obtain a positive clone lysate.

The prepared positive clone lysate was further verified at the flow level, and a candidate antibody that specifically recognized human PD-L1 was screened. The flow level verification method is as follows:

1) First collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer, count and adjust the cell suspension density to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add gradient dilutions of candidate antibody lysate and control antibody dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS buffer to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled flow-through antibody (GenScript), blow the cells evenly with a spray gun, and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

The results of binding affinity screening of anti-PD-L1 VHH lysate samples are shown in Figure 1.

It can be seen from Figure 1 that the VHH molecules of the indicated clones have a certain affinity for CHO cells expressing PD-L1. Since this detection experiment is only qualitative and semi-quantitative, it is impossible to confirm which clone number of the VHH antibody molecule has a better affinity, and further experimental confirmation is required.

The prepared positive clone lysate was further blocked and screened at the flow level, and a candidate antibody that specifically recognized human PD-L1 and blocked its binding to PD-1 protein was screened. The flow level verification method is as follows:

1) First collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody lysate and the control antibody dilution with a concentration gradient of 1:1, 1:5 and 1:25 respectively to the corresponding wells, blow the cells uniformly with a blow gun and place them at 4°C for incubation for 30 minutes. minute;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of PD-1-Fc protein diluent (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a discharge gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-human IgG Fc flow antibody (Abeam), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

The screening results of the blocking experiment of anti-PD-L1 antibody candidate molecules are shown in Figure 2. Through the preliminary screening by ELISA and FACS, it can be seen that the antibody lysate prepared from the screened antibody sequences contains antibodies that bind PD-L1 to PD1. In response to the antibody with blocking effect, the inventors screened 10 candidate molecules with good affinity and blocking activity.

Example 4 Production and Expression of Chimeric VHH-Fc Antibodies

The positive VHH candidate antibody obtained by screening is fused with the human IgG1 Fc segment, the C-terminus of the positive VHH gene sequence is connected to the N-terminus of the human IgG1 Fc segment gene sequence to construct a fusion expression vector, and the fusion expression vector plasmid is transformed into ExpiCHO cells, The VHH-Fc chimeric antibody protein fused to the Fc fragment was obtained by inducing expression.

The antibody was expressed using the ExpiCHO transient expression system, the medium was (Gibco, A29100-01), and the transfection kit was (Gibco, A29129). The specific method is as follows: Passage the ExpiCHO cells one day before transfection, mix 25 μg of the constructed plasmid with the transfection reagent in a 25 ml system, add dropwise to 25 ml of ExpiCHO cell culture, mix well, and express 18- After 22 hours, the feeding medium was added according to the instructions in the kit. After feeding, the cells were cultured at 32°C. On the 5th day after transfection, the second feed was added, and the cells were cultured at 32°C for 10 days. -12 days later, the expressed cell suspension was centrifuged at high speed to get the supernatant, the supernatant was filtered at 0.22 μm, purified by Protein A/G affinity purification method, and eluted with 100 mM glycinate (pH 3.0). The protein of interest was then neutralized with 1M Tris-HCl.

Example 5 Verification of Affinity Activity of Chimeric VHH-Fc Antibody at Cell Level

The obtained VHH-Fc candidate antibodies were evaluated, and their binding activity to PD-L1 protein on cells was detected by FACS. The specific methods are as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the cell suspension density to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and place them at 4°C to incubate for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 2, through the FACS experiment, the inventors screened two nanobody candidate molecules with high affinity, and the affinity was higher than or similar to that of the control antibody.

Table 2. EC50 of antibodies

clone number EC50(μg/mL) NB22D-21 0.39 NB22gb-10 0.39 KN035 (control) 0.39 Atezolizumab (control) 0.83

Example 6 Activity verification of chimeric VHH-Fc antibody blocking PD-1

The obtained VHH-Fc candidate antibodies were evaluated, and their blocking activity against PD-1/PD-L1 was detected by FACS. The specific methods are as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS to the corresponding well and resuspend the cells using a drain gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (streptavidin, eBioscience, 12-4317-87), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 3, through FACS experiments, the inventors verified that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 in Example 5 have both high blocking activities, and their blocking activities are higher than Control antibody or similar to control antibody.

Table 3. IC50 of antibodies

clone number IC50(μg/mL) NB22D-21 0.41 NB22gb-10 0.54 KN035 (control) 0.39 Atezolizumab (control) 0.86

Example 7 Activity verification of chimeric VHH-Fc antibody blocking CD80

The obtained VHH-Fc candidate antibodies were evaluated, and their blocking activity against CD80/PD-L1 was detected by FACS. The specific methods are as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled CD80 protein dilution solution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a discharge gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (eBioscience, 12-4317-87), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 4, through FACS experiments, the inventors verified that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 in Example 5 have high blocking activities at the same time, and their blocking activities are higher than Control antibody or similar to control antibody.

Table 4. IC50 of antibodies

clone number IC50(μg/mL) NB22D-21 0.7081 NB22gb-10 0.7651 KN035 (control) 0.6307 Atezolizumab (control) 0.9077

Example 8 Binding activity of chimeric VHH-Fc antibody on tumor cells

In order to evaluate the binding activity of VHH-Fc candidate antibody on human melanoma cell line A375 cells (ATCC, CRL-1619), FACS method was used to detect its binding activity to PD-L1 protein on cells. The specific method is as follows:

1) Digest A375 cells with Trypsin containing 0.25% EDTA, collect the cells by centrifugation at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add 100 μL of A375 cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of each candidate antibody and control antibody dilution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a discharge gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-biotin flow antibody (Abcam), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102). The detection results are shown in Figure 3.

It can be seen from Figure 3 that the binding activities of the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 on human melanoma cell line A375 cells are comparable to those of the control antibody.

Example 9 Activity verification of chimeric VHH-Fc antibody binding to mouse and monkey PD-L1

In order to evaluate the cross-binding activity of VHH-Fc candidate antibody to monkey and mouse PD-L1, FACS method was used to detect its binding activity to PD-L1 protein on cells. The specific method is as follows:

1) Collect the cultured mouse PD-L1-CHO cells and monkey PD-L1-CHO cells, centrifuge at 300g to remove the medium, resuspend the cells with the prepared FACS buffer, count and adjust the cell suspension density to 2 ×10 ⁶ /mL;

2) Add mouse PD-L1-CHO cells and monkey PD-L1-CHO cells to a 96-well round bottom plate at 100 μL per well, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a discharge gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-biotin flow antibody (Abcam), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

The results of human-mouse cross-reaction assay showed that the nanobody candidate molecule with clone number NB22D-21 had a certain binding activity to mouse PD-L1.

The detection results of human-monkey cross-reaction are shown in Table 5, from which it can be seen that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 have good activity in recognizing monkey PD-L1.

Table 5. EC50 values

clone number EC50(μg/mL) NB22D-21 0.25 NB22gb-10 0.20 KN035 (control) 0.21 Atezolizumab (control) 0.33

Example 10 Specific detection of chimeric VHH-Fc antibody binding to PD-L1

In order to confirm the specificity of the candidate molecule binding to PD-L1 protein, the ELISA method was used to detect the activity of the candidate molecule binding to other proteins of the B7 family. The specific methods are as follows:

One day before the experiment, 30 μL of B7-H1 (ie, PDL1), B7-H2, B7-H3, B7-H4, B7-DC (the above proteins were purchased from Yiqiao Shenzhou Company) were added to the ELISA plate at a final concentration of 2 μg/mL. , the product numbers are 10084-HNAH, 11559-H08H, 11188-H08H, 10738-H08H, 10292-H08H-B) and other protein dilutions, incubate overnight at 4°C; rinse the ELISA plate 3 times with PBST the next day, and then add The ELISA plate was blocked with 150 μL 5% PBSM and incubated at room temperature for 2 hours; then the plate was washed 3 times with PBST, and then 30 μL of the dilutions of candidate and control antibodies were added to the ELISA plate and incubated at room temperature for 1 hour; the plate was washed 3 times with PBST and added 1:7000 diluted anti-human IgG Fc-HRP secondary antibody, 30 μL per well, incubated at room temperature for 30 minutes; after washing the plate 6 times with PBST, TMB was added to develop color, and finally 2M HCl was added to stop the reaction, and the reaction was terminated by a microplate reader (Molecular Devices). , SpectreMax 190) at 450nM wavelength, the results are shown in Table 6 and Figure 4.

The results of the experimental verification of the specific binding reaction of the antibody molecule are shown in Table 6 and FIG. 4 .

Table 6. Specificity of candidate molecules for binding to PD-L1 protein

clone/protein B7-H1 B7-H2 B7-H3 B7-H4 B7-DC NB22D-21 + N/A N/A N/A N/A NB22-gb-10 + N/A N/A N/A N/A KN035 + N/A N/A N/A N/A Atezolizumab + N/A N/A N/A N/A

+: Indicates that the binding activity is detected;

N/A: Indicates that no binding activity was detected.

It can be seen from the results in Table 6 and Figure 4 that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 have no binding activity to B7 family molecules other than B7-H1, but only have binding activity to B7-H1 , this binding specificity is consistent with the control antibody.

Example 11 Verification of in vitro biological activity of chimeric VHH-Fc antibody

In order to confirm the ability of candidate molecules to stimulate T cell activation in vitro, the inventors established a mixed lymphocyte reaction system in vitro, the specific method is as follows:

Use Miltenyi sorting kit (Miltenyibiotec, 130-050-201) to sort CD14-positive monocytes and CD4-positive T cells in vitro, and induce culture with 100ng/mL IL-4 and 100ng/mL GM-CSF in vitro7 The monocytes were induced to become dendritic cells (DCs), CD4-positive T cells and DCs were mixed at a cell number ratio of 10:1, and serially diluted candidate antibodies and control antibodies were added to the cells, 37 After culturing for 5 days in a cell incubator at ℃, the cell supernatant was taken after culturing for 72 hours, and the IL-2 ELISA kit was used to detect the secretion of IL-2 in the culture supernatant after PBS dilution. The amount of IFN-γ secreted in the culture supernatant was detected using an IFN-γ ELISA kit.

By detecting the secretion amount of IL-2, the inventors obtained the verification result of the mixed lymphocyte reaction test of the antibody molecule, which is shown in FIG. 5 . It can be seen from Figure 5 that the nanobody candidate molecule of the present invention can activate immune response.

Example 12 Antibody Humanization Transformation

Antibody Modeling，采用同源建模方法建模，选取5-10个最优结构解，Loop区域一般使用同源建模方法建模，如CDR氨基酸序列比对结果显示低于50％同一性，则使用从头建模方法搭建CDR3结构模型。使用PDB BLAST调取序列最接近的10个抗体晶体结构模型(结构分辨率高于2.5埃)，对比自动建模模型，选取最优的结构模型。本发明人对NB22D-21分子进行了抗体人源化改造，得到了两个人源化的分子，这两个人源化改造后的分子编号分别为NB22D-21-huVH1和NB22D-21-huVH2。 In order to reduce the immunogenicity of the molecule in vivo, the inventors humanized the candidate molecule. Using Discovery Studio and

Antibody Modeling, using homology modeling method to model, select 5-10 optimal structural solutions, Loop region is generally modeled using homology modeling method, if the CDR amino acid sequence alignment result shows less than 50% identity, then A CDR3 structural model was constructed using ab initio modeling methods. Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence (structural resolution higher than 2.5 angstroms), compare the automatic modeling models, and select the optimal structural model. The inventors carried out antibody humanization transformation on the NB22D-21 molecule, and obtained two humanized molecules. The numbers of the two humanized molecules after the humanization transformation are NB22D-21-huVH1 and NB22D-21-huVH2, respectively.

Example 13 Binding of humanized derived molecules to human PD-L1-CHO cells

In order to detect the effect of humanization on the binding activity of antibody to human PD-L1 antigen, the inventors used FACS to detect candidate molecules and their humanized derivatives.

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the cell suspension density to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102). The results are shown in Figure 6.

As can be seen from Figure 6, the derived molecules obtained after humanization (i.e., NB22D-21-huVH1 and NB22D-21-huVH2) bind human PD-L1 with affinity and their parent molecules (i.e., NB22D-21) resemblance.

Example 14 Binding of humanized derivative molecules to murine PD-L1-CHO cells

In order to examine the effect of humanization on the cross-activity of the molecule binding to murine PD-L1 antigen, the inventors tested candidate molecules by FACS.

1) Collect the cultured mouse PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add 100 μL of mouse PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102). The results are shown in Figure 7 and Figure 8.

As can be seen from Figure 7, among the derived molecules obtained after humanization, the affinity of NB22D-21-huVH1 for binding to murine PD-L1 is better than that of the parent molecule (ie, NB22D-21).

Specifically, it can be seen from Figure 7 that compared with the parent molecule NB22D-21 and the control molecule KN035, the derivative molecule NB22D-21-huVH1 obtained after humanization transformation has a very good binding activity to mouse PD-L1 protein ; Select the flow peaks of KN035 and NB22D-21-huVH1 (as shown in Figure 8), from which it can be clearly seen that 20 μg/mL of KN035 and NB22D-21-huVH1 were used in combination with mouse PD-L1 overexpression cells, FACS assay data showed that KN035 could not recognize murine PD-L1 on the surface of CHO cells (Figure 8A), while NB22D-21-huVH1 could well recognize murine PD-L1 on the surface of CHO cells (Figure 8B) (Note: each The dividing line in the middle of the figure is the position of the set fluorescence intensity threshold).

Example 15 Activity verification of PD-1 blocking by humanized derivative molecules

The derived molecules obtained after humanization were evaluated, and their blocking activity against PD-1/PD-L1 was detected by FACS method. The specific methods are as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL.

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS to the corresponding well and resuspend the cells using a drain gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (streptavidin, eBioscience, 12-4317-87), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102). The results are shown in Figure 9.

As can be seen from Figure 9, the derived molecules obtained after humanization transformation (ie, NB22D-21-huVH1 and NB22D-21-huVH2 block the activity of PD-1 protein binding to PD-L1 and their parent molecules (ie, NB22D -21) is comparable to the positive control KN035.

Example 16 Validation of efficacy of NB22D-21 molecule in A375 mouse tumor model

In order to confirm the anti-tumor activity of the candidate antibody molecules in vivo, the inventors established a mouse model based on the human melanoma A375 cell line overexpressing human PD-L1. The specific methods are as follows:

Select NOD-SCID mice about 6 weeks old and similar in size and body weight, and divide the mice into 3 groups: control group, candidate antibody group and positive control antibody group, with 8 mice in each group. The human melanoma cell line PD-L1-A375 (prepared in Example 1) was cultured in vitro, 1 × 10 ⁷ PD-L1-A375 cells were mixed with 5 × 10 ⁶ PBMC cells, and the tail vein was injected into mice. Recorded as day 0. On the second day, 5 mg/mL or 10 mg/mL of candidate antibody or control antibody was injected into each group of mice, and then administered once every 7 days for 6 consecutive doses. The body weight and tumor size of mice were recorded weekly from day 7 until the tumor grew to 1500 mm ³ .

Example 17 The druggability modification of humanized molecules

In order to optimize the druggability of the molecule and avoid the influence of potential post-translational modification sites on protein folding, activity and function, the inventors have carried out druggability design of the antibody-derived molecule NB22D-21-huVH2 obtained after humanization transformation.

The druggability modification uses point mutation to randomly mutate the potential post-translational modification sites, constructs a druggability modified antibody library, and uses phage display technology to screen the druggable modified molecules. The VHH lysate was prepared after obtaining the single clone, and the blocking activity of the clone on PD-L1-CHO was detected by FACS.

Through preliminary screening, the inventors obtained 10 modified molecules with druggability: SY01-D21-3, SY01-D21-4, SY01-D21-5, SY01-D21-6, SY01-D21-8, SY01 -D21-17, SY01-D21-21, SY01-D21-24, SY01-D21-38 and SY01-D21-47 (abbreviated as 3, 4, 5, 6, 8, 17, 21, 24, 38 and 47, the parent molecule is NB22D-21-huVH2, abbreviated as D21-Vh2, and the isotype control (isotype) is human IgG1). The clones are SY01-D21-4, SY01-D21-8, SY01-D21-17, SY01-D21-24, SY01-D21-47, respectively.

The experimental results are shown in Figure 10. From the qualitative/semi-quantitative results of the cell lysate, it can be seen that the four medicines SY01-D21-4, SY01-D21-8, SY01-D21-17 and SY01-D21-24 The blocking activities of the sexually engineered candidate molecules were all similar to those of the parent molecule NB22D-21-huVH2. In addition, because the cell expression of SY01-D21-47 is not high, this clone was not selected for subsequent detection.

Example 18 Full-length construction and sample production of druggable candidate clones

The positive VHH candidate antibodies SY01-D21-4, SY01-D21-8, SY01-D21-17 and SY01-D21-24 obtained by screening were fused with human IgG1 Fc segment, and the C-terminus of the positive VHH gene sequence was used to connect to human IgG1 The fusion expression vector was constructed in the manner of the N-terminus of the Fc segment gene sequence, and the fusion expression vector plasmid was transformed into ExpiCHO cells to induce expression to obtain four VHH-Fc chimeric antibody proteins fused with the Fc segment, which were called NB22D-21-4, NB22D-21-8, NB22D-21-17 and NB22D-21-24.

The antibody was expressed using the ExpiCHO transient expression system, the medium was (Gibco, A29100-01), and the transfection kit was (Gibco, A29129). The specific method is as follows: Passage the ExpiCHO cells one day before transfection, mix 25μg of the constructed plasmid with the transfection reagent in a 25ml system, add dropwise to 25ml of ExpiCHO cell culture, mix well, and express 18- After 22 hours, the feeding medium was added according to the instructions in the kit. After feeding, the cells were cultured at 32°C. On the 5th day after transfection, the second feed was added, and the cells were cultured at 32°C for 10 days. -12 days later, the expressed cell suspension was centrifuged at high speed to get the supernatant, the supernatant was filtered at 0.22 μm, purified by Protein A/G affinity purification method, and eluted with 100 mM glycinate (pH 3.0). The protein of interest was then neutralized with 1M Tris-HCl.

Example 19 Activity verification of candidate antibody binding to human and monkey PD-L1 after druggability modification

Four VHH-Fc candidate antibodies obtained after druggability transformation were evaluated, and their binding activity to human and monkey PD-L1 proteins on cells was detected by FACS. The specific methods are as follows:

1) Collect the cultured human and monkey PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer, count and adjust the cell suspension density to 2×10 ⁶ cells/mL ;

2) Human and monkey PD-L1-CHO cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and place them at 4°C to incubate for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 7, comparing the binding activities of 4 candidate molecules on human and monkey PD-L1-CHO, the inventors screened 2 nanobody candidate molecules NB22D with high affinity for human and monkey PD-L1 -21-4 and NB22D-21-24.

Table 7. EC50 of antibodies

Example 20 Activity verification of PD-1 blocking by candidate antibody after druggability modification

The obtained VHH-Fc candidate antibodies were evaluated, and their blocking activity against PD-1/PD-L1 was detected by FACS. The specific methods are as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding wells, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL of FACS to the corresponding well and resuspend the cells using a drain gun;

8) Repeat step 7) twice, and centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (streptavidin, eBioscience, 12-4317-87), blow the cells evenly with a blow gun, and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 8, through FACS experiments, the inventors verified that the two nanobody candidate molecules with clone numbers NB22D-21-4 and NB22D-21-24 in Example 19 have both high blocking activity, and their blocking The activities were higher than NB22D-21-huVH2 antibody.

Table 8. IC50 of antibodies

clone number IC50(μg/mL) NB22D-21-huVH2 1.00 NB22D-21-4 0.30 NB22D-21-24 0.20

Example 21 Specific detection of PD-L1 binding of candidate antibodies after druggability modification

In order to confirm the specificity of the candidate molecule binding to PD-L1 protein after druggability modification, FACS method was used to detect the specificity of the candidate molecule binding to cells, which was divided into two parts. The specific methods of the first part are as follows:

1) Collect the cultured Jurkat and Raji cells, centrifuge at 300g to remove the supernatant, resuspend the cells with the prepared FACS buffer (buffer composition: 1*PBS+5%FBS+2%BSA), count the cells The density of the suspension was adjusted to 2×10 ⁶ /mL;

2) Jurkat and Raji cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) four times, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and place them at 4°C to incubate for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 9 below, according to the FACS test results, the inventors denoted positive and negative by "+" (indicates binding) and "-" (indicates no binding). In clones -21-24, the former had no non-specific binding on both cells, while the latter had high non-specific binding, and the control antibody showed no non-specific binding.

Table 9. Nonspecific binding of antibodies

clone/cell Jurkat Raji NB22D-21-huVH2 - - NB22D-21-4 - - NB22D-21-24 + + KN035 (control) - - Atezolizumab (control) - -

In the second part of the experiment, the inventors used the FACS method to detect the specificity of the NB22D-21-4 clone in binding to B7 family proteins. The specific method is as follows:

1) Collect the cultured B7-H2, B7-H4, B7-H5 cells, centrifuge at 300g to remove the supernatant, and wash the cells with the prepared FACS buffer (buffer composition: 1*PBS+5%FBS+2%BSA ), resuspended, counted and adjusted the density of cell suspension to 2×10 ⁶ cells/mL;

2) B7-H2, B7-H4, B7-H5 cells were added to a 96-well round bottom plate at 100 μL per well, and centrifuged at 300 g to remove the supernatant;

3) Add the candidate antibody dilution solution and the control antibody dilution solution of gradient dilution to the corresponding wells, blow the cells evenly with a drain gun, and incubate at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a drain gun;

5) Repeat step 4) four times, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abeam, ab98596), blow the cells evenly with a blow gun, and place them at 4°C to incubate for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 10) twice, add FACS buffer to the well, 200 μL per well, resuspend the cells, and detect by flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 10 below, according to the FACS test results, the inventors denoted positive and negative by "+" (indicates binding) and "-" (indicates no binding). There was no non-specific binding on the cells, and the control antibody showed no non-specific binding.

Table 10. Specificity of antibodies to B7 family proteins

clone/cell B7-H2 B7-H3 B7-H4 B7-H5 NB22D-21-huVH2 - - - - NB22D-21-4 - - - - KN035 (control) - - - -

Example 22 In vitro MLR activity verification of NB22D-21-4 antibody

In order to confirm the ability of candidate molecules to stimulate T cell activation in vitro, the inventors established a mixed lymphocyte reaction system in vitro, the specific method is as follows:

Use Miltenyi sorting kit (Miltenyibiotec, 130-050-201) to sort CD14-positive monocytes and CD4-positive T cells in vitro, and induce culture with 100ng/mL IL-4 and 100ng/mL GM-CSF in vitro7 The monocytes were induced to become dendritic cells (DCs), CD4-positive T cells and DCs were mixed at a cell number ratio of 10:1, and serially diluted candidate antibodies and control antibodies were added to the cells, 37 After culturing for 5 days in a cell incubator at ℃, the cell supernatant was taken after culturing for 72 hours, and the IL-2 ELISA kit was used to detect the secretion of IL-2 in the culture supernatant after PBS dilution. The amount of IFN-γ secreted in the culture supernatant was detected using an IFN-γ ELISA kit.

By detecting the secretion amounts of IFN-γ and IL-2, the inventors obtained the verification result of the mixed lymphocyte reaction test of antibody molecules, which is shown in FIG. 11 . It can be seen from Figure 11 that the nanobody candidate molecule NB22D-21-4 of the present invention can activate the immune response.

Example 23: Construction and expression of PD-L1/TGFβ bifunctional fusion proteins D21-4-T and D21-huVH2-T sequences

Based on NB22D-21-huVH1 (SEQ ID NO: 12), NB22D-21-huVH2 (SEQ ID NO: 11) and NB22D-21-4 (SEQ ID NO: 13) Nanobody molecules, the inventors constructed target A bifunctional fusion protein that binds PD-L1 and TGFβ (also known as PD-L1/TGFβtrap). The PD-L1/TGFβtrap design includes the above-mentioned PD-L1-targeting Nanobody, a human IgG1-Fc fragment (SEQ ID NO: 28) containing a hinge region, and a human TGFβRII ECD (SEQ ID NO: 31), and the specific designs are as follows ( Take NB22D-21-4 as an example):

The C-terminus of the NB22D-21-4 Nanobody is directly linked to a human IgG1-Fc fragment. The Fc terminal Lys was replaced with Ala to improve stability. TGFβRII ECD was fused to the end of IgG 1 Fc (SEQ ID NO: 28) through a (Gly ₄ Ser) ₄ Gly (SEQ ID NO: 27) flexible linker to form the final PD-L1/TGFβ trap bifunctional fusion protein molecule D21-4- T (SEQ ID NO: 31). In order to compare the function of TGFβRII, on the basis of NB22D-21-4 nanobody molecule, human IgG1-Fc fragment was fused to form D21-4 without TGFβtrap (SEQ ID NO: 40), whose DNA sequence is as (SEQ ID NO: 41) shown.

The nucleotide sequence (SEQ ID NO: 42) encoding the D21-4-T bifunctional fusion protein was optimized by Nanjing GenScript Company and constructed into the expression vector pcDNA3.1(+) . Plasmids were transiently transfected into EXPICHO-S cells for protein expression.

Based on the design of D21-4-T, the inventors also transiently expressed other PD-L1/TGFβtrap molecules listed in the following table for comparison of different IgG subtypes of the bifunctional fusion protein and the N-terminal truncated fragment of TGFβRII and in vivo experiments in mice. In addition, the inventors also transiently expressed M7824 (PD-L1/TGFβtrap bifunctional fusion protein developed by Merck, Germany, the sequence is derived from patent CN201580007865.3) and Avelumab (PD-L1 antibody that Merck has marketed in Germany) as experimental reference. Taste.

Table 11 Design and construction of VHH-L1-Fc-L2-TGFβRII fusion protein

Example 24: D21-4-T, D21-huVH2-T bifunctional fusion protein protein purification

Take the transiently transfected cell culture medium, centrifuge at 12000g for 20min, and take the supernatant for purification by affinity chromatography. The affinity chromatography medium is high affinity, high capacity Prism A (GE). The equilibration buffer was 1×PBS (138mM NaCl, 2.7mM KCl, 8mM Na ₂ HPO ₄ , 1.5mM KH ₂ PO ₄ , pH 7.4), first equilibrated for 5 times the column volume, and then loaded the cell supernatant with the flow rate Control the retention time on the column ≥ 2min. After loading, re-equilibrate with 1×PBS (pH 7.4) until the UV absorption of A280 drops to the baseline. The samples were eluted with 0.1M glycine (pH 3.0) elution buffer, and the elution peaks were collected according to the A280 UV absorption peak, and the collected samples were neutralized with 1M Tris.

The above neutralized samples were concentrated through an ultrafiltration concentrating tube and the solution was changed, the buffer solution was 1×PBS (pH 7.4), the centrifugal RCF≤3000, and the solution was changed 4-6 times. The collected protein was identified by SDS-PAGE and SEC-HPLC with a purity of ≥95%, and the endotoxin content was identified by dynamic turbidimetry to be less than 1 EU/mg.

Example 25: Octet detects the affinity of D21-4-T, D21-huVH2-T and PD-L1

PD-L1 with his tag (ACRO, Cat#PD1-H5221) was immobilized with NTA senor, 5ug/mL, and immobilization time was 600s; the antibody was serially diluted by 2 times, the initial concentration was 20nM, a total of 5 points; the affinity was measured (Association time 200s, dissociation time 800s); data fitting, calculating affinity.

The results are shown in Figure 13. Although the binding of D21-4-T to PD-L1 is slightly lower than that of parent D21-4, its affinity is still as high as 5.08E-10; the affinity of D21-huVH2-T to PD-L1 is 2.84 E-10.

Example 26: ELISA detection of the binding of D21-4-T, D21-huVH2-T and PD-L1

Recombinant PD-L1 (ACRO, Cat#PD1-H5221) was coated overnight on a 96-well plate at 100ng/mL (PBS, pH7.4); after blocking with 5% nonfat dry milk, washed three times with PBST; a 3-fold gradient was added Diluted D21-4-T, D21-huVH2-T, the initial concentration was 30nM, and 100uL/well was loaded. After incubation and washing, peroxidase-labeled donkey anti-human IgG antibody (Jackson ImmunoResearch, Cat#709-066-098) was added, and the color was developed by conventional methods and the OD450 was read on a PerkinElmer _Enspire instrument. M7824 and Avelumab were serially diluted at the same molar concentration as reference.

The results are shown in FIG. 14 , the EC50 values of effective binding concentrations of D21-4-T and PD-L1 were 0.04262 nM, respectively, and the EC50 values of D21-huVH2-T and PD-L1 were 0.03178 nM. The EC50 of both D21-4-T and D21-huVH2-T with PD-L1 was slightly higher than that of the reference M7824.

Example 27: Detection of specific binding of D21-4-T, D21-huVH2-T to dual targets by ELISA

Recombinant PD-L1 (ACRO, Cat#PD1-H5221) was coated overnight on 96-well plate at 100ng/mL (PBS, pH7.4); after blocking with 5% nonfat milk powder, washed three times with PBST; D21-4-T, D21-huVH2-T, the initial concentration was 50nM, 3-fold serial dilution, 100uL/well was loaded. After incubation and washing, TGFβ1 (Sino Biological, Cat#10804-H08H) was added at a concentration of 50 nM and loaded at 100 uL/well. After incubation and washing, detection was performed with a peroxidase-labeled TGFβ1 antibody (R&D Systems, Cat#MB100B), and color was developed by conventional methods. Equimolar dilutions of M7824 were used as reference.

The results are shown in FIG. 15 , D21-4-T and D21-huVH2-T have the activity of simultaneously binding PD-L1 and TGFβ1, and their binding ability is comparable to that of the control substance M7824.

Example 28: ELISA detection of the ability of D21-4-T and D21-huVH2-T to block the binding of PD-1/PD-L1

Recombinant PD-L1 (ACRO, Cat#PD1-H5221) was coated overnight on 96-well plates at 100ng/ml (PBS, pH7.4); after blocking with 5% nonfat dry milk, washed 3 times with PBST; D21-4 -T, D21-huVH2-T and reference products M7824 and Avelumab were diluted with equimolar concentration gradient and mixed with recombinant human PD-1 carrying mouse IgG1 Fc Tag (ACRO, Cat#PD1-H5255) at a fixed concentration of 200ng/mL, Incubate for 2 hours at 25°C, then add to the above 96-well plate; after incubation and washing, add biotinylated goat anti-mouse IgG antibody (Jackson ImmunoResearch, Cat#115-066-071); add Streptavidin after incubation and washing - HRP (Thermo, Cat#434323), developed by conventional methods.

The results are shown in Figure 16, D21-4-T and D21-huVH2-T have very good blocking activity on PD1/PD-L1 binding, even slightly higher than M7824.

Example 29: FACS detection of the ability of D21-4-T and D21-huVH2-T to block the binding of PD-1/PD-L1

Take log-phase CHOZN-PD-L1 cells (stably transfected cell line with high expression of human PD-L1), rinse twice with DPBS/1% FBS, and add cells to a 96-well U-shaped culture plate after counting, adding 2 × cells to each well 10 ⁵ (30 μL); add 1 μg recombinant PD-1 carrying mouse IgG1 Fc Tag (ACRO, Cat#PD1-H5255) to each well, incubate at 4°C for 15 min; add 20 μL of D21-4-T, D21-huVH2- T (initial concentration of 9 μM followed by 2.5-fold serial dilution), incubated at 4°C for 30 min; rinsed twice with DPBS/1% FBS, and added 0.5 μg of FITC-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch, Cat#115-096- 071), incubate at 4°C for 30 min after mixing; rinse twice with DPBS/1% FBS, add 50 μL of DPBS/1% FBS to resuspend, and perform flow cytometry detection.

The results are shown in Figure 17, D21-4-T and D21-huVH2-T can completely block the mutual binding of PD-1/PD-L1, and their blocking activity is even slightly higher than that of the reference product M7824.

Example 30: PD-L1 target-mediated endocytosis analysis of D21-4-T, D21-huVH2-T

The core of the design of PD-L1/TGFβtrap bifunctional fusion protein, in addition to blocking PD-1/PD-L1 signaling pathway and neutralizing TGFβ, another important mechanism of action is clearance through PD-L1 receptor-mediated endocytosis Therefore, it is very necessary to verify the endocytosis of the PD-L1/TGFβtrap bifunctional fusion protein of the present invention. In this experiment, in addition to comparing D21-4-T, D21-huVH2-T with the reference product M7824, non-specific IgG1 was used as a negative control, and the IgG4 subtype D21-4-T-G4 of D21-4-T was also included ( SEQ ID NO:36), IgG1 mutant D21-4-T-mG1 (SEQ ID NO:37) with ADCC, ADCP and CDC removed.

The PD-L1 target-mediated antibody endocytosis process was evaluated as follows:

1. Antibody preparation: Dilute the antibody to be tested with CD fusion to a final concentration of 160nM;

2. Preparation of pHrodo: Dilute pHrodo (Thermo, Cat#Z25611) and the mother solution by 12.5 times, namely take 75μL of pHrodo mother solution and mix with 862.5μL of CD fusion for use;

3. Antibody labeling: Mix 185 μL of the standby antibody dilution solution with 185 μL of the standby pHrodo, respectively, and incubate in a 37°C incubator for 10 minutes;

4. Cell preparation: Take log-phase CHOZN-PD-L1 cells, count them, and put the cells into a centrifuge tube, centrifuge at 300g for 5 minutes, remove the supernatant, adjust the cell density using CD fusion according to the counting results, and spread 10 μL on a 96-well plate (final concentration 10 ⁵ cell/well) 300g centrifugation for 5min;

5. Add 50 μL of the labeled complex Abs-pHrodo to the cell plate, mix by pipetting, and then incubate on ice for 20 minutes. At this time, take out a plate as the 0-h sample, rinse with DPBS/1% FBS, and put it on ice for a while. Measurement. The rest of the well plates were incubated at 37°C in a 5% CO2 incubator, taken out at 1h, 2h, 4h, 6h, and 8h, rinsed with DPBS/1%FBS, and then stored on ice for testing;

6. Rinse twice with DPBS/1%FBS, resuspend in 50μL DPBS/1%FBS, and perform flow cytometry.

The results are shown in Figure 18, D21-4-T, D21-huVH2-T and their different IgG variants can increase the fluorescence signal value with the prolongation of cell incubation time, indicating endocytosis. As shown in Figure 18A, D21-4-T had the most obvious endocytosis effect, and the endocytosis effect was stronger than that of M7824; while replacing the Fc part with IgG4 or mut IgG1, the endocytosis effect was significantly weakened.

Example 31: Mixed lymphocyte reaction (MLR) to detect the effects of D21-4-T and D21-huVH2-T on T cell proliferation and activation

To study the effect of the PD-L1/TGFβtrap bifunctional fusion protein of the present invention on the proliferation and activation of T cells, the specific experimental procedures are as follows:

1. Preparation of DC cells: Take PBMC cells and use RPMI1640 complete medium (containing 10% FBS+4.5g/D-Glucose+2.383g/L HEPES+L-Glutamine+1.5g/L Sodium Bicarbonate+110mg/L Sodium Pyruate +50nMβ-ME) complete medium to resuspend cells, divide into T75 flasks, and incubate for 2h in 37°C, 5%CO ₂ incubator. After the cells adhered, remove the upper lymphocyte suspension, add RPMI1640 medium to rinse the cell surface, aspirate the supernatant, add 15mL induction medium (RPMI1640 complete medium+100ng/mL GM-CSF+75ng/mL IL-4) ), placed in a 37°C, 5% CO ₂ incubator for 5 days. The cells were collected, centrifuged at 300g for 5 min to remove the supernatant, and resuspended in RPMI1640 complete medium according to the counting result to adjust the density to 10 ⁵ cells/mL;

2. Isolation of T cells: Take fresh PBMC, and use CD4 ⁺ T cell negative selection kit (Miltenyi, Cat#130-096-533) to isolate CD4 ⁺ T cells, and adjust the density by resuspending in RPMI1640 complete medium according to the counting result. to 10 ⁶ cells/mL;

3. Cell mixing: Mix the prepared DC and T cell suspensions 1:1 by volume;

4. Cell administration: add 50 μL of RPMI 1640 complete medium (containing 10% FBS + final concentration of 50 μM β-mercaptoethanol) to the blank control, and add 50 μL of antibody RPMI1640 complete medium with a concentration of 40 μg/mL in other groups; Cell mixed suspension;

5. Cell culture: the above-treated cells were cultured for 5 days in a 37°C, 5% CO ₂ incubator;

6. Detection: Take the above cultured cells, 300g heart for 5min, take the supernatant, use IL2 detection kit (R&D Systems, Cat#D2050) and IFNγ detection kit (R&D Systems, Cat#DIF50C) to express IL2 and IFNγ respectively Level detection.

The results are shown in Figure 19, D21-4-T and D21-huVH2-T increased the secretion of IL2 and IFNγ from CD4 ⁺ T cells, and its effect was better than that of M7824.

Example 32: Antitumor activity of D21-4-T, D21-huVH2-T in vivo

The in vivo antitumor activity of the PD-L1/TGFβtrap bifunctional fusion protein of the present invention was carried out in PD-L1 humanized mouse BALB/c-hPDL1 (Jicui Yaokang, China). The experimental design is as follows: using the high-expressing human PD-L1 cell line EMT6-hPD-L1, cultured in RPMI-1640 complete medium (90%RPMI1640+10%FBS+200μg/mL hygromycin B) for expansion; before inoculation The medium was changed on 1 day, and the cells were harvested on the day of inoculation; the harvested cells were washed once with the bulk medium (without serum and other supplementary components) to prepare a cell suspension with a density of 3x10 ⁶ cells/mL. Inoculate 100 μL of the suspension with a 1 mL syringe subcutaneously in each mouse, that is, the inoculation amount is 3×10 ⁵ cells/Mouse, and there are eight mice in each group. Tumor volumes were measured twice a week during the experiment. On Day 5, the tumors of the modeled mice all grew, and the average tumor volume in each group was about 100 mm ³ , and the administration was started. On Day5, Day8, Day11, Day14, a total of 4 intraperitoneal injections were administered, of which the test antibodies D21-huVH2-T and D21-4-T were given 188ug each time, the reference antibody M7824 was given 300ug each time, and the test antibody was given 300ug each time. Equivalent to the number of moles of the reference antibody administered per dose.

As shown in Figure 20, when the experiment reached Day 35, the tumor volumes of the administration groups D21-huVH2-T, D21-4-T and M7824 showed significant differences compared with the normal saline group, and the results showed that the test antibody D21-huVH2- Both T and D21-4-T had good tumor suppressive effects. There was no significant difference in tumor suppressive effect between the test antibody and the reference antibody. However, due to the small molecular weight of the tested antibody, it is only about 63% of the similar drugs M7824 and SHR-1701 under development. The dosage can be reduced or the molar concentration ratio of the drug can be increased at the same dosage to improve the efficacy of the drug.

Claims (50)

A bifunctional fusion protein targeting PD-L1 and TGFβ, comprising: (a) human TGFβ receptor II (TGFβRII) that binds TGFβ, or a fragment thereof in an N-terminal truncated form; and (b) a Nanobody that binds PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein: (i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G; (ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and (iii) the sequence of CDR3 is shown as SEQ ID NO: 3; The bifunctional fusion protein according to claim 1, which is shown in the general formula (I): VHH-L1-Fc-L2-TGFβRII (I) Wherein, VHH is a Nanobody that binds to PD-L1, wherein the heavy chain variable region of the Nanobody comprises CDR1, CDR2 and CDR3 sequences, wherein: (i) The sequence of CDR1 is shown in formula RTDX ₁ NINX ₂ MH, wherein X ₁ is R or S; X ₂ is T or G; (ii) the sequence of CDR2 is shown in the formula TIFIDX ₃ NTI, wherein X ₃ is G or L; and (iii) the sequence of CDR3 is shown as SEQ ID NO: 3; Wherein, L1 and L2 are connecting peptides, and each is present or absent; wherein, Fc is the Fc domain of a human IgG antibody; Wherein, TGFβRII is human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ. The bifunctional fusion protein according to claim 2, wherein the sequence of the heavy chain variable region CDR1 of the Nanobody is consistent with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4. The bifunctional fusion protein according to claim 2, wherein the sequence of the heavy chain variable region CDR2 of the Nanobody is consistent with the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5. The bifunctional fusion protein according to claim 2, wherein the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region of the Nanobody are respectively the same as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID The sequences shown in NO: 3 or SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 3 are identical. The bifunctional fusion protein according to any one of claims 1-5, wherein the heavy chain variable region of the Nanobody comprises a framework region, and the FR region comprises FR1, FR2, FR3 and FR4, and is associated with CDR1, CDR2 and CDR3 are spaced on the heavy chain variable region to form a structure from N-terminus to C-terminus of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Wherein, FR1 is selected from the sequence shown in SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22; FR2 is selected from the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 19; FR3 is selected from the sequence shown in SEQ ID NO: 19 ID NO: 16 or the sequence shown in SEQ ID NO: 20; FR4 is selected from the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 21; The bifunctional fusion protein according to claim 2, wherein the heavy chain variable region of the Nanobody VHH contains as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO : The identical amino acid sequence shown in any one of 13. The bifunctional fusion protein according to claim 7, wherein the heavy chain variable region of the Nanobody VHH is the same as SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: The sequence shown in 13 has one or more amino acid additions, deletions and/or substitutions and retains the amino acid sequence of the ability to specifically bind to PD-L1, or at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% homology and amino acid sequences that retain the ability to specifically bind PD-L1. The bifunctional fusion protein according to claim 8, wherein the addition, deletion and/or substitution of one or more amino acids does not exceed five. The bifunctional fusion protein according to claim 9, wherein the addition, deletion and/or substitution of one or more amino acids does not exceed three. The bifunctional fusion protein according to claim 2, wherein: in the bifunctional fusion protein, the connecting peptides L1 and L2 are respectively present or absent, and the general sequence formulas are (G ₄ S) _n , (SG ₄ ) _n , G ₄ (SG ₄ ) _n or (G ₄ S) _n G, where n is an integer from 0-6. The bifunctional fusion protein according to claim 11, wherein n is 3-5. The bifunctional fusion protein according to claim 11, wherein in the bifunctional fusion protein, the connecting peptide L1 does not exist, and the connecting peptide L2 is (G ₄ S) ₄ G (SEQ ID NO: 27). The bifunctional fusion protein according to claim 2, wherein: in the bifunctional fusion protein, Fc is selected from the Fc domain of human IgG1, IgG2, IgG3 or IgG4. The bifunctional fusion protein according to claim 14, wherein the human constant region Fc segment is IgG1 Fc, the sequence of which is shown in SEQ ID NO: 28. The bifunctional fusion protein according to claim 14, wherein the human constant region is an IgG1 Fc mutant whose sequence is shown in SEQ ID NO: 29. The bifunctional fusion protein according to claim 14, wherein the human constant region Fc segment is IgG4 Fc, the sequence of which is shown in SEQ ID NO: 30. The bifunctional fusion protein according to claim 2, wherein the human TGFβRII capable of binding TGFβ is its extracellular domain sequence (ECD1-136), and the sequence is shown in SEQ ID NO: 31. The bifunctional fusion protein according to claim 2, wherein the N-terminal truncated form fragment of the human TGFβRII is selected from the deletion of 26 or less consecutive amino acids on the N-terminal of SEQ ID NO: 31. The bifunctional fusion protein according to claim 19, wherein the N-terminal truncated form fragment of human TGFβRII is selected from the deletion of 14-21 consecutive amino acids at the N-terminal of SEQ ID NO:31. The bifunctional fusion protein according to claim 20, wherein the N-terminal truncated form fragment of the human TGFβRII is ECD (15-136) (SEQ ID NO: 32) that lacks 14 amino acids at the N-terminal, ECD (20-136) (SEQ ID NO: 33) with a deletion of the N-terminal 19 amino acids, or ECD (22-136) with a deletion of the N-terminal 21 amino acids (SEQ ID NO: 34). The bifunctional fusion protein according to any one of claims 18-21, wherein the human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ is combined with SEQ ID NO:31-SEQ ID NO:34 The sequence homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A bifunctional fusion protein targeting and binding PD-L1 and TGFβ, the fusion protein is shown in general formula (I): VHH-L1-Fc-L2-TGFβRII (I) Wherein, VHH is a Nanobody that binds PD-L1, wherein the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region of the Nanobody are respectively the same as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or The sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 3 are identical; Wherein, the L1 linking peptide does not exist, and the general sequence formula of the L2 linking peptide is (G ₄ S) _n , (SG ₄ ) _n , G ₄ (SG ₄ ) _n or (G ₄ S) _n G, and n is 3- An integer of 5. wherein, Fc is the Fc domain of a human IgG1 or IgG4 antibody; Wherein, TGFβRII is human TGFβRII or its N-terminal truncated form fragment capable of binding TGFβ. A bifunctional fusion protein targeting and binding PD-L1 and TGFβ, the fusion protein is shown in general formula (I): VHH-L1-Fc-L2-TGFβRII (I) Wherein, VHH is a Nanobody that binds to PD-L1, wherein the heavy chain variable region of the Nanobody VHH contains any of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 A consensus amino acid sequence shown; Wherein, the L1 linking peptide does not exist, the L2 linking peptide has the general formula (G ₄ S) _n G, and n is an integer of 3-5; wherein, Fc is the Fc domain of a human IgG1 or IgG4 antibody; Wherein, TGFβRII is human TGFβRII capable of binding TGFβ or its N-terminal truncated form fragment, the sequence of which is shown in any one of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 . A bifunctional fusion protein that targets and binds PD-L1 and TGFβ, its amino acid sequence is such as SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:43 or SEQ ID NO:44. The bifunctional fusion protein according to claim 25, wherein the amino acid sequence of the bifunctional fusion protein is the same as that of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% homology as set forth in SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:44 , 99% or 100%. A nucleic acid molecule encoding the bifunctional fusion protein of any one of claims 1-26. The nucleic acid molecule of claim 27, which is DNA or RNA, with or without intronic sequences. The nucleic acid molecule of claim 28, which is a cDNA molecule. The nucleic acid molecule according to claim 28, whose nucleotide sequence is shown in SEQ ID NO:42. An expression vector containing the nucleic acid molecule of any one of claims 27-30. A host cell obtained by transforming or transfecting a prokaryotic or eukaryotic host cell with the vector of claim 31 . The host cell of claim 32, which is a bacterial, yeast or mammalian cell. The host cell according to claim 33, which is Chinese hamster ovary cells (CHO cells), NSO myeloma cells, COS cells or SP2 cells. The host cell of claim 34, which is a CHO cell. The method for preparing the bifunctional fusion protein of any one of claims 1-26, comprising: (a) introducing a recombinant expression vector carrying a gene encoding a bifunctional fusion protein targeting binding to PD-L1 and TGFβ into a mammalian host cell, and culturing the recombinant host cell under conditions conducive to production of the fusion protein; and (b) recovering the fusion protein. The method of claim 36, wherein the recovery method comprises centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The method according to claim 36, wherein the method is recovered by a purification method of Protein A affinity column, ion exchange, hydrophobicity, chromatographic focusing, size exclusion or any combination thereof. A pharmaceutical composition, comprising a therapeutically effective amount of the bifunctional fusion protein targeting PD-L1 and TGFβ according to any one of claims 1-26 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable carrier includes any and all carriers that are physiologically compatible with solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like . The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is formulated into a solution, a microemulsion, a liposome or a lyophilized powder for injection. The pharmaceutical composition of claim 39, wherein the route of administration of the pharmaceutical composition includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal/spinal or other parenteral administration routes. Use of the bifunctional fusion protein targeting and binding to PD-L1 and TGFβ according to any one of claims 1-26 in the preparation of a medicament for treating cancer or for inhibiting tumor growth. The use of claim 43, wherein the method comprises administering to a subject in need of treatment a therapeutically effective amount of the fusion protein of any one of claims 1-26. The use according to claim 44, wherein the subject is a human or a non-human animal. The use according to claim 45, wherein the non-human animals include non-human primates, sheep, dogs, mice, rats, cats, cattle, horses, chickens, amphibians and reptiles. The use according to claim 43, characterized in that: the cancer or tumor is selected from the group or parts of: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia , thyroid, endometrium, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, Dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. The use according to claim 47, wherein the subject is a human or a non-human animal. The present invention can also be used to treat metastatic cancers, especially cancers that express PD-L1. The use according to any one of claims 43-48, wherein the administration dose of the bifunctional fusion protein targeting PD-L1 and TGFβ is 0.1 mg/kg-100 mg/kg, or 0.5 mg/kg-50 mg/kg, or 1 mg/kg-25 mg/kg, or 2 mg/kg-10 mg/kg, or 5 mg/kg-10 mg/kg. The use according to claim 49, wherein the drug is administered on a regimen of once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or every Once every 3-6 months.

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