WO2023072213A1 - Pd-l1 binding molecule and application thereof - Google Patents

Abstract

A PD-L1 binding molecule and an application thereof, relating to the technical field of monoclonal antibodies. Provided are a preparation method for the PD-L1 binding molecule, a use of the PD-L1 binding molecule, and a kit, immune conjugate, and pharmaceutical composition containing the PD-L1 binding molecule.

Description

A kind of PD-L1 binding molecule and its application

priority statement

This application claims the priority of the Chinese invention patent application with the application number 202111274164.X, the application date is October 29, 2021, and the invention name is a PD-L1 binding molecule and its application, the entire content of which is incorporated herein by reference middle.

technical field

The invention relates to the technical field of monoclonal antibodies, in particular to a PD-L1 binding molecule and its application.

Background technique

Programmed cell death 1 ligand 1 (PD-L1), also known as surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274), was established by Chinese scholar Professor Chen Lieping in 1999 as a B7 family The third member, B7-H1, was discovered for the first time. PD-L1 protein is widely expressed in activated T, B cells and macrophages. The interaction between PD-L1 and the PD-1 protein on T cells can inhibit the activation of T cells, cause the apoptosis of T cells, and play a negative regulatory role in the immune response. In the tumor microenvironment, tumor cells and tumor-associated antigen presenting cells (APCs) highly express PD-L1, and tumor-infiltrating lymphocytes highly express PD-1 under long-term stimulation of tumor antigens. The combination of PD-L1 and PD-1 can induce T cell apoptosis, incapacity, and exhaustion, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+ T cells, and achieving tumor immune escape.

The successful application of monoclonal antibodies in cancer detection and biological targeted therapy has caused a revolution in tumor treatment. However, the molecular mass of traditional monoclonal antibody (150kD) is too large to penetrate tissue, resulting in a low effective concentration in the tumor area and insufficient therapeutic effect; traditional antibodies are highly immunogenic, and engineered antibodies are difficult to Reach the original affinity. In addition, many factors such as long development cycle, high production cost and insufficient stability of fully humanized traditional antibodies limit their clinical application and popularization.

Nanobodies are currently the smallest antibody molecules, and their molecular weight is 1/10 of that of ordinary antibodies. In addition to the antigen reactivity of monoclonal antibodies, nanobodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, and strong targeting , Humanization is simple, and the preparation cost is low, which almost perfectly overcomes the defects of traditional antibody such as long development cycle, low stability, and harsh storage conditions.

Although some Nanobodies binding to PD-L1 have been disclosed in the art, there is still a need in the art for Nanobodies with better binding affinity and specificity.

Contents of the invention

One of the objectives of the present invention is to provide an antibody that can specifically bind to PD-L1 and its application.

Specifically, the present application solves the technical problems in this field through the following technical solutions.

1. A PD-L1 binding molecule comprising at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising any one of the following (i) to (vi) CDR1, CDR2, and CDR3:

(i) CDR1 as shown in SEQ ID NO: 29, CDR2 as shown in SEQ ID NO: 30, and CDR3 as shown in SEQ ID NO: 31;

(ii) CDR1 as shown in SEQ ID NO: 23, CDR2 as shown in SEQ ID NO: 24, and CDR3 as shown in SEQ ID NO: 25;

(iii) CDR1 as shown in SEQ ID NO: 26, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 28;

(iv) CDR1 as shown in SEQ ID NO: 20, CDR2 as shown in SEQ ID NO: 21, and CDR3 as shown in SEQ ID NO: 22;

(v) CDR1 as set forth in SEQ ID NO: 32, CDR2 as set forth in SEQ ID NO: 33, and CDR3 as set forth in SEQ ID NO: 34; or

(vi) CDR1 as shown in SEQ ID NO:35, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:37.

2. The PD-L1 binding molecule according to item 1, wherein the immunoglobulin single variable domain is VHH.

3. The PD-L1 binding molecule as described in item 2, the amino acid sequence of the VHH is as SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 9 shown in any one.

4. The PD-L1 binding molecule according to any one of items 1 to 3, said PD-L1 binding molecule further comprising an immunoglobulin Fc region;

Optionally, the C-terminus of the immunoglobulin single variable domain is linked to the N-terminus of the immunoglobulin Fc region.

5. The PD-L1 binding molecule as described in item 4, the amino acid sequence of the Fc region of the immunoglobulin is shown in the 120th-346th amino acid of SEQ ID NO:44.

6. The PD-L1 binding molecule as described in any one of items 1 to 5, said PD-L1 binding molecule comprising an amino acid sequence as shown in any one of SEQ ID NO: 38-43.

7. An isolated polynucleotide encoding the PD-L1-binding molecule according to any one of items 1 to 6.

8. An expression vector comprising the polynucleotide according to item 7.

9. A host cell comprising the expression vector of item 8 or the polynucleotide of item 7 integrated in its genome.

10. A method for preparing the PD-L1 binding molecule described in any one of items 1 to 6, comprising culturing the host cell described in item 9 under conditions that allow the production of the PD-L1 binding molecule and recovering and isolating the obtained The PD-L1 binding molecule.

11. A phage displaying Nanobodies, wherein the PD-L1 binding molecule according to any one of items 1-6 is displayed on the surface of the phage.

12. A kit for detecting PD-L1, comprising the PD-L1 binding molecule described in any one of items 1 to 6 or the phage displaying Nanobody described in item 11.

13. The kit according to item 12, further comprising a solid-phase carrier, and the PD-L1 binding molecule or the phage displaying the nanobody is immobilized on the solid-phase carrier.

14. The kit as described in item 13, which also includes a detectable label that can be linked to the PD-L1 binding molecule; and/or a PD-L1 standard or a PD-L1 conjugate standard and/or a substrate corresponding to a detectable label; and/or an ELISA reagent;

Preferably, said detectable label is attached to said PD-L1 binding molecule or present in a kit separately.

15. A method for detecting the presence of PD-L1 in a test sample for therapeutic purposes or non-therapeutic purposes, using the PD-L1 binding molecule described in any one of items 1 to 6 or the display nanometer described in item 11 The phage of the antibody is used as the detection antibody of PD-L1, and the presence of PD-L1 in the sample to be tested is detected by enzyme-linked immunosorbent assay.

16. The method as described in item 15, which specifically comprises the following steps:

The sample to be tested is coated on a solid phase carrier, and the PD-L1 binding molecule carrying or not carrying a detectable marker or the phage displaying the Nanobody is used as a detection antibody to detect the presence of PD-L1 .

17. An immunoconjugate comprising a therapeutic agent and a PD-L1 binding molecule as described in any one of items 1 to 6 conjugated to the therapeutic agent;

Preferably, the therapeutic agent comprises a toxin, radioisotope, drug or cytotoxic agent.

18. A bispecific or multispecific antibody, comprising the PD-L1 binding molecule described in any one of items 1 to 6, and another or several other antibodies functionally linked to the PD-L1 binding molecule. Antibodies or antibody fragments with antigen-binding properties.

19. A pharmaceutical composition comprising the PD-L1 binding molecule described in any one of items 1-6; preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier or diluent.

20. The PD-L1 binding molecule as described in any one of items 1 to 6, the immunoconjugate as described in item 17, the bispecific or multispecific antibody as described in item 18, and the pharmaceutical composition as described in item 19 Use in the preparation of medicines for treating PD-L1-mediated diseases;

The PD-L1-mediated disease is preferably cancer, more preferably a cancer with high expression of PD-L1. The cancers include but not limited to lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, Head and neck cancer is preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma.

21. A method for diagnosing, treating, preventing or alleviating a disease, disease or condition related to PD-L1 in a subject, which comprises administering a therapeutically effective amount of any one of items 1 to 6 to the above subject. The PD-L1 binding molecule and/or the pharmaceutical composition as described in item 19.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Figure 1. Electropherogram of total RNA extracted in Example 2.

Fig. 2. Electrophoresis diagram of the second round of nested PCR in Example 2.

Figure 3. Diagram of flow cytometry detection results in Example 5. Among them, a shows the flow cytometry detection results of R1014, R1015, R1016, R1017, R1018, R1019, R1020; b shows the flow cytometry detection results of R1021, R1022, R1023, R1024, R1025, R1026, R1027 Figure; c shows the results of flow cytometry detection of R1028; d shows the results of flow cytometry detection of R1148, R1149, R1150.

[Corrected 08.12.2022 under Rule 91]
Figure 4a. Binding curve of fusion protein R1015 in Example 6.
Figure 4b. Binding curve of fusion protein R1016 in Example 6.
Figure 4c. Binding curve of fusion protein R1019 in Example 6.
Figure 4d. Binding curve of fusion protein R1148 in Example 6.
Figure 4e. The binding curve of the fusion protein R1149 in Example 6.
Figure 4f. The binding curve of the fusion protein R1150 in Example 6.
Figure 4g. Binding curve of fusion protein R0999 in Example 6.
Figure 4h. The binding curve of the fusion protein R0516 in Example 6.
Figure 4i. Binding curve of fusion protein R0323 in Example 6.

Figure 5 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human PD-1 using detection method (A) in Example 7. Among them, a shows the binding curve and IC50 value of R1148, R1149, R1150 blocking membrane expressed human PD-L1 protein and free human PD-1; b shows R1014, R1015, R1016, R1017, R1018, R1019, R1027 blocking Binding curves and IC50 values of membrane-expressed human PD-L1 protein and free human PD-1.

Figure 6 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human PD-1 using detection method (B) in Example 7. Among them, a shows the binding curve and IC50 value of R1148, R1149, R1150 blocking membrane expressed human PD-L1 protein and free human PD-1; b shows R1014, R1015, R1016, R1017, R1018, R1019, R1027 blocking Binding curves and IC50 values of membrane-expressed human PD-L1 protein and free human PD-1.

Figure 7 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human CDSO using detection method (A) in Example 9.

Figure 8 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human CDSO using detection method (B) in Example 9.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

As used herein, the term "Nanobody" refers to a heavy chain antibody lacking a light chain (e.g. derived from camels), a single domain antibody obtained by cloning its variable region (VHH), which is a minimally functional antigen-binding antibody. Fragment, the relative molecular mass (Mr) is only about 15000. Nanobodies have the characteristics of small molecular weight, strong stability, good solubility, easy expression, and low immunogenicity. VHH usually contains three hypervariable regions, called "complementarity determining regions (CDRs)", and these three CDRs are CDR1, CDR2, and CDR3, respectively.

As used herein, "immunoglobulin Fc region" or "Fc region" refers to the crystallizable fragment of an immunoglobulin antibody following papain digestion. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE is present in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4).

As used herein, the "detection antibody" refers to an antibody specifically anti-PD-L1, with a detectable label.

As used herein, the "detectable marker" refers to a marker located on the detection antibody and used to determine the presence or absence and the amount of PD-L1 in the sample to be detected. Such as: enzymes, fluorescent labels, nuclides, quantum dots, colloidal gold, etc. Preferably, the marker is selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

As used herein, the "substrate corresponding to the detectable label" refers to a color that can be catalyzed by the label of the detection antibody to display the recognition signal of the binding of the detection antibody to PD-L1. Described substrate is such as: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP); and so on.

As used herein, the term "amino acid" refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C ); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine ( Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S ), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

1. Anti-PD-L1 Nanobody

The invention provides a nanobody, and the nanobody is selected from a camel-derived natural single-domain heavy chain antibody library.

The nanobody of the present invention can specifically bind to PD-L1 with high affinity. In one embodiment, a Nanobody of the invention has a heavy chain variable region (VHH) as set forth in SEQ ID NO: 1-19. In a preferred embodiment, a Nanobody of the invention has a heavy chain variable region (VHH) as set forth in SEQ ID NO: 7, 3, 6, 2, 8 and 9. Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 7, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 29, 30 and 31, respectively (based on IMGT numbering). Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 3, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 23, 24 and 25, respectively (based on IMGT numbering). Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 6, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 26, 27 and 28, respectively (based on IMGT numbering). Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 2, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 20, 21 and 22, respectively (based on IMGT numbering). Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 8, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 32, 33 and 34, respectively (based on IMGT numbering). Where the sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 9, the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 35, 36 and 37, respectively (based on IMGT numbering). It should be pointed out that the numbering method of the antibody sequence is not limited to the IMGT method, and other methods can also be used to number the antibody sequence, such as Kabat, Chothia, Martin, AHo and other methods. The CDR sequences of antibodies may differ where different numbering schemes are used. Unless otherwise specified, the antibody numbers in this application are numbered using the IMGT method.

The present invention uses the IMGT numbering system to mark the CDR region, and the CDR region marked by other methods also belongs to the protection scope of the present invention.

The invention also includes variants, derivatives and analogs of said Nanobodies. As used herein, the terms "variants", "derivatives" and "analogues" refer to polypeptides that substantially retain the same biological function or activity of the Nanobodies of the invention. The polypeptide variants, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues The base may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence ( Such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a propolypeptide sequence, or a fusion polypeptide). These variants, derivatives and analogs are within the purview of those skilled in the art as defined herein.

In addition, other amino acid sequences that do not substantially affect the activity, expression level and stability of the Nanobody of the present invention can be added to the amino-terminal or carboxyl-terminal of the Nanobody.

Preferably, these added amino acid sequences are favorable for expression (such as signal peptide), favorable for purification (such as 6XHis sequence), or other sequences that can promote the activity, expression amount or stability of the Nanobody.

The invention also includes DNA molecules encoding the Nanobodies of the invention or variants, derivatives thereof. The DNA molecules can be all artificially synthesized, or can be obtained by PCR amplification.

In order to further increase the expression level of the host cell, the coding sequence of the Nanobody of the present invention can be modified, for example, using codons preferred by the host cell to eliminate sequences that are not conducive to gene transcription and translation.

After obtaining the DNA sequence encoding the Nanobody of the present invention or its variants and derivatives, it is cloned into a suitable expression vector, and then transformed into a suitable host cell. Finally, the transformed host cells are cultured, and the novel nanobody of the present invention is obtained by separation and purification.

In some specific embodiments, said Nanobody is fused to an immunoglobulin Fc region.

In some specific embodiments, the C-terminus of the Nanobody is fused to the N-terminus of the Fc region of an immunoglobulin.

In some specific embodiments, the immunoglobulin Fc region is from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE.

In some specific embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown in amino acid 120 to amino acid 346 of SEQ ID NO:44.

2. Polynucleotide

The present invention provides an isolated polynucleotide encoding the aforementioned Nanobody.

As used herein, the term "polynucleotide" is generally RNA or DNA, polynucleotides may be single-stranded or double-stranded, but are preferably double-stranded DNA. Unless otherwise stated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as expressly indicated the sequence of.

3. Expression vector

The present invention provides an expression vector, which comprises the aforementioned polynucleotide.

As used herein, the term "expression vector" includes plasmids, cloning vectors, viral vectors, and the like. Various vectors known in the art can be used. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the Nanobody of the present invention is operably linked to the expression control sequence to form an expression vector.

4. Host cells

The present invention provides a host cell comprising the aforementioned expression vector or the aforementioned polynucleotide integrated in its genome.

As used herein, the term "host cell" includes both prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Host cells for expressing Nanobodies include Escherichia coli, yeast cells, insect cells, COS cells, CHO cells, and the like. Preferably, the host cell is a eukaryotic cell, more preferably a CHO cell.

After the transformed host cell is obtained, the cell can be cultured under conditions suitable for expressing the Nanobody of the present invention, thereby expressing the Nanobody; and then the expressed Nanobody can be isolated.

5. Phage

The present invention provides a phage displaying Nanobody, and the surface of the phage displays the aforementioned Nanobody.

Phage display technology is used in the construction of the nanobody library, and the DNA sequence of the foreign protein or polypeptide is inserted into the appropriate position of the structural gene of the phage coat protein, so that the foreign gene is expressed with the expression of the coat protein, and at the same time, the foreign protein is Phage reassembly and display on the phage surface.

6. Kit

Based on the nanobody obtained in the present invention, the present invention provides a kit for detecting PD-L1, and the kit can be used for the detection of PD-L1.

The kit contains: the nanobody of the present invention or a phage (phagemid) displaying the nanobody. In some specific embodiments, the sample to be tested can be coated on a solid phase carrier, and the Nanobody of the present invention can be used as a detection antibody for detection. The Nanobody can be linked to a detectable label, or can be linked to a Another antibody (anti-antibody) that detects the marker binds, so as to know the presence of PD-L1 in the sample to be tested. It should be understood that after obtaining the Nanobody of the present invention, various methods known in the art can be adopted to detect PD-L1, and these methods are all included in the present invention.

After the detection antibody used in the kit of the present invention is determined, various markers that are routinely used in the art for detection in combination with the detection antibody can be used as detectable markers. The present invention has no particular limitation on the marker used, as long as it can bind to the detection antibody and can accurately indicate the presence or absence and the amount of PD-L1 in the sample to be detected after appropriate treatment are available. For example, the marker can be selected from (but not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or Glucoamylase. For example, the detection antibody is labeled with horseradish peroxidase (HRP). Antibody labeling methods are well known in the art, for example, the simple sodium periodate method or the glutaraldehyde two-step method for HRP-labeling antibodies.

When using some of the above-mentioned enzyme markers, it is also necessary to use some substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be indicated by means of color development and the like. Described substrate is for example: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP).

In order to eliminate false positives and false negatives, a quality control (control) should be set up during the detection process. The quality control product can be, for example, a PD-L1 standard product. In addition, in order to obtain quantitative results, multiple standards containing known concentrations of PD-L1 can be set during the detection process. A conventional method can be used for the setting method of the standard. Using the standard, the standard curve is set as follows: use the OD value detection result of the standard as the vertical axis (Y axis), and the standard concentration as the horizontal axis (X axis) to draw the quantitative standard curve of the PD-L1 kit. Therefore, according to the OD value obtained by testing the sample to be tested, the concentration of PD-L1 in the sample to be tested can be calculated using the standard curve.

In addition, in order to make the kit of the present invention more convenient for detection, the kit preferably also contains other auxiliary reagents, and the auxiliary reagents are some reagents routinely used in enzyme-linked immunoassays. The properties and methods of their formulation are well known to those skilled in the art. The reagents include: chromogen, washing solution, stop solution, and sensitizing diluent.

The coating antibody is coated on a solid phase carrier. The present invention has no particular limitation on the solid phase carrier used, as long as it can be coupled (linked) with the coated antibody. For example, the solid phase carrier is selected from: microtiter plates (also called multi-well plates, such as 96-well plates) or microspheres.

In an example of the present invention, the solid phase carrier used is a microtiter plate (elisa plate), and the microtiter plate is a polystyrene plate with a size of 12×8 detachable strips.

Because the nanobody used in the kit of the present invention has extremely excellent binding properties (high specificity) to PD-L1. According to the above method, as long as an antigen control of known concentration is set, a concentration standard curve is made, and the PD-L1 content in the sample to be tested can be obtained by comparing the concentration standard curve.

7. Immunoconjugates

The present invention provides an immunoconjugate comprising a therapeutic agent and the aforementioned Nanobody conjugated to the therapeutic agent;

Preferably, the therapeutic agent comprises a toxin, radioisotope, drug or cytotoxic agent.

As used herein, the term "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

8. Bispecific or multispecific antibodies

The present invention provides a bispecific or multispecific antibody comprising the aforementioned Nanobody, and an antibody or antibody fragment functionally linked to the Nanobody having another or several other antigen-binding properties.

As used herein, the term "bispecific or multispecific antibody" refers to a molecule that can bind to two or more different epitopes of the same antigen or that can bind to two or more different antigens.

9. Pharmaceutical composition

The present invention provides a pharmaceutical composition, which includes a PD-L1 binding molecule (such as the aforementioned nanobody, immunoconjugate, bispecific or multispecific antibody);

Preferably, the pharmaceutical composition includes a pharmaceutically acceptable excipient, carrier or diluent.

As used herein, the term "pharmaceutical composition" is in a form that permits the biological activity of the active ingredients to be effective, and does not contain additional ingredients that would be unacceptably toxic to the subject to which the composition will be administered. In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable excipient, carrier or diluent, specifically, any and all solvents, dispersion media, coatings, antibacterial agents that are physiologically compatible. Agents and antifungal agents, isotonic and absorption delaying agents, etc., are used to prolong the shelf life or potency of antibodies.

Beneficial effects include:

The present invention has creatively developed PD-L1 binding molecules with high affinity and strong specificity to PD-L1, such as PD-L1 Nanobody, which can block the binding of human PD-L1 protein to free human PD-1 and free human CD80 , the nanobody and the phage displaying the nanobody can be used to prepare a kit for detecting PD-L1, and the PD-L1 nanobody and its immunoconjugate, bispecific or multispecific antibody, and pharmaceutical composition are prepared Promising applications in drugs for the treatment of PD-L1-mediated diseases.

Example

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to the conditions described in J. Sambrook et al., edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer suggested conditions. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Embodiment 1: the preparation of material

The genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1), mouse PD-L1 (NP_068693.1), human PD-1 (NP_054862.1) and human CD80 (NP_005182.1) The target gene was cloned into plasmids containing His, human Fc or mouse Fc, and then transfected into HEK293 cells, a transient expression system, for transient expression to obtain hPD-L1-His (R0201 for short), cynoPD-L1-His, mPD - L1-His, hPD-L1-hFc, hPD-1-hFc (abbreviated as R0479), hPD-1-mFc (abbreviated as R0323) and hCD80-hFc secreted protein.

The target genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1) and mouse PD-L1 (NP_068693.1) were cloned into the above plasmids containing His, human Fc or mouse Fc, and then Transfected into CHO cells for membrane protein expression, and then obtained cell lines overexpressing PD-L1 membrane protein, namely CHO-hPD-L1, CHO-cynoPD-L1 and CHO-mPD-L1.

According to the sequence of Nanobody No. 56 in the application number 201510465481.8 and the sequence of the variable region of Avelumab on the Drug Bank website, the anti-standard antibodies of the mouse IgG1 subtype were constructed, and their abbreviations are respectively R0999 and R0516.

Example 2: Construction of Nanobody Library

The construction of the phage library displaying nanobodies was completed by Chengdu Apak Biotechnology Co., Ltd. Mix 1.5 mg of human PD-L1 antigen-mFc (purchased from Sino Biologics) with Freund's adjuvant in equal volume, and immunize an alpaca by intradermal and subcutaneous multi-point injection on the back, once a week, and immunize 4 times in total , to stimulate B cells to express antigen-specific nanobodies. After the 4 times of immunization, 50ml of alpaca peripheral blood was extracted, and lymphocytes were separated by lymphocyte separation medium. Total RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen), and the integrity of RNA was detected by 1% agarose electrophoresis, see Figure 1 for details. Alpaca total cDNA was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

Amplification of Nanobody genes was performed by 2 rounds of nested PCR. The first round of nested PCR system is shown in Table 1:

Table 1

in:

The NPR-19001 primer sequence is: 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO: 45);

The NPR-19002 primer sequence is: 5'-GTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 46);

Reaction program: 98°C, 52min; 98°C for 10s, 55°C for 30s, 72°C for 45s, a total of 25 cycles; 72°C, 7min; after the reaction, gel electrophoresis and gel tapping to recover the target VHH fragment of about 700bp.

The second round of nested PCR used conventional methods to design primers and reaction procedures to amplify the target gene fragments, and performed 2% agarose electrophoresis.

The vector and the target fragment (i.e. the VHH fragment after the second round of nested PCR amplification and purification) were respectively digested with SfiI, digested overnight at 50°C and recovered. T4 ligase was used to carry out the ligation reaction at 4° C. for 16 h at the ligation molar ratio of Vector:VHH=1:3. The vector carrying the target gene fragment was transformed into Escherichia coli competent E.coli TG1 cells by electroporation, and the calculated bacterial library capacity was 2.55×10 ⁹ cfu. The helper phage M13KO7 was added according to the MOI of 20 times to continue the culture, and the standard purification method of PEG-NaCl was used to purify twice to construct the phage library. The titer of the identified phage library was 1.41×10 ¹³ cfu/mL.

Example 3: Phage panning of Nanobodies

The hPD-L1-His antigen was coated on the microtiter plate, screened and affinity enriched on six 96-well microtiter plates using a solid-phase screening scheme, and eluted with Gly-HCl at a concentration of 0.2M and pH=2.2 Specifically bound phages were then immediately neutralized with Tris-HCl neutralization buffer, and three rounds of panning were repeated. The ELISA method was used to screen positive clones that combined with human PD-L1, and the nucleotide sequence was sequenced. The nucleotide sequences were translated into amino acid sequences by software and then compared, and classified into 19 categories according to CDR3 differences and amino acid differences in the phylogenetic tree. The best-performing sequences of each category are shown in Table 2 below.

Table 2

The amino acid sequences of the 19 Nanobodies are shown in Table 3 below.

table 3

The amino acid sequences (based on IMGT numbering) of the CDRs of Nanobodies are shown in Table 4 below.

Table 4

The screening method is as follows: First, the hPD-L1-His and mPD-L1-hFc antigens were diluted to 2 μg/mL with 0.05M carbonate buffer (pH 9.6), coated at 100 μL/well overnight at 4°C; Wash 3 times with PBST, add 300 μL 5% skim milk to each well, block at 37°C for 1 hour; wash 3 times with PBST, add 100 μL/well of phages that have undergone three rounds of panning, incubate for 45 minutes at 37°C; wash 5 times with PBST , respectively added horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (diluted 1:1W with PBS), 100 μL/well, incubated at 37°C for 45min; washed the plate 5 times with BST. Add 100 μL of TMB chromogenic solution to each well for color development, and react at 37°C for 5 min; add 50 μL of stop solution to each well to stop the reaction, and measure the optical density at 450 nm.

Embodiment 4: Preparation of fusion protein

The target gene sequences encoding nanobodies were cloned into plasmids containing mouse IgG1 Fc, and then transfected into transient expression system HEK293 cells for transient expression to obtain corresponding secreted proteins (ie, fusion proteins, C-terminal of VHH and Fc The N-terminal connection of the region), the abbreviations of each fusion protein are R1014, R1015, R1016, R1017, R1018, R1019, R1148, R1149, R1150, R1020, R1021, R1022, R1023, R1024, R1025, R1026, R1027, R1028 and R1029 . The fusion protein contains two identical peptide chains, the VHH sequence of each fusion protein is shown in the above table 3, and the full-length amino acid sequence of one of the chains of the partial fusion protein is shown in any of SEQ ID NO: 38-43 in the following table 5 amino acid sequence. The amino acid sequence of one CH2-CH3 of the immunoglobulin Fc region is shown in the 120th-346th amino acid of SEQ ID NO: 44 (Table 5 italics).

table 5

Example 5: Detection of binding activity of fusion protein to membrane-expressed human PD-L1 protein

The combination of the fusion protein obtained in Example 4 and R0999, R0516 obtained in Example 1, and CHO-hPD-L1 cells was detected by flow cytometry (FCM). The detection method is as follows:

Add the fusion protein to be detected and the standard antibody at an appropriate dilution factor to 2E5/well CHO-hPD-L1 cells, incubate at 2-8°C for 30 minutes, wash once with 200 μl/well 1×PBS, and add 1:1 to 100 μl/well 500 diluted E-anti-hIgG fluorescent secondary antibody (diluted with 1×PBS containing 3% BSA), incubated at 2-8°C for 30min, washed once with 200μl/well 1×PBS, then washed with 100μl/well 1×PBS Resuspended for flow cytometry detection.

The flow cytometry results are shown in Figure 3. The results show that the EC50 of R1015, R1016, R1019, R1148, R1149, and R1150 are 0.148nM, 0.247nM, 0.773nM, 0.445nM, 0.497nM, and 0.512nM, respectively, indicating that the 6 This fusion protein has good binding activity to membrane-expressed human PD-L1 protein.

Embodiment 6: Detection of fusion protein affinity

Biomembrane interferometry (BLI) was used to detect the positively binding fusion proteins (R1015, R1016, R1019, R1148, R1149, R1150) obtained in Example 5 and the reference antibodies obtained in Example 1 (referred to as R0999 and R0516), human PD- 1 protein (referred to as R0323) protein and human PD-L1 protein (referred to as R0201) affinity.

The detection method is as follows: 5 μg/ml of R0201 is solidified on the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe of the molecular interaction analyzer ForteBIO (Octet OK ^e ), and the probe is balanced for 90 seconds, respectively, with The analyte diluted at an appropriate concentration binds for 180s, then dissociates for 300s. After the probe is regenerated and neutralized in 10mM glycine, follow this method for the next cycle of equilibrium, binding, dissociation and regeneration neutralization. All cycle speeds were set at 1000 rpm and the experimental temperature was 30°C.

The test results are shown in Table 6 below, and the binding curves are shown in Figure 4. The results show that: R1015, R1016, R1019, R1148, R1149, R1150 have high affinity with human PD-L1 protein.

Table 6

protein abbreviation	kon(1/Ms)	koff(1/s)	KD(M)
R1015	7.69E+05	8.85E-05	1.15E-10
R1016	8.44E+05	1.18E-04	1.40E-10
R1019	6.48E+05	<1.0E-07	<1.0E-12
R1148	8.26E+05	9.34E-05	1.13E-10
R1149	4.10E+05	7.54E-05	1.84E-10
R1150	3.94E+05	4.87E-05	1.24E-10
R0999	8.57E+05	2.20E-05	2.57E-11
R0516	4.61E+05	4.67E-05	1.01E-10
R0323	1.42E+05	2.12E-04	7.78E-08

Example 7: Fusion proteins block the binding of membrane-expressed human PD-L1 protein and free human PD-1

The fusion proteins obtained in Example 4 (R1015, R1016, R1019, R1148, R1149, R1150) and the R0999, R0516 obtained in Example 1, blocking cell CHO-hPD-L1 and free human were detected by flow cytometry (FCM). Binding of PD-1 protein. Detection methods (A) and (B) are as follows:

The detection method (A) is as follows:

Add the fusion protein to be detected and the standard antibody at an appropriate dilution factor to 2E5/well CHO-hPD-L1 cells, incubate on ice for 30 minutes in the dark, then add hPD-1-hFc protein to a 96-well V-type plate (according to 50 μL/well Add), incubate on ice in the dark for 30min; centrifuge (300g/5min), discard the supernatant, add 200μL FCM buffer to wash once, centrifuge (300g/5min), discard the supernatant, add 1:500 diluted FCM at 100μl/well PE-anti-hIgG fluorescent secondary antibody was incubated on ice for 30 minutes in the dark; after centrifugation and washing, 100 μL of 1×PBS was added to resuspend and tested on the machine.

The detection results are shown in Figure 5, and the results showed that the IC50 of R1148, R1149 and R1150 were 6.431nM, 6.330nM and 8.813nM, respectively, indicating that these three fusion proteins can block membrane expression of human PD-L1 protein and free human PD- 1 in combination.

The detection method (B) is as follows:

Add hPD-1-hFc protein to 2E5/well CHO-hPD-L1 cells, incubate on ice in the dark for 30 minutes, then add the fusion protein to be detected and the standard antibody at an appropriate dilution to a 96-well V-type plate (according to 50 μL/well Add), incubate on ice in the dark for 30min; centrifuge (300g/5min), discard the supernatant, add 200μL FCM buffer to wash once, centrifuge (300g/5min), discard the supernatant, add 1:500 diluted FCM at 100μl/well PE-anti-hIgG fluorescent secondary antibody was incubated on ice for 30 minutes in the dark; after centrifugation and washing, 100 μL of 1×PBS was added to resuspend and tested on the machine.

The detection results are shown in Figure 6, and the results showed that the IC50 of R1148, R1149 and R1150 were 6.072nM, 10.64nM and 13.98nM, respectively, indicating that these three fusion proteins can block membrane expression of human PD-L1 protein and free human PD- 1 in combination.

Example 8: Fusion protein binding epitope analysis

The positive binding fusion proteins (R1148, R1149 and R1150) obtained in Example 7 and the binding regions of the reference antibodies obtained in Example 1 (referred to as R0999 and R0516) and human PD-L1 protein (referred to as R0201) were grouped by competition ELISA . The detection method is as follows:

5 μg/ml of R0201 was immobilized onto the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe of the molecular interaction analyzer ForteBIO (Octet OK ^e ), equilibrated the probe for 90 s, and respectively mixed with 150 nM of the first The antibody binds for 180s, and then binds with 150nM of the first antibody for 180s. After the probe is regenerated and neutralized in 10mM glycine, the next cycle of balance, binding and regenerated neutralization is carried out according to this method.

The test results are shown in Table 7 below, and the results show that: R1148, R0999 and R0516 epitopes are consistent; R1149 and R1150 epitopes are consistent, and R0999 and R0516 epitopes are inconsistent.

Table 7

AB#	R1148	R1149	R1150	R0999	R0516
R1148	3.07%	107.45%	110.88%	0.06%	1.30%
R1149	110.09%	4.24%	4.45%	111.57%	87.67%
R1150	109.96%	4.48%	4.45%	113.42%	88.32%
R0999	3.96%	106.86%	108.81%	1.13%	2.87%
R0516	14.32%	110.05%	115.77%	9.08%	7.79%

Example 9: Fusion Protein Blocks the Binding of Membrane Expressed Human PD-L1 Protein and Free Human CD80

Flow cytometry (FCM) was used to detect the fusion proteins (R1148, R1149 and R1150) and the combination of R0999, R0516, blocking cells CHO-hPD-L1 and free human CD80 protein obtained in Example 1. Detection methods (A) and (B) are as follows:

The detection method (A) is as follows:

Add the fusion protein to be detected and the standard antibody at an appropriate dilution factor to 2E5/well CHO-hPD-L1 cells, incubate on ice in the dark for 30 minutes, then add hCD80 protein to a 96-well V-shaped plate (according to 50 μL/well), and store on ice. Incubate in the dark for 30 minutes; centrifuge (300g/5min), discard the supernatant, add 200μL FCM buffer to wash once, centrifuge (300g/5min), discard the supernatant, add 1:500 diluted PE-anti- hIgG fluorescent secondary antibody, incubated on ice in the dark for 30 minutes; after centrifugation and washing, add 100 μL 1×PBS to resuspend, and detect on the machine.

The test results are shown in Figure 7, and the results showed that: R1148, R1149 and R1150 can block the binding of membrane-expressed human PD-L1 protein and free human CD80, and the blocking effect is equivalent to that of the standard antibody.

The detection method (B) is as follows:

Add hCD80 protein to 2E5/well CHO-hPD-L1 cells, incubate on ice in the dark for 30 minutes, then add the fusion protein to be detected and the standard antibody at an appropriate dilution to a 96-well V-plate (according to 50 μL/well), ice Incubate in the dark for 30 minutes; centrifuge (300g/5min), discard the supernatant, add 200μL FCM buffer to wash once, centrifuge (300g/5min), discard the supernatant, add 1:500 diluted PE-anti- hIgG fluorescent secondary antibody, incubated on ice in the dark for 30 minutes; after centrifugation and washing, add 100 μL 1×PBS to resuspend, and detect on the machine.

The detection results are shown in Figure 8, and the results showed that: R1148, R1149 and R1150 can block the binding of membrane-expressed human PD-L1 protein and free human CD80, and the blocking effect is equivalent to that of the standard antibody.

To sum up, in this embodiment of the present invention, a phage-displayed nanobody library was constructed, and positive clones that bind to human PD-L1 were screened out by ELISA method to obtain PD-L1-binding molecules that specifically bind to PD-L1, such as FBP002-1128, FBP002-1136, FBP002-1136, FBP002-1369, FBP002-1389, FBP002-1471, FBP002-2002, FBP002-2056, FBP002-2058, FBP002-1191, FBP002-1217, FBP002-1118, F BP002-1134, FBP002- 1153, FBP002-1184, FBP002-1224, FBP002-1249, FBP002-1075, FBP002-1095, R1015, R1016, R1019, R1148, R1149, R1150, R1014, R1017, R1018, R1020, R 1021, R1022, R1023, R1024, R1025, R1026, R1027, R1028, R1029, these PD-L1 binding molecules can block the binding of human PD-L1 protein to free human PD-1 and free human CD80.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (20)

1. A PD-L1 binding molecule, characterized in that, the PD-L1 binding molecule comprises at least one immunoglobulin single variable domain, and the at least one immunoglobulin single variable domain comprises a group selected from the following (i) CDR1, CDR2 and CDR3 of any of (vi):

   (i) CDR1 as shown in SEQ ID NO: 29, CDR2 as shown in SEQ ID NO: 30, and CDR3 as shown in SEQ ID NO: 31;

   (ii) CDR1 as shown in SEQ ID NO: 23, CDR2 as shown in SEQ ID NO: 24, and CDR3 as shown in SEQ ID NO: 25;

   (iii) CDR1 as shown in SEQ ID NO: 26, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 28;

   (iv) CDR1 as shown in SEQ ID NO: 20, CDR2 as shown in SEQ ID NO: 21, and CDR3 as shown in SEQ ID NO: 22;

   (v) CDR1 as set forth in SEQ ID NO: 32, CDR2 as set forth in SEQ ID NO: 33, and CDR3 as set forth in SEQ ID NO: 34; or

   (vi) CDR1 as shown in SEQ ID NO:35, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:37.
2. The PD-L1 binding molecule according to claim 1, wherein the immunoglobulin single variable domain is VHH.
3. The PD-L1 binding molecule according to claim 2, wherein the amino acid sequence of the VHH is such as SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO : 8 or SEQ ID NO: 9 shown in either.
4. The PD-L1 binding molecule according to any one of claims 1 to 3, wherein the PD-L1 binding molecule further comprises an immunoglobulin Fc region;

   Optionally, the C-terminus of the immunoglobulin single variable domain is linked to the N-terminus of the immunoglobulin Fc region.
5. The PD-L1 binding molecule according to claim 4, wherein the amino acid sequence of the Fc region of the immunoglobulin is as shown in amino acids 120-346 of SEQ ID NO:44.
6. The PD-L1 binding molecule according to any one of claims 1-5, wherein the PD-L1 binding molecule comprises an amino acid sequence as shown in any one of SEQ ID NO: 38-43.
7. An isolated polynucleotide, characterized in that the polynucleotide encodes the PD-L1 binding molecule according to any one of claims 1-6.
8. An expression vector, characterized in that the expression vector comprises the polynucleotide according to claim 7.
9. A host cell, characterized in that the host cell comprises the expression vector of claim 8 or the polynucleotide of claim 7 integrated in its genome.
10. A method for preparing the PD-L1 binding molecule according to any one of claims 1 to 6, characterized in that it comprises culturing the host cell according to claim 9 under conditions that allow the production of the PD-L1 binding molecule and The PD-L1 binding molecule is recovered and isolated.
11. A phage, characterized in that the PD-L1 binding molecule according to any one of claims 1-6 is displayed on the surface of the phage.
12. A kit for detecting PD-L1, characterized in that it comprises the PD-L1 binding molecule according to any one of claims 1-6 or the phage according to claim 11.
13. The kit according to claim 12, wherein the kit further comprises a solid phase carrier, and the PD-L1 binding molecule or phage is immobilized on the solid phase carrier.
14. The kit according to claim 13, wherein the kit also includes a detectable label that can be linked to the PD-L1 binding molecule; and/or a PD-L1 standard or a PD-L1 conjugate and/or a substrate corresponding to a detectable label; and/or an ELISA reagent;

    Preferably, said detectable label is attached to said PD-L1 binding molecule or present in a kit separately.
15. A method for detecting the presence of PD-L1 in a sample to be tested, characterized in that the PD-L1 binding molecule according to any one of claims 1 to 6 or the phage according to claim 11 is used as the detection of PD-L1 The antibody is used to detect the presence of PD-L1 in the sample to be tested by an enzyme-linked immunosorbent assay.
16. The method according to claim 15, characterized in that it specifically comprises the following steps:

    The sample to be tested is coated on a solid phase carrier, and the PD-L1 binding molecule or the phage with or without a detectable marker is used as a detection antibody to detect the presence of PD-L1.
17. An immunoconjugate, characterized in that it comprises a therapeutic agent and the PD-L1 binding molecule according to any one of claims 1-6 conjugated to the therapeutic agent;

    Preferably, the therapeutic agent comprises a toxin, radioisotope, drug or cytotoxic agent.
18. A bispecific or multispecific antibody, characterized in that it comprises the PD-L1 binding molecule according to any one of claims 1 to 6, and another antibody functionally linked to the PD-L1 binding molecule Or antibodies or antibody fragments with several other antigen-binding properties.
19. A pharmaceutical composition, characterized in that it comprises the PD-L1 binding molecule according to any one of claims 1-6;

    Preferably, the pharmaceutical composition includes a pharmaceutically acceptable excipient, carrier or diluent.
20. The PD-L1 binding molecule according to any one of claims 1 to 6, the immunoconjugate according to claim 17, the bispecific or multispecific antibody according to claim 18, and the drug according to claim 19 Use of the composition in the preparation of medicines for treating PD-L1-mediated diseases.

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