CN113509562B

CN113509562B - Formulations comprising PD-L1 binding polypeptide compositions and methods of making the same

Info

Publication number: CN113509562B
Application number: CN202110435346.4A
Authority: CN
Inventors: 须涛; 孙艳; 杨艳玲; 顾奕; 李芳芳
Original assignee: Suzhou Zhihe Biomedical Technology Co ltd
Current assignee: Suzhou Zhihe Biomedical Technology Co ltd
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2023-04-07
Anticipated expiration: 2041-04-22
Also published as: CN113509562A; WO2022222978A1

Abstract

The present application relates to a composition comprising: (i) A conjugate at a concentration of about 0.1mg/mL to about 50mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator; (ii) a buffer at a concentration of about 10mM to about 80 mM; and (iii) a stabilizer and/or an osmotic pressure regulator; wherein the pH of the composition is not less than 5. The compositions of the present application are capable of maintaining the physical, chemical and biological stability of the conjugate during storage. The application also provides application of the composition in detecting PD-L1.

Description

Formulations comprising PD-L1 binding polypeptide compositions and methods of making the same

Technical Field

The application relates to the field of biomedicine, in particular to a composition containing an anti-PD-L1 antibody.

Background

Programmed death-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The first members of this family, CD28 and ICOS, were found to enhance T cell proliferation by addition of monoclonal antibodies (Hutloff et al (1999), nature 397. Two cell surface glycoprotein ligands of PD-1, PD-L1 and PD-L2, have been identified which have been shown to down-regulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al (2000), J Exp Med15 192, 1027-34 latchman et al (2001), nat Immunol 2 (261-8), cater et al (2002), eur J Immunol 32, 634-43 ohigashi et al (2005), clin Cancer Res 11. Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1 but not to other CD28 family members (Blank et al 2004). Expression of PD-L1 has been found in several murine and human cancers, including human lung, ovarian, colon, melanoma and various myelomas 20 (Iwai et al (2002), PNAS 99 12293-7, ohigashi et al (2005), clin Cancer Res11: 2947-53). It has been shown that PD-L1, which is highly expressed in tumor cells, plays an important role in immune escape of tumors by increasing apoptosis of T cells. The detection of the expression of PD-L1 in a patient can be used for the diagnosis of tumors, or provide clinical diagnosis basis for the anti-PD-1 or anti-PD-L1 immunotherapy of tumors. However, previously reported antibodies against PD-L1 have not been successfully used to date to effectively detect and/or diagnose cancer early.

Emission Computed Tomography (ECT) has been used for the diagnosis of tumors. ECT includes Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), which provides high resolution tumor imaging and can be quantitatively analyzed from images.

The anti-PD-L1 antibody is easy to degrade in a solution state, has poor physical stability, is easy to form precipitated particles or aggregates, and is difficult to obtain a stable protein preparation. At present, no formulation based on PD-L1 antibodies suitable for ECT detection has been reported.

Disclosure of Invention

The purpose of the present invention is to provide a PD-L1 antibody-based preparation suitable for ECT detection, which can maintain stable physicochemical properties during storage, and use thereof.

In one aspect, the present application provides a composition comprising:

(i) A conjugate at a concentration of about 0.1mg/mL to about 50mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator;

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Stabilizers and/or osmo-regulators;

wherein the pH of the composition is not less than 5.

In certain embodiments, wherein the pH of the composition is from about 5.0 to 6.0.

In certain embodiments, wherein the pH of the composition is about 5.2 to 5.8.

In certain embodiments, wherein the pH of the composition is about 5.4 to 5.6.

In certain embodiments, wherein the concentration of the stabilizing agent is from about 100mM to about 300mM.

In certain embodiments, wherein the concentration of the stabilizing agent is from about 140mM to about 220mM.

In certain embodiments, wherein the concentration of the stabilizing agent is from about 150mM to about 200mM.

In certain embodiments, wherein the stabilizing agent comprises a mixture of one or more of sucrose, trehalose, mannitol, glycine, methionine, arginine.

In certain embodiments, wherein the stabilizing agent comprises sucrose.

In certain embodiments, wherein the sucrose concentration is from about 150mM to about 200mM.

In certain embodiments, wherein the stabilizing agent further comprises methionine.

In certain embodiments, wherein the methionine concentration is from about 0.2mM to about 20mM.

In certain embodiments, wherein the conjugate concentration is about 0.2mg/mL to about 40mg/mL.

In certain embodiments, wherein the conjugate concentration is about 0.5mg/mL to about 20mg/mL.

In certain embodiments, wherein the conjugate concentration is about 1mg/mL to about 10mg/mL.

In certain embodiments, wherein the buffering agent comprises a mixture of one or more of acetate, citrate, histidine, glycine, succinate.

In certain embodiments, wherein the buffer is at a concentration of about 10mM to 80mM.

In certain embodiments, wherein the buffer is at a concentration of about 40mM to 60mM.

In certain embodiments, wherein the tonicity modifier comprises a mixture of one or more of sodium chloride, magnesium chloride, dextrose, sorbitol, sodium citrate.

In certain embodiments, wherein the osmolality adjusting agent concentration is from about 100mM to about 300mM.

In certain embodiments, wherein the osmolality adjusting agent concentration is from about 120mM to about 240mM.

In certain embodiments, wherein the PD-L1 binding polypeptide comprises an antibody or antigen binding fragment thereof.

In certain embodiments, wherein the antibody comprises a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and/or a human antibody.

In certain embodiments, wherein the antigen binding fragment comprises a Fab fragment, a F (ab') ₂ A fragment, fd fragment, fv fragment, dAb fragment, isolated Complementarity Determining Region (CDR), scFv and/or VHH.

In certain embodiments, wherein the antibody or antigen binding fragment thereof comprises at least one variable domain.

In certain embodiments, wherein the variable domain comprises a VHH domain.

In certain embodiments, wherein the variable domain comprises: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 4; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

In certain embodiments, wherein the variable domain comprises: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 11; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

In certain embodiments, wherein the variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs 1-2, 6-10, and 12-16.

In certain embodiments, wherein the variable domain comprises at least one lysine residue.

In certain embodiments, wherein the variable domain comprises 3 lysine residues.

In certain embodiments, wherein the chelating agent is selected from: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N, N', N "-triacetic acid), NETA ([ 4- [2- (di-carboxymethylamino) -ethyl ] -7-carboxymethyl- [1,4,7] triazonane-1-yl } -acetic acid), p-NH2-Bn-NOTA (2-S- (4-aminobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA (2- (p-isothiocyanatobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid).

In certain embodiments, wherein the PD-L1 binding polypeptide and chelator are linked by lysine.

In certain embodiments, wherein the chelator is directly attached to the PD-L1 binding polypeptide through a primary amine of an amino acid of the PD-L1 binding polypeptide.

In certain embodiments, wherein the chelator is p-SCN-Bn-NOTA.

In certain embodiments, wherein the p-SCN-Bn-NOTA is directly attached to the PD-L1 binding polypeptide by reaction of an isocyanuric group with an amino group at the lysine terminus of the PD-L1 binding polypeptide.

In certain embodiments, the composition further comprises at least one detectable label.

In certain embodiments, wherein the detectable label is bound to the conjugate.

In certain embodiments, wherein the detectable label is selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic particle, and an enzyme.

In certain embodiments, wherein the radionuclide is selected from: ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ sr or other gamma-, beta-, or positron emitters.

In certain embodiments, wherein the radionuclide is ⁶⁷ Ga or ⁶⁸ Ga。

In certain embodiments, the detectable label is detectable by positron emission tomography.

In certain embodiments, the composition comprises:

(i) A conjugate at a concentration of about 1mg/mL to about 10mg/mL, the conjugate comprising a PD-L1 binding polypeptide and a chelator;

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

(iv) Sodium chloride as an osmolality adjusting agent at a concentration of about 120mM to about 240 mM;

wherein the pH of the formulation is from about 5.2 to about 5.8.

In certain embodiments, the composition comprises:

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

(iii) Sucrose as a stabilizer at a concentration of about 150mM to about 200 mM;

wherein the pH of the composition is from about 5.2 to about 5.8.

In certain embodiments, the pH of the composition is from about 5.4 to about 5.6.

In certain embodiments, the pH of the composition is about 5.5.

In certain embodiments, the sucrose is at a concentration of about 160mM to about 180mM.

In certain embodiments, it further comprises from about 0.2mM to about 20mM methionine as a stabilizer.

In certain embodiments, the PD-L1 binding polypeptide is a VHH.

In certain embodiments, the VHH comprises the amino acid sequence set forth in SEQ ID NO. 10.

In certain embodiments, the chelator is p-SCN-Bn-NOTA.

In certain embodiments, the composition further comprises a detectable label that is ⁶⁷ Ga or ⁶⁸ Ga。

In another aspect, the present application further provides a PD-L1 detection method, including: an effective amount of the foregoing composition is administered.

In certain embodiments, wherein said administering is performed in vitro or ex vivo.

In certain embodiments, wherein said administering comprises any method of contacting a cell, organ, or tissue with said combination.

In certain embodiments, the method further comprises detecting or quantifying the detectable label.

In certain embodiments, the method further comprises obtaining an image of a cell, organ or tissue expressing PD-L1.

In another aspect, the present application also provides a method of detecting and/or diagnosing a disease associated with PD-L1, the method comprising: administering to the subject the aforementioned composition.

In certain embodiments, the method further comprises ECT imaging the subject.

In certain embodiments, wherein the disease associated with PD-L1 comprises a tumor.

In certain embodiments, the tumor is overexpressing PD-L1.

In another aspect, the present application also provides the use of the aforementioned composition for the preparation of a medicament for the detection of PD-L1.

In certain embodiments, the drug is a PD-L1 imaging agent.

In another aspect, the present application also provides a PD-L1 imaging agent, which PD-L1 imaging agent comprises the aforementioned composition.

In another aspect, the present application also provides a kit comprising the aforementioned composition.

In certain embodiments, wherein the composition and detectable label are present in separate dosage forms.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIG. 1 shows a schematic structural diagram of the conjugate SNA002 of the present application;

FIG. 2 shows the results of UV280 measurements of the formulations of example 1 of the present application under different storage conditions;

FIG. 3 shows the results of SEC measurements on formulations of example 1 of the present application under different storage conditions;

FIG. 4 shows the results of RP-HPLC measurements (the rate of change of Average DAR) of the formulations of example 1 of the present application under different storage conditions;

FIG. 5 shows the results of a conjugate of the formulation of example 1 of the present application in an ELISA assay at different storage conditions with T0 as reference;

FIG. 6 shows the results of UV280 concentration measurements at different storage conditions for the formulations of example 2 of the present application;

FIG. 7 shows the results of SEC measurements for the formulations of example 2 of the present application under different storage conditions;

FIG. 8 results of RP-HPLC measurements of the formulations of example 2 of the present application under different storage conditions;

FIG. 9 results of ELISA assays using conjugates of the formulation of example 2 of the present application with T0 as reference under different storage conditions.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "conjugate" generally refers to any substance formed from two or more separate moieties joined together. For example, a conjugate may comprise a substance formed by linking one polypeptide to another polypeptide or polypeptides. For example, a conjugate can be a substance formed by the conjugation of a small molecule (e.g., a chelator) with a large molecule such as a carrier or polyamine polymer, particularly a protein (e.g., an antibody). In some embodiments, a small molecule may be conjugated at one or more active sites of a macromolecule.

In the present application, the term "PD-L1 binding polypeptide" means any polypeptide capable of specifically binding to PD-L1. In some embodiments, the binding polypeptide is an antibody. In other embodiments, the binding polypeptide is, for example, an antibody mimetic, a cell surface receptor, a cytokine, or a growth factor. Such as the single domain antibodies of the present application that specifically bind PD-L1. "PD-L1 binding polypeptide" may alternatively refer to monovalent polypeptides that bind PD-L1 (i.e., polypeptides that bind to one epitope of PD-L1), as well as to bivalent or multivalent binding polypeptides (i.e., binding polypeptides that bind to more than one epitope). A "PD-L1 binding polypeptide" of the present application may comprise at least one immunoglobulin single variable domain that binds PD-L1, such as a VHH.

In the present application, the term "chelating agent" generally refers to an organic molecule capable of forming a complex with a metal ion. Chelators are commonly used to label proteins or peptides. The end product of the metal ion conjugate is used for radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities. Non-limiting examples of chelating agents include DTPA (diethylenetriamine pentaacetic anhydride) and its derivatives, NOTA and its derivatives such as NODA-GA, DOTA and its derivatives, TETA and its derivatives, DTTA or NETA. These and other chelating agents are readily available from commercial sources.

In the present application, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Milleretal (2003) journal. Of immunology170: 4854-4861). The antibody may be murine, human, humanized, chimeric, or derived from other species.

In the present application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that comprises the amino acids responsible for specific binding between an antibody and an antigen. The portion of the antigen specifically recognized and bound by the antibody is referred to as an "epitope" as described above. The antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. The Fd fragment, for example, has two VH regions and typically retains some of the antigen binding function of the entire antigen binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments with VL, VH, constant light Chain (CL) and CH1 domains; (2) F (ab') ₂ A fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge of the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH Domains of a Single arm of an antibody, (5) dAb fragments (Ward et al, "Binding Activities of a reprintaire of Single Immunoglobulin Variable domain Secreted From Escherichia coli," Nature 341; (6) an isolated Complementarity Determining Region (CDR); (7) Single chain Fv (scFv), e.g.derived from a scFv-library. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that are syntheticSuch that it is prepared as a Single Protein Chain in which the VL and VH regions pair to form monovalent molecules (known as Single Chain Fv (scFv)) (see, e.g., huston et al, "Protein Engineering of Antibody Binding Sites: recovery of Specific Activity in an Anti-Digoxin site-Chain Fv analog Produced in Escherichia coli," Proc. Natl. Acad. Sci.USA 85; and (8) VHH, "VHH" relates to the variable antigen-binding domain of heavy chain antibodies from camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen v.k. Et al, 2000, the EMBO journal,19, 921-930, muydermans S, 2001, j Biotechnol, 74, 277-302 and reviewed in Vanlandschoot p. Et al, 2011, antiviral Research 92, 389-407). VHH may also be referred to as Nanobody (Nb) and/or single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is assessed in the same manner as for intact antibodies.

In the present application, the term "variable domain" generally refers to a variable domain of an antibody capable of specifically binding an epitope of an antigen. For example, antibody variable domains VH and VL (VH and VL domains). Another example of a variable domain is a "VHH domain" (or simply "VHH"). "VHH domains", also known as heavy chain single domain antibodies, VHHs, vs _H H domains, VHH antibody fragments and VHH antibodies, variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e. "antibodies lacking light chain") (Hamers-Casterman C, atarhouch T, muylermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: naturally occuring antibodies void of light chains "; nature 363,446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from a heavy chain variable domain (which is referred to herein as a "VH domain") present in conventional 4 chain antibodies, and a light chain variable domain (which is referred to herein as a "VL domain") present in conventional 4 chain antibodies. The VHH domain specifically binds to an epitope without the need for an additional antigen binding domain (as opposed to the VH or VL domain in conventional 4 chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). VHH domainsIs a small, stable and highly efficient antigen recognition unit formed by a single immunoglobulin domain.

"variable domains" typically have the same general structure, each domain comprising 4 Framework (FR) regions highly conserved in sequence, wherein the FR regions comprise four "framework regions" of "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", three "complementarity determining regions" or "CDRs" of the FR region "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3" are linked. The general structure or sequence of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Antibody variable domains confer specificity for an antigen on an antibody by virtue of having an antigen binding site.

In the present application, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "V _H H domain, VHH antibody fragment, VHH antibody and

and>

the domain "(" Nanobody "is a trademark of Ablynx n.v. company, ghent, belgium) may be used interchangeably.

For example, as shown in FIG. 2 of Riechmann and Muyledermans, J.Immunol.methods 231,25-38 (1999), the amino acid residues employed for VHH domains in Camelidae are numbered according to the general numbering of VH domains given by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, md., publication No. 91). According to the numbering, FR1 comprises amino acid residues at positions 1-30, CDR1 comprises amino acid residues at positions 31-35, FR2 comprises amino acids at positions 36-49, CDR2 comprises amino acid residues at positions 50-65, FR3 comprises amino acid residues at positions 66-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113. It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering, as is well known in the art for VH and VHH domains (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

In the present application, the term "sequence identity" generally refers to a nucleic acid or amino acid sequence in which two or more aligned sequences are identical when aligned using a sequence alignment program. The term "% sequence identity" as used herein generally refers to the level of nucleic acid or amino acid sequence identity between two or more aligned sequences, when aligned using a sequence alignment program. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988; biocontrol, information and Genome Projects, smith, D.W., ed., academic Press, new York,1993; computer Analysis of Sequence Data, part I, griffin, A.M., and Griffin, H.G., eds., humana Press, new Jersey,1994; sequence Analysis in Molecular Biology,20von Heinje, G., academic Press,1987 and Sequence Analysis Primer, gribskov, M.and Devereux, J., eds., M Stockton Press, new York,1991.

In the present application, amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. In comparing two amino acid sequences, the term "amino acid difference" generally refers to the specified number of amino acid residues at a position in the reference sequence compared to the other sequence, insertion, deletion or substitution. In the case of a substitution, the substitution will preferably be a conservative amino acid substitution, meaning that the amino acid residue is replaced with another amino acid residue that is chemically similar in structure and that has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (i) - (v) is replaced with another amino acid residue within the same group: (i) a less aliphatic non-polar or weakly polar residue: ala, ser, thr, pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: asp, asn, glu and Gln; (iii) polar positively charged residues: his, arg and Lys; (iv) larger aliphatic apolar residues: met, leu, ile, val and Cys; and (v) aromatic residues: phe, tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, gln, or Glu; met is substituted by Leu, tyr or Ile; phe is substituted by Met, leu, or Tyr; ser substituted by Thr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu.

In the present application, the term "detectable label" generally refers to a moiety having a detectable physical or chemical property, which label can produce a signal that can be detected by visual or instrumental means. Examples of labels for polypeptides include (but are not limited to) the following: radioisotopes or radionuclides, fluorescent labels (e.g., FITC, rhodamine (rhodamine), lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups (which can be detected by labeled avidin (e.g., molecules containing streptavidin moieties) and fluorescent labels or enzymatic activity that can be detected by optical methods or calorimetry), and predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

In the present application, the term "buffer" or "buffer solution" generally refers to an aqueous solution comprising a mixture of an acid (typically a weak acid, such as acetic acid, citric acid, the imidazolium (imidazolium) form of histidine) and its conjugate base (e.g., acetate or citrate, such as sodium acetate, sodium citrate, or histidine), or a base (typically a weak base, such as histidine) and its conjugate acid (e.g., protonated histidine salt). The pH of the "buffer solution" changes only slightly when a small amount of strong acid or base is added, due to the "buffering effect" imparted by the "buffer".

In the present application, the term "buffer" generally refers to an acid or base component (typically a weak acid or base) of a buffer or buffer solution. Buffering agents help to maintain the pH of a given solution at or near a predetermined value, and buffering agents are typically selected to complement their predetermined value. Suitably, the buffering agent is a single compound which produces the desired buffering action, particularly when mixed with (and suitably capable of undergoing proton exchange with) an appropriate amount (depending on the desired predetermined pH) of its corresponding "acid/base conjugate", or if the desired amount of its corresponding "acid/base conjugate" is formed in situ — this can be achieved by adding a strong acid or base until the desired pH is reached. For example, in a sodium acetate buffer system, a solution of sodium acetate (basic) may be used and then acidified, e.g., with hydrochloric acid, or added to acetic acid (acidic), sodium hydroxide, or sodium acetate solution until the desired pH is reached.

In the present application, "stabilizer" generally refers to a component that helps maintain the structural integrity of the biopharmaceutical drug, particularly during freezing and/or lyophilization and/or storage (particularly when exposed to stress). This stabilization may occur for a variety of reasons, although in general such stabilizers may act as osmolytes to mitigate protein denaturation. As used herein, a stabilizer can be a sugar alcohol (e.g., inositol, sorbitol), a disaccharide (e.g., sucrose, maltose), a monosaccharide (e.g., dextrose (D-glucose)), or various forms of the amino acid lysine (e.g., lysine monohydrochloride, acetate, or monohydrate) or a salt (e.g., sodium chloride).

In the present application, the terms "stability" and "stable" generally refer to the resistance of an antibody in a formulation to aggregation, degradation, or fragmentation under given manufacturing, preparation, shipping, and storage conditions. A "stable" formulation retains biological activity under given manufacturing, preparation, shipping, and storage conditions. Stability of an antibody can be assessed by the degree of aggregation, degradation, or fragmentation as compared to a reference formulation, as measured by high pressure size exclusion chromatography (HP-SEC), static Light Scattering (SLS), fourier transform infrared spectroscopy (FTIR), circular Dichroism (CD), urea unfolding technique (urea unfolding technique), endogenous tryptophan fluorescence, differential scanning calorimetry, and/or ANS binding technique. The overall stability of formulations comprising human PD-L1 antibodies can be assessed by different immunological assays, including, for example, ELISA and radioimmunoassay using isolated antigenic molecules.

In the present application, the term "administration (administer)" and similar terms are generally not limited to bodily administration, and suitable methods include in vitro, ex vivo, or in vivo methods. For example, any method of administration known to those skilled in the art for contacting a cell, organ or tissue with a composition may be employed. For example, the compounds may be introduced into the body of a subject in need of treatment by any route of introduction or delivery. In some embodiments, the compositions of the present application may be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.

In the present application, the terms "ex vivo" and "in vitro" are interchangeable, and generally refer to activities performed in a controlled environment in cells, tissues and/or organs that have been removed from a subject.

In the present application, the term "diagnosing" generally refers to detecting a disease or condition, or determining the state or extent of a disease or condition. The term "diagnosing" may also include detecting a predisposition for a disease or condition, determining the therapeutic effect of a drug treatment, or predicting a response pattern to a drug treatment.

In the present application, the term "treatment" generally means: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to the disease, disorder, and/or condition, but has not yet been diagnosed as having the disease; (ii) Inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) alleviating the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition.

In the present application, the terms "tumor" and "cancer" are used interchangeably and generally refer to neoplastic or malignant cell growth. The tumor of the present application may be benign or malignant. The tumor of the present application may be solid or non-solid.

In the present application, "high expression" of a tumor antigen or tumor antigen generally means that the expression level of the tumor antigen in cells from a diseased region within a specific tissue or organ, such as a solid tumor, of a patient is abnormal relative to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by high expression of tumor antigens can be identified by standard assays known in the art.

In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys, etc.

In this application, "positron emission tomography" or "PET" refers generally to non-invasive nuclear medicine techniques that produce three-dimensional images of the location of tracers in the body. The method detects gamma ray pairs emitted indirectly by positron emitting radionuclides (tracers) introduced into the body on biologically active molecules. PET imaging tools have a wide variety of uses and are helpful in drug development both preclinical and clinical. Exemplary applications include direct visualization of in vivo saturation of a target; monitoring uptake in normal tissue to anticipate toxicity or patient-to-patient variation; quantifying the diseased tissue; tumor metastasis; and monitoring changes in drug potency over time, or changes in resistance over time.

In this application, the term "about" generally refers to a variation in the range of 0.5% -10% above or below a specified value, such as a variation in the range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

In this application, the terms "comprises," "comprising," and variations thereof, including "comprises," "comprising," and other variations thereof, are intended to cover a generic term for use herein, which comprises or includes other components, elements, values, steps, or the like.

Detailed Description

Composition comprising a metal oxide and a metal oxide

In one aspect, the present application provides a composition that may comprise:

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Stabilizers and/or osmo-regulators;

wherein the pH of the composition is not less than 5.

In certain embodiments, the pH of the composition may be from about 5.0 to about 6.0. For example, the pH of the composition may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0.

In certain embodiments, the pH of the composition may be about 5.5.

For example, the composition may comprise:

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Stabilizers and/or osmo-regulators;

wherein the composition may have a pH of about 5.5.

In certain embodiments, the concentration of the stabilizing agent therein may be from about 100mM to about 300mM. For example, the concentration of the stabilizer may be about 110mM to about 280mM, about 120mM to about 260mM, about 130mM to about 240mM, about 140mM to about 220mM.

In certain embodiments, the concentration of the stabilizing agent therein may be from about 150mM to about 200mM.

In certain embodiments, wherein the stabilizing agent may comprise a mixture of one or more of sucrose, trehalose, mannitol, glycine, methionine, arginine.

In certain embodiments, wherein the stabilizing agent may comprise sucrose.

In certain embodiments, the sucrose concentration may be from about 150mM to about 200mM. For example, the sucrose concentration may be about 6% (w/v) (about 175 mM).

For example, the composition may comprise:

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Sucrose at a concentration of about 150mM to about 200 mM;

wherein the composition may have a pH of about 5.5.

In certain embodiments, wherein the methionine concentration is from about 0.2mM to about 20mM. For example, the methionine concentration may be from about 0.3mM to about 18mM,0.4mM to about 116mM, 0.5mM to about 14mM,0.6mM to about 12mM, 0.7mM to about 10mM,0.8mM to about 8mM. As another example, the methionine concentration may be from about 0.2mM to about 10mM, 0.2mM to about 5mM,0.2mM to about 4mM,0.2mM to about 3mM,0.2mM to about 2mM,0.2mM to about 1mM.

In certain embodiments, the methionine concentration may be from about 0.8mM to about 8mM. For example, the methionine concentration may be about 1.2mg/mL (about 8 mM).

In certain embodiments, the methionine concentration may be from about 0.2mM to about 1mM. For example, the methionine concentration may be about 0.12mg/mL (about 0.8 mM).

For example, the composition may comprise:

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Sucrose at a concentration of about 150mM to about 200mM, methionine at a concentration of about 0.8mM to about 8 mM;

wherein the composition may have a pH of about 5.5.

In certain embodiments, wherein the conjugate concentration may be about 0.2mg/mL to about 40mg/mL. For example, the conjugate concentration can be about 0.3mg/mL to about 30mg/mL, about 0.4mg/mL to about 25mg/mL, about 0.5mg/mL to about 20mg/mL, about 0.6mg/mL to about 18mg/mL, about 0.7mg/mL to about 16mg/mL, about 0.8mg/mL to about 14mg/mL, about 0.9mg/mL to about 12mg/mL, about 1.0mg/mL to about 10mg/mL.

In certain embodiments, wherein the conjugate concentration may be about 1mg/mL to about 10mg/mL. For example, the conjugate concentration can be about 1mg/mL, about 2mg/mL, about 3mg/mL, about 4mg/mL, about 5mg/mL, about 7mg/mL, about 8mg/mL, about 9mg/mL, about 10mg/mL.

For example, the composition may comprise:

(i) A conjugate at a concentration of about 1mg/mL to about 10mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator;

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

wherein the composition may have a pH of about 5.5.

In certain embodiments, wherein the buffer may comprise a mixture of one or more of acetate, citrate, histidine, glycine, succinate. For example, the buffer may be acetate, and for example, the acetate may be sodium acetate.

For example, the composition may comprise:

(ii) Sodium acetate at a concentration of about 10mM to about 80 mM; and

wherein the composition may have a pH of about 5.5.

In certain embodiments, the buffer may be present in a concentration of about 10mM to 80mM. For example, the buffer is at a concentration of about 10mM to 80mM, about 20mM to 70mM, about 30mM to 60mM, about 40mM to 60mM.

In certain embodiments, the buffer may be present at a concentration of about 40mM to 60mM. For example, the buffer is at a concentration of about 40mM, about 41mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM.

For example, the composition may comprise:

(ii) Sodium acetate at a concentration of about 40mM to about 60 mM; and

wherein the composition may have a pH of about 5.5.

In certain embodiments, the osmotic pressure regulator may include one or more of sodium chloride, magnesium chloride, glucose, sorbitol, and sodium citrate. For example, the osmolality adjusting agent may be sodium chloride.

In certain embodiments, the osmolality adjusting agent concentration may be from about 100mM to about 300mM. For example, the osmolality regulator concentration may be from about 110mM to about 270mM, from about 120mM to about 240mM, from about 130mM to about 210mM, from about 140mM to about 180mM.

In certain embodiments, wherein the osmolality adjusting agent concentration may be from about 140mM to about 180mM. For example, the osmolality adjusting agent may be at a concentration of about 140mM, about 145mM, about 150mM, about 155mM, about 160mM, about 165mM, about 170mM, about 175mM, about 180mM. For another example, the osmolality adjusting agent concentration can be about 150mM. As another example, the osmolality regulator concentration can be about 0.9% (w/v) (about 154 mM).

In certain embodiments, the composition may comprise:

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

wherein the pH of the formulation is from about 5.2 to about 5.8.

For example, the composition may comprise:

(ii) Sodium acetate at a concentration of about 40mM to about 60 mM; and

(iii) Sodium chloride at a concentration of about 140mM to about 180 mM;

wherein the composition may have a pH of about 5.5.

In certain embodiments, wherein the PD-L1 binding polypeptide can comprise an antibody or antigen binding fragment thereof.

In certain embodiments, the antibody may comprise a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and/or a human antibody.

In certain embodiments, whereinThe antigen binding fragments may include Fab fragments, F (ab') ₂ A fragment, fd fragment, fv fragment, dAb fragment, isolated Complementarity Determining Region (CDR), scFv and/or VHH.

In certain embodiments, wherein the antibody or antigen binding fragment thereof may comprise at least one variable domain. For example, the antibody or antigen binding fragment thereof may comprise 1,2 or 3 variable domains.

In certain embodiments, wherein the variable domain may comprise a VHH domain. For example, the antibody or antigen-binding fragment thereof may comprise 1 VHH domain.

In certain embodiments, wherein the variable domain may comprise:

CDR1 comprising an amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 3;

CDR2 comprising the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 4, for example comprising the amino acid sequence shown in SEQ ID NO. 11; and

CDR3 comprising the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions relative to SEQ ID NO. 5.

For example, wherein the variable domain may comprise: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 4; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

For example, wherein the variable domain may comprise: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 11; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

In certain embodiments, wherein the variable domain may comprise an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID No. 1.

In certain embodiments, wherein the variable domain may comprise an amino acid sequence set forth in any one of SEQ ID NOs 1-2, 6-10, and 12-16.

In certain embodiments, wherein the variable domain may comprise at least one lysine residue. For example, the variable domain may comprise 1,2, 3, 2, 4, 5, 6, 7, 8, 9 lysine residues.

In certain embodiments, wherein the variable domain may comprise 3 lysine residues.

For example, the composition may comprise:

(i) A conjugate at a concentration of about 1mg/mL to about 10mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator; the PD-L1 binding polypeptide is a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOs 1-2, 6-10, and 12-16;

(ii) Sodium acetate at a concentration of about 40mM to about 60 mM; and

wherein the composition may have a pH of about 5.5.

In certain embodiments, wherein the chelating agent may be selected from: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N, N', N "-triacetic acid), NETA ([ 4- [2- (di-carboxymethylamino) -ethyl ] -7-carboxymethyl- [1,4,7] triazononane-1-yl } -acetic acid), p-NH2-Bn-NOTA (2-S- (4-aminobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA (2- (p-isothiocyanatobenzyl) -1,4,7-1,4,7-triacetic acid).

In certain embodiments, wherein the PD-L1 binding polypeptide and the chelator are linked by lysine.

In certain embodiments, wherein the chelator is directly attachable to the PD-L1 binding polypeptide through a primary amine of an amino acid of the PD-L1 binding polypeptide.

In certain embodiments, wherein the chelator may be p-SCN-Bn-NOTA.

In certain embodiments, wherein the p-SCN-Bn-NOTA may be directly attached to the PD-L1 binding polypeptide by reaction of an isothiocyante group with an amino group at the lysine terminus of the PD-L1 binding polypeptide.

For example, the composition may comprise:

(i) A conjugate at a concentration of about 1mg/mL to about 10mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator; the PD-L1 binding polypeptide is a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOs 1-2, 6-10, and 12-16; the chelator may be p-SCN-Bn-NOTA; the p-SCN-Bn-NOTA may be directly attached to the PD-L1 binding polypeptide by reaction of an isocyanuric group with an amino group at the lysine terminus of the PD-L1 binding polypeptide;

(ii) Sodium acetate at a concentration of about 40mM to about 60 mM; and

wherein the composition may have a pH of about 5.5.

In certain embodiments, the VHH binds to 1,2 or 3 p-SCN-Bn-NOTA.

In certain embodiments, the composition may further comprise at least one detectable label.

In certain embodiments, wherein the detectable label is bound to the conjugate.

In certain embodiments, wherein the detectable label may be selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic particle, and an enzyme.

The conjugates of the present application can be applied to Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) depending on the label. High resolution tumor imaging can be provided in tumor diagnosis and quantitative analysis can be performed by the image. The SPECT imaging may also include SPECT/CT imaging, and the PET imaging may also include PET/CT imaging, which may provide superior imaging results.

In certain embodiments, wherein the radionuclide may be selected from: ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ sr or other gamma-, beta-, or positron emitters.

In certain embodiments, wherein the radionuclide may be ⁶⁷ Ga or ⁶⁸ Ga。

In certain embodiments, the composition may comprise:

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

wherein the pH of the composition may be from about 5.2 to about 5.8.

In certain embodiments, the pH of the composition may be from about 5.4 to about 5.6.

In certain embodiments, the pH of the composition may be about 5.5.

In certain embodiments, the sucrose concentration may be about 160mM to about 180mM.

In certain embodiments, it may further comprise from about 0.2mM to about 20mM methionine as a stabilizer.

In certain embodiments, the PD-L1 binding polypeptide can be a VHH.

In certain embodiments, the VHH may comprise the amino acid sequence set forth in SEQ ID NO. 10.

In certain embodiments, the chelating agent can be p-SCN-Bn-NOTA.

In certain embodiments, the composition may further comprise a detectable label that is a detectable label ⁶⁷ Ga or ⁶⁸ Ga。

For example, the composition may comprise:

(i) A conjugate at a concentration of about 1mg/mL to about 10mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator; the PD-L1 binding polypeptide is VHH, and the VHH comprises an amino acid sequence shown in SEQ ID NO. 10; the chelating agent is p-SCN-Bn-NOTA; the p-SCN-Bn-NOTA is directly attached to the PD-L1 binding polypeptide by reaction of an isocyanuric group with an amino group at the lysine terminus of the PD-L1 binding polypeptide; wherein the conjugate may be with ⁶⁷ Ga or ⁶⁸ Ga is combined;

(ii) Sodium acetate at a concentration of about 40mM to about 60 mM; and

(iii) Sucrose at a concentration of about 160mM to about 180mM, methionine at a concentration of about 0.8mM to about 8 mM;

wherein the composition may have a pH of about 5.5.

Detection/diagnostic uses

In another aspect, the present application further provides a PD-L1 detection method, which may include: an effective amount of the foregoing composition is administered.

In certain embodiments, wherein the administering may be performed in vitro or ex vivo.

In certain embodiments, wherein said administering may comprise any method of contacting a cell, organ, or tissue with said combination.

For example, the method may comprise:

a) Contacting a biological sample (e.g., a cell, organ, or tissue) and a control sample with a composition of the present application under conditions that enable the formation of a complex between the conjugate of the present application and PD-L1;

b) Detecting the formation of the complex by detecting the presence of the complex,

wherein a difference in complex formation between the biological sample and the control sample is indicative of the presence of PD-L1 and/or the expression level of PD-L1 in the sample.

In another aspect, the present application also provides a method of detecting and/or diagnosing a disease associated with PD-L1, which may comprise: administering to the subject the aforementioned composition.

In certain embodiments, the method may further comprise detecting or quantifying the detectable label.

In certain embodiments, the method may further comprise ECT imaging the subject. In some embodiments, the ECT imaging may be SPECT imaging. In some embodiments, the ECT imaging may be PET imaging. Imaging techniques and apparatus for scanning by SPECT or PET are well known in the art and any such known ECT imaging techniques and apparatus may be used.

For example, the method may comprise:

a) Administering to a subject in need thereof a composition described herein; and

b) Obtaining a radiological image of at least a portion of the subject to detect the presence or absence of the composition;

wherein the presence and location of the composition above background is indicative of the presence and location of PD-L1 or a tumor that expresses PD-L1.

In certain embodiments, wherein the disease associated with PD-L1 may comprise a tumor.

In certain embodiments, the tumor may overexpress PD-L1. Non-limiting examples include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, and osteosarcoma. Examples of other cancers that may be detected and/or diagnosed using the methods of the present application include, but are not limited to: bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, carcinoma of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the ureter, tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal column tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of said cancers. The invention can also be used to detect and/or diagnose metastatic cancers, particularly those expressing PD-L1 (Iwai et al (2005) Int Immunol 17: 133-144).

The present application also provides methods of treating a subject having cancer, which can include:

a) Administering a composition described herein to a subject in need thereof, and obtaining an image (static or dynamic) of at least a portion of the subject to determine the presence of PD-L1 in one or more tumors; and if the subject has a level of PD-L1 in one tumor or in several tumors that is equal to or higher than the level in need of treatment with a PD-1 or PD-L1 antagonist, then,

b) Administering to the subject an anti-tumor therapy, e.g., an agent that inhibits the interaction between PD-1 and PD-L1 (PD-1 or a PD-L1 antagonist).

The present application also provides a method of monitoring the progress of an anti-tumor therapy against a PD-L1-expressing tumor in a subject, which method can comprise:

a) Administering a composition described herein to a subject in need thereof at a first point in time and obtaining an image of at least a portion of the subject to determine the size of the tumor;

b) Administering an anti-tumor therapy to the subject;

c) Administering the composition to the subject at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point;

wherein the size and location of the tumor at each time point is indicative of the progression of the disease.

Kit and imaging agent

In certain embodiments, the drug is a PD-L1 imaging agent.

In certain embodiments, the PD-L1 imaging agent comprises a PD-L1 binding polypeptide (e.g., an anti-PD-L1 antibody comprising an amino acid sequence set forth in any one of SEQ ID NOS: 1-2, 6-10, and 12-16), a chelator p-SCN-Bn-NOTA, and a radionuclide ⁶⁷ Ga or ⁶⁸ Ga。

In certain embodiments, the composition and detectable label may be present in separate dosage forms.

In certain embodiments, the kit may include a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container may contain a composition described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is a conjugate described herein or a derivative or precursor thereof. The article may further comprise a second container. The second container comprises a detectable label as described herein. It may further include other items necessary from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present application encompasses the following embodiments:

1. a composition, comprising:

(ii) A buffer at a concentration of about 10mM to about 80 mM; and

(iii) Stabilizers and/or osmo-regulators;

wherein the composition has a pH of not less than 5.

2. The composition of embodiment 1, wherein the pH of the composition is about 5.0-6.0.

3. The composition according to any of embodiments 1-2, wherein the pH of the composition is about 5.2-5.8.

4. The composition according to any of embodiments 1-3, wherein the pH of the composition is about 5.4-5.6.

5. The composition according to any one of embodiments 1-4, wherein the concentration of the stabilizing agent is from about 100mM to about 300mM.

6. The composition according to any one of embodiments 1-5, wherein the concentration of the stabilizing agent is from about 140mM to about 220mM.

7. The composition according to any one of embodiments 1-6, wherein the concentration of the stabilizing agent is from about 150mM to about 200mM.

8. The composition according to any of embodiments 1-7, wherein the stabilizing agent comprises a mixture of one or more of sucrose, trehalose, mannitol, glycine, methionine, arginine.

9. The composition according to any of embodiments 1-8, wherein the stabilizing agent comprises sucrose.

10. The composition of embodiment 9, wherein the sucrose concentration is about 150mM to about 200mM.

11. The composition according to any one of embodiments 1-10, wherein the stabilizing agent further comprises methionine.

12. The composition of embodiment 11, wherein the methionine concentration is about 0.2mM to about 20mM.

13. The composition according to any one of embodiments 1-12, wherein the conjugate concentration is about 0.2mg/mL to about 40mg/mL.

14. The composition according to any one of embodiments 1-13, wherein the conjugate concentration is about 0.5mg/mL to about 20mg/mL.

15. The composition according to any one of embodiments 1-14, wherein the conjugate concentration is about 1mg/mL to about 10mg/mL.

16. The composition according to any of embodiments 1-15, wherein the buffering agent comprises one or more of acetate, citrate, histidine, glycine, succinate in admixture.

17. The composition according to any one of embodiments 1-16, wherein the buffer is at a concentration of about 10mM to 80mM.

18. The composition according to any one of embodiments 1-17, wherein the buffer is at a concentration of about 40mM to 60mM.

19. The composition according to any of embodiments 1-18, wherein the osmolality adjusting agent comprises one or more of sodium chloride, magnesium chloride, glucose, sorbitol, sodium citrate in admixture.

20. The composition according to any one of embodiments 1-19, wherein the osmolality regulator concentration is from about 100mM to about 300mM.

21. The composition according to any one of embodiments 1-20, wherein the osmolality regulator concentration is from about 120mM to about 240mM.

22. The composition of any one of embodiments 1-21, wherein the PD-L1 binding polypeptide comprises an antibody or antigen-binding fragment thereof.

23. The composition according to any one of embodiments 1-22, wherein the antibody comprises a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, and/or a human antibody.

24. The composition of any one of embodiments 1-23, wherein the antigen-binding fragment comprises a Fab fragment, a F (ab') 2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated Complementarity Determining Region (CDR), a scFv, and/or a VHH.

25. The composition of any one of embodiments 22-24, wherein the antibody or antigen-binding fragment thereof comprises at least one variable domain.

26. The composition of embodiment 25, wherein said variable domain comprises a VHH domain.

27. The composition according to any one of embodiments 25-26, wherein said variable domain comprises: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 4; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

28. The composition according to any one of embodiments 25-27, wherein said variable domain comprises: CDR1 having the amino acid sequence shown in SEQ ID NO. 3; CDR2 having the amino acid sequence shown in SEQ ID NO. 11; and CDR3 having the amino acid sequence shown in SEQ ID NO. 5.

29. The composition according to any one of embodiments 25-28, wherein said variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs 1-2, 6-10, and 12-16.

30. The composition according to any one of embodiments 25-29, wherein said variable domain comprises at least one lysine residue.

31. The composition according to any one of embodiments 25-30, wherein said variable domain comprises 3 lysine residues.

32. The composition according to any one of embodiments 1-31, wherein the chelating agent is selected from the group consisting of: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N, N', N "-triacetic acid), NETA ([ 4- [2- (di-carboxymethylamino) -ethyl ] -7-carboxymethyl- [1,4,7] triazononane-1-yl } -acetic acid), p-NH2-Bn-NOTA (2-S- (4-aminobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA (2- (p-isothiocyanatobenzyl) -1,4,7-1,4,7-triacetic acid).

33. The composition according to any one of embodiments 1-32, wherein the PD-L1 binding polypeptide and chelator are linked by lysine.

34. The composition according to any one of embodiments 1-33, wherein the chelating agent is directly attached to the PD-L1 binding polypeptide through a primary amine of an amino acid of the PD-L1 binding polypeptide.

35. The composition according to any one of embodiments 1-34, wherein the chelating agent is p-SCN-Bn-NOTA.

36. The composition of embodiment 35, wherein the p-SCN-Bn-NOTA is directly attached to the PD-L1 binding polypeptide by reaction of an isocyanuric group with an amino group at the lysine terminus of the PD-L1 binding polypeptide.

37. The composition of any one of embodiments 1-36, further comprising at least one detectable label.

38. The composition of embodiment 37, wherein the detectable label is bound to the conjugate.

39. The composition of any one of embodiments 37-38, wherein said detectable label is selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic particle, and an enzyme.

40. The composition of embodiment 39, wherein said radionuclide is selected from the group consisting of: : ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ sr or other gamma-, beta-, or positron emitters.

41. The composition of embodiment 40, wherein the radionuclide is 67Ga or 68Ga.

42. The composition of any one of embodiments 37-41, wherein the detectable label is detectable by positron emission tomography.

43. The composition according to any one of embodiments 1-42 comprising

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

wherein the pH of the formulation is from about 5.2 to about 5.8.

44. The composition according to any one of embodiments 1-42, comprising:

(ii) Sodium acetate as a buffer at a concentration of about 40mM to 60 mM;

wherein the pH of the composition is from about 5.2 to about 5.8.

45. The composition of embodiment 44, having a pH of about 5.4 to about 5.6.

46. The composition of any of embodiments 44-45, having a pH of about 5.5.

47. The composition according to any one of embodiments 44-46, wherein the sucrose is at a concentration of about 160mM to about 180mM.

48. The composition according to any one of embodiments 44-47, further comprising from about 0.2mM to about 20mM methionine as a stabilizer.

49. The composition of any one of embodiments 1-48, wherein said PD-L1 binding polypeptide is a VHH.

50. The composition according to any one of embodiments 1-49, wherein said VHH comprises the amino acid sequence set forth in SEQ ID NO 10.

51. The composition of any one of embodiments 1-50, wherein said chelator is p-SCN-Bn-NOTA.

52. The composition of any one of embodiments 1-51, further comprising a detectable label, the detectable label being ⁶⁷ Ga or ⁶⁸ Ga。

53. A PD-L1 detection method, the method comprising: administering an effective amount of the composition of any one of embodiments 1-52.

54. The method of embodiment 53, wherein said administering is performed in vitro or ex vivo.

55. The method of embodiment 54, wherein said administering comprises any method of contacting a cell, organ, or tissue with said combination.

56. The method of any one of embodiments 53-55, further comprising detecting or quantifying the detectable label.

57. The method of any one of embodiments 53-56, further comprising obtaining an image of a cell, organ or tissue expressing PD-L1.

58. A method of detecting and/or diagnosing a disease associated with PD-L1, the method comprising: administering to the subject a composition of any one of embodiments 1-52.

59. The method of embodiment 58, further comprising detecting or quantifying the detectable label.

60. The method according to any one of embodiments 58-59, further comprising ECT imaging the subject.

61. The method according to any one of embodiments 58-60, wherein the disease associated with PD-L1 comprises a tumor.

62. The method of embodiment 61, wherein the tumor is overexpressing PD-L1.

63. Use of a composition according to any one of embodiments 1-52 in the preparation of a medicament for the detection of PD-L1.

64. The use of embodiment 63, the medicament is a PD-L1 imaging agent.

A PD-L1 imaging agent, said PD-L1 imaging agent comprising a composition according to any one of embodiments 1-52.

66. A kit comprising a composition according to any one of embodiments 1-52.

67. The kit of embodiment 66, wherein the composition and detectable label are present in separate dosage forms.

Without wishing to be bound by any theory, the following examples are intended only to illustrate the compositions, preparation methods, uses, etc. of the present application and are not intended to limit the scope of the invention of the present application. In the present application, an anti-PD-L1 antibody is as disclosed in PCT/CN2019/093225, which is incorporated herein by reference in its entirety.

Examples

Materials and reagents

Instrument

SHIMADZU HPLC OF LabSolution version 6.72SP

Denovix DS-11 spectrophotometer

TSKgel G2000SWXL, internal diameter 7.8mm X30cm, 5 μm, part number 0008540

MabPac RP，2.1mm ID×100mm，4μm，PN088647

Thermo ProPac CEX-10,4X250mm, product number 054993, serial number 032566

Integrated electronic multichannel pipettor, 1250. Mu.L, 300. Mu.L and 50. Mu.L

Spectra Max Plus enzyme-labeling instrument

Microplate washer 405TS

Thermo Orion star A215 pH/conductivity meter

Method

Unless otherwise indicated, all methods, steps, techniques and operations not specifically recited may be, and have been, performed in a manner known per se to those of skill in the art. Reference is also made, for example, to standard manuals, the general prior art and other references cited therein.

SEC methods:

the chromatographic column is TSKgel G2000SWXL 7.8mm multiplied by 300mm,5 mu m, silica gel filler; the mobile phase is 50mM PB, 300mM NaCl, pH7.2; ultraviolet detection wavelength is 280nm; the flow rate is 0.6ml/min; the column temperature is 25 ℃, isocratic elution is carried out for 25min, and 50 mu g of sample is injected.

RP-HPLC method:

the chromatographic column is Thermo Scientific ^TM MAbPac ^TM RP,4 μm, 2.1X 100mm, mobile phase A0.1% TFA in UPW, mobile phase B0.1% TFA in 90% ACN; ultraviolet detection wavelength is 280nm; the flow rate is 0.6ml/min; the column temperature is 80 deg.C, gradient elution is carried out, and elution time is 31min. Sample introduction 10. Mu.L.

UV280 concentration detection method:

the method is based on the analysis that both sdAb and Nota-Lys have characteristic absorption at 280nm, and the OD value of the coupling product sdabC at 280nm is the sum of the OD values of Nota-Lys and sdAb. The concentration of sdAbC was determined from the molar extinction coefficients at 280nm of sdAb and Nota-Lys and the DAR value.

Binding activity method:

PDL1-Fc protein is coated in a 96-hole enzyme label plate, after the 96-hole enzyme label plate is sealed, a reference substance and a test solution which are diluted in a gradient manner are respectively added for antibody incubation, after the incubation is finished, an enzyme-labeled secondary antibody is added for incubation, and finally, after TMB color development and reaction termination, the absorbance value A450 at the wavelength of 450nm is read on an enzyme label instrument. The A450 value is inversely related to the coupling stock concentration. Taking the protein concentration of the sample as an X axis and the A450 value as a Y axis, drawing a curve by using a four-parameter fitting regression model in SoftMax Pro computer software to obtain the EC50 of each detection sample, and reporting the relative activity of the sample by calculating the ratio of the EC50 of the standard substance to the EC50 of the sample.

Example 1

1.1 preformulation formulations

In this example, the conjugate is SNA002-NOTA formed by the combination of an anti-PD-L1 single domain antibody (VHH) having the amino acid sequence shown in SEQ ID NO:10 and p-SCN-Bn-NOTA comprising 3 lysine residues, and the p-SCN-Bn-NOTA is directly attached to the VHH through the reaction of its isothiocyanate group with the amino group at the lysine terminus of the VHH, and the structural schematic diagram is shown in FIG. 1. Wherein the p-SCN-Bn-NOTA can coordinate with a radioactive element (e.g., ⁶⁷ ga or ⁶⁸ Ga) is connected.

As shown in Table 1, SNA002-NOTA was formulated into 8 different buffers at 3mg/mL or 10mg/mL. The pre-formulation is stored at 70 + -10 deg.C, 25 + -2 deg.C/60 + -5% RH and 40 + -2 deg.C/75 + -5% RH for up to four weeks. The pre-formulations were also evaluated by shaking (stirring) at 300rpm for 72 hours at room temperature, freezing at-70 ± 10 ℃ and thawing at 25 ℃ (freeze/thaw, fz/Th) for a maximum of 5 cycles. Samples were tested by SEC, RP-HPLC, UV280 concentration and binding ELISA.

Table 1 preformulation formula

1.2 results of the experiment

The formulation of example 1 of this application appeared as a clear, colorless solution under different storage conditions, with all samples being substantially free of visible particulates.

The results of UV280 measurement under different storage conditions for the formulation of example 1 of the present application are shown in fig. 2, the results of SEC measurement are shown in fig. 3, and the rate of change of the Average drug/antibody ratio (Average DAR) (results of RP-HPLC measurement) is shown in fig. 4. The results of the formulations in example 1 of the present application in the ELISA assay with the conjugate as reference at different storage conditions with T0 are shown in figure 5.

The above results show that under different conditions, there was no significant difference between SEC and RP-HPLC detection for each condition sample, and no increase in residual NOTA was seen for all samples. The F2 and F7 formulation varied minimally under all conditions tested. The relative activity of F3 and F8 is reduced after being stored for 4 weeks at 40 ℃, and the relative antigen binding force of other samples is not obviously changed. Either formulation 2 or formulation 7 was used for the next formulation confirmation study.

Example 2

2.1 formulation

Methionine was added as an antioxidant to F7 in example 1. As shown in Table 2, SNA002-NOTA was formulated into 4 different formulations at pH 5.5 and 2mg/mL. The preparation is stored at-70 + -10 deg.C, 5 + -3 deg.C and 40 + -2 deg.C/75 + -5% RH for up to 12 weeks. The formulations were also evaluated by shaking at 300rpm for 66 hours at room temperature and freezing/thawing (freezing at-70 ± 10 ℃ and thawing at 25 ℃) for up to 5 cycles. Samples were tested by SEC, RP-HPLC, UV280 concentration and binding ELISA.

Table 2 formulation tabulation of the formulations

2.2 results of the experiment

The results of the formulation in example 2 of the present application in the UV350 nm measurement in the sample under stirring conditions are shown in table 3.

TABLE 3

Formulation of preparation	350nm(Turbidity)	280nm(FIO)
			F1	1.2951	7.6049
F2	1.7457	8.7881
			F3	0.0441	5.4667
F4	1.7025	8.6107

The results showed that F2 and F3 differ only in L-methionine, except that F3 was not turbid and all were elevated. Subsequent validation experiments conducted comparative studies using only F2 and F3.

The results of the UV280 concentration measurement under different storage conditions of the formulation of example 2 of the present application are shown in fig. 6, the results of the SEC measurement are shown in fig. 7, and the results of the RP-HPLC measurement are shown in fig. 8. The results of the formulations of example 2 of the present application in an ELISA assay with T0 as reference under different storage conditions are shown in figure 9.

The results show that F1, F2 and F4 appeared cloudy under stirring, and UV350 turbidity, concentration, binding activity and SEC purity were measured separately, with a significant reduction in formulation for each formulation except F3. Therefore F1 and F4 stopped the study after 4 weeks. No significant difference was found between F2 and F3 by several key analytical methods, except that F3 was the clearest appearance in the candidate after stirring. F3 has an excipient methionine over F2, which is considered a stabilizer that protects proteins from conformational instability, aggregation due to thermal and shock (agitation) stresses, and chemical degradation.

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Claims

1. A composition consisting of:

(i) A conjugate at a concentration of 1mg/mL to 10mg/mL, the conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelator;

(ii) Sodium acetate as a buffer at a concentration of 40mM to 60 mM;

(iii) Sucrose as a stabilizer at a concentration of 160mM to 180 mM; and (iv) methionine as a stabilizer at a concentration of 0.2mM to 1 mM;

wherein the composition has a pH of 5.4 to 5.6; the PD-L1-binding polypeptide is VHH, which is the amino acid sequence shown in SEQ ID No. 10, the chelator is p-SCN-Bn-NOTA, which is directly attached to the PD-L1-binding polypeptide by reaction of an isothiocyante group with the amino group at the lysine terminus of the PD-L1-binding polypeptide.

2. The composition of claim 1, further comprising at least one detectable label.

3. The composition of claim 2, wherein the detectable label is bound to the conjugate.

4. The composition of claim 2, wherein the detectable label is selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic particle, and an enzyme.

5. The composition of claim 4, wherein the radionuclide is selected from the group consisting of: ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ sr or other gamma-, beta-, or positron emitters.

6. The composition of claim 5, wherein the radionuclide is ⁶⁷ Ga or ⁶⁸ Ga。

7. The composition of claim 2, wherein the detectable label is detectable by positron emission tomography.

8. A method of PD-L1 detection for non-diagnostic therapeutic purposes, the method comprising: administering an effective amount of the composition of any one of claims 1-7, wherein the administration is performed in vitro or ex vivo.

9. The method of claim 8, further comprising detecting or quantifying the detectable label.

10. A kit comprising the composition of any one of claims 1-7.

11. The kit of claim 10, wherein the composition and detectable label are present in separate dosage forms.