WO2022222978A1

WO2022222978A1 - Preparation comprising pd-l1 binding polypeptide composition, preparation method therefor and use thereof

Info

Publication number: WO2022222978A1
Application number: PCT/CN2022/088054
Authority: WO
Inventors: 须涛; 孙艳; 杨艳玲; 顾奕; 李芳芳
Original assignee: 苏州智核生物医药科技有限公司
Priority date: 2021-04-22
Filing date: 2022-04-21
Publication date: 2022-10-27
Also published as: CN113509562A; CN113509562B

Abstract

A composition, comprising: (i) a conjugate the concentration of which is from approximately 0.1 mg/mL to approximately 50 mg/mL, the conjugate comprising a programmed death-ligand 1 (PD-L1) binding polypeptide and a chelator; (ii) a buffer the concentration of which is from approximately 10 mM to approximately 80 mM; and (iii) a stabilizer and/or osmotic pressure regulator. The pH value of the composition is not lower than five. The composition can maintain the physical stability, chemical stability and biological stability of the conjugate during storage. The composition can also be applied in the detection of PD-L1.

Description

Formulation comprising PD-L1 binding polypeptide composition and preparation method and use thereof

technical field

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

Background technique

Programmed death-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. The original members of this family, CD28 and ICOS, were discovered by their ability to enhance T cell proliferation upon addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). Two cell surface glycoprotein ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al (2000), J Exp Med 15 192:1027-34; 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 11:2947- 53). Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind PD-1 but not 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 myeloma20 (Iwai et al. (2002), PNAS 99:12293-7; Ohigashi et al. ( 2005), Clin Cancer Res 11:2947-53). Existing results show that PD-L1 highly expressed by tumor cells plays an important role in tumor immune escape by increasing the apoptosis of T cells. Detection of PD-L1 expression in patients can be used for tumor diagnosis, or to provide clinical diagnosis basis for tumor anti-PD-1 or anti-PD-L1 immunotherapy. However, previously reported antibodies against PD-L1 have so far not been successfully used to effectively detect and/or diagnose cancer at an early stage.

Emission Computed Tomography (ECT) has been used for tumor diagnosis. ECT includes Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), which provide high-resolution imaging of tumors and allow quantitative analysis of the images.

Anti-PD-L1 antibodies are easily degraded in solution state, have poor physical stability, and are prone to form precipitated particles or aggregates, making it difficult to obtain stable protein preparations. Currently, no PD-L1 antibody-based preparations suitable for ECT detection have been reported.

SUMMARY OF THE INVENTION

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

In one aspect, the application provides a composition comprising:

(i) a conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelating agent at a concentration of from about 0.1 mg/mL to about 50 mg/mL;

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

(iii) stabilizers and/or osmotic pressure regulators;

Wherein, the pH value of the composition is not lower than 5.

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

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

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

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

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

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

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

In certain embodiments, wherein the stabilizer comprises sucrose.

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

In certain embodiments, wherein the stabilizer further comprises methionine.

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

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

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

In certain embodiments, wherein the conjugate concentration is from about 1 mg/mL to about 10 mg/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 concentration of the buffer is about 10 mM to 80 mM.

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

In certain embodiments, wherein the osmotic pressure regulator comprises a mixture of one or more of sodium chloride, magnesium chloride, dextrose, sorbitol, and sodium citrate.

In certain embodiments, wherein the osmolyte concentration is from about 100 mM to about 300 mM.

In certain embodiments, wherein the osmolyte concentration is from about 120 mM to about 240 mM.

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

In certain embodiments, wherein the antibodies comprise monoclonal antibodies, polyclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies and/or human antibodies.

In certain embodiments, wherein the antigen-binding fragment comprises a Fab fragment, F(ab') ₂ 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 set forth in SEQ ID NO:3; CDR2 having the amino acid sequence set forth in SEQ ID NO:4; and CDR2 having the amino acid sequence set forth in SEQ ID NO:5 CDR3 showing the amino acid sequence.

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

In certain embodiments, wherein the variable domain comprises the 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 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]triaznonan-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 the chelator are linked through a lysine.

In certain embodiments, 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.

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

In certain embodiments, wherein the p-SCN-Bn-NOTA is directly attached to the PD-L1-binding polypeptide by reacting an isothiocyanate group with the 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 associated with the conjugate.

In certain embodiments, wherein the detectable label is selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic particles, and enzymes.

In certain embodiments, 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.

In certain embodiments, wherein the radionuclide ^is67Ga ^or68Ga .

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

In certain embodiments, the composition comprises:

(i) a conjugate comprising a PD-L1 binding polypeptide and a chelating agent at a concentration of about 1 mg/mL to about 10 mg/mL;

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

(iv) sodium chloride at a concentration of from about 120 mM to about 240 mM as an osmotic pressure regulator;

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

In certain embodiments, the composition comprises:

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

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

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 concentration of sucrose is from about 160 mM to about 180 mM.

In certain embodiments, it also contains about 0.2 mM to about 20 mM methionine as a stabilizer.

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

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

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

In certain embodiments, the composition further comprises a detectable label, the detectable label ^being67Ga ^or68Ga .

In another aspect, the present application also provides a PD-L1 detection method, the method comprising: administering an effective amount of the aforementioned composition.

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

In certain embodiments, wherein the administering comprises any method of contacting a cell, organ or tissue with the 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 for detecting and/or diagnosing a disease associated with PD-L1, the method comprising: administering the aforementioned composition to a subject.

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 highly expresses PD-L1.

On the other hand, the present application also provides the use of the aforementioned composition in the preparation of a medicament for detecting 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, the PD-L1 imaging agent comprising the aforementioned composition.

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

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

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this 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 to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Fig. 1 shows the structural representation of conjugate SNA002 in the present application;

Figure 2 shows the results of UV280 determination of the formulation in Example 1 of the present application under different storage conditions;

Figure 3 shows the results of the SEC determination of the formula in Example 1 of the present application under different preservation conditions;

Figure 4 shows the results (change rate of Average DAR) measured by RP-HPLC of the formulation in Example 1 of the present application under different preservation conditions;

Figure 5 shows the results of the ELISA assay of the formulation in Example 1 of the present application using T0 as a reference conjugate under different storage conditions;

Figure 6 shows the results of the UV280 concentration determination of the formulation in Example 2 of the present application under different storage conditions;

Figure 7 shows the results of the SEC determination of the formulation in Example 2 of the present application under different preservation conditions;

The results of the RP-HPLC determination of the formula in Fig. 8 in Example 2 of the application under different preservation conditions;

Figure 9. The results of the ELISA assay of the conjugate of the formulation in Example 2 of the present application under different storage conditions with T0 as a reference.

Detailed ways

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "conjugate" generally refers to any substance formed by the joining together of two or more separate moieties. For example, a conjugate may comprise a substance formed by linking one segment of polypeptide to another segment or segments of polypeptide. For example, a conjugate can be a substance in which a small molecule (eg, a chelating agent) is joined to a macromolecule such as a carrier or a polyamine polymer, especially a protein (eg, an antibody). In some embodiments, a small molecule can 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 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. For example, the single domain antibody of the present application that specifically binds to PD-L1. "PD-L1 binding polypeptide" may alternatively refer to monovalent polypeptides that bind PD-L1 (ie, a polypeptide that binds to one epitope of PD-L1), as well as bivalent or multivalent binding polypeptides (ie, a binding polypeptide that binds to more than one epitope) ). The "PD-L1 binding polypeptide" of the present application may comprise at least one immunoglobulin single variable domain such as VHH that binds PD-L1.

In this application, the term "chelating agent" generally refers to organic molecules capable of forming complexes with metal ions. Chelating agents are often used to label proteins or peptides. The final product of the metal ion conjugate is used in radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities. Non-limiting examples of chelating agents include DTPA (diethylenetriaminepentaacetic 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 this application, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) , and antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule comprising the amino acids responsible for specific binding between the antibody and an antigen. The portion of an antigen that is specifically recognized and bound by an antibody is referred to as an "epitope" as described above. An antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it need not comprise both. Fd fragments, for example, have two VH regions and generally retain some antigen-binding function of the intact 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 fragments, with divalent fragments consisting of a hinge region. Bivalent fragment of two Fab fragments linked by a sulfur bridge; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragment with VL and VH domains of the antibody one-arm, (5) dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is hereby incorporated by reference in its entirety), which has a VH domain; ( 6) Isolated Complementarity Determining Regions (CDRs); (7) Single-chain Fvs (scFvs), eg from scFV-libraries. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows it to be prepared as a single protein in which the VL and VH domains are paired to form a monovalent molecule chain (referred to as single-chain Fv (scFv)) (see, eg, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl . Acad. Sci. USA 85: 5879-5883 (1988)); and (8) VHH, "VHH" refers to variable antigens from heavy chain antibodies of the family Camelidae (camelids, dromedaries, llamas, alpacas, etc.) Binding domains (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and reviewed in Vanlandschoot P. et al., 2011, Antiviral Research 92 , 389-407). VHHs may also be referred to as Nanobodies (Nb) and/or single domain antibodies. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the function of the fragments is assessed in the same manner as intact antibodies.

In this application, the term "variable domain" generally refers to the variable domain of an antibody capable of specifically binding an epitope. 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, _VHH domains, VHH antibody fragments, and VHH antibodies, are antigens known as "heavy chain antibodies" (ie, "antibodies lacking light chains") Binding to variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446- 448 (1993)). The term "VHH domain" is used to relate the variable domains to the heavy chain variable domains (which are referred to herein as "VH domains") present in conventional 4-chain antibodies and to those present in conventional 4-chain antibodies Light chain variable domains (which are referred to herein as "VL domains") are distinguished. The VHH domain binds specifically to an epitope without the need for additional antigen binding domains (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.

"Variable domains" usually have the same general structure, and each domain contains 4 framework (FR) regions with highly conserved sequences, wherein the FR regions include "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and four "framework regions" of "framework region 4" or "FR4", FR regions "complementarity determining region 1" or "CDR1", "complementarity determining region" 2" or "CDR2", and the three "complementarity determining regions" or "CDRs" of "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 antigen specificity to an antibody by having an antigen-binding site.

In this application, the terms "heavy chain single domain antibody", "VHH domain", "VHH", " _VHH domain", "VHH antibody fragment", "VHH antibody" and

and"

Domain"("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) is used interchangeably.

For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domains of Camelidae are based on the VH domains given by Kabat et al. Numbering by general numbering ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). According to this numbering, FR1 contains amino acid residues at positions 1-30, CDR1 contains amino acid residues at positions 31-35, FR2 contains amino acids at positions 36-49, and CDR2 contains amino acid residues at positions 50-65 FR3 contains amino acid residues at positions 66-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113. It should be noted, however, that, as is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number 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 amino acid residues in the actual sequence.

In this application, the term "sequence identity" generally refers to nucleic acid or amino acid sequences in which two or more aligned sequences are identical when aligned using a sequence alignment program. The term "% sequence identity" 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, identity can be determined using the BLAST program of the NCBI database. For determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics 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 this application, amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as well known and agreed in the art. When comparing two amino acid sequences, the term "amino acid difference" generally refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in the reference sequence compared to another sequence. In the case of substitutions, the substitutions will preferably be conservative amino acid substitutions, meaning that an amino acid residue is replaced by another amino acid residue that is chemically similar in structure and which affects the function, activity or other biological properties of the polypeptide Little or essentially no effect. Said conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably one amino acid residue within the following groups (i)-(v) replaced by another amino acid residue within the same group: (i) lesser Aliphatic non-polar or weakly polar residues: 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 non-polar 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 by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Val by Ile or Leu.

In this application, the term "detectable label" generally refers to a moiety having a detectable physical or chemical property that produces 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 (eg, FITC, rhodamine, lanthanide phosphors), enzymatic labels (eg, horseradish peroxidation) enzymes, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups (which can be passed through a labeled avidin (eg, a molecule containing a streptavidin moiety) and Fluorescent labels or enzymatic activity detectable by optical or calorimetric methods) and predetermined polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

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

In this application, the term "buffer" generally refers to the acid or base component (usually a weak acid or base) of a buffer or buffer solution. Buffers help maintain the pH of a given solution at or near a predetermined value, and are typically selected to complement its predetermined value. Suitably, a buffer is a single compound that produces the desired buffering effect, especially when the buffer is mixed with an appropriate amount (depending on the desired predetermined pH) of its corresponding "acid/base conjugate" ( and suitably capable of proton exchange), 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) can be used first and then acidified, for example with hydrochloric acid, or added to a solution of acetic acid (acidic), sodium hydroxide or sodium acetate until the desired pH.

In this application, "stabilizer" generally refers to a component that helps maintain the structural integrity of a biopharmaceutical drug, especially during freezing and/or lyophilization and/or storage (especially when exposed to stress ( stress)). This stabilizing effect can occur for a variety of reasons, although generally such stabilizers act as osmotic agents to reduce protein denaturation. As used in this application, stabilizers can be sugar alcohols (eg, inositol, sorbitol), disaccharides (eg, sucrose, maltose), monosaccharides (eg, dextrose (D-glucose)) , or various forms of the amino acid lysine (eg, lysine monohydrochloride, acetate or monohydrate) or salts (eg, sodium chloride).

In this application, the terms "stability" and "stable" generally refer to the resistance of an antibody in a formulation to aggregation, degradation or fragmentation under given conditions of manufacture, preparation, transportation, and storage. A "stable" formulation retains biological activity under given conditions of manufacture, preparation, transportation, and storage. Compared with the reference formulation, such as by high pressure size exclusion chromatography (HP-SEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea expansion technique (urea) Antibody stability can be assessed by the degree of aggregation, degradation, or fragmentation as measured by unfolding techniques), endogenous tryptophan fluorescence, differential scanning calorimetry, and/or ANS binding techniques. 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 this application, the term "administer" and similar terms are generally not limited to in vivo 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 compound can 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 this application, the terms "ex vivo" are used interchangeably with "in vitro" and generally refer to an activity in a controlled environment in cells, tissues and/or organs that have been removed from a subject.

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

In this application, the term "treating" generally refers to: (i) preventing the occurrence of a disease, disorder or condition in a patient who may be susceptible to a disease, disorder and/or condition but has not been diagnosed with the disease; (ii) inhibiting the disease , disease or condition, i.e. arresting its development; and (iii) alleviating the disease, disorder or condition, i.e. causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subsided.

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

In this application, "high expression" of a tumor antigen or tumor antigen generally refers to the level of expression of the tumor antigen in cells from a diseased area within a particular tissue or organ of a patient, such as a solid tumor, relative to normal cells from that tissue or organ abnormal expression levels. 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 this 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, and the like.

In this application, "positron emission tomography" or "PET" generally refers to a non-invasive nuclear medicine technique that produces a three-dimensional image of the location of a tracer in the body. The method detects pairs of gamma rays indirectly emitted by positron-emitting radionuclides (tracers) on bioactive molecules introduced into the body. PET imaging tools have a wide variety of uses and aid in drug development both preclinically and clinically. Exemplary applications include direct visualization of target saturation in vivo; monitoring uptake in normal tissue to anticipate toxicity or patient-to-patient variation; quantifying diseased tissue; tumor metastasis; and monitoring drug efficacy over time, or resistance over time The change.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, eg, about 0.5%, about 1%, about 1.5%, about 2% above or below the specified value , 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%.

In this application, the term "comprising" and its variants, including other forms such as "comprising", "including", etc., generally refers to the inclusion of other components, elements, values, steps, and the like.

Detailed description of the invention

combination

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

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

(iii) stabilizers and/or osmotic pressure regulators;

Wherein, the pH value of the composition is not lower than 5.

In certain embodiments, wherein the pH of the composition may be about 5.0-6.0. 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, wherein the pH of the composition may be about 5.5.

For example, the composition can include:

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

(iii) stabilizers and/or osmotic pressure regulators;

Wherein, the pH of the composition may be about 5.5.

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

In certain embodiments, wherein the concentration of the stabilizer may be from about 150 mM to about 200 mM.

In certain embodiments, the stabilizer may include a mixture of one or more of sucrose, trehalose, mannitol, glycine, methionine, and arginine.

In certain embodiments, wherein the stabilizer may comprise sucrose.

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

For example, the composition can include:

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

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

Wherein, the pH of the composition may be about 5.5.

In certain embodiments, wherein the stabilizer further comprises methionine.

In certain embodiments, wherein the methionine concentration is from about 0.2 mM to about 20 mM. For example, the methionine concentration can be about 0.3 mM to about 18 mM, 0.4 mM to about 16 mM, 0.5 mM to about 14 mM, 0.6 mM to about 12 mM, 0.7 mM to about 10 mM, 0.8 mM to about 8 mM. As another example, the methionine concentration can be about 0.2 mM to about 10 mM, 0.2 mM to about 5 mM, 0.2 mM to about 4 mM, 0.2 mM to about 3 mM, 0.2 mM to about 2 mM, 0.2 mM to about 1 mM.

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

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

For example, the composition can include:

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

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

Wherein, the pH of the composition may be about 5.5.

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

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

For example, the composition can include:

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

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

Wherein, the pH of the composition may be about 5.5.

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

For example, the composition can include:

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

Wherein, the pH of the composition may be about 5.5.

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

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

For example, the composition can include:

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

Wherein, the pH of the composition may be about 5.5.

In certain embodiments, wherein the osmotic pressure regulator may include a mixture of one or more of sodium chloride, magnesium chloride, dextrose, sorbitol, and sodium citrate. For example, the osmotic pressure regulator can be sodium chloride.

In certain embodiments, wherein the osmolyte concentration can be from about 100 mM to about 300 mM. For example, the osmolyte concentration can be about 110 mM to about 270 mM, about 120 mM to about 240 mM, about 130 mM to about 210 mM, about 140 mM to about 180 mM.

In certain embodiments, wherein the osmolyte concentration can be from about 140 mM to about 180 mM. For example, the osmolyte concentration can be about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM. As another example, the osmolyte concentration can be about 150 mM. As another example, the osmolyte concentration can be about 0.9% (w/v) (about 154 mM).

In certain embodiments, the composition may comprise:

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

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

For example, the composition can include:

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

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

Wherein, the pH of the composition may be about 5.5.

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

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

In certain embodiments, wherein the antigen-binding fragments may comprise Fab fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions (CDRs), scFv and/or VHHs.

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 domains may comprise:

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

CDR2, which comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence with one or more amino acid residue substitutions, deletions or additions relative to SEQ ID NO:4, such as comprising the amino acid sequence shown in SEQ ID NO:11; and

A CDR3 comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence with 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 CDR2 having the amino acid sequence shown in SEQ ID NO:5 CDR3.

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 CDR2 having the amino acid sequence shown in SEQ ID NO:5 CDR3.

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

In certain embodiments, wherein the variable domain may comprise the 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 can include:

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

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

Wherein, the pH of the composition may be about 5.5.

In certain embodiments, wherein the chelating agent may be selected from the group consisting of: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,10-tetraacetic acid) 4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N′,N″-tri acetic acid), NETA ([4-[2-(di-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triaznonan-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 the chelator can be linked via a lysine.

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

In certain embodiments, wherein the p-SCN-Bn-NOTA can be directly attached to the PD-L1 binding via an isothiocyanate group reacting with the amino group at the lysine terminus of the PD-L1 binding polypeptide peptide.

For example, the composition can include:

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

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

Wherein, the pH of the composition may be about 5.5.

In certain embodiments, the VHH binds 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 can be associated with the conjugate.

In certain embodiments, wherein the detectable label may be selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic particles, and enzymes.

The conjugates of the present application can be applied to Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) according to different labels. In tumor diagnosis, high-resolution tumor imaging can be provided and quantitative analysis can be performed through the images. The SPECT imaging may further include SPECT/CT imaging, and the PET imaging may further include PET/CT imaging, which may provide better imaging effects.

In certain embodiments, wherein the radionuclide can 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 ^be67Ga ^or68Ga .

In certain embodiments, the composition may comprise:

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

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 concentration of the sucrose may be from about 160 mM to about 180 mM.

In certain embodiments, it may also contain about 0.2 mM to about 20 mM 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 may be p-SCN-Bn-NOTA.

In certain embodiments, the composition may further comprise a detectable label, the detectable label ^being67Ga ^or68Ga .

For example, the composition can include:

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

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

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

Wherein, the pH of the composition may be about 5.5.

Detection/Diagnostic Use

On the other hand, the present application also provides a PD-L1 detection method, the method may comprise: administering an effective amount of the aforementioned composition.

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

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

For example, the method may include:

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

b) detecting the formation of complexes,

wherein the 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 for detecting and/or diagnosing a disease associated with PD-L1, the method may comprise: administering the aforementioned composition to a subject.

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 can be SPECT imaging. In some embodiments, the ECT imaging can be PET imaging. Imaging techniques and devices for scanning by SPECT or PET are well known in the art and any such known ECT imaging techniques and devices may be used.

For example, the method may include:

a) administering a composition described herein to a subject in need thereof; 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 PD-L1 expressing tumor.

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 (eg, metastatic malignant melanoma), bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, Gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumor and osteosarcoma. Examples of other cancers that can 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, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer , Fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder carcinoma, ureteral carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell Cancer, T-cell lymphoma, environment-induced cancer, including asbestos-induced cancer, and combinations of such cancers. The present invention can also be used to detect and/or diagnose metastatic cancer, particularly metastatic cancer expressing PD-L1 (Iwai et al. (2005) Int Immunol 17: 133-144).

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

a) administering a composition described herein to a subject in need thereof, and obtaining images (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 PD-L1 levels in one tumor or in several tumors that are equal to or higher than the levels required to be treated with PD-1 or a PD-L1 antagonist, then,

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

The present application also provides a method of monitoring the progress of anti-tumor therapy for a PD-L1-expressing tumor in a subject, the method may include:

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

b) administering anti-tumor therapy to the subject;

c) administering the composition to the subject at one or more subsequent time points, and obtaining images of at least a portion of the subject at each time point;

The size and location of the tumor at each time point were indicative of disease progression.

Kits and Imaging Agents

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 (eg, an anti-PD comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-2, 6-10, and 12-16) -L1 antibody), the chelator p-SCN-Bn-NOTA and the radionuclide ⁶⁷ Ga or ⁶⁸ Ga.

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

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

Without being bound by any theory, the following examples are only intended to illustrate the composition, preparation method and use of the present application, and are not intended to limit the scope of the invention of the present application. In this application, anti-PD-L1 antibodies are as disclosed in PCT/CN2019/093225, which is incorporated herein by reference in its entirety.

Example

Materials and Reagents

instrument

SHIMADZU HPLC with LabSolution Version 6.72SP

Denovix DS-11 Spectrophotometer

TSKgel G2000SWXL, inner diameter 7.8mm×30cm, 5μm, part number 0008540

MabPac RP, 2.1mm ID×100mm, 4μm, PN088647

Thermo ProPac CEX-10, 4x250mm, Product No. 054993, Serial No. 032566

Integrated Electronic Multichannel Pipettes, 1250µL, 300µL and 50µL

Spectra Max Plus Microplate Reader

Microplate Washer 405TS

Thermo Orion star A215 pH/Conductivity Meter

method

Unless otherwise indicated, all methods, steps, techniques and operations not specifically recited can and have been performed in a manner known per se, which would be understood by those skilled in the art. Reference is also made to eg standard handbooks, general prior art and other references cited therein.

SEC method:

The chromatographic column is TSKgel G2000SWXL 7.8mm×300mm, 5μm, silica gel packing; mobile phase is 50mM PB, 300mMNaCl, pH7.2; UV detection wavelength is 280nm; flow rate is 0.6ml/min; The elution time was 25min, and 50μg was injected.

RP-HPLC method:

The chromatographic column is Thermo Scientific ^TM MAbPac ^TM RP, 4 μm, 2.1×100 mm, mobile phase A is 0.1% TFA in UPW, mobile phase B is 0.1% TFA in 90% ACN; UV detection wavelength is 280 nm; flow rate is 0.6 ml/min; The column temperature was 80°C, gradient elution was performed, and the elution time was 31 min. Inject 10 μL.

UV280 concentration detection method:

This method is based on the characteristic absorption of both sdAb and Nota-Lys at 280nm. The OD value of the coupling product sdAbC at 280nm is the sum of the OD values of Nota-Lys and sdAb. According to the molar extinction coefficient and DAR value of sdAb and Nota-Lys at 280 nm, the concentration of sdAbC can be obtained.

Binding Active Methods:

The 96-well microtiter plate was coated with PDL1-Fc protein. After blocking, the reference substance and the test solution were added to the gradient dilution for antibody incubation. After the end, the enzyme-labeled secondary antibody was added for incubation. Finally, the color was developed by TMB and the reaction was terminated. After that, read the absorbance value A450 at the wavelength of 450nm on the microplate reader. The A450 value is inversely correlated with the concentration of the coupling stock solution. Taking the sample protein concentration as the X axis and the A450 value as the Y axis, use the four-parameter fitting regression model in the SoftMax Pro computer software to draw a curve to obtain the EC50 of each test sample, and report the sample by calculating the EC50 ratio of the standard and the sample. relative activity.

Example 1

1.1 Pre-formulation formula

In this example, the conjugate is SNA002-NOTA, which is formed by combining an anti-PD-L1 single-domain antibody (VHH) with p-SCN-Bn-NOTA, and the amino acid sequence of the VHH is as shown in SEQ ID NO: 10, and the VHH contains 3 lysine residues, the p-SCN-Bn-NOTA is directly attached to the VHH by reacting its isothiocyanate group with the amino group at the lysine terminus of the VHH. A schematic diagram of the structure of the VHH is shown in Figure 1. Wherein, the p-SCN-Bn-NOTA can be linked to a radioactive element (eg, ⁶⁷ Ga or ⁶⁸ Ga) through coordination.

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

Table 1 Pre-formulation formula

1.2 Experimental results

The formulation in Example 1 of the present application appears as a transparent and colorless solution under different storage conditions, and all samples are substantially free of visible particles.

The results of UV280 measurement of the formulation in Example 1 of the present application under different storage conditions are shown in Figure 2, and the results of SEC measurement are shown in Figure 3. The rate of change of the average drug/antibody ratio (Average DAR) (RP-HPLC The measurement results) are shown in FIG. 4 . Figure 5 shows the results of the ELISA assay of the formulation in Example 1 of the present application using T0 as a reference under different storage conditions.

The above results showed that under different conditions, there was no significant difference in the SEC and RP-HPLC detection of the samples under different conditions, and no residual NOTA increased in all samples. The F2 and F7 formulation formulations showed minimal variation across all test conditions. The relative activity of F3 and F8 decreased at 40°C for 4 weeks, and the relative antigen binding capacity of other samples did not change significantly. Use Recipe 2 or Recipe 7 for the next round of formulation confirmation studies.

Example 2

2.1 Preparation formula

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

Table 2 Formulation list of preparations

2.2 Experimental results

Table 3 shows the results of the formulation in Example 2 of the present application in UV 350nm measurement in the sample under stirring conditions.

table 3

制剂配方Formulation	350nm(Turbidity)350nm(Turbidity)	280nm(FIO)280nm (FIO)
F1F1	1.29511.2951	7.60497.6049
F2F2	1.74571.7457	8.78818.7881
F3F3	0.04410.0441	5.46675.4667
F4F4	1.70251.7025	8.61078.6107

The results showed that except F3 had no turbidity, all others were elevated, and the difference between F2 and F3 was only L-methionine. Subsequent confirmation experiments only used F2 and F3 for comparative studies.

The results of UV280 concentration measurement of the formulation in Example 2 of the present application under different storage conditions are shown in Figure 6, the results of SEC measurement are shown in Figure 7, and the results of RP-HPLC measurement are shown in Figure 8. Figure 9 shows the results of the ELISA assay of the formulation in Example 2 of the present application using T0 as a reference conjugate under different storage conditions.

The results showed that F1, F2 and F4 were turbid under stirring conditions. The UV350 turbidity, concentration, binding activity and SEC purity were detected respectively. Except for F3, the rest of the formulations were significantly reduced. Therefore F1 and F4 were discontinued after 4 weeks. By several key analytical methods, no significant differences were found between F2 and F3, except that F3 was the clearest appearance among the candidates after stirring. F3 has one more excipient, methionine, than F2, which is thought to be a stabilizer that protects the protein from conformational instability, aggregation caused by thermal and shock (stirring) stress, and chemical degradation.

Claims

A composition comprising:

(i) a conjugate comprising a programmed death ligand 1 (PD-L1) binding polypeptide and a chelating agent at a concentration of from about 0.1 mg/mL to about 50 mg/mL;

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

(iii) stabilizers and/or osmotic pressure regulators;

Wherein, the pH value of the composition is not lower than 5.
The composition of claim 1, wherein the pH of the composition is about 5.0-6.0.
The composition of any of claims 1-2, wherein the composition has a pH of about 5.2-5.8.
The composition of any one of claims 1-3, wherein the composition has a pH of about 5.4-5.6.
The composition of any one of claims 1-4, wherein the concentration of the stabilizer is from about 100 mM to about 300 mM.
The composition of any one of claims 1-5, wherein the concentration of the stabilizer is from about 140 mM to about 220 mM.
The composition of any one of claims 1-6, wherein the concentration of the stabilizer is from about 150 mM to about 200 mM.
The composition of any one of claims 1-7, wherein the stabilizer comprises a mixture of one or more of sucrose, trehalose, mannitol, glycine, methionine, and arginine.
The composition of any one of claims 1-8, wherein the stabilizer comprises sucrose.
The composition of claim 9, wherein the sucrose concentration is from about 150 mM to about 200 mM.
The composition of any one of claims 1-10, wherein the stabilizer further comprises methionine.
The composition of claim 11, wherein the methionine concentration is from about 0.2 mM to about 20 mM.
The composition of any one of claims 1-12, wherein the conjugate concentration is from about 0.2 mg/mL to about 40 mg/mL.
The composition of any one of claims 1-13, wherein the conjugate concentration is from about 0.5 mg/mL to about 20 mg/mL.
The composition of any one of claims 1-14, wherein the conjugate concentration is from about 1 mg/mL to about 10 mg/mL.
The composition of any one of claims 1-15, wherein the buffer comprises a mixture of one or more of acetate, citrate, histidine, glycine, succinate.
The composition of any one of claims 1-16, wherein the concentration of the buffer is about 10 mM to 80 mM.
The composition of any one of claims 1-17, wherein the concentration of the buffer is about 40 mM to 60 mM.
The composition of any one of claims 1-18, wherein the osmotic pressure regulator comprises a mixture of one or more of sodium chloride, magnesium chloride, dextrose, sorbitol, and sodium citrate.
19. The composition of any one of claims 1-19, wherein the osmotic pressure regulator is at a concentration of about 100 mM to about 300 mM.
20. The composition of any one of claims 1-20, wherein the osmolyte concentration is from about 120 mM to about 240 mM.
The composition of any one of claims 1-21, wherein the PD-L1 binding polypeptide comprises an antibody or antigen-binding fragment thereof.
The composition of any one of claims 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.
The composition of any one of claims 1-23, wherein the antigen-binding fragments comprise Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions (CDRs) ), scFv and/or VHH.
The composition of any one of claims 22-24, wherein the antibody or antigen-binding fragment thereof comprises at least one variable domain.
The composition of claim 25, wherein the variable domain comprises a VHH domain.
The composition of any one of claims 25-26, 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 the CDR3 having the amino acid sequence shown in SEQ ID NO:5.
The composition of any one of claims 25-27, 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 the CDR3 having the amino acid sequence shown in SEQ ID NO:5.
The composition of any one of claims 25-28, wherein the variable domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-2, 6-10, and 12-16.
The composition of any one of claims 25-29, wherein the variable domain comprises at least one lysine residue.
The composition of any one of claims 25-30, wherein the variable domain comprises 3 lysine residues.
The composition of any one of claims 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]triazane- 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).
The composition of any one of claims 1-32, wherein the PD-L1 binding polypeptide and the chelator are linked by lysine.
The composition of any one of claims 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.
The composition of any one of claims 1-34, wherein the chelating agent is p-SCN-Bn-NOTA.
The composition of claim 35, wherein the p-SCN-Bn-NOTA is directly attached to the PD-SCN by reacting an isothiocyanate group with the amino group of the lysine terminus of the PD-L1 binding polypeptide L1 binding polypeptide.
The composition of any one of claims 1-36, further comprising at least one detectable label.
The composition of claim 37, wherein the detectable label is associated with the conjugate.
The composition of any one of claims 37-38, wherein the detectable label is selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic particles, and enzymes.
The composition of claim 39, wherein the radionuclide is selected from the group consisting of: 110In , 111In , 177Lu , 18F , 52Fe , 62Cu , 67Cu , 67Ga , 68Ga , 68Ge , 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.
The composition of claim 40, wherein the radionuclide is67Ga or68Ga .
The composition of any one of claims 37-41, wherein the detectable label is detectable by positron emission tomography.
The composition of any one of claims 1-42, comprising:

(i) a conjugate comprising a PD-L1 binding polypeptide and a chelating agent at a concentration of about 1 mg/mL to about 10 mg/mL;

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

(iii) sodium chloride at a concentration of about 120 mM to about 240 mM as an osmotic pressure regulator;

wherein the pH of the formulation is from about 5.2 to about 5.8.
The composition of any one of claims 1-42, comprising:

(i) a conjugate comprising a PD-L1 binding polypeptide and a chelating agent at a concentration of about 1 mg/mL to about 10 mg/mL;

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

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

wherein the pH of the composition is from about 5.2 to about 5.8.
The composition of claim 44 having a pH of from about 5.4 to about 5.6.
The composition of any of claims 44-45, which has a pH of about 5.5.
The composition of any one of claims 44-46, the sucrose at a concentration of about 160 mM to about 180 mM.
The composition of any one of claims 44-47, further comprising about 0.2 mM to about 20 mM methionine as a stabilizer.
The composition of any one of claims 1-48, wherein the PD-L1 binding polypeptide is VHH.
The composition of any one of claims 1-49, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO: 10.
The composition of any one of claims 1-50, wherein the chelating agent is p-SCN-Bn-NOTA.
The composition of any one of claims 1-51, further comprising a detectable label, the detectable label being67Ga or68Ga .
A PD-L1 detection method, comprising: administering an effective amount of the composition of any one of claims 1-52.
The method of claim 53, wherein the administering is performed in vitro or ex vivo.
The method of claim 54, wherein the administering comprises any method of contacting a cell, organ or tissue with the combination.
The method of any of claims 53-55, further comprising detecting or quantifying the detectable label.
The method of any one of claims 53-56, further comprising obtaining an image of a cell, organ or tissue expressing PD-L1.
A method of detecting and/or diagnosing a disease associated with PD-L1, the method comprising: administering to a subject the composition of any one of claims 1-52.
59. The method of claim 58, further comprising detecting or quantifying the detectable label.
The method of any one of claims 58-59, further comprising subjecting the subject to ECT imaging.
The method of any one of claims 58-60, wherein the disease associated with PD-L1 comprises a tumor.
The method of claim 61, wherein the tumor highly expresses PD-L1.
Use of the composition of any one of claims 1-52 in the preparation of a medicament for detecting PD-L1.
The use of claim 63, wherein the drug is a PD-L1 imaging agent.
A PD-L1 imaging agent comprising the composition of any one of claims 1-52.
A kit comprising the composition of any one of claims 1-52.
The kit of claim 66, wherein the composition and the detectable label are present in separate dosage forms.