Polypeptides that specifically bind pdl1 and uses thereof

Abstract

The present application provides a polypeptide specifically binding to PDL1 and its use. Specifically, the present application relates to a polypeptide or an active fragment thereof capable of specifically binding to PDL1, and a fusion protein comprising the polypeptide or the active fragment thereof. The present application also provides a CAR-T cell capable of expressing the fusion protein, and the CAR-T cell is used to specifically kill tumor cells, such as lung cancer, multiple myeloma or acute myeloid leukemia. The CAR-T of the present application can be used as a therapeutic drug for tumor diseases, and provides a new method for the prevention and treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to peptides that specifically bind to PDL1 and their preparation and application. Background Technology

[0002] T cells are a type of lymphocyte that play a crucial role in cell-mediated immunity. They differ from other lymphocytes (such as B cells and natural killer cells (NK cells)) in that they possess the T cell receptor (TCR) on their cell surface. T helper cells, also known as CD4+ cells... + T or CD4 T cells express CD4 glycoprotein on their surface. Helper T cells are activated upon exposure to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, these cells proliferate rapidly and secrete cytokines that regulate immune responses. Cytotoxic T cells, also known as CD8... + T cells, or CD8 T cells, express CD8 glycoprotein on their cell surface. + T cells are activated upon exposure to peptide antigens presented by MHC class I molecules. Memory T cells are a subset of T cells that persist long-term and respond to relevant antigens, thus providing the immune system with a memory of past infections and / or tumor cells.

[0003] Chimeric antigen receptor (CAR) modified T cells, also known as CAR-T cells, are T cells genetically engineered under in vitro culture conditions to express exogenous anti-tumor genes. The CAR gene is an artificially designed gene fragment that encodes a protein primarily consisting of an extracellular recognition domain and an intracellular signal transduction domain: the former is a specific antibody fragment used to target and recognize specific molecules on the tumor surface; the latter is used to initiate an immune cell response after specific recognition, exerting cellular immunity. After genetic modification, T cells can produce chimeric antigen receptors on their surface. CARs are proteins that allow T cells to recognize specific proteins (antigens) on tumor cells. Genetically engineered CAR T cells can be grown in the laboratory until their numbers reach billions. These expanded CAR cells can then be infused into patients.

[0004] Programmed cell death ligand 1 (PDL1) possesses IgV and IgC-like regions, a transmembrane region, and a cytoplasmic tail. The cytoplasmic tail is involved in intracellular signal transduction, while IgV and IgC participate in intercellular signal transduction. PDL1 interacts with its receptor PD1 on T cells, playing a crucial role in the negative regulation of the immune response. This molecule exhibits a broad tissue expression profile, showing high expression in some tumor cell lines, and numerous studies have indicated its association with tumor immune escape mechanisms. The tumor microenvironment can induce widespread expression of PDL1 on tumor cells, promoting tumor development and growth, and inducing apoptosis of anti-tumor T cells.

[0005] After PD1 binds to PDL1, it transmits inhibitory signals, which can inhibit lymphocyte proliferation and activity, and inhibit CD4. + T cell differentiation into Th1 and Th17 cells and inhibition of inflammatory cytokine release all play a role in negative immune regulation. Under normal circumstances, the binding of PDL1 and PD1 can maintain peripheral lymphocytes' immune tolerance to self-antigens through the above-mentioned functions, thereby preventing the occurrence of autoimmune diseases. However, in the development and progression of tumors, PDL1 expressed by tumor cells binding to PD1 can promote tumor immune escape through its inhibitory effect on lymphocytes. Summary of the Invention

[0006] In view of this, the present invention provides a polypeptide that specifically binds to PDL1 and its uses. Specifically, the present invention relates to a polypeptide or an active fragment thereof that specifically binds to PDL1, and a fusion protein comprising the polypeptide or the active fragment thereof. The present invention also provides CAR-T cells capable of expressing the fusion protein, utilizing CAR-T cells to specifically kill tumor cells, such as multiple myeloma or acute myeloid leukemia. The CAR-T of the present invention can serve as a therapeutic agent for tumor diseases, providing a new method for the prevention and treatment of tumors.

[0007] Specifically, the present invention provides a polypeptide or an active fragment thereof capable of binding to PDL1, wherein the polypeptide has an amino acid sequence of SQGINHLRGVLQSSG (SEQ ID NO: 1) or a variant sequence of the amino acid sequence, wherein the variant sequence has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0008] By way of example, for the polypeptide or active fragment of the present invention, the variant sequence is an amino acid sequence in which 1, 2 or 3 amino acids are added, deleted or replaced in the amino acid sequence of SEQ ID NO: 1 while retaining the activity of binding PDL1.

[0009] The present invention also provides a fusion protein comprising the polypeptide or its active fragment described herein. Exemplarily, the fusion protein of the present invention further comprises a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., an epitope targeting human BCMA) or an additional antibody active fragment (e.g., an antibody or antibody active fragment targeting an epitope targeting human BCMA, or a ligand capable of binding to human BCMA).

[0010] The present invention also provides an antibody-drug conjugate comprising the polypeptide or its active fragment described herein, preferably, the conjugate being conjugated with pseudomonas extoxin (PE), more preferably PE24.

[0011] For the antibody-drug conjugate of the present invention, exemplarily, the drug is selected from the following: radiolabeled substances, 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digitalisin, haptens, immunogenic proteins, nucleic acid molecules having sequences complementary to the target, or any combination thereof; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or any combination thereof.

[0012] This invention also provides polynucleotides encoding the polypeptides or their active fragments, or fusion proteins described herein.

[0013] Preferably, the polynucleotide encoding the polypeptide of the present invention or its active fragment is as shown in SEQ ID NO: 2 (AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA) or its degenerate sequence or complementary sequence.

[0014] The present invention also provides isolated CAR-T cells or CAR-NK cells, wherein the CAR-T cells or CAR-NK cells are capable of expressing the polypeptides or active fragments thereof described in the present invention; wherein the CAR-T cells or CAR-NK cells are capable of expressing the fusion proteins described in the present invention; wherein the CAR-T cells or CAR-NK cells are capable of expressing the antibody-drug conjugates described in the present invention; and wherein the CAR-T cells or CAR-NK cells contain the polynucleotides described in the present invention.

[0015] The present invention also provides a vector comprising the polynucleotides described herein. Exemplarily, the vector is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Vesner-Medy virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0016] The present invention also provides an immune effector cell comprising the polynucleotides described in the present invention or the vectors described in the present invention, preferably wherein the immune effector cell is a T lymphocyte or a natural killer cell.

[0017] The present invention also provides a pharmaceutical composition comprising the polypeptide or an active fragment thereof described herein, the fusion protein described herein, the antibody-drug conjugate described herein, CAR-T cells or CAR-NK cells described herein, or immune effector cells described herein, and optionally, a pharmaceutically acceptable carrier.

[0018] The present invention also provides a method for preparing CAR-T cells or CAR-NK cells as described in the present invention, or containing immune effector cells as described in the present invention, comprising introducing the vector of the present invention into T lymphocytes or natural killer cells.

[0019] The present invention also provides the use of the above-mentioned polypeptide or its active fragment, the above-mentioned fusion protein, the above-mentioned antibody-drug conjugate, the above-mentioned CAR-T cell or CAR-NK cell, or the above-mentioned immune effector cell in the preparation of a medicament for treating and / or preventing cancer, wherein, by way of example, the cancer is lung cancer, multiple myeloma, and acute myeloid leukemia, preferably lung cancer.

[0020] The present invention also provides nucleotides encoding chimeric antigen receptors (CARs) comprising nucleotides encoding (1) an extracellular antigen-binding domain, (2) a transmembrane domain and (3) an intracellular signal transduction domain, characterized in that it further comprises nucleotides encoding a polypeptide of the present invention or an active fragment thereof.

[0021] For example, in the CAR of the present invention, the transmembrane domain is derived from one or more transmembrane domains selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD3ε, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154, ICOS and PD1.

[0022] For example, in the CAR of the present invention, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain and is selected from one or more of the following: CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C and B7-H3.

[0023] Furthermore, the CAR described in this invention may also exemplary include a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. Preferably, the hinge domain is derived from CD8α.

[0024] Furthermore, in the CAR described in this invention, the extracellular antigen-binding domain is targeted at tumor cell surface antigens.

[0025] For the polypeptides or their active fragments, fusion proteins, CARs, etc., described in this invention, variations thereof are also considered, such as their identity sequences or humanized sequences. Exemplarily, the identity sequence refers to a sequence that shares approximately 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more identity with the original sequence or reference sequence (e.g., SEQ ID NO: 1, 3, or 5). 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1% or above, 99.2% or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above.

[0026] For the polynucleotides of this invention, the invention also considers their degenerate or complementary sequences. Exemplarily, the degenerate sequence has homology with the original sequence or reference sequence of approximately 60% or more, approximately 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, or 88% or more. % or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1% or above, 99.2% or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above. Detailed Implementation

[0027] In vitro experiments have confirmed that the polypeptide of the present invention can bind to human PDL1 ligand, and CAR-T cells containing the polypeptide of the present invention can significantly enhance tumor cell killing activity.

[0028] Further explanation of chimeric antigen receptors

[0029] In some embodiments, the CAR of the present invention may include linker residues added between the various domains for appropriate spacing and conformation of the molecule, such as linkers comprising amino acid sequences that connect the VH and VL domains and provide spacer region functions compatible with the interactions of the two sub-binding domains, such that the resulting polypeptide maintains specific binding affinity for the target molecule. The CAR of the present invention may contain one, two, three, four, or five or more linkers. In particular embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any suitable amino acid length.

[0030] Exemplary examples of connectors include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible connectors known in the art, such as the Whitlow connector. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as links between domains of a fusion protein or some of those domains (e.g., the CAR described herein).

[0031] In a particular embodiment, the binding domain of the CAR is followed by one or more "spacer regions" or "spacer polypeptides," which act as linkers to decouple the antigen-binding domain from the effector cell surface, enabling proper cell-to-cell contact, antigen binding, and activation. In some embodiments, the spacer region is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer region may include naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge region amino acid sequences. In one embodiment, the spacer region includes the CH2 and CH3 domains of IgG1 or IgG4.

[0032] In some embodiments, the binding domain of a CAR may be followed by one or more "hinge domains" that decouple the antigen-binding domain from the effector cell surface to enable proper cell-cell contact, antigen binding, and activation. A CAR may include one or more hinge domains between its binding domain and transmembrane domain (TM). The hinge domain may be of natural, synthetic, semi-synthetic, or recombinant origin. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for the CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8α, CD4, CD28, PD1, CD152, and CD7), which may be wild-type hinge regions from these molecules or may be modified. In another embodiment, the hinge domain includes a PD1, CD152, or CD8α hinge region.

[0033] The transmembrane domain is part of the CAR, fusing an extracellular binding portion and an intracellular signaling domain to anchor the CAR to the plasma membrane of immune effector cells. The TM domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from the α, β, or ζ chain of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In one embodiment, the CAR of the present invention comprises a TM domain derived from CD8α or CD28.

[0034] In a particular embodiment, the CAR of the present invention includes an intracellular signaling domain. An "intracellular signaling domain" refers to a region of a protein that participates in transducing information about the binding of an effective extracellular binding domain to the tumor-specific surface antigen PDL1 into the immune effector cell to induce effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity, said cytotoxic activity including the release of cytotoxic factors to the target cell to which the CAR is bound) or other cellular responses induced by antigen binding to the extracellular CAR domain. The term "effective function" refers to the specialized function of an immune effector cell. For example, the effector function of a T cell may be cytolytic activity or an assisting or activating activity including cytokine secretion. The term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specialized function.

[0035] The CAR of the present invention comprises one or more co-stimulatory signaling domains to enhance the efficacy, expansion, and / or memory formation of T cells expressing the CAR receptor. As used herein, the term "co-stimulatory signaling domain" refers to the intracellular signaling domain of the CAR molecule that provides a second signal required for the effective activation and function of T lymphocytes upon binding to the antigen.

[0036] protein

[0037] The terms “protein,” “peptide fragment,” and “peptide” are used interchangeably unless otherwise stated and are used in their conventional sense as an amino acid sequence. Proteins are not limited to a specific length; for example, they may comprise full-length protein sequences or fragments of full-length proteins and may include post-translational modifications of peptides (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other modifications known in the art, including both naturally occurring and non-naturally occurring modifications.

[0038] In various embodiments, the CAR peptides or proteins of the present invention comprise a signal (or leader) sequence at the N-terminus of a protein that guides protein transfer during or after translation. The peptides can be prepared using a variety of well-known recombinant and / or synthetic techniques. Specifically, the peptides of the present invention comprise the CAR of this disclosure, or sequences having one or more (e.g., 1-20, 1-10, or 1-5) amino acid deletions, additions, and / or substitutions of the CAR disclosed herein.

[0039] Nucleic acid

[0040] As used herein, the term "polynucleotide" refers to mRNA, RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention comprise polynucleotides or variants having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity with any reference sequence described herein (e.g., SEQ ID NO: 2, 4, or 6), typically wherein the variant retains at least one biological activity of the reference sequence.

[0041] On the other hand, the polynucleotide sequence described in this invention may be a polynucleotide or its complementary sequence that has hybridized with the polynucleotide sequence of SEQ ID NO: 2 under strict conditions and encodes an active polypeptide.

[0042] The "strict conditions" described herein can be any of low-strict, medium-strict, or high-strict conditions, preferably high-strict conditions. For example, "low-strict conditions" can be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; "medium-strict conditions" can be 40°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide; and "high-strict conditions" can be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, and 52% formamide. Those skilled in the art should understand that higher temperatures yield polynucleotides with higher homology. Furthermore, those skilled in the art can choose a combination of factors affecting hybridization strictness, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, to achieve the appropriate strictness.

[0043] In addition, hybridizable polynucleotides can also be, by way of example, those that, when calculated using the same search software such as FASTA or BLAST with the system's default parameters, have approximately 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, or 90% of the content of the polynucleotide encoding SEQ ID NO: 5. Polynucleotides with 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of the same identity.

[0044] Nucleotide sequence identity can be determined using the BLAST algorithm rules of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90:5873, 1993). The programs BLASTN and BLASTX, based on the BLAST algorithm rules, have been developed (Altschul SF, et al: J MolBiol 215:403, 1990). When using BLASTN to analyze base sequences, the parameters can be set to score=100 and wordlength=12; similarly, when using BLASTX to analyze amino acid sequences, the parameters can be set to score=50 and wordlength=3. When using BLAST and GappedBLAST programs, the system can be configured with default parameter values ​​for each program.

[0045] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of well-established techniques known and available in the art. To express a desired polypeptide or protein, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Other exemplary vectors include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacteriophages (e.g., λ phage or M13 phage), and animal viruses. Examples of animal viral vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivoid papillomaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; and Lenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequence of the chimeric protein disclosed herein can be ligated into such expression vectors for expression of the chimeric protein in mammalian cells. The “control elements” or “regulatory sequences” present in the expression vector are the vector’s untranslated regions (e.g., origin of replication, promoter, enhancer, translation initiation signal (SD sequence or Kozak sequence) introns, polyadenylated sequences, 5′ and 3′ untranslated regions) that interact with host cell proteins for transcription and translation. The strength and specificity of these elements or sequences can vary. Depending on the vector system and host used, any number of suitable transcriptional and translational elements or sequences can be used, including broadly expressive promoters and inducible promoters.

[0046] Regarding ADC

[0047] Antibody-drug conjugate (ADC) technology is a target-directed technology that allows for the selective killing or inhibition of the growth or division of cancer cells. Typically, ADCs work by using antibodies to target cancer cells and then releasing a toxic substance (i.e., a drug) into the cells, thereby inducing cell death. Because ADC technology allows for the precise delivery of drugs to target cancer cells and release under specific conditions while minimizing collateral damage to healthy cells, it increases the efficacy of therapeutic or targeted antibodies and reduces the risk of adverse reactions.

[0048] The basic structure of an antibody-drug conjugate can be either "antibody-linker-drug active molecule" or "antibody-drug active molecule" (without a linker). For conjugates with a linker, the linker allows the drug to exert its effect on target cancer cells, for example, after separation from the antibody (e.g., via enzyme-mediated hydrolysis) and after the drug reaches the target cells. The linker also functions by connecting the antibody and the drug. The efficacy and toxicity of the antibody-drug conjugate thus depend in part on the stability of the linker; therefore, the linker plays an important role in drug safety.

[0049] Linkers in antibody-drug conjugates can be broadly classified as either non-cleavable or cleavable. Many non-cleavable linkers utilize a thioether attached to the antibody, the thioether containing the antibody's cysteine ​​residue. The drug with this side-conjugation typically cannot separate from the antibody in vivo and may experience reduced efficacy. In the widely used thiol-maleimide method, the antibody-drug conjugate is unstable, which can cause the drug to separate from the conjugate before or after it reaches the target cell. Cleavable linkers can be, for example, hydrolyzed by lysosomal enzymes. Cleavable linkers can contain disulfide bonds, such as those containing the antibody's cysteine ​​residue. Disulfide linkers that allow dissociation via thiol exchange reactions rely to some extent on the uptake of the antibody-drug conjugate into the target cell and the exposure of the disulfide to the cytosol as a reducing environment. However, because various types of thiols (e.g., albumin and glutathione) are present in the blood, the drug may separate from the antibody before reaching its target.

[0050] To replace chemically unstable linkers, such as hydrazone and disulfide linkers, which are poorly stable under physiological extracellular conditions, there is a need for linkers that are stable under physiological extracellular conditions. Furthermore, there is a need for linkers with high plasma stability to improve therapeutic applicability, since drugs should be released only into the cells targeted by the proteins they are linked to, rather than outside the cells.

[0051] Existing literature has reported novel methods for preparing antibody-drug conjugates; for example, see U.S. Patent Publication No. 2012 / 0308584. Further improvements are possible.

[0052] The CAR or antibody of the present invention can still be conjugated to the antigen-binding domain with active drug molecules, such as erlotinib, lymphokines, botulinum toxin, affinity ligands, radiolabeled substances, immunomodulatory compounds, anticancer agents, ribozymes, etc.

[0053] carrier

[0054] In a particular embodiment, cells (e.g., immune effector cells, such as T cells) are transduced using a retroviral vector (e.g., a lentiviral vector) encoding CAR and the polypeptide of the present invention or an active fragment thereof.

[0055] Retroviruses are a common tool for gene delivery. In particular implementations, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) into cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy, which is then covalently integrated into the host genome. Once integrated into the host genome, the virus is called a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes required to produce new viral particles.

[0056] Exemplary retroviruses suitable for particular implementations include, but are not limited to: Moloney mouse leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), mouse stem cell virus (MSCV), and Rouss sarcoma virus (RSV), and lentiviruses.

[0057] As used herein, the term "lentivirus" refers to a group (or genus) comprising a number of retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); viscena-maedivirus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In a particular embodiment, the lentivirus is used to deliver a CAR-containing polynucleotide into cells.

[0058] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, for example, inserted into, the vector nucleic acid molecule. Vectors may include sequences that guide autonomous replication within the cell, or sequences sufficient to allow integration into the host cell's DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, granules, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0059] As will be apparent to those skilled in the art, the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer. Nucleic acid molecules include virus-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically comprise a variety of viral components and sometimes also include host cell components other than nucleic acids.

[0060] The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid primarily derived from retroviruses that contains structural and functional genetic elements or portions thereof. The term "lentivirus" refers to the genus of the family Retroviridae, which can effectively infect non-periodic and post-mitotic cells; they can transfer significant amounts of genetic information into the host cell's DNA, making them one of the most efficient methods of gene delivery vectors.

[0061] Therefore, in a preferred embodiment, the present invention relates to a method of transfecting cells with an expression vector encoding a CAR. For example, in some embodiments, the vector contains additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly-A signals, and / or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by transposon sequences, such that transposases are present to allow the coding sequence to be integrated into the genome of the transfected cells.

[0062] In some embodiments, genetically transformed cells are further transfected with a transposase, which promotes the integration of the CAR coding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or protein such that the transposase does not undergo long-term expression in the transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA containing a cap and a poly-A tail). Any transposase system can be used according to embodiments of the invention. However, in some embodiments, the transposase is a salmon-type Tel-like transposase (SB). In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB 100X transposases (see, for example, Mates et al., 2009, Nat Genet. 41(6): 753-61 or US9228180, which are incorporated herein by reference). For example, the method may include electroporation of cells having mRNA encoding SB 10, SB 11, or SB 100X transposases.

[0063] Sequence variants:

[0064] Sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments, and / or CARs (e.g., those defined by percentage sequence identity) are also included within the scope of this invention, maintaining similar binding properties of the invention. These variants exhibit alternative sequences but maintain substantially the same binding properties, such as target specificity, because the particular sequence provided is known to be a functional analog or functionally similar. Sequence identity refers to the percentage of identical nucleotides or amino acids when sequence alignment is performed.

[0065] As used in this paper, “sequence identity” refers to the degree of sequence similarity based on nucleotide-nucleotide or amino acid-amino acid sequences within a comparison window. Therefore, the “percentage of sequence identity” can be calculated as follows: Compare two best-aligned sequences within the comparison window, determine the number of positions on both sequences that contain the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to generate the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the percentage of sequence identity. This includes nucleotides or polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any reference sequence described herein, wherein the polypeptide variant typically retains at least one biological activity of the reference polypeptide.

[0066] Those skilled in the art will understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or proteins as described herein. Some of these polynucleotides have minimal homology or sequence identity with the nucleotide sequence of any natural gene. Nevertheless, the present invention specifically considers polynucleotides that vary due to differences in codon usage. Deletions, substitutions, and other variations in sequences falling within the aforementioned sequence identity are also included in the present invention.

[0067] Protein sequence modifications that can occur through substitution are also included within the scope of this invention. Substitution, as defined herein, is a modification of the amino acid sequence of a protein, whereby one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the primary protein. Substitutions can be made, preferably without significantly altering the function of the protein. As with additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering protein function. This is especially true when the modification involves a “conserved” amino acid substitution, where one amino acid replaces another amino acid with similar properties. Such “conserved” amino acids can be natural or synthetic amino acids that can be substituted due to size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. Typically, many amino acids can be substituted with conserved amino acids without adversely affecting the function of the protein.

[0068] Generally, the nonpolar amino acids Gly, Ala, Val, Ile, and Leu; the nonpolar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu represent conserved amino acid groups. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be substituted for each other, even though they belong to different groups.

[0069] Substitutional variants involve removing at least one amino acid residue from an antibody molecule and inserting a different residue at its position. For substitutional mutagenesis to occur, the most interesting sites include hypervariable regions, but FR alterations are also considered. If such substitution leads to changes in biological activity, more extensive alterations can be introduced, and products can be screened.

[0070] Gene-modified cells and immune cells

[0071] In particular embodiments, the present invention contemplates genetically modified cells to express the CAR of the present invention for use in cancer or related conditions. As used herein, the terms “genetically modified” or “genetically engineered” refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are used interchangeably. As used herein, the term “gene therapy” refers to the introduction of additional genetic material in the form of DNA or RNA into the total genetic material of a cell, which restores, corrects, or modifies gene expression, or is used to express therapeutic peptides (e.g., CARs or ADCs). In particular embodiments, the CAR of the present invention is introduced into and expressed in immune effector cells to redirect them to a target antigen of interest.

[0072] "Immune cells" or "immune effector cells" are any cells of the immune system that have one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC).

[0073] The immune effector cells of the present invention can be autologous or non-autologous (“non-self”, such as allogeneic, syngeneic, or allogeneic). As used herein, “autologous” refers to cells derived from the same subject, which is a preferred embodiment of the invention. As used herein, “allogeneic” refers to cells of the same species as the subject or patient but genetically different. As used herein, “syngeneic” refers to cells that are genetically identical but derived from different subjects. As used herein, “alienic” refers to cells from a different species. In a preferred embodiment, the cells of the present invention are autologous or allogeneic.

[0074] Exemplary immune effector cells used with the CAR of the present invention include T lymphocytes. The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3- and CD56-positive non-major histocompatibility complex (MHC) cells, which are restricted natural killer (NK)-like T lymphocytes. T cells can be T helper cells (Th), such as T helper cell 1 (Th1) or T helper cell 2 (Th2). T cells can be helper T cells or cytotoxic T cells or any other T cell subset. Other exemplary T cell populations suitable for particular embodiments include naive T cells and memory T cells.

[0075] For example, when reintroduced into a patient after autologous cell transplantation, the CAR-modified T cells of the present invention described herein can recognize and kill tumor cells. CIK cells, compared to other T cells, can exhibit enhanced cytotoxic activity, and therefore represent a preferred embodiment of the immune cells of the present invention.

[0076] As those skilled in the art will understand, other cells can also be used as immune effector cells with the CAR described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitor cells of effector cells, which can be induced to differentiate into immune effector cells in vivo or in vitro.

[0077] This invention provides a method for preparing immune effector cells expressing the CAR of this invention. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from an individual, such that the immune effector cells express one or more CARs as described herein. In some embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. These cells can then be directly re-administered to the individual. In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express a CAR. In this regard, the immune effector cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express the CAR of this invention).

[0078] In a particular embodiment, the cell source is obtained from the subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In a particular embodiment, the CAR-modified immune effector cells comprise T cells. T cells can be obtained from a number of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from blood units collected from the subject using any technique or combination of techniques known to those skilled in the art, such as by sedimentation and antibody-conjugated bead methods. In one embodiment, cells from an individual's circulating blood are obtained via component blood collection. Component blood products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected via component blood collection can be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing. Cells can be washed with PBS or another suitable solution free of calcium, magnesium, and most divalent cations. As will be understood by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as using a semi-automatic flow-through centrifuge (e.g., Cobe2991 cell processor, Baxter CytoMate, etc.). After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In some embodiments, unwanted components of the blood sample can be removed from the cells directly resuspended in the culture medium.

[0079] In some implementations, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting mononuclear cells (e.g., by PERCOLL™ gradient centrifugation). Specific T cell subsets can be further isolated using positive or negative selection techniques. One method that can be used is cell sorting and / or selection using a mixture of monoclonal antibodies targeting cell surface markers present on negatively selected cells via negative magnetic immunoadhesion or flow cytometry.

[0080] PBMCs can be directly genetically modified to express CAR using the methods of this invention. In some embodiments, T lymphocytes are further isolated after PBMC isolation, and in some embodiments, cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before or after genetic modification and / or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying each associated cell surface antigen in these types of CD8+ cells.

[0081] Immune effector cells (e.g., T cells) can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In a particular embodiment, immune effector cells (e.g., T cells) are genetically modified with the chimeric antigen receptor of the present invention (e.g., transduced with a viral vector containing nucleic acid encoding a CAR), and then activated and expanded in vitro. In various embodiments, methods described, for example, in U.S. Patent Nos. 5,858,358; 6,905,681; 7,067,318; 7,232,566; 5,883,223; 6,797,514 and 6,867,041, can be used to activate and expand T cells before or after genetic modification to express a CAR.

[0082] In another embodiment, for example, a mixture of one, two, three, four, five or more different expression vectors can be used as a donor population for genetically modifying immune effector cells, wherein each vector encodes a different chimeric antigen receptor protein (e.g., a CAR variant sequence) as described in this invention. The resulting modified immune effector cells form a mixed population of modified cells, a subset of which express more than one different CAR protein.

[0083] In one embodiment, the present invention provides a method for storing immune effector cells expressing genetically modified mouse, human, or humanized CAR proteins that target tumor antigens, comprising cryopreserving the immune effector cells so that the cells remain viable upon thawing. A subset of the CAR protein-expressing immune effector cells can be cryopreserved using methods known in the art to provide a permanent source of such cells for future treatment of patients with cancer. If needed, the cryopreserved transformed immune effector cells can be thawed, grown, and expanded to obtain more such cells.

[0084] Compositions and Formulations

[0085] The compositions of the present invention may comprise one or more polypeptides, polynucleotides, carriers containing such polynucleotides, genetically modified immune effector cells, etc., as considered herein. The compositions include, but are not limited to, pharmaceutical compositions. A “pharmaceutical composition” means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution, which is administered alone or in combination with one or more other therapeutic agents to cells or animals. It should also be understood that, if desired, the compositions of the present invention may also be administered in combination with other pharmaceutical agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are practically no limitations on other components that may be included in the compositions, provided that additional components do not adversely affect the composition’s ability to deliver the intended therapy.

[0086] The term “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissue contact with reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications and in proportion to a reasonable benefit / risk ratio.

[0087] As used herein, “pharmaceuticalally acceptable carriers, diluents, or excipients” include, but are not limited to, any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that have been approved by the U.S. Food and Drug Administration or the China Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus gum; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable oils; paraffin wax; organosilicon; bentonite; silicic acid; zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and any other compatible substances used in pharmaceutical preparations.

[0088] In a particular embodiment, the composition of the present invention comprises an amount of the CAR-expressing immune effector cells of the present invention. As used herein, the term “amount” refers to an “effective amount” of genetically modified therapeutic cells (e.g., T cells) that achieve a beneficial or desired preventive or therapeutic outcome (including clinical outcomes).

[0089] "Prophylactic effective dose" refers to the amount of genetically modified therapeutic cells that effectively achieve the desired preventative outcome. It is usually, but not always, less necessary because the preventative dose is used in subjects before or in the early stages of disease, so the prophylactic effective dose is less than the therapeutic effective dose. The term "prophylaxis" does not necessarily mean the complete prohibition or prevention of a specific medical condition. Prophylaxis also refers to reducing the risk of developing a particular medical condition or worsening its symptoms.

[0090] The “therapeutic effective amount” of genetically modified therapeutic cells can vary depending on various factors, such as disease state, age, sex, and individual weight, as well as the ability of stem cells and progenitor cells to elicit a desired response in an individual. Therapeutic effective amount is also a amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the virus or transduced therapeutic cells. The term “therapeutic effective amount” includes the amount that effectively “treats” a subject (e.g., a patient). When indicating a therapeutic amount, the precise amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. It can generally be specified that pharmaceutical compositions containing T cells described herein can be administered in quantities of 10... 2 Up to 1010 Cells / kg body weight, preferably 10 5 Up to 10 6 The dosage is administered at 1 cell / kg body weight (inclusive of all integer values ​​within these ranges). The number of cells will depend on the end use of the composition and the cell type contained therein. For the uses provided herein, cells are typically in volumes of 1 L or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Therefore, a cell density greater than 10 is generally desired. 6 cells / ml, typically greater than 10 7 cells / ml, typically 10 8 Cells / ml or higher. A clinically relevant number of immune cells can be allocated to multiple infusions, with the cumulative number of infusions equal to or exceeding 10. 5 10 6 10 7 10 8 10 9 10 10 10 11 Or 10 12 Cells. In some embodiments of the invention, particularly because all infused cells will be redirected to a specific target antigen, a lower number of cells can be administered. CAR-expressing cell compositions can be administered multiple times at doses within these ranges. For the patient receiving treatment, the cells can be allogeneic, syngeneic, allogeneic, or autologous.

[0091] Generally, compositions comprising activated and expanded cells as described herein can be used to treat and prevent diseases in immunocompromised individuals. In particular, compositions comprising the CAR-modified T cells of the present invention are used to treat B-cell malignancies. The CAR-modified T cells of the present invention can be administered alone or as pharmaceutical compositions in combination with carriers, diluents, excipients, and / or other components (e.g., IL-2) or other cytokines or cell populations. In particular embodiments, the pharmaceutical compositions of the present invention comprise a quantity of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0092] The pharmaceutical compositions of the present invention comprising a population of immune effector cells (e.g., T cells) expressing CAR may include: a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration.

[0093] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may include one or more of the following: sterile diluents (e.g., water for injection), saline solutions (preferably physiological saline, Ringer's solution, isotonic sodium chloride), fixed oils (e.g., synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and buffers, such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0094] In a particular embodiment, the compositions of the present invention comprise an effective amount of CAR-expressing immune effector cells alone, or in combination with one or more therapeutic agents. Thus, the CAR-expressing immune effector cell compositions can be administered alone or in combination with other known cancer treatments such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents are acceptable in the art as standard treatment for specific disease states (e.g., specific cancers) as described herein. Exemplary therapeutic agents of consideration include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapy agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents.

[0095] The terminology and expressions used herein are descriptive rather than restrictive, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, those skilled in the art may employ modifications and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims. Attached Figure Description

[0096] Figure 1 : Schematic diagram of the structure of pShort-P8C phage particle.

[0097] Figure 2 Competitive ELISA assay of PP01 peptide against PD1.

[0098] Figure 3 : Construction diagram of Mesothelin scFv CAR-PP01 fusion protein.

[0099] Figure 4 Secretory PDL1 inhibitory peptides promote the in vitro cell-killing effect of CAR-T.

[0100] To illustrate the present invention more clearly, the following embodiments are described in detail. However, these embodiments are merely exemplary descriptions of the present invention and should not be construed as limiting the present application.

[0101] Example 1: Construction of a phage display peptide library

[0102] Following the method reported by Sidhu, a random peptide library with an amino acid length of 8-15 was constructed. The specific construction method is as follows: 1) A mutant of the M13 phage P8 protein was inserted into the pShort phage particle (obtained through pBluescript modification), and a linker sequence and a mutation site sequence (including a terminator sequence) were inserted at the C-terminus of the P8 protein. The resulting new phage particle was named pShort-P8C. Figure 1 2) Total gene synthesis containing (NNK) 8-15 The mutation primers (Shanghai Sangon Biotech) are shown in Table 1; 3) pShort-P8C was electroporated into CJ236 competent bacteria, cultured overnight, and ssDNA was extracted using the M13ssDNA extraction kit. The ssDNA was then mutated using the Kunkel mutation method (NNK). 8-15 4) The Kunkel reaction product cccDNA was electroporated into SS320 supercompetent bacteria containing M13K07 helper phage. After resuscitation with SOC, the bacteria were cultured in a shaker for 1 hour (37°C, 220 rpm). Then, the bacteria were transferred to 1L 2YT / Carb / Kan medium and cultured overnight in a shaker (37°C, 220 rpm, approximately 20 hours). 5) The phages were isolated and purified using the PEG8000 / NaCl precipitation method. The phages were resuspended in PBST buffer to obtain a final concentration of 10. 13 A phage display random peptide library of pfu / ml was stored at -80°C.

[0103] Table 1: Mutation primers for random peptide libraries

[0104]

[0105] Example 2: Bioscreening and confirmation of anti-PDL1 phage

[0106] The target antigen PDL1-his (purchased from Acro Biosystems) was coated onto a maxi-sorp 96-well plate. A peptide library and bacteria were added sequentially, and the plate underwent 3-5 rounds of high-throughput screening cycles (incubation, binding, elution, and amplification). High-affinity positive phages were gradually enriched. Single clones were then picked from the corresponding solid plates, positive clones were amplified, and the resulting phages were collected. The specificity of the phages was determined by Phage-ELISA, and finally, DNA sequencing was performed to obtain the genetic information.

[0107] The His-tagged PDL1 protein (PDL1-His) was coated into Maxisorp 96-well plates at 4°C. After gently pouring out the coating buffer, 1% polyvinyl alcohol (PVA) solution was added, and the plates were blocked at room temperature for 1 hour. 100 μL of phage library (10 μL / well) was added to each well. 13 The phages were bound for 2 hours. Next, each well was washed 8 times with PT buffer (0.05% Tween 20 dissolved in PBS buffer), followed by incubation with 100 μL of 100 mM HCl for 5 minutes to elute the bound phages. The phage eluent was transferred to a 1.5 mL centrifuge tube, and the pH was adjusted by adding 1 M Tris-HCl (pH 8.0). Half of the phage eluent was added to 1 mL of E. coli NEB5-alpha F' in good growth condition and mixed well (E. coli NEB 5-alpha F' was cultured in 2xYT medium containing 10 μg / mL tetracycline until OD600 = 0.8), and incubated at 37°C for 1 hour. Then, 10 μg / mL of HCl was added to the centrifuge solution. 10 PFU M13K07 helper phage was cultured for another hour. Infected E. coli were inoculated into 50 mL of 2xYT medium containing 50 μg / mL carbenicillin and 25 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. The next day, phages were collected by precipitation with PEG8000 / NaCl solution, resuspended in PBS solution, and used for the next round of screening. Before adding the helper phage, 10 μL of bacterial culture was diluted and plated onto LB agar plates containing 50 μg / mL carbenicillin and incubated overnight at 37°C. The number of colonies produced by the phage eluent from wells coated with PDL1-His and the number of colonies produced by the phage eluent from wells coated only with PVA were counted to calculate the enrichment rate.

[0108] Through the above screening steps, enrichment was obtained after the third round of screening (the number of clones in the PDL1-His wells was 147 times that in the PVA wells). Several single clones were selected from the enriched bacteria, and the phage specificity was determined by Phage-ELISA. The results are shown in Table 2 below, where an OD value > 4 indicates overflow on the microplate reader.

[0109] Table 2: Anti-PDL1 recombinant phage monoclonal ELISA screening

[0110]

[0111] Select the clones with relative OD from the above clones. 450 Nine large monoclonal strains were obtained, and their polypeptide sequences were determined by sequencing. The sequencing results showed that the sequences of these nine monoclonal strains were completely identical; therefore, the polypeptide was named PP01, and its sequence information is as follows:

[0112] PP01 DNA seq: AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA (SEQ ID NO: 2)

[0113] PP01 protein seq: SQGINHLRGVLQSSG (SEQ ID NO: 1)

[0114] Meanwhile, to verify the specificity of the PP01 clone and to exclude the influence of the recombinant protein His tag, PP01 phage was used to perform binding assays (Phage-ELISA) on various proteins. The results are as follows:

[0115]

[0116]

[0117] The ELISA results above indicate that the PP01 clone is a PDL1-specific phage clone and can be used as a candidate strain for anti-PDL1 antibodies.

[0118] Example 3: Affinity assay of peptides and their competitive inhibition of PDL1-PD1 binding

[0119] PP01 peptide was synthesized using a solid-phase synthesis method, and the synthesized PP01 peptide was analyzed by HPLC and mass spectrometry, showing that the peptide purity was >95%.

[0120] The binding of the peptide to PDL1-His protein was determined using an ELISA method. Specifically, 2 μg / ml of PP01 peptide was coated into Maxisorp 96-well plates (100 μL / well) and incubated overnight at 4°C. After pouring out the coating solution, 1% polyvinyl alcohol (PVA) solution was added, and the plates were blocked at room temperature for 1 hour. Each well was washed three times with PT buffer, and then 100 μL of different concentrations of PDL1-His protein was added, followed by incubation for 1 hour at room temperature. Each well was washed three times with PT buffer and three times with PBS buffer, and then 50 μL of TMB was added, followed by incubation for 5-10 minutes. The reaction was terminated by adding 1M phosphate, and the OD450 reading was recorded on a microplate reader. The experimental results showed that the PP01 peptide can recognize and bind to PDL1-His protein.

[0121] The competitive inhibitory effect of PP01 peptide on the binding of PDL1-PD1 was detected using a competitive ELISA assay. Specifically, 5 μg / ml PD1-hFc was coated into Maxisorp 96-well plates (100 μL / well) and incubated overnight at 4°C. After pouring out the coating solution, 1% polyvinyl alcohol (PVA) solution was added and the plates were blocked at room temperature for 1 hour. Each well was washed three times with PT buffer, and then 200 μL of a mixture of 1 μg / ml PDL1-his and different concentrations of PP01 peptide was added and incubated at room temperature for 1 hour. Each well was washed three times with PT buffer, and then 100 μL of HRP-labeled mouse anti-his antibody was added and incubated at room temperature for 1 hour. Each well was washed three times with PT buffer and three times with PBS buffer, and then 50 μL of TMB was added and incubated for 5-10 minutes. The reaction was terminated by adding 1M phosphate, and the OD450 reading was recorded on a microplate reader. Experimental results (see...) Figure 2 This indicates that the PP01 peptide can competitively inhibit PDL1-PD1 binding, with an IC50 of 31 ng / ml.

[0122] Example 4: Secretory PDL1 inhibitory peptides promote the in vitro cell-killing effect of CAR-T.

[0123] The PP01 polypeptide gene was expressed in tandem with the CAR gene to investigate whether secreted PP01 could promote the killing effect of CAR-T cells. The constituent elements of the CAR-PP01 lentivirus are shown in [reference needed]. Figure 3 .

[0124] 1. Preparation of lentiviral vector: 1) Gene synthesis of Mesothelin scFv-CAR-P2A-PP01 DNA sequence (its amino acid sequence and DNA sequence are shown below); 2) Insertion of the fragment into the PWPXLD-kana vector using homologous recombination or enzyme digestion ligation; transformation of the recombinant vector into Escherichia coli strain Stbl3, screening with kanamycin, and sequencing of single clones to obtain the correct recombinant plasmid; then expansion culture of host bacteria containing the recombinant plasmid, and use an endotoxin removal kit to obtain sterile endotoxin-free plasmid, i.e., PWPXLD plasmid vector containing CAR gene fragment; 3) Simultaneously, transformation of lentiviral packaging helper plasmids psPax2 and PMD2.0G into DH5α, screening with ampicillin, and extraction of plasmids.

[0125] 2. Preparation of CAR-expressing lentivirus (Lenti-CAR): 1) Inoculate 3x10 6 1) Place 293T cells in a culture dish; 2) After 24 hours, mix the viral plasmids (CAR-PWPXLD: 9 μg, psPax2: 9 μg, and PMD2.0G: 4.5 μg), add 0.45 mL of sterile water and 50 μL of 2.5 M CaCl2 solution, then add 500 μL of 2×BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4) dropwise, vortexing the solution; incubate at room temperature for 30 minutes; then add the mixture to the 293T culture medium and mix gently; 3) After 18 hours, replace the culture medium with DMEM medium containing 2% FBS; 4) After 48 hours, collect the culture supernatant, centrifuge to remove cell debris, and filter the supernatant through a 0.45 μm filter; then add one-third volume of TAKARA lentiviral concentrate ( Concentrator (product number 631231), mix well and let stand overnight at 4°C. Centrifuge at 4°C and 1500g for 45 minutes, resuspend the precipitate in PBS to obtain the virus solution, aliquot and store at -80°C.

[0126] 3. Preparation of (Mesothelin scFv-CAR-P2A-PP01)-T cells (CART-PP01): 1) On Day 0, peripheral blood was collected, and lymphocytes (PBMCs) and plasma were separated; CD3+ T cells were sorted from PBMCs; the cell suspension was adjusted to a concentration of 1×10⁻⁶. 6 1) Incubate at 12-well plate with 5 μg / ml of fibronectin solution; add dynabeads (Thermo Fisher) for stimulation; 2) On Day 0, add fibronectin solution (5 μg / ml) to the 12-well plate. 2), coat overnight at 4℃; 3) On Day 1, discard the fibronectin solution in the 12-well plate, block with 2% BSA for 30 minutes; remove the blocking solution, and rehydrate at 750 μL / 4.5 cm. 2 Add virus solution, incubate at 37°C for 4-6 hours; collect stimulated T cells, and take 10 6 Add the cells to each well and incubate at 37°C with 5% CO2; 4) On Day 3, adjust the T cell concentration to 5 × 10⁻⁶ cells / well. 5 Replace all culture medium with fresh medium at / ml; 5) On Day 5, adjust the T cell concentration to 5×10⁹ / ml. 5 / ml, the expression of CAR and PP01 peptide in T cells was detected by flow cytometry.

[0127] Mesothelin scFv-CAR-T cells were prepared using the methods described in steps 1, 2, and 3 above.

[0128] In vitro cell killing experiments were conducted using lactate dehydrogenase (LDH) as the detection index. Specifically, T cells were mixed with target cells MSTO-211H (human lung cancer cells), K562, and A549 at a ratio of 5:1 and cultured in DMEM complete medium at 37°C and 5% CO2 for 20 hours. The LDH content in the CART-PP01 experimental group, CART cell group, spontaneous release group of target cells, and solution background correction group was detected by a microplate reader at specific wavelengths, and the actual target cell killing ratio was calculated. Experimental results (see...) Figure 4 The CAR-T-PP01 experimental group (labeled as "CART-PP001") indicates that secretory PDL1 inhibitory peptides can promote the anti-tumor effect of CAR-T.

[0129] Mesothelin scFv antibody DNA sequence

[0130] GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGGCATCAGCAACTACCTGGCCTGGTTTCAACAGAAACCAGGAAAAGCTCCGAAGTCCCTGATTTACGCCGCCAGCAGCCTGCAGTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCGGATCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTTTCTTCTCTCCTCTGACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGAGGAGGAGGTTCCGGAGGAGGAGGTAGCGGCGGAGGGGGTTCTCAGGTTCAGCTGGTGGAGTCTGGCGGTGGCGTGGTGCAGCCAGGGAGGTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCACCTTCAGCAGCTACGGCATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGTGATCAGCTACGACGGCAGCAACAAGTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCCGTGACAATTCCAAAAACACACTGTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCTCATTACTCTTACGTTCCGTGGTCTTACTCTGGTTACTACTACTACTACGGATTCGACATTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG(SEQ ID NO:4)

[0131] Amino acid sequence of Mesothelin scFv antibody

[0132] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGFFSPLTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSHYSYVPWSYSGYYYYYGFDIWGQGTLVTVSS(SEQ ID NO:3)

[0133] Mesothelin scFv-CAR-P2A-PP01 DNA sequence

[0134]

[0135] Mesothelin scFv-CAR-P2A-PP01 amino acid sequence

[0136] (SEQ ID NO: 5)

[0137] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide capable of binding PDL1, wherein the sequence of the polypeptide is set forth by the amino acid sequence of SQGINHLRGVLQSSG (SEQ ID NO: 1).

2. An antibody-drug conjugate comprising as an antibody moiety a polypeptide of claim 1.

3. The antibody-drug conjugate of claim 2, conjugated with a pseudomonas exotoxin (PE).

4. The antibody-drug conjugate of claim 3, wherein the pseudomonas exotoxin is PE24.

5. The antibody-drug conjugate of claim 2, wherein the drug is selected from the group consisting of a radiolabel, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digitoxin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to the target, or a combination of any of the foregoing; or an immunomodulatory compound, an anti-cancer agent, an anti-viral agent, an anti-bacterial agent, and an anti-parasitic agent, or a combination of any of the foregoing.

6. The antibody-drug conjugate of claim 5, wherein the radiolabel is 32 P or 35 S, the antibacterial agent is an antifungal agent.

7. A polynucleotide encoding the polypeptide of claim 1.

8. The polynucleotide of claim 7, encoding the polypeptide of claim 1, set forth by SEQ ID NO: 2 (AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA) or a degenerate sequence thereof.

9. Isolated CAR-T or CAR-NK cells characterized in that, the CAR-T cell or CAR-NK cell is capable of expressing the polypeptide of claim 1; or the CAR-T cell or CAR-NK cell is capable of expressing the antibody-drug conjugate of any one of claims 2-6; or the CAR-T cell or CAR-NK cell comprises the polynucleotide of claim 7 or 8.

10. The isolated CAR-T cell or CAR-NK cell of claim 9, wherein the expressed polypeptide is secreted extracellularly and / or the polypeptide and the CAR are expressed in the same reading frame.

11. A vector comprising the polynucleotide of claim 7 or 8.

12. The vector of claim 11, wherein the vector is an expression vector.

13. The vector of claim 12, wherein the vector is a viral vector.

14. The vector of claim 13, wherein the vector is a retroviral vector.

15. An immune effector cell comprising the polynucleotide of claim 7 or 8 or comprising the vector of any one of claims 11-14.

16. The immune effector cell of claim 15, wherein the immune effector cell is a T lymphocyte or a natural killer cell.

17. A pharmaceutical composition comprising the polypeptide of claim 1, comprising the antibody-drug conjugate of any one of claims 2-6, comprising the CAR-T cell or CAR-NK cell of claim 9 or 10, or comprising the immune effector cell of claim 15 or 16, and optionally, a pharmaceutically acceptable carrier.

18. A method of making the CAR-T cell or CAR-NK cell of claim 9 or 10, or the immune effector cell of claim 15 or 16, comprising introducing the vector of any one of claims 11-14 into a T lymphocyte or a natural killer cell.

19. Use of the polypeptide of claim 1, the antibody-drug conjugate of any one of claims 2-6, the CAR-T cell or CAR-NK cell of claim 9 or 10, or the immune effector cell of claim 15 or 16 in the manufacture of a medicament for the treatment of cancer.

20. The use of claim 19, wherein the cancer is lung cancer or multiple myeloma and acute myeloid leukemia.

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