CN115124599A - Polypeptides that specifically bind to PDL1 and uses thereof - Google Patents

Abstract

The present invention provides polypeptides that specifically bind to PDL1 and uses thereof. Specifically, the invention 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 invention also provides a CAR-T cell capable of expressing the fusion protein, and uses the CAR-T cell to specifically kill tumor cells, such as lung cancer, multiple myeloma or acute myelogenous leukemia. The CAR-T can be used as a therapeutic drug for tumor diseases, and provides a new method for preventing and treating tumors.

Description

Polypeptides that specifically bind to PDL1 and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a polypeptide specifically binding to PDL1 and preparation and application thereof.

Background

T cells are a type of lymphocyte, and play an important role in cell-mediated immunity. It differs from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T Cell Receptor (TCR) on the cell surface. T helper cell, also known as CD4 ⁺ T or CD 4T cells expressing CD4 glycoprotein on their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, such cells rapidly proliferate and secrete cytokines that can modulate the immune response. Cytotoxic T cells, also known as CD8 ⁺ T cells, or CD8T cells, express CD8 glycoprotein on the cell surface. CD8 ⁺ T cells are activated when exposed to peptide antigens presented by MHC class I molecules. Memory T cells are a subpopulation of T cells that persist and respond to associated antigens, thus providing the immune system with memory against past infections and/or tumor cells.

A chimeric antigen receptor (abbreviated as CAR) modified T cell is CAR-T, which is a T cell modified by a chimeric antigen receptor (abbreviated as CAR) by using a genetic engineering means under in vitro culture conditions to express exogenous anti-tumor genes. The CAR gene is an artificially designed gene fragment, and its encoded protein mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is a specific antibody fragment used for targeted recognition of tumor surface specific molecules; the latter is used for starting immune cell response after specific recognition and playing a role in cellular immunity. After genetic engineering, 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 grow in the laboratory until their numbers reach billions. The expanded CART cells can then be infused into the patient.

Programmed cell death ligand 1 (PDL 1) has IgV and IgC-like regions, a transmembrane region and a cytoplasmic tail, wherein the cytoplasmic tail is involved in intracellular signal transduction, and IgV and IgC are involved in intercellular signal transduction. PDL1 interacts with the receptor PD1 on its T cells and plays an important role in the negative regulation of immune responses; the molecule has a wide tissue expression spectrum and high expression on some tumor cell lines, and a plurality of researches show that the molecule is related to an immune escape mechanism of tumors. The microenvironment of the tumor part can induce the expression of PDL1 on the tumor cells, and the expression is wide, and the expressed PDL1 is beneficial to the generation and the growth of tumors and induces the apoptosis of anti-tumor T cells.

PD1 can be combined with PDL1 to transmit inhibitory signal, and can inhibit proliferation and activity of lymphocyte, and inhibit CD4 ⁺ T cells differentiate to Th1 and Th17 cells, and release of inflammatory cytokines is inhibited, which play a role in immune negative regulation. Under normal conditions, the combination of PDL1 and PD1 can maintain the immune tolerance of peripheral lymphocytes to self-antigens through the above-mentioned actions, thereby preventing the occurrence of autoimmune diseases. However, in the development of tumor, PDL1 expressed by tumor cells can be combined with PD1 to promote the immune escape of tumor through the inhibitory action on lymphocyte.

Disclosure of Invention

In view of the above, the present invention provides polypeptides that specifically bind to PDL1 and uses thereof. Specifically, the invention 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 invention also provides a CAR-T cell capable of expressing the fusion protein, which is used to specifically kill tumor cells, such as multiple myeloma or acute myelogenous leukemia. The CAR-T can be used as a therapeutic drug for tumor diseases, and provides a new method for preventing and treating tumors.

Specifically, the present invention provides a polypeptide or an active fragment thereof that binds 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 a sequence that differs from SEQ ID NO: 1 amino acid sequence that is at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

Illustratively, for the polypeptide of the invention or an active fragment thereof, wherein the variant sequence is as set forth in SEQ ID NO: 1 and retains the activity of binding to PDL1 by adding, deleting or substituting 1, 2 or 3 amino acids in the amino acid sequence of 1.

The invention also provides a fusion protein comprising the polypeptide of the invention or an active fragment thereof. Illustratively, the fusion proteins of the invention further comprise a tag sequence (e.g., Poly-His, Hemagglutenin, c-Myc, GST, Flag-tag, etc.) or IgG1-Fc protein sequence, or an epitope tag (e.g., an epitope directed against human BCMA) or an additional antibody active fragment (e.g., an antibody or antibody active fragment directed against an epitope of human BCMA, or a ligand capable of binding to human BCMA).

The present invention also provides an antibody-drug conjugate comprising a polypeptide according to the invention or an active fragment thereof, preferably said conjugate is conjugated to a Pseudomonas Exotoxin (PE), more preferably PE 24.

For the antibody-drug conjugates of the present invention, illustratively, the drug is selected from the following: a radioactive label, ³² P、 ³⁵ S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; or immunomodulatory compounds, anti-cancer agents, anti-viral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing.

The invention also provides a polynucleotide for encoding the polypeptide or the active fragment and the fusion protein thereof,

preferably, the polynucleotide encoding the polypeptide of the invention or an active fragment thereof is as set forth in SEQ ID NO: 2(AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA) or a degenerate or complementary sequence thereof.

The invention also provides an isolated CAR-T cell or CAR-NK cell capable of expressing a polypeptide or an active fragment thereof according to the invention; the CAR-T cell or CAR-NK cell is capable of expressing a fusion protein of the invention; the CAR-T cell or CAR-NK cell is capable of expressing the antibody-drug conjugate of the invention; the CAR-T cell or CAR-NK cell comprises a polynucleotide of the invention.

The invention also provides a vector comprising a polynucleotide of the invention. Illustratively, the vector is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from the group consisting of human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-mei virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

The invention also provides an immune effector cell comprising a polynucleotide of the invention or comprising a vector of the invention, preferably wherein the immune effector cell is a T lymphocyte or a natural killer cell.

The invention also provides a pharmaceutical composition comprising a polypeptide or active fragment thereof of the invention, comprising a fusion protein of the invention, comprising an antibody-drug conjugate of the invention, comprising a CAR-T cell or CAR-NK cell of the invention, or comprising an immune effector cell of the invention, and optionally, a pharmaceutically acceptable carrier.

The invention also provides a method of making a CAR-T cell or CAR-NK cell of the invention, or comprising an immune effector cell of the invention, comprising introducing a vector of the invention into a T lymphocyte or a natural killer cell.

The invention also provides the use of a polypeptide as described above or an active fragment thereof, a fusion protein as described above, an antibody-drug conjugate as described above, a CAR-T cell or CAR-NK cell as described above, or an immune effector cell as described above, in the manufacture of a medicament for the treatment and/or prevention of cancer, illustratively lung cancer, multiple myeloma and acute myeloid leukemia, preferably lung cancer.

The invention also provides a nucleotide encoding a Chimeric Antigen Receptor (CAR) comprising nucleotides encoding (1) an extracellular antigen-binding domain, (2) a transmembrane domain, and (3) an intracellular signaling domain, characterized in that it further comprises a nucleotide encoding a polypeptide of the invention or an active fragment thereof.

Illustratively, in the CAR of the invention, the transmembrane domain is derived from a transmembrane domain selected from one or more of the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8 alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD80, CD86, CD134, CD137, CD152, CD154, ICOS and PD 1.

Illustratively, in the CAR of the invention, the intracellular signaling domain comprises a costimulatory signaling domain and is selected from the group consisting of: one or more of CD2, CD3 ζ, CD3 γ, CD3 δ, CD3 ε, 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.

In addition, in the CAR of the present invention, a hinge domain between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain may also be illustratively included. Preferably, the hinge domain is derived from CD8 a.

Further, in the CAR of the invention, wherein the extracellular antigen-binding domain is directed against a tumor cell surface antigen.

Variants thereof, such as their identity sequences or humanized sequences and the like, are also contemplated by the present invention for the polypeptides, or active fragments thereof, fusion proteins, CARs, and the like, described herein. Illustratively, the identity sequence refers to about 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, 90% or more, 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, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99% or more, of the identity to the original sequence or a reference sequence (e.g., SEQ ID NO: 1, 5), 99.7% or more, 99.8% or more, or 99.9% or more.

Degenerate or complementary sequences are also contemplated by the present invention for the polynucleotides of the present invention. Illustratively, the degenerate sequence has a homology of about 60% or more, about 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, 90% or more, 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, with the original or reference sequence, Or 99.9% or more.

Detailed Description

In vitro experiments prove that the polypeptide can be combined with a human PDL 1ligand, and the CAR-T cell containing the polypeptide can obviously improve the killing activity of tumor cells.

Further description of chimeric antigen receptors

In certain embodiments, the CARs of the invention may comprise linker residues between the various domains added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence that connects the VH domain and the VL domain and provides a spacer function that is compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the target molecule. The CAR of the invention may comprise one, two, three, four, or five or more linkers. In particular embodiments, the linker 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 length of amino acids.

Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymer; alanine-serine polymers; other flexible joints are known in the art, such as a wheatstone joint. Glycine and glycine-serine polymers are relatively unstructured and thus can serve as a link between domains of a fusion protein or some of the domains (e.g., a CAR as described herein).

In particular embodiments, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides", corresponding to linkers, that move the antigen binding domain away from the surface of the effector cell to enable proper cell-to-cell contact, antigen binding and activation. In certain 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 CH 3. The spacer region may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer region comprises the CH2 and CH3 domains of IgG1 or IgG 4.

In some embodiments, the binding domain of the CAR may be followed by one or more "hinge domains" that distance the antigen binding domain from the surface of the effector cell to enable appropriate cell-to-cell contact, antigen binding and activation. The CAR may comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be of natural, synthetic, semi-synthetic or recombinant origin. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8 a, CD4, CD28, PD1, CD152, and CD7), which may be wild-type hinge regions from these molecules, or may be altered. In another embodiment, the hinge domain comprises a PD1, CD152, or CD8 a hinge region.

The "transmembrane domain" is a portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR on the plasma membrane of the immune effector cell. The TM domain may be derived from natural, synthetic, semi-synthetic or recombinant sources. The TM domain may be derived from the α, β or ζ chain of a T cell receptor, CD3 ∈, CD3 ζ, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD 1. In one embodiment, the CAR of the invention comprises a TM domain derived from CD8 a or CD 28.

In particular embodiments, the CAR of the invention comprises an intracellular signaling domain. By "intracellular signaling domain" is meant information involved in the binding of an effective extracellular binding domain to the tumor specific surface antigen PDL1 to the interior of an immune effector cell to elicit effector cell function (e.g., activation, cytokine production, proliferation, and cytotoxic activity including release of cytotoxic factors to target cells to which the CAR binds) or other cellular response elicited by the antigen-binding extracellular CAR domain. The term "effector function" refers to a specialized function of an immune effector cell. For example, the effector function of a T cell may be cytolytic activity or help or activity including cytokine secretion. The term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specialized function.

The CARs of the invention comprise one or more costimulatory 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, providing the secondary signal required for effective activation and function of T lymphocytes upon binding to antigen.

Protein

"protein", "polypeptide fragment" and "polypeptide" are used interchangeably unless specified to the contrary, and are used according to conventional meanings, i.e., as amino acid sequences. Proteins are not limited to a particular length, e.g., they may comprise full-length protein sequences or fragments of full-length proteins, and may include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other modifications known in the art, including both naturally occurring and non-naturally occurring.

In various embodiments, the CAR polypeptides or proteins of the invention comprise a signal (or leader) sequence at the N-terminus of the protein that can direct protein transfer upon or after translation. Polypeptides may be prepared using a variety of well-known recombinant and/or synthetic techniques. The polypeptides of the invention specifically include the CARs of the disclosure, or sequences having one or more (e.g., 1-20, 1-10, or 1-5) amino acid deletions, additions, and/or substitutions to the CARs disclosed herein.

Nucleic acids

The term "polynucleotide" as used herein refers to mRNA, RNA, genomic RNA (grna), positive strand RNA (+), negative strand RNA (-), genomic DNA (gdna)), complementary DNA (cdna), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, a polynucleotide of the invention includes a polynucleotide or variant 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 to any of the reference sequences 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.

In another aspect, the sequence of the polynucleotide of the invention may be one that hybridizes under stringent conditions to the sequence defined by SEQ ID NO: 2, and encodes an active polypeptide, or a complement thereof;

the "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions, and preferably high stringency conditions. Illustratively, "low stringency conditions" can be conditions of 30 ℃,5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 52% formamide; "moderate stringent conditions" can be 40 degrees C, 5 x SSC, 5 x Denhardt liquid, 0.5% SDS, 52% formamide conditions; the "high stringency conditions" may be 50 ℃ in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures will result in polynucleotides with higher homology. In addition, one skilled in the art can select the result of combining multiple factors, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., that affect the stringency of hybridization to achieve the corresponding stringency.

In addition, the polynucleotide hybridizable to the polynucleotide encoding SEQ ID NO: 5, has about 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, 90% or more, 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, identity.

The identity of nucleotide sequences can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA90:5873,1993). The programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (AltschulSF, et: J Mol Biol 215:403,1990). When BLASTN is used to analyze a nucleotide sequence, the parameters are set to score 100 and workength 12; when BLASTX is used to analyze an amino acid sequence, the parameters are set to score 50 and Wordlength 3; when BLAST and GappedBLAST programs are used, default parameter values can be set for a system using each program.

Polynucleotides may be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide or protein, the nucleotide sequence encoding the polypeptide may be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC)), phages (e.g., lambda 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, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV 40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; lenti4/V5-DESTTM, pLenti6/V5-DESTTM 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 proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric proteins in mammalian cells. "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector (e.g., origins of replication, promoters, enhancers, translational initiation signal (SD sequence or Kozak sequence) introns, polyadenylation sequences, 5 'and 3' untranslated regions) that interact with host cell proteins for transcription and translation. The strength and specificity of such elements or sequences may vary. Depending on the vector system and host used, any number of suitable transcription and translation elements or sequences may be used, including broadly expressing promoters and inducible promoters.

Related ADC

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 function by targeting cancer cells with antibodies and then releasing toxic substances (i.e., drugs) in the cells, thereby triggering cell death. ADC technology increases the efficacy of therapeutic or targeting antibodies and reduces the risk of adverse reactions, as it allows for accurate drug delivery to target cancer cells and release under specific conditions, while minimizing collateral damage to healthy cells.

The basic structure of an antibody-drug conjugate can be "antibody-linker-pharmaceutically active molecule" or "antibody-pharmaceutically active molecule" (no linker). For conjugates with linkers, the linkers allow the drug to exhibit an effect on the target cancer cell, e.g., after separation from the antibody (e.g., by enzyme-mediated hydrolysis) and after the drug reaches the target cell. The linker also serves a functional role by linking the antibody and the drug. The efficacy and toxicity of the antibody-drug conjugates thus depends in part on the stability of the linker, which therefore plays an important role in drug safety.

The linker of the antibody-drug conjugate can be broadly classified as non-cleavable or cleavable. Many non-cleavable linkers are attached to an antibody using a thioether, which comprises the cysteine of the antibody. Drug conjugates typically cannot be separated from the antibody in vivo and may also suffer from reduced efficacy. In the case of the widely used thiol-maleimide approach, the antibody-drug conjugate is unstable, which may result in the separation of the drug from the conjugate before or after it reaches the target cell. The cleavable linker may be, for example, hydrolyzed by a lysosomal enzyme. The cleavable linker may comprise a disulfide bond, e.g. including cysteine of an antibody. Disulfide linkers that allow dissociation via a thiol exchange reaction rely to some extent on uptake of the antibody-drug conjugate into the target cell and 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 blood, the drug may separate from the antibody before reaching its target.

In order to replace chemically labile linkers that are poorly stable in physiological extracellular conditions, such as hydrazone and disulfide-like linkers, 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, as the drug should only be released into the cells targeted by the protein to which the drug is attached, not outside the cells.

Novel methods for preparing antibody-drug conjugates have been reported in the literature, see, for example, U.S. patent publication No. 2012/0308584. Further improvements are possible.

The CAR of the invention or the antibody of the invention may still be conjugated on the antigen binding domain side with a pharmaceutically active molecule, such as erlotinib, lymphokines, botulinum toxin, affinity ligands, radiolabels, immunomodulatory compounds, anti-cancer agents, ribozymes, and the like.

Carrier

In particular embodiments, a cell (e.g., an immune effector cell, such as a T cell) is transduced with a retroviral vector (e.g., a lentiviral vector) encoding a CAR and a polypeptide of the invention, or an active fragment thereof.

Retroviruses are a common tool for gene delivery. In particular embodiments, the retrovirus is used to deliver a polynucleotide encoding a Chimeric Antigen Receptor (CAR) to a cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into linear double-stranded DNA copies, followed by covalent integration of its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus". The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoding structural proteins and enzymes required for the production of new viral particles.

Exemplary retroviruses suitable for use in particular embodiments include, but are not limited to: moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), Feline Leukemia Virus (FLV), Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)), and lentiviruses.

As used herein, the term "lentivirus" refers to a group (or genus) that contains many retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-madivirus (VMV) virus; 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 particular embodiments, the lentivirus is used to deliver a polynucleotide comprising the CAR to a cell.

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, e.g., inserted into, a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication in the cell, or may include sequences sufficient to permit integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication defective retroviruses and lentiviruses.

It will be apparent to those skilled in the art that the term "viral vector" is used broadly to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate nucleic acid transfer, including virus-derived nucleic acid elements that generally facilitate transfer or integration of a nucleic acid molecule into the genome of a cell. Viral particles typically include various viral components and sometimes host cell components in addition to nucleic acids.

The term viral vector may 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 derived primarily from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof, primarily derived from a retrovirus. The term "lentivirus" refers to a genus of the family retroviridae that is capable of efficiently infecting non-periodic and post-mitotic cells; they can transmit significant amounts of genetic information into the DNA of the host cell, so that they are one of the most efficient methods of gene delivery vectors.

Thus, in a preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding a CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that facilitate expression of the CAR, e.g., a promoter, an enhancer, a poly-a signal, and/or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by transposon sequences such that a transposase is present to allow integration of the coding sequence into the genome of the transfected cell.

In some embodiments, the genetically transformed cell is further transfected with a transposase that promotes integration of the CAR-encoding sequence into the genome of the transfected cell. 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 long term expression of the transposase does not occur in the transgenic cell. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA comprising a cap and a poly-a tail). Any transposase system may be used in accordance with embodiments of the present 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, e.g., Mates et al, 2009, Nat Genet.41 (6): 753-61 or US9228180, which are incorporated herein by reference). For example, the method can include electroporating cells having mRNA encoding SB 10, SB 11, or SB 100X transposase.

Sequence variants:

also included within the scope of the invention are sequence variants (e.g., those defined by percent sequence identity) of the claimed nucleic acids, proteins, antibodies, antibody fragments, and/or CARs that maintain similar binding properties of the invention. These variants show alternative sequences but retain essentially the same binding properties such as target specificity, since the particular sequences provided are known to be functional analogs or functional analogs. Sequence identity relates to the percentage of identical nucleotides or amino acids when aligned.

As used herein, the statement "sequence identity" refers to the degree to which nucleotide-nucleotide based or amino acid-amino acid based sequences are identical over a comparison window. Thus, the "percentage of sequence identity" can be calculated by: comparing the two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is present on the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are 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 to any reference sequence described herein, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide or protein as described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in the sequences that fall within the described sequence identity are also encompassed by the present invention.

Modifications of the protein sequence which may occur by substitution are also included within the scope of the present invention. Substitutions as defined herein are modifications of the amino acid sequence of a protein whereby one or more amino acids are replaced by the same number of (different) amino acids, thereby producing a protein containing a different amino acid sequence than the primary protein. Substitutions may be made which preferably do not significantly alter the function of the protein. As with the addition, the replacement may be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of the protein. This is particularly true when the modification involves a "conservative" amino acid substitution of one amino acid for another with similar properties. Such "conservative" amino acids may be natural or synthetic amino acids that may be substituted for size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. In general, many amino acids can be substituted for conservative amino acids without adversely affecting the function of the protein.

In general, the nonpolar amino acids Gly, Ala, Val, Ile and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu represent a conserved group of amino acids. 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 if they belong to different groups.

Substitutional variants remove at least one amino acid residue from the antibody molecule and insert a different residue in its place. For the generation of substitutional mutagenesis, the positions of most interest include the hypervariable regions, but FR alterations are also contemplated. If such substitutions result in a change in biological activity, a greater number of changes can be introduced and the product screened.

Genetically modified gene cells and immune cells

In particular embodiments, the invention contemplates cells genetically modified to express a CAR of the invention for use in cancer or a related disorder. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in 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 in a cell that restores, modifies, or modifies the expression of a gene, or is used to express a therapeutic polypeptide (e.g., CAR or ADC). In particular embodiments, the CARs of the invention are introduced into and expressed in immune effector cells in order to redirect them to a target antigen of interest.

An "immune cell" or "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and/or CDC).

The immune effector cells of the invention may be autologous or non-autologous ("non-self", e.g., allogeneic, syngeneic or xenogeneic). As used herein, "autologous" refers to cells from the same subject, which are a preferred embodiment of the present 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 from different subjects. As used herein, "allogeneic" refers to cells from different species. In a preferred embodiment, the cells of the invention are autologous or allogeneic.

Exemplary immune effector cells for use with the CARs of the invention include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is 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 CD 3-and CD 56-positive non-Major Histocompatibility Complex (MHC), which are restricted Natural Killer (NK) -like T lymphocytes. The T cell may be a T helper cell (Th), such as T helper 1(Th1) or T helper 2(Th 2). The T cells may be helper T cells or cytotoxic T cells or any other T cell subset. Other exemplary T cell populations suitable for use in particular embodiments include naive T cells and memory T cells.

For example, T cells modified with the inventive CARs described herein can recognize and kill tumor cells when reintroduced back into the patient after autologous cell transplantation. CIK cells may have enhanced cytotoxic activity compared to other T cells and therefore represent a preferred embodiment of the immune cells of the invention.

As understood by the skilled person, other cells may also be used as immune effector cells with the CARs 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, wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.

The invention provides methods of making immune effector cells expressing a CAR of the invention. In one embodiment, the method comprises transfecting or transducing an immune effector cell isolated from the individual, such that the immune effector cell expresses one or more CARs as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. These cells can then be directly re-administered to the individual. In a further embodiment, the immune effector cell is first activated and stimulated to proliferate in vitro, and then genetically modified to express the 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 CARs of the invention).

In particular embodiments, 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 cell comprises a T cell. T cells can be obtained from a number of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells may be obtained from a blood unit collected from a subject using any technique or combination of techniques known to those skilled in the art, for example, by sedimentation and antibody-conjugated bead-based methods. In one embodiment, the cells from the circulating blood of the individual are obtained by apheresis. The constituent blood products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis collection may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing. The cells may be washed with PBS or another suitable solution that is free of calcium, magnesium, and most divalent cations. As understood by one of ordinary skill in the art, the washing step can be accomplished by methods known to those of skill in the art, such as by 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 buffer. In certain embodiments, undesired components that make up the blood sample can be removed in cells that are directly resuspended in culture medium.

In certain embodiments, T cells are isolated from Peripheral Blood Mononuclear Cells (PBMCs) by lysing erythrocytes and depleting monocytes (e.g., by PERCOLLTM gradient centrifugation). Specific T cell subsets can be further isolated by positive or negative selection techniques. One method that may be used is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells.

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

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 (or differentiated in the case of progenitor cells) in vitro prior to genetic modification. In particular embodiments, immune effector cells (e.g., T cells) are genetically modified (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) with the chimeric antigen receptors of the invention, and then activated and amplified in vitro. In various embodiments, for example, U.S. patent No. 5,858,358; 6,905,681, No. 6,905,681; 7,067,318 No; 7,232,566 No; 5,883,223, No. 5,883,223; the methods described in nos. 6,797,514 and 6,867,041, activate and expand T cells before or after being genetically modified to express a CAR.

In another embodiment, for example, a mixture of one, two, three, four, five or more different expression vectors, each vector encoding a different chimeric antigen receptor protein (e.g., CAR variant sequence) as in the present invention, can be used to genetically modify a donor population of immune effector cells. The resulting modified immune effector cells form a mixed population of modified cells, wherein a portion of the modified cells express more than one different CAR protein.

In one embodiment, the invention provides a method of storing immune effector cells expressing a genetically modified murine, human, or humanized CAR protein targeted to a tumor antigen, comprising cryopreserving the immune effector cells such that the cells remain viable when thawed. A portion of immune effector cells expressing the CAR protein can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with cancer. If desired, cryopreserved transformed immune effector cells can be thawed, grown, and expanded to obtain more such cells.

Compositions and formulations

The compositions of the invention may comprise one or more polypeptides, polynucleotides, vectors comprising the polynucleotides, genetically modified immune effector cells, and the like, as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. "pharmaceutical composition" refers to a composition formulated in a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, either alone or in combination with one or more other therapeutic modalities. It is also understood that the compositions of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or various other pharmaceutically active agents, if desired. There is virtually no limitation on the other components that may also be included in the composition, provided that the additional components do not adversely affect the ability of the composition to deliver the intended therapy.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the chinese 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; gum tragacanth; malt; gelatin; talc; cocoa butter, wax, animal and vegetable oil, paraffin, organic silicon, bentonite, silicic acid and 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and any other compatible materials used in pharmaceutical formulations.

In particular embodiments, the compositions of the invention comprise an amount of an immune effector cell of the invention that expresses a CAR. As used herein, the term "amount" refers to an "effective amount" of genetically modified therapeutic cells (e.g., T cells) to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical result.

By "prophylactically effective amount" is meant an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Typically, but not necessarily, the prophylactically effective amount is less than the therapeutically effective amount because the prophylactic dose is used in the subject prior to or at an early stage of the disease. The term preventing does not necessarily mean completely prohibiting or preventing a particular medical condition. The term preventing also refers to reducing the risk of the occurrence of a medical condition or worsening of a symptom.

The "therapeutically effective amount" of the genetically modified therapeutic cells may vary depending on various factors, such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount such that the therapeutically beneficial effect outweighs any toxic or detrimental effects of the virus or the transduced therapeutic cells. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, 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, extent of infection or metastasis, and patient (subject) condition. It may be generally stated that a pharmaceutical composition comprising T cells as described herein may be in the range of 10 ² To 10 ¹⁰ Individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Doses of individual cells per kg body weight (including all integer values within these ranges) are administered. The number of cells will depend on the end use of the composition and the cell type contained therein. For the uses provided herein, the cells are typically 1L or less in volume and may be 500mL or less, even 250mL or 100mL or less. Thus, the desired cell density is generally greater than 10 ⁶ Individual cells/ml, usually greater than 10 ⁷ Individual cell/ml, usually 10 ⁸ Individual cells/ml or higher. Clinically relevant numbers of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ 、10 ¹⁰ 、10 ¹¹ Or 10 ¹² And (4) cells. In some embodiments of the invention, a lower number of cells may be administered, particularly because all infused cells will be redirected to a particular target antigen. The CAR-expressing cell composition can be administered multiple times at doses within these ranges. For patients receiving treatment, the cells may be allogeneic, syngeneic, allogeneic or autologous.

In general, compositions comprising cells activated and expanded as described herein are useful for treating and preventing diseases that occur in immunocompromised individuals. In particular, compositions comprising the CAR-modified T cells of the invention are useful for treating B cell malignancies. The CAR-modified T cells of the invention can be administered alone, or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and/or with other components (e.g., IL-2) or other cytokines or groups of cells. In particular embodiments, the pharmaceutical compositions of the invention comprise an amount of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

A pharmaceutical composition of the invention comprising a population of immune effector cells (e.g., T cells) that express a CAR can comprise: buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids (e.g., glycine); an antioxidant; chelating agents (e.g., EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and a preservative. The compositions of the present invention are preferably formulated for parenteral administration, for example intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

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 mono-or diglycerides which may be used as a solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and buffers such as acetates, citrates or phosphates, and agents for regulating the osmotic pressure, such as sodium chloride or glucose. The parenteral formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In particular embodiments, the compositions of the invention comprise an effective amount of an immune effector cell expressing a CAR alone, or in combination with one or more therapeutic agents. Thus, the immune effector cell composition expressing the CAR can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, and the like. The compositions may also be administered in combination with an antibiotic. Such therapeutic agents are accepted in the art as standard treatments for particular disease states (e.g., particular cancers) as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active and auxiliary agents.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Drawings

FIG. 1: schematic structure of pShort-P8C phagemid.

FIG. 2: competitive ELISA experiments with PD1 for PP01 polypeptide.

FIG. 3: construction scheme for Mesothelin scFv CAR-PP01 fusion protein.

FIG. 4: the secreted PDL1 inhibitory polypeptide promotes the in vitro cell killing effect of CAR-T.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Example 1: construction of phage display polypeptide library

A library of random polypeptides with amino acid lengths of 8-15 was constructed by reference to the method reported by Sidhu. The specific construction method comprises the following steps: 1) the mutant of M13 phage P8 protein is inserted into pShort phagemid (obtained by pBluescript modification), and the C terminal of P8 protein is inserted with linker sequence and mutation site sequence (containing terminator sequence), and the formed new phagemid is named pShort-P8C (FIG. 1); 2) gene total synthesis gene (NNK) _8-15 The sequence of the mutant primer (Shanghai worker) is shown in Table 1; 3) pShort-P8C was electroporated into CJ236 competent bacteria, overnight cultured, and after extraction of ssDNA using M13ssDNA extraction kit (NNK) by Kunkel mutagenesis _8-15 Introducing a random polypeptide library into the C end of the P8 protein; 4) electrically transferring a Kunkel reaction product cccDNA into SS320 super competent bacteria containing M13K07 helper phage (helper phase), culturing for 1 hour (the temperature is 37 ℃, the rotating speed is 220rpm) in a shaking table after SOC is suspended, then transferring the bacteria into 1L2YT/Carb/Kan culture medium, and culturing overnight (the temperature is 37 ℃, the rotating speed is 220rpm, and about 20 hours) in the shaking table; 5) separating and purifying phage by PEG8000/NaCl precipitation method, suspending phage in PBST buffer solution to obtain final concentration of 10 ¹³ The pfu/ml phage display random polypeptide library was stored at-80 ℃.

Table 1: mutation primer of random polypeptide library

Example 2: biopanning of anti-PDL 1 phage and confirmation of anti-PDL 1 phage

Coating a target antigen PDL1-his (purchased from Acro Biosystems) on a maxi-sorp 96 pore plate, sequentially adding a polypeptide library and bacteria, and performing 3-5 rounds of high-throughput screening cycles (incubation, combination, elution and amplification) to gradually enrich the high-affinity positive phage; and then selecting monoclonal on a corresponding solid plate, amplifying positive clones, collecting phages generated by the monoclonal antibodies, determining the specificity of the phages through Phage-ELISA reaction, and finally performing DNA sequencing to obtain gene information.

His-tagged PDL1 protein (PDL1-His) was coated into Maxisorp 96-well plates at 4 ℃. After pouring off the coating buffer gently, 1% polyvinyl alcohol solution (PVA) was added and blocked at room temperature for 1 hour. Add 100. mu.L phage library per well (10) ¹³ pfu/ml) for 2 hours. Subsequently, each well was washed 8 times with PT buffer (0.05% Tween 20 in PBS buffer), and then incubated with 100. mu.L of 100mM HCl for 5 minutes to elute bound phage. The phage eluate was transferred to a 1.5mL centrifuge tube and neutralized with 1M Tris-HCl (pH 8.0) to adjust the pH. Half of the phage eluate was added to 1mL of E.coli NEB5-alpha F 'having a good growth state, mixed (E.coli NEB5-alpha F' was cultured in 2XYT medium containing 10. mu.g/mL tetracycline until OD600 was 0.8), and cultured at 37 ℃ for 1 hour. Then 10 is added ¹⁰ pfu M13K07 helper phage were cultured for an additional 1 hour. Infected Escherichia coli was inoculated into 50mL of 2XYT medium containing 50. mu.g/mL carbenicillin and 25. mu.g/mL kanamycin, and cultured overnight at 37 ℃ and 200 rpm. The next day, phage were collected by PEG8000/NaCl solution precipitation, resuspended in PBS solution and used for the next round of screening. Before adding the helper phage, 10. mu.L of the bacterial culture solution is diluted and smeared on an LB plate containing 50. mu.g/mL carbenicillin and incubated overnight at 37 ℃. The number of colonies generated by the phage eluate from the PDL1-His coated wells and the number of colonies generated by the phage eluate from the PVA coated wells were counted, respectively, and used to calculate the enrichment ratio.

Enrichment was obtained after round 3 panning by the screening procedure described above (number of clones in PDL1-His well was 147 times higher than in PVA well). Several single clones were picked from the enriched bacteria and the specificity of the Phage was determined by Phage-ELISA reaction. The results are shown in table 2 below, where the microplate reader reads as overflow when the OD value > 4.

Table 2: anti-PDL 1 recombinant phage monoclonal ELISA screening

Selection of relative OD from the above clones ₄₅₀ The larger monoclonal 9 strain, and the polypeptide sequence was determined by sequencing. The sequencing result shows that the 9 monoclonal sequences are completely consistent, so the polypeptide is named as PP01, and the sequence information is as follows:

PP01 DNA seq：AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA(SEQ ID NO：2)

PP01 protein seq: SQGINHLRGVLQSSG (SEQ ID NO: 1)

Meanwhile, in order to verify the specificity of the PP01 clone and eliminate the influence of the recombinant protein His tag, the PP01 Phage was selected to perform binding assay (Phage-ELISA) on various proteins, and the results are as follows:

from the above Elisa results, it was found that PP01 was a PDL1 specific phage clone and was a candidate for anti-PDL 1 antibody.

Example 3: affinity assay for polypeptides and competitive inhibition thereof of binding of PDL1-PD1

The PP01 polypeptide is synthesized by a solid phase synthesis method, and HPLC and mass spectrometry analysis are carried out on the synthesized PP01 polypeptide, so that the purity of the polypeptide is more than 95 percent.

The binding of the polypeptide to the PDL1-His protein was determined using ELISA. Specifically, 2. mu.g/ml PP01 polypeptide was coated onto Maxisorp 96-well plates (100. mu.L/well) overnight at 4 ℃. After pouring out the coating solution, 1% polyvinyl alcohol solution (PVA) was added and blocked at room temperature for 1 hour. After washing each well 3 times with PT buffer, 100. mu.L of PDL1-His protein was added at various concentrations for binding for 1 hour. After washing each well 3 times with PT buffer, 100. mu.L of HRP-labeled murine anti-6 XHis antibody was added and incubated for 1 hour at room temperature. Each well was washed 3 times with PT buffer and 3 times with PBS buffer, and after incubation for 5-10 min with 50. mu.L of TMB, the reaction was stopped by addition of 1M phosphoric acid and read on an microplate reader with OD 450. The experimental result shows that the PP01 polypeptide can recognize and bind to the PDL1-His protein.

Competitive ELISA assays were used to test the Competitive inhibitory effect of PP01 polypeptide on binding of PDL1-PD 1. Specifically, 5. mu.g/ml PD1-hFc was coated onto Maxisorp 96-well plates (100. mu.L/well) overnight at 4 ℃. After pouring out the coating solution, 1% polyvinyl alcohol solution (PVA) was added and blocked at room temperature for 1 hour. After washing each well 3 times with PT buffer, 200. mu.L of a mixture of 1. mu.g/ml PDL1-his and various concentrations of PP01 polypeptide was added and incubated at room temperature for 1 hour. After washing each well 3 times with PT buffer, 100 μ L of HRP-labeled murine anti-his antibody was added and incubated for 1 hour at room temperature. Each well was washed 3 times with PT buffer and 3 times with PBS buffer, and after incubation for 5-10 minutes with 50. mu.L of TMB, the reaction was stopped by addition of 1M phosphoric acid and read on an microplate reader with OD 450. The experimental results (see fig. 2) show that PP01 polypeptide can competitively inhibit PDL1-PD1 binding, and IC50 is 31 ng/ml.

Example 4: the secreted PDL1 inhibitory polypeptide promotes the in vitro cell killing effect of CAR-T.

The PP01 polypeptide gene and the CAR gene are expressed in series, and whether the secretory PP01 can promote the killing effect of CAR-T is studied, wherein the constitutional elements of CAR-PP01 lentivirus are shown in figure 3.

1. Preparation of lentiviral vector: 1) the Mesothelin scFv-CAR-P2A-PP01DNA sequence (the amino acid sequence and DNA sequence of which are shown below) was gene synthesized; 2) inserting the fragment into a PWPXLD-kana vector by using a homologous recombination or enzyme digestion ligation method; transforming the recombinant vector into an escherichia coli strain Stbl3, screening by kanamycin, and sequencing the monoclonal to obtain correct recombinant plasmid; then expanding and culturing host bacteria containing recombinant plasmids, and obtaining sterile endotoxin-free plasmids, namely PWPXLD plasmid vectors containing CAR gene segments by using an endotoxin removal kit; 3) meanwhile, lentiviral vector packaging helper plasmids psPax2 and PMD2.0G are transformed into DH5 alpha, ampicillin screening is carried out, and plasmids are extracted.

2. Preparation of CAR-expressing lentivirus (Lenti-CAR): 1) inoculation 3X10 ⁶ The 293T cells in the culture dish; 2) after 24 hours, the viral plasmid (CAR-PWPXLD: 9 μ g, psPax 2: 9. mu.g, and PMD2.0G: 4.5. mu.g) were mixed, 0.45mL of sterile water and 50. mu.L of 2.5MCaCl were added ₂ The solution was then added dropwise 500. mu.L of 2 XBBS (50mM BES,280mM NaCl,1.5mM Na) ₂ HPO4), keeping the solution vortex mixed; standing at room temperature for 30 minutes; then adding the mixed solution into a 293T culture medium, and gently and uniformly mixing; 3) after 18 hours, the medium was changed to DMEM medium containing 2% FBS; 4) collecting culture medium supernatant after 48 hr, centrifuging to remove cell debris, and filtering the supernatant with 0.45 μm filter; then one third volume of TAKARA lentivirus concentration reagent(s) is added

Concentrator, trade name 631231), mixed and left to stand overnight at 4 ℃. Centrifuging at 4 deg.C for 45 min at 1500g, resuspending the precipitate with PBS to obtain virus solution, packaging, and storing at-80 deg.C.

3. Preparation of (Mesothelin scFv-CAR-P2A-PP01) -T cells (CART-PP 01): 1) collecting peripheral blood at Day 0, and separating lymphocyte PBMC and plasma; selecting CD3+ T cells from the PBMCs; adjusting the cell suspension to a concentration of 1X 10 ⁶ Culturing in 12-well plate; adding dynabeads magnetic beads (Thermo Fisher) for stimulation; 2) in Day 0, in 12-well plates, a solution of fibrinectin (5. mu.g/cm) was added ² ) Coating overnight at 4 ℃; 3) in Day 1, the fibrinectin solution in 12-well plates was discarded and blocked with 2% BSA for 30 min; removing the blocking solution at 750 μ L/4.5cm ² Adding virus liquid, placing in an incubator at 37 ℃, and standing for 4-6 hours; collecting stimulated T cells, and collecting 10 ⁶ Adding into each well, placing in 37 deg.C incubator with 5% CO ₂ Culturing; 4) in Day 3, T cell concentration was adjusted to 5X 10 ⁵ Ml, change fresh medium completely; 5) in Day 5, T cell concentration was adjusted to 5X 10 ⁵ Perml, flow cytometry detection of T cells for CAR and PP01 PolypeptidesThe expression of (1).

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

An in vitro killing experiment is carried out by taking Lactate Dehydrogenase (LDH) as a 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 in DMEM complete medium at 37 deg.C with 5% CO ₂ The culture is carried out for 20 hours in the environment, the LDH content of the CART-PP01 experimental group, the CART cell group, the target cell spontaneous release group and the solution background correction group is respectively detected by specific wavelength of an enzyme-labeling instrument, and the actual target cell killing ratio is calculated. The results of the experiment (see FIG. 4, where the CAR-T-PP01 panel was identified as "CART-PP 001") indicate that the secreted PDL1 inhibitory polypeptide is capable of promoting the anti-tumor effects of CAR-T.

Mesothelin scFv antibody DNA sequence

GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGGCATCAGCAACTACCTGGCCTGGTTTCAACAGAAACCAGGAAAAGCTCCGAAGTCCCTGATTTACGCCGCCAGCAGCCTGCAGTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCGGATCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTTTCTTCTCTCCTCTGACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGAGGAGGAGGTTCCGGAGGAGGAGGTAGCGGCGGAGGGGGTTCTCAGGTTCAGCTGGTGGAGTCTGGCGGTGGCGTGGTGCAGCCAGGGAGGTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCACCTTCAGCAGCTACGGCATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGTGATCAGCTACGACGGCAGCAACAAGTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCCGTGACAATTCCAAAAACACACTGTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCTCATTACTCTTACGTTCCGTGGTCTTACTCTGGTTACTACTACTACTACGGATTCGACATTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG(SEQ ID NO：4)

Mesothelin scFv antibody amino acid sequences

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGFFSPLTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSHYSYVPWSYSGYYYYYGFDIWGQGTLVTVSS(SEQ ID NO：3)

Mesothelin scFv-CAR-P2A-PP01DNA sequence

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCATGCTGCTAGACCGGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGGCATCAGCAACTACCTGGCCTGGTTTCAACAGAAACCAGGAAAAGCTCCGAAGTCCCTGATTTACGCCGCCAGCAGCCTGCAGTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCGGATCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTTTCTTCTCTCCTCTGACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGAGGAGGAGGTTCCGGAGGAGGAGGTAGCGGCGGAGGGGGTTCTCAGGTTCAGCTGGTGGAGTCTGGCGGTGGCGTGGTGCAGCCAGGGAGGTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCACCTTCAGCAGCTACGGCATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGTGATCAGCTACGACGGCAGCAACAAGTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCCGTGACAATTCCAAAAACACACTGTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCTCATTACTCTTACGTTCCGTGGTCTTACTCTGGTTACTACTACTACTACGGATTCGACATTTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGCAGACAACTACTCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCTCCGGCGCCACCAACTTTTCCCTGCTGAAGCAGGCCGGCGATGTGGAGGAAAACCCCGGACCCATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGAGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGATGA(SEQ ID NO：6)

Mesothelin scFv-CAR-P2A-PP01 amino acid sequence

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGFFSPLTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSHYSYVPWSYSGYYYYYGFDIWGQGTLVTVSSQTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNSSQGINHLRGVLQSSG(SEQ ID NO：5)

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Claims (15)

1. A polypeptide or an active fragment thereof that binds to PDL1, wherein the polypeptide has an amino acid sequence of SQGINHLRGVLQSSG (SEQ ID NO: 1) or a variant sequence of said amino acid sequence, wherein said variant sequence has a sequence identical to SEQ ID NO: 1 amino acid sequence that is at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

2. The polypeptide or active fragment thereof of claim 1, wherein the variant sequence is the amino acid sequence set forth in SEQ ID NO: 1 and retains the activity of binding to PDL1 by adding, deleting or substituting 1, 2 or 3 amino acids in the amino acid sequence of 1.

3. A fusion protein comprising the polypeptide of any one of claims 1-2 or an active fragment thereof.

4. The fusion protein of claim 4, further comprising a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an epitope tag (e.g., an epitope for human BCMA) or an antibody active fragment of an add-on (e.g., an antibody or antibody active fragment for an epitope for human BCMA, or a ligand capable of binding to human BCMA).

5. An antibody-drug conjugate comprising as an antibody moiety the polypeptide of any one of claims 1-2 or an active fragment thereof, preferably said conjugate is conjugated to Pseudomonas Exotoxin (PE), more preferably PE 24.

6. The antibody-drug conjugate of claim 5, wherein the drug is selected from the group consisting of: a radioactive label, ³² P、 ³⁵ S, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, streptavidin, digoxigenin, a hapten, an immunogenic protein, a nucleic acid molecule having a sequence complementary to a target, or a combination of any of the foregoing; or immunomodulatory compounds, anti-cancer agents, anti-viral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing.

7. A polynucleotide encoding the polypeptide of claim 1 or an active fragment thereof, the fusion protein of claim 3,

preferably, the polynucleotide encoding the polypeptide of claim 1 is as set forth in SEQ ID NO: 2(AGTCAGGGCATCAACCATTTACGTGGTGTCCTACAGTCCTCAGGA) or a degenerate or complementary sequence thereof.

8. An isolated CAR-T cell or CAR-NK cell, wherein said CAR-T cell or CAR-NK cell is capable of expressing the polypeptide or active fragment thereof of any of claims 1-2, preferably the expressed polypeptide or active fragment thereof is secreted extracellularly and/or the expression of the polypeptide or active fragment thereof and the expression of the CAR are in the same reading frame; the CAR-T cell or CAR-NK cell is capable of expressing the fusion protein of any one of claims 3-4; the CAR-T cell or CAR-NK cell is capable of expressing the antibody-drug conjugate of any one of claims 5-6; the CAR-T cell or CAR-NK cell comprising the polynucleotide of claim 7.

9. A vector comprising the polynucleotide of claim 7.

10. The vector of claim 9, wherein the vector is an expression vector, such as a viral vector, preferably a retroviral vector, such as a lentiviral vector, preferably selected from the group consisting of human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-mei virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV) and Simian Immunodeficiency Virus (SIV).

11. An immune effector cell comprising the polynucleotide of claim 7 or comprising the vector of claim 9 or 10.

12. The immune effector cell of claim 11, wherein the immune effector cell is a T lymphocyte or a natural killer cell.

13. A pharmaceutical composition comprising the polypeptide or active fragment thereof of any one of claims 1-2, comprising the fusion protein of any one of claims 3-4, comprising the antibody-drug conjugate of any one of claims 5-6, comprising the CAR-T cell or CAR-NK cell of claim 8, or comprising the immune effector cell of claim 11 or 12, and optionally, a pharmaceutically acceptable carrier.

14. A method of making a CAR-T cell or CAR-NK cell of claim 8, or comprising an immune effector cell of claim 11 or 12, comprising introducing the vector of claim 9 or 10 into a T lymphocyte or a natural killer cell.

15. Use of the polypeptide of any one of claims 1-2 or an active fragment thereof, the fusion protein of any one of claims 3-4, the antibody-drug conjugate of any one of claims 5-6, the CAR-T cell or CAR-NK cell of claim 8, or the immune effector cell of claim 11 or 12 in the manufacture of a medicament for the treatment and/or prevention of cancer, illustratively lung cancer or multiple myeloma and acute myelogenous leukemia.

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