Recombinant antibodies, genetically modified cells, and uses thereof in treating cancers

Abstract

This article discloses a recombinant antibody specific for programmed death 1 (PD-1), chimeric antigen receptors (CARs), and genetically modified cells configured to express CARs on their surface and secrete recombinant antibodies specific for PD-1. Furthermore, this article also discloses a method for treating cancer, comprising administering the aforementioned genetically modified cells to subjects with cancer.

Description

[Technical Field]

[0001] This disclosure generally relates to the field of disease treatment. More specifically, this disclosure relates to a novel antibody specific for programmed death 1 (PD-1), a novel chimeric antigen receptor T (CAR-T), and their use in cancer treatment. [Previous Technology]

[0002] CAR-T therapy refers to a therapy that uses genetic engineering to modify a patient's T cells to express artificial T cell receptors (TCRs) on their cell surfaces. These artificial TCRs help to reorient the modified T cells to recognize and eliminate target cells (e.g., cancer cells) that express specific target antigens (e.g., tumor-associated antigens (TAAs)). Since 2017, the U.S. Food and Drug Administration has approved six CAR-T therapies, including: tisagenlecleucel for B-cell acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin lymphoma (NHL); axicabtagene ciloleucel for NHL and follicular lymphoma; brexucabtagene autoleucel for mantle cell lymphoma (MCL) and ALL; lisocabtagene maraleucel for NHL; idecabtagene vicleucel for multiple myeloma; and ciltacabtagene autoleucel for multiple myeloma. However, the widespread clinical application of CAR-T technology is still limited by factors such as life-threatening toxicity, limited efficacy against solid tumors (due to the immunosuppressive tumor microenvironment (TME) and physical tumor barriers that restrict the penetration and infiltration of CAR-T cells), the development of tumor resistance, and antigen escape.

[0003] In view of the above, there is still a need to develop a novel secretory PD-1 antibody that can be used for CAR-T therapy to overcome TME and achieve the goal of treating cancer. [Summary of the Invention]

[0004] The present invention is intended to provide a simplified summary of this disclosure so that the reader can have a basic understanding of it. This present invention is not a complete overview of this disclosure, and it is not intended to identify important / critical elements of the embodiments of the invention or to define the scope of the invention.

[0005] As specifically implemented and broadly described in this disclosure, the first aspect of this disclosure relates to a recombinant antibody. According to some embodiments of this disclosure, the recombinant antibody is specific for PD-1. The structure of the recombinant antibody includes a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain includes a first heavy chain complementarity determining region (CDR-H1), a second heavy chain CDR (CDR-H2), and a third heavy chain CDR (CDR-H3); and the VL domain includes a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2), and a third light chain CDR (CDR-L3).

[0006] According to one embodiment of the present disclosure, the CDR-H1, CDR-H2, and CDR-H3 of the recombinant antibody respectively contain the amino acid sequences "GFTFSSYTMS" (sequence number: 1), "TISGGGANIYYPDSVKG" (sequence number: 2), and "PYYAIDF" (sequence number: 3); the CDR-L1, CDR-L2, and CDR-L3 of the recombinant antibody respectively contain the amino acid sequences "KASQDVGSAVA" (sequence number: 4), "WASTRHT" (sequence number: 5), and "QQYSTYTWT" (sequence number: 6).

[0007] According to various embodiments of this disclosure, the VH and VL domains of the recombinant antibody respectively contain amino acid sequences that are at least 85% identical to the sequences of sequence numbers 7 and 8. According to some preferred embodiments, the VH domain contains amino acid sequences that are 100% identical to the sequences of sequence numbers 7, 12, 13, or 14 (i.e., contains amino acid sequences that are 100% identical to the sequences of sequence numbers 7, 12, 13, or 14); the VL domain contains amino acid sequences that are 100% identical to the sequences of sequence numbers 8 or 15 (i.e., contains amino acid sequences that are 100% identical to the sequences of sequence numbers 8 or 15).

[0008] According to an exemplary embodiment, the recombinant antibody disclosed herein is in the form of a single-chain variable fragment (scFv). Optionally or additionally, the scFv further comprises a crystallizable region fragment (Fc region) of an immunoglobulin (e.g., IgG).

[0009] According to one embodiment of this disclosure, the recombinant antibody is secreted by immune cells or stem cells. Preferably, the immune cell is a T cell.

[0010] This article also discloses a single cell (e.g., a T cell) configured to express a chimeric antigen receptor (CAR) and the aforementioned recombinant antibody. The single cell is transfected with a nucleic acid comprising, from its 5' to 3' end: a first coding sequence encoding a first single-stranded variable region fragment (scFv) specific to a first antigen; a second coding sequence encoding a hinge and transmembrane (HTM) domain of a first protein; a third coding sequence encoding a co-stimulatory molecule; a fourth coding sequence encoding a cytoplasmic domain of a second protein; an internal ribosome entry site (IRES) or a linker sequence encoding a 2A peptide; and a fifth coding sequence encoding a second scFv containing the recombinant antibody disclosed herein; wherein the first to fourth coding sequences collectively encode the CAR, located on the cell membrane of the single cell; and the recombinant antibody, secreted from the single cell after expression.

[0011] According to one embodiment of this disclosure, the first scFv encoded by the first coding sequence is specific to the TAA antigen; the HTM domain encoded by the second coding sequence is the HTM domain of cluster of differentiation 8 (CD8); the co-stimulatory molecule encoded by the third coding sequence is 4-1BB; and the cytoplasmic domain encoded by the fourth coding sequence is the cytoplasmic domain of the CD3 zeta chain (CD3ζ). In some exemplary embodiments, the HTM domain of CD8 contains the amino acid sequence number 9; the 4-1BB co-stimulatory molecule contains the amino acid sequence number 10; and the cytoplasmic domain of CD3ζ contains the amino acid sequence number 11.

[0012] According to one embodiment of this disclosure, the CDR-H1, CDR-H2, and CDR-H3 of the second scFv encoded by the fifth coding sequence respectively contain the following amino acid sequences: "GFTFSSYTMS" (sequence number: 1), "TISGGGANIYYPDSVKG" (sequence number: 2), and "PYYAIDF" (sequence number: 3); the CDR-L1, CDR-L2, and CDR-L3 of the second scFv respectively contain the following amino acid sequences: "KASQDVGSAVA" (sequence number: 4), "WASTRHT" (sequence number: 5), and "QQYSTYTWT" (sequence number: 6). According to some exemplary embodiments, the VH and VL domains of the second scFv respectively contain amino acid sequences that are at least 85% identical to the sequences of sequence numbers: 7 and 8. According to some embodiments, the VH domain of the second scFv contains an amino acid sequence with sequence number 7, 12, 13 or 14; and the VL domain of the second scFv contains an amino acid sequence with sequence number 8 or 15.

[0013] Optionally, in addition to the first, second, third, fourth, and fifth coding sequences and the linker sequence, the nucleic acid further includes a sixth coding sequence linked to the 3' end of the fifth coding sequence. In these embodiments, the sixth coding sequence may encode a fragment crystallizable region (Fc segment) that produces an immunoglobulin. Depending on the intended use, the immunoglobulin may be immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). According to an exemplary embodiment, the immunoglobulin is IgG, for example, IgG1 or IgG4.

[0014] According to one embodiment of this disclosure, the isolated cell is transfected with an expression vector containing the aforementioned nucleic acid. This expression vector can be a viral vector; for example, a lentiviral vector, adenovirus vector, retroviral vector, adeno-associated virus vector, or Syndex virus vector. In an exemplary embodiment, the expression vector is a lentiviral vector. After transfection, the isolated cell can express CAR on its surface and secrete recombinant antibodies extracellularly.

[0015] Therefore, another aspect of this disclosure relates to the use of genetically modified cells (i.e., isolated cells transfected with the aforementioned nucleic acids) for cancer treatment.

[0016] According to certain embodiments of this disclosure, the genetically modified cell contains the aforementioned nucleic acid and is therefore configured to express the CAR and the recombinant antibody of this disclosure, wherein the CAR is located on the cell membrane of the genetically modified cell, and the recombinant antibody of this disclosure is secreted outside the genetically modified cell after expression. Preferably, the genetically modified cell is a genetically modified immune cell, such as a genetically modified T cell, a genetically modified natural killer (NK) cell, or a genetically modified macrophage.

[0017] The genetically modified immune cells can recognize and specifically bind to cancer cells via CAR for cancer treatment. Therefore, this article discloses a method for treating cancer in a subject. The method involves administering an effective dose of the genetically modified immune cells to the subject to alleviate or improve cancer symptoms.

[0018] Depending on the treatment purpose, the cancer may be stomach cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or squamous cell carcinoma of the head and neck.

[0019] Subjects who can be treated with the genetically modified immune cells and / or methods disclosed herein are mammals; preferably humans.

[0020] After reading the following embodiments, those skilled in the art to which this invention pertains will be able to easily understand the basic spirit and other inventive objectives of this invention, as well as the technical means and implementation methods adopted by this invention.

Implementation Method

[0030] To make the description of this disclosure more detailed and complete, illustrative descriptions of embodiments and specific examples of the present invention are provided below; however, this is not the only form of implementing or using the specific examples of the present invention. The embodiments cover features of multiple specific examples and methods and steps for constructing and operating these specific examples, as well as their order. However, other specific examples may also be used to achieve the same or equivalent functions and order of steps.

[0031] I. Definition of Terms

[0032] Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meaning as understood and commonly used by those skilled in the art to which this invention pertains. Furthermore, unless conflicting with the context, singular nouns used herein encompass their plural forms, and vice versa. Moreover, in this specification and the claims, expressions such as "at least one" and "one or more" have the same meaning, both representing a total of one, two, three, or more. Furthermore, in this specification and the claims, "at least one of A, B, and C," "at least one of A, B, or C," and "at least one of A, B, and / or C" refer to a total of only A, only B, only C, both A and B, both B and C, both A and C, and all three A, B, and C.

[0033] Although the numerical ranges and parameters used to define the broader scope of this disclosure are approximate values, the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "approximately" means that the actual value falls within the acceptable standard error of the average value, as determined by those skilled in the art to which this invention pertains. Except for experimental examples, or unless explicitly stated otherwise, it is understood that all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, and the like) are modified with "approximately". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying claims are approximate values ​​and are subject to change as needed. At least these numerical parameters should be understood as the indicated significant digits and values ​​obtained by applying general rounding. Here, a range of values ​​is expressed as a distance from one endpoint to another or between two endpoints; unless otherwise stated, all ranges of values ​​herein include the endpoints.

[0034] The term "nucleic acid" refers to a polynucleotide, such as deoxyribonucleic acid (DNA), and where appropriate, ribonucleic acid (RNA). Nucleic acids include, but are not limited to, single-stranded and double-stranded polynucleotides. For example, polynucleotides may include DNA, single-stranded DNA, cDNA, and mRNA. This term also includes DNA or RNA analogs made from nucleotide analogs and may be applied to single-stranded (sense or antisense) and double-stranded polynucleotides. Furthermore, the term further includes modified nucleic acids, including modified DNA and modified RNA, for example, DNA and RNA containing one or more non-natural nucleotides or nucleosides. The term "nucleic acid" is used herein to refer to deoxynucleotides or ribonucleotides and their polymers, whether in single-stranded or double-stranded form. The term also covers nucleic acids containing known nucleotide analogs or modified backbone groups or bonds, whether synthetic, naturally occurring or non-natural, and / or having binding properties similar to a reference nucleic acid, and / or being metabolized in a manner similar to a reference nucleotide. Examples of such nucleotide analogues include, but are not limited to: thiophosphates, phosphoramidates, methylphosphates, chiral methylphosphates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0035] As used herein, “recombinant antibody” means an antibody expressed and isolated from one or more cells or cell lines transfected with an expression vector (usually two expression vectors) containing the antibody coding sequence, wherein the coding sequence of the antibody is not a sequence native to the cell.

[0036] The term "antibody (Ab)" has the meaning known in the fields of cell biology and biochemistry, encompassing monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific or multivalent antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, and biologically active antibody fragments. The term "antibody fragment" can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Examples of antibody fragments include antigen-binding fragments (Fab), Fab', F(ab')₂, scFv, domain antibodies (dAb), bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific binding to the polypeptide antigen. According to certain embodiments of this disclosure, the antibody is in the form of scFv.

[0037] The term "single-chain variable fragment (scFv)" is understood in the fields of cell biology and biochemistry as a fusion protein consisting of the variable domains of the heavy chain (VH) and light chain (VL) of an immunoglobulin linked by a short linker peptide (usually composed of serine and / or glycine). Although the constant domain is removed and a linker is introduced, the scFv retains the antigen specificity of the original immunoglobulin.

[0038] The term "complementarity determining region (CDR)" as used herein refers to a highly variable region in an antibody molecule, which forms a surface complementary to the three-dimensional structure of the antigen. From the N-terminus to the C-terminus, each antibody heavy and light chains contain three CDRs (i.e., CDR-1, CDR-2, and CDR-3). Therefore, the antigen binding site consists of six CDRs, including three located in the variable domains of the heavy chain (i.e., CDR-H1, CDR-H2, and CDR-H3) and three located in the variable domains of the light chain (i.e., CDR-L1, CDR-L2, and CDR-L3).

[0039] The term "variable domain" refers to the amino-terminal domain of the heavy or light chain in an antibody. These domains are typically the most variable parts of an antibody and contain antigen-binding sites. "Variability" refers to the significant differences in the sequence of certain parts of the variable domain between antibodies. This variability is related to the binding and specificity of the antibody to its specific antigen. However, this variability is not uniformly distributed throughout the variable domain, but is concentrated in three segments within the variable domains of the light and heavy chains, called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved parts of the variable domain are called framework regions (FRs). The natural heavy and light chain variable domains each contain four FR regions, mainly forming a β-sheet configuration, and are connected by three CDRs. CDRs usually form a loop structure, connecting the β-sheet, and sometimes also participate in the formation of the β-sheet structure. The CDRs in each chain are tightly bound together through the FR regions and together with the CDRs on the other chain, constitute the antigen-binding site of the antibody.

[0040] As described herein, variations in the amino acid sequence of an antibody (particularly minor variations in the FR sequence) or the nucleotide sequence of a nucleic acid also fall within the scope of the inventive concepts contained in this disclosure and the patent application, provided that the amino acid / nucleotide sequence variation retains at least 85% identical sequence, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequence. The antibodies disclosed herein can be specifically modified to alter specific properties unrelated to their physiological activity. For example, specific amino acid residues in the antibody FR can be altered and / or deleted without affecting their physiological activity in this study (e.g., their ability to treat cancer). Specifically, conserved amino acid substitutions are considered. Conserved substitutions refer to substitutions occurring within a family of amino acid residues associated with the side chain. The amino acids encoded by the genetic code can generally be classified into the following groups: (1) acidic: aspartic acid, glutamic acid; (2) basic: lysine, arginine, histamine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) non-polar: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. A better family is: serine and threonine are aliphatic hydroxyl families; asparagine and glutamic acid are acetylamine-containing families; alanine, valine, leucine, and isoleucine are aliphatic families; phenylalanine, tryptophan, and tyrosine are aromatic families. For example, it is reasonable to expect that replacing leucine with isoleucine or an amino acid alone, replacing aspartic acid with glutamic acid, replacing threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not have a significant impact on the binding or properties of the resulting molecule, especially if the substitution does not involve amino acid residues within antigen-binding sites (i.e., CDRs). Whether amino acid changes result in functional peptides can be easily determined by measuring the specific activity of the peptide derivative. Those skilled in the art can readily prepare protein / peptide fragments or analogs. Preferred amino and carboxyl terms of the fragments or analogs are located near the boundaries of the functional domains.

[0041] "Sequence identity percentage (%)" is defined as the percentage of amino acid residues / nucleotides in a candidate sequence that are identical to the amino acid residues / nucleotides of a specific polypeptide / polynucleotide sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum sequence identity percentage, without considering any conserved substitutions as part of the identical sequence. The alignment performed to determine the sequence identity percentage can be carried out in various ways known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. For this purpose, sequences between two amino acid / nucleotide sequences are aligned using the computer programs Blastp (protein-protein BLAST) / Blastn (nucleotide-nucleotide BLAST) available online at the National Center for Biotechnology Information (NCBI). The percentage of amino acid sequences / nucleotides identical to those of a given amino acid sequence / nucleic acid A and a given amino acid sequence / nucleic acid B (which can also be expressed as a given amino acid sequence / nucleic acid B having the same percentage of amino acid sequences / nucleotides as a given amino acid sequence / nucleic acid A) is calculated by the following formula: where X is the number of amino acid residues / nucleic acids that are scored as identical matches by the sequence alignment program BLAST in the alignment of A and B, and where Y is the total number of amino acid residues / nucleic acids in A or B, whichever is shorter.

[0042] As used herein, the term "link" means any way in which two components are connected by a direct link or by an indirect link between the two components.

[0043] The terms "treat," "treating," and "treatment" used herein are used interchangeably and encompass the partial or complete prevention, improvement, relief, and / or control of cancer-related symptoms, complications, or conditions. The term "treating" refers to the application or administration of the chimeric antigen receptor T-cell (CAR-T) disclosed herein to a subject suffering from cancer-related symptoms, complications, or conditions, with the aim of partially or completely reducing, improving, alleviating, delaying onset, inhibiting progression, reducing severity, and / or reducing the incidence of one or more cancer-related symptoms, complications, or characteristics. Cancer-related symptoms, complications, and / or conditions include, but are not limited to: nausea, vomiting, loss of appetite, constipation, fatigue, muscle weakness, increased thirst, bone pain or fractures, swelling or lumps, bleeding, cough, fever, night sweats, coma, and pain. Treatment may also be administered to subjects exhibiting only the aforementioned symptoms, complications, and / or early signs of the condition to reduce the risk of developing a full set of symptoms, complications, and / or conditions. A treatment is generally considered "effective" if it alleviates one or more of the symptoms or clinical indicators as defined herein. Furthermore, a treatment is also considered "effective" if it slows or halts the progression of symptoms, symptoms, or the condition.

[0044] The term "effective amount" as used herein refers to the amount of an ingredient sufficient to produce the desired response. For therapeutic purposes, an effective amount also refers to the amount at which any toxic or harmful effects of the ingredient are offset by its therapeutic benefits. An effective amount is not necessary to cure a disease or condition, but should be able to delay, inhibit, or prevent the onset of the disease or condition, or improve its symptoms. The effective amount may be divided into one, two, or multiple doses, administered once, twice, or more over a specified period, depending on the appropriate dosage form. The specific effective or adequate dose will depend on several factors, including: the specific disease or condition being treated, the subject's physical condition (e.g., weight, age, or sex), the mammal or animal species being treated, the duration of treatment, the nature of any concomitant treatments, and the specific dosage form and structure of the compound or its derivatives used. Effective amounts may be expressed in different units, such as grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg), or in the number of cells per kilogram of body weight (cells / kg). Those with general technical knowledge can calculate the human equivalent dose (HED) of a drug (e.g., CAR-T cells as disclosed herein) based on dosages determined in animal models. For example, the industry guidance issued by the U.S. Food and Drug Administration (FDA) entitled "Estimating the maximum safe starting dose in a preliminary clinical trial of treatment in healthy adult volunteers" can be consulted to estimate the maximum safe starting dose applicable to human subjects.

[0045] The term "subject" refers to an animal, including a human, that can be treated with the CAR-T cells and / or methods of the present invention. Unless specifically indicated by sex, the term "subject" means male and female.

[0046] II. Summary of the Invention

[0047] PD-1 and its ligand PD-L1 are known to play a key role in tumor immunosuppression. The mechanism involves inhibiting the activation of immune cells (e.g., T cells) and enhancing the immune tolerance of tumor / cancer cells, allowing them to evade immune surveillance. Therefore, this disclosure aims to provide a monoclonal antibody with binding affinity for PD-1 and inhibitory / neutralizing activity, a nucleic acid encoding CAR and monoclonal antibody production, and a genetically modified cell configured to express a monoclonal antibody specific for CAR and PD-1. When this nucleic acid is introduced into an immune cell (e.g., T cells), the cell will simultaneously express CAR and monoclonal antibody, enabling the immune cell (e.g., CAR-T cells) to have binding specificity and cytotoxicity against cancer cells and to inhibit the immunosuppressive factor PD-1 in the tumor microenvironment. The inhibitory effect of this monoclonal antibody on PD-1 can enhance the antitumor response of CAR-T cells in solid tumors. Therefore, this disclosure also provides CAR-expressing immune cells (e.g., CAR-T cells) and their use in cancer therapy.

[0048] (i) Monoclonal antibody

[0049] The first aspect of this disclosure relates to an anti-PD-1 monoclonal antibody (mAb) named "2B6". According to some embodiments of this disclosure, mAb 2B6 has binding affinity and specificity for PD-1 and can be used to block the binding between PD-1 and PD-L1, thereby inhibiting the immunosuppressive response induced by the PD-1 / PD-L1 pathway.

[0050] According to certain embodiments of this disclosure, the mAb 2B6 disclosed herein can be manufactured by conventional immunization methods (i.e., immunizing animals with specific peptides to induce the animals to produce specific peptide antibodies). As is well known in the art, the mAb 2B6 disclosed herein can also be prepared by phage display of scFv libraries or by recombinant DNA technology (also known as DNA cloning technology; i.e., constructing and transfecting recombinant DNA encoding the production of a specific antibody into host cells to express the antibody).

[0051] Structurally, mAb 2B6 contains three CDRs (i.e., CDR-H1, CDR-H2, and CDR-H3) in its VH domain and three CDRs (i.e., CDR-L1, CDR-L2, and CDR-L3) in its VL domain. According to certain embodiments of this disclosure, the CDR-H1, CDR-H2, and CDR-H3 of mAb 2B6 respectively contain the following amino acid sequences: "GFTFSSYTMS" (sequence number: 1), "TISGGGANIYYPDSVKG" (sequence number: 2), and "PYYAIDF" (sequence number: 3); and the CDR-L1, CDR-L2, and CDR-L3 respectively contain the following amino acid sequences: "KASQDVGSAVA" (sequence number: 4), "WASTRHT" (sequence number: 5), and "QQYSTYTWT" (sequence number: 6).

[0052] For example, the amino acid sequences of the VH and VL domains of mAb 2B6 are sequence numbers 7 and 8, respectively, as detailed below. The CDRs (i.e., CDR-H1, CDR-H2, CDR-H3 of the VH domain and CDR-L1, CDR-L2, CDR-L3 of the VL domain) are marked in bold and arranged in order.

[0053] Sequence number: 7 (VH domain of mAb 2B6) EVKLVESGGGLVKPGGSLKLSCAAAGFTFSSYTMSWVRQTPAKRLEWVATISGGGANIYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCVSPYYAIDFWGQGTSVTVSS

[0054] Serial Number: 8 (VL field of mAb 2B6) DTVMTQSDKFMSTSVGDRVSITCKASQDVGSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSTYTWTFGGGTKLEIK

[0055] Since the binding affinity and specificity of an antibody are primarily determined by its CDR sequence, it is understood that the frame region (FR) sequences of the VH and VL domains can be altered without affecting the binding affinity and / or specificity of the antibody (e.g., by substitution with conserved or non-conserved amino acid residues). Preferably, the FR sequence is a conserved substitution made by one or more amino acids with similar properties; for example, replacing leucine (a nonpolar amino acid residue) with isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (other nonpolar amino acid residues); replacing aspartic acid (an acidic amino acid residue) with glutamic acid (another acidic amino acid residue); or replacing lysine (a basic amino acid residue) with arginine or histamine (other basic amino acid residues).

[0056] Based on the principle of conserved substitution, those skilled in the art can substitute amino acid residues in the FR sequences of the VH and VL domains of mAb 2B6 without affecting its activity and / or function (i.e., binding to PD-1 and / or blocking the binding between PD-1 and PD-L1). Therefore, antibodies containing substituted amino acids in the FR sequences of the VH and VL domains are also included within the scope of this disclosure. According to some embodiments, the VH domain of mAb 2B6 contains an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence at sequence number 7, and its VL domain contains an amino acid sequence that is at least 85% identical to the sequence at sequence number 8. According to certain preferred embodiments, the VH and VL domains of mAb 2B6 each contain an amino acid sequence that is at least 90% identical to the sequences of sequence numbers 7 and 8. More preferably, the VH and VL domains each contain an amino acid sequence that is at least 95% identical to the sequences of sequence numbers 7 and 8.

[0057] According to certain embodiments of this disclosure, the VH and VL domains of mAb 2B6 have been modified to resemble human antibodies to reduce their immunogenicity in human subjects. Therefore, this disclosure also provides different humanized VH and VL sequences, including: 2B6 Hd VH (sequence number: 12), 2B6 HdB1 VH (sequence number: 13), 2B6 HuB1 VH (sequence number: 14), and 2B6 Hd VL (sequence number: 15).

[0058] Serial number: 12 (2B6 Hd VH) EVQLVESGGGLVQPGGSLRLSCKASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARPYYAIDFWGQGTLVTVSS

[0059] Serial number: 13 (2B6 HdB1 VH) EVQLVESGGGLVQPGGSLRLSCKASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSPYYAIDFWGQGTLVTVSS

[0060] Serial number: 14 (2B6 HuB1 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSPYYAIDFWGQGTLVTVSS

[0061] Serial number: 15 (2B6 Hd VL) DIQMTQSPSSSLSASVGDRVTITCKASQDVGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYTWTFGQGTKVEIK

[0062] Depending on its intended use, the mAb 2B6 or humanized 2B6 disclosed herein may be prepared in the form of a complete antibody (e.g., IgG, IgA, IgM, IgD, or IgE) or in the form of an antibody fragment (e.g., scFv, Fab, Fab', F(ab')₂, or a bivalent antibody). According to certain exemplary embodiments of the present disclosure, the mAb of the present disclosure is produced in the form of scFv (i.e., 2B6 scFv).

[0063] (ii) This disclosure can encode nucleic acids that generate CAR and mAb 2B6 scFv.

[0064] The second aspect of this disclosure aims to provide a nucleic acid that can encode a CAR and the mAb 2B6 scFv described in Part (i) above. Referring to Figure 1, which is a schematic diagram of the nucleic acid construct of this disclosure, the nucleic acid sequentially includes, from its 5' end to its 3' end: a first, second, third, and fourth coding sequence, collectively encoding a CAR; an IRES or a linker sequence encoding a 2A peptide; and a fifth coding sequence encoding the mAb 2B6 scFv of this disclosure. Specifically, the first to fifth coding sequences respectively encode: an antigen (e.g., the first scFv), the HTM domain of a first protein (e.g., the HTM domain of CD8), a co-stimulatory molecule (e.g., a 4-1BB molecule), the cytoplasmic domain of a second protein (e.g., the cytoplasmic domain of CD3ζ), and the mAb 2B6 scFv of this disclosure. Please note that the IRES or the linker sequence encoding the 2A peptide is positioned between the CAR coding sequence and the mAb coding sequence, so that the nucleic acid can independently express the CAR and the mAb during translation, thus appearing as two separate proteins rather than a fusion protein.

[0065] As intended, the selected antigen or first scFv is specific to any expressed / overexpressed and / or tumor / cancer cell-associated antigens, such as alpha-fetoprotein (AFP), CA19-9, CA125, carcinoembryonic antigen (CEA), cancer / testis antigen 1B (CTAG1B, also known as "NY-ESO-1"), epithelial tumor antigen (ETA), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), folate receptor-alpha (FR-α), human epidermal growth factor receptor-1 (HER1), HER2, HER3, HER4, mucin 1 (MUC1), melanoma-associated antigen (MAA), etc. MAGE), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), B7 homolog 3 protein (B7-H3), stage-specific embryonic antigen-4 (SSEA-4), tyrosinase, mucin-associated Tn antigen, sialic acid Tn antigen, Globo H, ganglioside GD2, CD5, CD19, CD20, CD22, CD23, CD27, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD133, CD134, CD137, CD138, or CD319. According to an exemplary embodiment, the first scFv is specific to Globo H. In another exemplary embodiment, a scFv is specific to B7-H3.

[0066] According to one embodiment of this disclosure, the first and second proteins are differentiation group 8 (CD8) and CD3 zeta chain (CD3ζ), respectively. Therefore, the HTM domain of CD8 contains the amino acid sequence number 9; the 4-1BB co-stimulatory molecule contains the amino acid sequence number 10; and the cytoplasmic domain of CD3ζ contains the amino acid sequence number 11. Depending on the desired purpose, the hinge domain of the HTM domain of the first protein may also be derived from CD28, IgG1, or IgG4, for example, the hinge domain of CD28, IgG1, or IgG4; and / or the transmembrane domain of the HTM domain of the first protein may also be derived from CD3ζ, the CD8α chain (CD8α), CD4, CD28, or the B7 family inducible costimulatory (ICOS) molecule, for example, the transmembrane domain of CD3ζ, CD8α, CD4, CD28, or ICOS.

[0067] It is understood that, in addition to the 4-1BB molecule, the CAR disclosed herein may also contain other costimulatory molecules, such as CD27, CD28, or OX40 (CD134). The 4-1BB molecule in the CAR disclosed herein may be replaced by other costimulatory molecules, such as CD27, CD28, or OX40 (CD134).

[0068] The fifth coding sequence that can encode the 2B6 scFv disclosed herein includes a VH domain coding segment and a VL domain coding segment. As the names suggest, the VH domain coding segment can encode the VH domain that generates the 2B6 scFv disclosed herein, and the VL domain coding segment can encode the VL domain that generates the 2B6 scFv disclosed herein. According to one embodiment of the present disclosure, the mAb 2B6 encoded by the fifth coding sequence has VH and VL regions with sequence numbers 7 and 8, respectively; in this embodiment, the VH and VL domain coding segments correspond to nucleic acid sequences with sequence numbers 16 and 17, respectively. According to another embodiment, the humanized 2B6 encoded by the fifth coding sequence has VH and VL regions with sequence numbers (2B6 Hd VH) and 15 (2B6 Hd VL), respectively; in this embodiment, the corresponding VH and VL domain coding segments are sequence numbers 18 and 21, respectively. According to a further embodiment of this disclosure, the fifth coding sequence encodes a humanized 2B6, whose VH and VL fields are sequence numbers 13 (2B6 HdB1 VH) and 15 (2B6 Hd VL), respectively; the corresponding VH and VL field encoded segments are sequence numbers 19 and 21, respectively. In another alternative embodiment, the fifth coding sequence encodes a humanized 2B6, whose VH and VL fields are sequence numbers 14 (2B6 HuB1 VH) and 15 (2B6 Hd VL), respectively; the corresponding VH and VL field encoded segments are sequence numbers 20 and 21, respectively.

[0069] According to one embodiment of this disclosure, the CAR coding sequence (i.e., the first to fourth coding sequences) and the second scFv coding sequence are separated by an IRES or a linker sequence that encodes a 2A peptide. An IRES is a sequence that recruits ribosomes and allows cap-independent translation. In practice, the IRES acts as a linker to connect two coding sequences in a bicistronic vector, enabling simultaneous translation of both proteins in the cell. A 2A peptide, also known as a "2A autocliptic peptide," is a class of peptides approximately 18 to 22 amino acid residues long that can induce ribosomal skipping during protein translation in cells, thereby generating multiple proteins from a single open reading frame (ORF). In an example embodiment of this disclosure, the CAR coding sequence and the 2B6 scFv coding sequence are linked by a linker sequence that encodes a 2A peptide. Examples of commonly used 2A peptides in this art include, but are not limited to, T2A (EGRGSLLTCGDVEENPGP; sequence number: 22), P2A (ATNFSLLKQAGDVEENPGP; sequence number: 23), E2A (QCTNYALLKLAGDVESNPGP; sequence number: 24), and F2A (VKQTLNFDLLKLAGDVESNPGP; sequence number: 25). In another specific embodiment, the CAR coding sequence and the second scFv coding sequence are linked via IRES.

[0070] Optionally or additionally, the N-terminus of the second scFv may further include a signal peptide (also known as a "message sequence" or "lead sequence"). As is known in the art, a signal peptide is a peptide of about 15 to 50 amino acid residues in length that functions to guide proteins into the secretion pathway. Examples of signaling peptides suitable for use in the second scFv of this disclosure include, but are not limited to: tissue plasminogen activator (tPA), IgK, IgG, CD33, metalloproteinase inhibitor 1 (TIMP1), chronodroitin sulfate proteoglycan 4 (CSPG4), calreticulin (CARL), Dickkopf-related protein 3 (DKK3), 60S acidic ribosomal protein P2 (RPLP2), complement C1s (C1S), cathepsin Z (CTSZ), nucleobinin-2 (NUCB2), and protein disulfide isomerase. The signal peptides used include PDIA1, protein disulfide-isomerase A3 (PDIA3), endoplasmic reticulum protein, hypoxia upregulated protein 1 (HYOU1), trypsinogen 2, serum albumin, and serine protease inhibitor H1. According to one specific embodiment, the signal peptide used is derived from the IgG light chain. Those skilled in the art can select a suitable signal peptide according to actual needs.

[0071] Preferably, the second scFv of this disclosure further includes an immunoglobulin Fc region (referred to as "2B6 scFv-Fc") located at its C-terminus. Preferably, this Fc region includes glycine (G) and / or serine (S) residues, such as (G₄S)₃ (Sequence number: 26). Therefore, the nucleic acid of this disclosure may further include a sixth coding sequence encoding the Fc region that produces the immunoglobulin (e.g., the Fc region of IgG). Depending on the intended purpose, the selected immunoglobulin may be IgG, IgA, IgM, IgD, or IgE. According to some preferred embodiments, the immunoglobulin used is IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant region of the immunoglobulin contains a mutation that reduces the binding affinity of the immunoglobulin to the Fc receptor or reduces the function of the Fc effector. For example, its immunoglobulin constant region may contain mutations to eliminate glycosylation sites within the immunoglobulin heavy chain constant region. In other embodiments, the immunoglobulin constant region may contain one or more mutations, deletions, and / or insertions at amino acid positions L234, L235, G236, G237, N297, or P331 corresponding to IgG1 amino acids. In one specific embodiment, the immunoglobulin constant region has a mutation at position N297 of IgG1. In other alternative embodiments, the immunoglobulin constant region contains one or more mutations, deletions, and / or insertions at amino acid positions corresponding to L281, L282, G283, G284, N344, or P378 of IgG1. According to various embodiments of this disclosure, the immunoglobulin used is IgG1 or IgG4.

[0072] According to embodiments of this disclosure, the second, third, and fourth coding sequences correspond to nucleotide sequences numbered 27, 28, and 29, respectively. It is understood that the second, third, and / or fourth coding sequences of this disclosure can be modified to include one or more degenerate nucleotides, as long as the proteins they encode (i.e., CD8 HTM, 4-1BB molecules, and / or CD3ζ cytoplasmic domains) retain the desired activity or function. The term "degenerate nucleotide sequence" (also known as "nucleotide degeneration") refers to a nucleotide sequence containing one or more degenerate codons; these degenerate codons, although having different triplet combinations, can encode the same amino acid (e.g., both GAU and GAC can encode aspartic acid Asp). Therefore, nucleic acid sequences containing degraded nucleotides are intended to be included within the scope of this disclosure, provided that the variations in their nucleotide sequences are at least 85% identical to the sequences of sequence numbers 27, 28, and 29, for example, sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences of sequence numbers 27, 28, and 29 are also considered to be included within the scope of this disclosure.

[0073] As stated above, the nucleic acid sequence of this disclosure may be modified to include one or more degenerate nucleotides, provided that the encoded protein (i.e., 2B6 scFv) can maintain its intended activity or function. Therefore, nucleic acid sequences containing degenerate nucleotides are also within the scope of this disclosure, provided that the variations in their nucleotide sequences are at least 85% identical to the sequences of sequence numbers 18–21, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences of sequence numbers 18–21.

[0074] This disclosure also provides expression vectors containing the above-described nucleic acids. According to some embodiments, the expression vector is a viral vector, such as a lentiviral vector, adenovirus vector, retroviral vector, adeno-associated virus vector, or sindbis viral vector. In some exemplary embodiments, the expression vector is a lentiviral vector.

[0075] (iii) Cells expressing CAR and their use in cancer treatment

[0076] The nucleic acid or expression vector described in Part (ii) of this disclosure can be used to transfect cells (e.g., T cells) to generate genetically modified cells (e.g., CAR-T cells). Specifically, the nucleic acid or expression vector can be introduced into cells via transfection methods known in the art, preferably immune cells (e.g., T cells, NK cells, or macrophages). Transfection methods may include chemical transfection (e.g., calcium phosphate transfection, liposome transfection, or non-liposome transfection) or physical transfection (e.g., microinjection, electroporation, or bioballistic particle delivery). In addition, if the expression vector is a viral vector (e.g., a lentiviral vector), it can first be introduced into host cells (e.g., HEK293T cells) via transfection to generate a virus (e.g., lentivirus), and then the virus can be used to infect target cells (e.g., T cells, NK cells, or macrophages) to achieve gene expression.

[0077] The resulting cells (e.g., CAR-T cells) have the following characteristics: (a) expressing CAR on the cell surface, which enables the cells to specifically recognize targets and destroy cancer cells; and (b) producing and secreting anti-PD-1 antibodies (i.e., anti-PD-1 scFv or anti-PD-1 scFv-Fc), which can reduce immunosuppression in the tumor microenvironment, thereby enhancing the antitumor response of CAR-expressing cells (e.g., CAR-T cells) in solid tumors.

[0078] Therefore, another aspect of this disclosure relates to a genetically modified cell (i.e., a cell expressing CAR) and the use of such a cell in cancer treatment.

[0079] Depending on the application, the cells modified by the nucleic acid or expression vector disclosed herein may be T cells, NK cells, or macrophages. In some preferred embodiments, the genetically modified cells are T cells (i.e., CAR-T cells). Methods of treating cancer include administering an effective dose of genetically modified cells (e.g., CAR-T cells, CAR-NK cells, or CAR-macrophages) to a subject to alleviate or improve cancer symptoms.

[0080] According to some embodiments, the subject is a mouse, which may be transferred with approximately 1×10⁴ to 1×10⁸ CAR-T cells (e.g., 1×10⁴, 1.5×10⁴, 2×10⁴, 2.5×10⁴, 3×10⁴, 3.5×10⁴, 4×10⁴, 4.5×10⁴, 5×10⁴, 5.5×10⁴, 6×10⁴, 6.5×10⁴, 7×10⁴). 7.5×10⁴, 8×10⁴, 8.5×10⁴, 9×10⁴, 9.5×10⁴, 1×10⁵, 1.5×10⁵, 2×10⁵, 2.5×10⁵, 3×10⁵, 3.5×10⁵, 4×10⁵, 4.5×10⁵, 5×10⁵, 5.5×10⁵, 6×10⁵, 6.5×10⁵, 7×10⁵, 7.5×10⁵, 8×10⁵ 8.5×10⁵, 9×10⁵, 9.5×10⁵, 1×10⁶, 1.5×10⁶, 2×10⁶, 2.5×10⁶, 3×10⁶, 3.5×10⁶, 4×10⁶, 4.5×10⁶, 5×10⁶, 5.5×10⁶, 6×10⁶, 6.5×10⁶, 7×10⁶, 7.5×10⁶, 8×10⁶, 8.5×10⁶, 9×10⁶ 9.5×10⁶, 1×10⁷, 1.5×10⁷, 2×10⁷, 2.5×10⁷, 3×10⁷, 3.5×10⁷, 4×10⁷, 4.5×10⁷, 5×10⁷, 5.5×10⁷, 6×10⁷, 6.5×10⁷, 7×10⁷, 7.5×10⁷, 8×10⁷, 8.5×10⁷, 9×10⁷, 9.5×10⁷, or 1×10⁸). A preferred dose is about 1×10⁵ to 1×10⁷ CAR-T cells, more preferably about 5×10⁵ to 1×10⁶ CAR-T cells. In one specific embodiment, approximately 6 × 10⁵ CAR-T cells are sufficient to provide protective and / or therapeutic effects in subjects in mice.

[0081] Generally speaking, for human CAR-T therapy, each dose requires approximately 1×10⁶ to 1×10⁷ CAR-T cells / kg body weight (e.g., 1×10⁶, 1.5×10⁶, 2×10⁶, 2.5×10⁶, 3×10⁶, 3.5×10⁶, 4×10⁶, 4.5×10⁶, 5×10⁶, 5.5×10⁶, 6×10⁶, 6.5×10⁶, 7×10⁶, 7.5×10⁶, 8×10⁶, 8.5×10⁶, 9×10⁶, 9.5×10⁶ or 1×10⁷). Understandably, the number of CAR-T cells administered to human subjects can vary depending on clinical factors such as age, sex, underlying diseases, treatment plan, pretreatment regimen, and infection status. Those with ordinary knowledge in the field or medical professionals can adjust or optimize the number of CAR-T cells transplanted according to the desired purpose.

[0082] The genetically modified cells can be autologous cells (i.e., taken from the cancer patient themselves), allogeneic cells (i.e., taken from another individual of the same species as the cancer patient), or xenogeneic cells (i.e., taken from a donor of a different species than the cancer patient). Preferably, the genetically modified cells are taken from a patient receiving treatment / medication to avoid transplant rejection. When the genetically modified cells are allogeneic or xenogeneic, the method further includes administering immunosuppressive therapy to the patient before, during, or after the administration of the genetically modified cells to suppress the patient's immune response to these cells. The immunosuppression can be achieved by any agent and / or method known to those skilled in the art to prevent transplant rejection, such as gamma radiation or immunosuppressants.

[0083] Depending on the therapeutic purpose, the immunosuppressants used may be: glucocorticoids (e.g., prednisone, budesonide, prednisolone, dexamethasone, or hydrocortisone); JAK inhibitors (janus kinase inhibitors, e.g., tofacitinib); calcineurin inhibitors (e.g., cyclosporine or tacrolimus); mTOR inhibitors (e.g., sirolimus or everolimus); and inhibitors of inosine monophosphate dehydrogenase. IMDH inhibitors, such as azathioprine, leflunomide, or mycophenolate; biologics or monoclonal antibodies (e.g., abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab). Or daclizumab, or any other known immunosuppressant that can inhibit or slow the immune response, such as methotrexate or mecaptopurine. Clinicians or those with ordinary knowledge in this field can determine the appropriate type and method of immunosuppressant based on the subject's physiological condition.

[0084] The genetically modified cells may be administered to the subject via any suitable route, such as intravenous, intraperitoneal, intra-arterial, or intratumoral injection. Preferably, the genetically modified cells are administered to the subject via intravenous injection.

[0085] It is understood that the methods disclosed herein can be used alone or in combination with other therapies that can prevent or treat cancer, such as immunotherapy (e.g., PD-1 or PD-L1 inhibitor therapy), surgery, chemotherapy, and / or radiation therapy. Depending on the intended / therapeutic purpose, this method can be applied to the subject before, during, or after administration of other therapies.

[0086] Non-limiting examples of cancers that can be treated with the methods and / or pharmaceutical compositions disclosed herein include: gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, and squamous cell carcinoma of the head and neck.

[0087] Essentially, the subject who may be treated by the methods and / or pharmaceutical compositions disclosed herein is a mammal, such as a human, mouse, rat, guinea pig, hamster, monkey, pig, dog, cat, horse, sheep, goat, cow, and rabbit. Preferably, the subject is a human.

[0088] Several experimental examples are presented below to illustrate certain aspects of the present invention, so as to facilitate the implementation of the present invention by those skilled in the art, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art, after reading the description presented herein, can fully utilize and practice the present invention without excessive interpretation. All published documents cited herein are considered to be part of this specification in their entirety.

[0089] Example

[0090] Materials and Methods

[0091] Preparation of mAb 2B6

[0092] Two BALB / c mice were infected with the extracellular region of PD-1 to generate anti-PD-1 fusion tumors. After screening by enzyme-linked immunosorbent assay (ELISA), a fusion tumor cell line capable of producing anti-PD-1 antibodies was identified, clone 2B6, for subsequent research. The VH and VL domains of mouse anti-PD-1 antibody clone 2B6 were sequenced and subcloned into an expression vector. The specific steps for preparing anti-PD-1 antibody clone 2B6 are as follows: Expression vectors encoding the VH and VL domains that produce anti-PD-1 2B6 antibodies were transfected into FreeStyle™ 293 cells using polyethyleneimine (PEI) as the transfection agent at a ratio of 1.25 μg plasso DNA per 1 × 10⁶ cells. After transfection, the cells were acclimatized in FreeStyle™ 293 expression medium and cultured in tissue culture flasks. The culture supernatant was collected when the viability reached 90%. The collected culture supernatant was first filtered through 10-micron and 0.2-micron filter membranes to remove impurities. Next, the antibody was purified using Protein A affinity chromatography, with phosphate-buffered saline (PBS) as the adsorption buffer and 200 mM glycine buffer (pH 2.5) as the elution buffer. The pH of the elution buffer was adjusted to approximately 6.0-7.0 by adding 50 mM Tris buffer (pH 9.0). The antibody solution was then replaced with PBS using a dialysis membrane (molecular weight 10,000), and filtered through a 0.22-micron membrane filter for sterilization. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and converting the measured value using a conversion factor of 1.45 equaling 1 mg / ml.

[0093] The mAb prepared as described above is named "mAb 2B6", and the VH and VL sequences are summarized in Table 1.

[0094] Table 1. VH and VL sequences of mAb 2B6 name amino acid sequence Serial Number mAb 2B6 VH EVKLVESGGGLVKPGGSLKLSCAAAGFTFSSYTMSWVRQTPAKRLEWVATISGGGANIYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCVSPYYAIDFWGQGTSVTVSS 7 mAb 2B6 VL DTVMTQSDKFMSTSVGDRVSITCKASQDVGSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSTYTWTFGGGTKLEIK 8 *CDR sequences (including CDR-H1, CDR-H2, and CDR-H3 in the VH domain, and CDR-L1, CDR-L2, and CDR-L3 in the VL domain) are marked in bold and arranged in order.

[0095] Humanized mAb 2B6

[0096] (A) Selecting human variable domain framework region sequences

[0097] Mouse-derived monoclonal antibodies may induce strong immunogenicity and drug-resistant antibodies in humans. Therefore, humanization of mouse monoclonal antibodies is an important step in drug development.

[0098] To prepare the humanized antibody 2B6 4D5 (abbreviated as 2B6-HdHd), the human receptor framework selected was chosen from a framework validated in clinical trials. The human heavy and light chain framework regions of VH subset III (IGHV3) and VLκ subset I (IGKV1) have been shown to perform well in clinical applications and have been successfully applied to various humanized antibodies.

[0099] To prepare the humanized antibody 2B6 IMGT VH (abbreviated as 2B6-HuHd), the human germline VH sequence most homologous to the mAb 2B6 framework region was identified using the IMGT database (International Immunogene Information System®). Homology searches were performed using BLAST or similar methods, with the variable region sequence of mAb 2B6 as the query sequence. The results showed that the human germline gene IGH3-23*04 (VH) was most similar to the mAb 2B6 heavy chain framework sequence.

[0100] These two sets of heavy chain sequences (human 4D5 and human IMGT) and one set of light chains (human 4D5) were used as templates for constructing humanized antibodies against human PD-1.

[0101] (B) Reversion mutation

[0102] Transplanting the CDR into the human frame results in variable domains (VH and VL) from different sources. These chimeric domains may not be optimal sequence combinations, and therefore the antibody affinity may not be optimal. To improve binding affinity, certain amino acids can be reverted to their original species sequences. These key amino acid residues can sometimes affect antibody binding in the core and interfacial regions of the antibody. Based on this principle, three reversion mutations were introduced into the humanized 2B6 HdB1 (VH) and 2B6 HuB1 (VH) (at positions 73, 93, and 94 in the frame region, respectively).

[0103] The amino acid sequences of the humanized VH and VL domains prepared as described above are summarized in Table 2.

[0104] Table 2. Amino acid sequences of humanized VH and VL domains. name amino acid sequence Serial Number 2B6 Hd VH EVQLVESGGGLVQPGGSLRLSCKASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARPYYAIDFWGQGTLVTVSS 12 2B6 HdB1 VH EVQLVESGGGLVQPGGSLRLSCKASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSPYYAIDFWGQGTLVTVSS 13 2B6 HuB1 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISGGGANIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSPYYAIDFWGQGTLVTVSS 14 2B6 Hd VL DIQMTQSPSSSLSASVGDRVTITCKASQDVGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYTWTFGQGTKVEIK 15

[0105] Exhibition of humanized antibodies

[0106] To confirm the changes in antibody affinity after humanization of mouse antibodies, variable regions of the humanized light and heavy chains were prepared using nucleic acid synthesis. The mouse or humanized variable regions were constructed into a human chimeric antibody mammalian expression vector, which was then introduced into host cells to prepare recombinant antibody-expressing cells. FreeStyle™ 293 or Expi™ 293 cells were used as host cells. The antibody expression vector was transfected using polyethyleneimine (PEI) at a ratio of approximately 1.25 μg of antibody expression vector per 1 × 10⁶ cells. 30 ml of transient expression culture supernatant containing human IgG antibody was prepared using the following method. Antibody-producing cells were cultured in FreeStyle™ 293 expression medium for acclimatization at 8% CO₂, 37°C, and in an orbital shaking culture flask at 135 rpm. The supernatant was collected on day 6 post-transfection. The supernatant was collected and filtered through a 0.2-micron filter membrane. Protein A was then used for affinity purification, with PBS as the adsorption buffer and 200 mM glycine buffer (pH 2.5) as the elution buffer. The pH of the elution buffer was adjusted to approximately 6.0-7.0 by adding 50 mM Tris buffer (pH 9.0). The antibody solution was replaced with PBS using a dialysis membrane (molecular weight 10,000), and then filtered through a 0.22-micron membrane filter for sterilization to obtain the purified antibody. The absorbance at 280 nm was measured, and the concentration of the purified antibody was determined by converting the measured value according to the conversion factor of 1.45 (1 mg / ml). (See Table 3 for details).

[0107] Table 3. Performance levels and yields of each antibody sample

[0108] Converting intact IgG to the scFv-Fc configuration

[0109] The heavy chain variable region (VH) and light chain variable region (VL) of the humanized 2B6 antibody HuB1 Hd were cloned into the scFv(VH-linker (Gly₄Ser)₃-VL)-Fc configuration by polymerase chain reaction (PCR). Recombinant humanized 2B6 HuB1-scFv-Fc (derived from the scFv configuration of 2B6(HuB1 Hd), Pembro-scFv-Fc (derived from the scFv configuration of Pembrolizumab), and Nivo-scFv-Fc (derived from the scFv configuration of Nivolumab) were all expressed in FreeStyle™ 293 cells (as shown in Figure 5A). All purified scFv-Fc antibodies were quantified by OD260 / 280, and their yields are listed in Table 4. The results showed that the yield of 2B6 HuB1-scFv-Fc was significantly higher than that of Pembro-scFv-Fc and Nivo-scFv-Fc.

[0110] Table 4. Performance level and yield of each specified antibody

[0111] The binding affinity of humanized antibodies and scFv-Fc antibodies to PD-1 was measured by ELISA.

[0112] The ELISA plate was coated with PD-1-hFc (1 µg / mL), and non-specific binding sites were blocked with blocking buffer (5% skim milk powder dissolved in PBS). Next, antibody diluted from 66.67 nM to 2 × 10⁻⁴ nM (4-fold dilution) was added to the reaction wells. After adding goat anti-human Kappa chain HRP IgG (dilution ratio 1:3,000), the binding affinity with PD-1-hFc was measured, and the binding curve and dissociation constant (KD) were calculated using a non-linear fitting method for single-point specific binding.

[0113] CAR plasmid construction

[0114] In this study, a lentiviral expression vector (pLVX-EF1a-IRES) based on human immunodeficiency virus (HIV)-1 was used. DNA fragments encoding Globo H scFv, the CD8 hinge and transmembrane domain, the co-stimulatory molecule 4-1BB, the CD3ζ domain, T2A, the signal peptide (sequence number: 30), humanized 2B6 HuB1, and the human IgG4 Fc domain were synthesized and assembled into a CAR gene cassette. The assembled gene cassette was inserted into the pLVX-EF1a-IRES vector via EcoRI and BamHI restriction enzyme sites.

[0115] The plasmid prepared as described above is named "Globo H / PD-12B6 scFv-Fc CAR plasmid" (see Figure 2).

[0116] This study also provides three positive control plasmids, including: (1) the plasmid encoding the production of Globo H CAR (named "Globo H CAR plasmid"); (2) the plasmid encoding the production of Globo H CAR and Pembro scFv-Fc (named "Globo H / Pembro scFv-Fc CAR plasmid"); and (3) the plasmid encoding the production of Globo H CAR and Nivo scFv-Fc (named "Globo H / Nivo scFv-Fc CAR plasmid") (see Figure 2).

[0117] Globo H / PD-12B6 scFv-Fc CAR plasmids, Globo H / Pembro scFv-Fc CAR plasmids, and Globo H / Nivo scFv-Fc CAR plasmids were expressed in FreeStyle™ 293 cells. All purified scFv-Fc antibodies were quantified by OD260 / 280, and their PD-1 binding affinity was also determined using the methods described above.

[0118] On the other hand, humanized 2B6 HuB1 was also fused with B7-H3 scFv (sequence number: 31). The resulting plasmid was named "B7-H3 / PD-12B6 scFv-Fc CAR plasmid," which contained DNA fragments encoding the generation of B7-H3 scFv (sequence number: 32), the CD8 hinge and transmembrane domain, the co-stimulatory molecule 4-1BB, the CD3ζ domain, T2A, the signal peptide, humanized 2B6 HuB1, and the human IgG4 Fc domain. In this study, the plasmid encoding the generation of B7-H3 CAR (named "B7-H3 CAR plasmid") was used as a positive control.

[0119] Lentivirus Preparation

[0120] One × 10⁷ 293T cells were seeded in a 15 cm culture dish. Before transfection, the culture medium was replaced with fresh DMEM containing 10% fetal bovine serum (FBS). pMD.G (6 µg), R8.91 (15 µg), and transplasts (20 µg) were mixed with polyethylenimine (PEI) at a volume ratio of 1:2.5 and incubated at room temperature for 20 minutes. This mixture was then added to the 293T cells. Sixteen hours later, the culture medium was replaced with DMEM containing 2% FBS. At 48 and 72 hours post-transfection, the supernatant containing viral particles was collected, mixed with a concentration reagent, and incubated overnight. The mixture was then centrifuged at 1500 × g for 30 minutes at 4°C. The lentivirus particles were resuspended in the culture medium. Viral titer was determined by infection of Jurkat cells and flow cytometry analysis.

[0121] The lentiviruses prepared as described above are named "Globo H / PD-12B6 scFv-Fc CAR virus" and "B7-H3 / PD-12B6 scFv-Fc CAR virus" respectively.

[0122] This study also provides four lentiviruses as positive controls, including: (1) a lentivirus carrying the Globo H CAR plasmid (named "Globo H CAR virus"); (2) a lentivirus carrying the Globo H / Pembro scFv-Fc CAR plasmid (named "Globo H / Pembro scFv-Fc CAR virus"); (3) a lentivirus carrying the Globo H / Nivo scFv-Fc CAR plasmid (named "Globo H / Nivo scFv-Fc CAR virus"); and (4) a lentivirus carrying the B7-H3 CAR plasmid (named "B7-H3 CAR virus").

[0123] CAR-T cell preparation

[0124] (i) Isolation of peripheral blood mononuclear cells (PBMCs)

[0125] A blood sample (10 ml) isolated from a healthy donor was diluted with 1-fold phosphate-buffered saline (PBS; 10 ml) or balanced salt buffer. 15 ml of Lymphoprep medium was added to a centrifuge tube, and the diluted blood sample (total 20 ml) was carefully layered onto the Lymphoprep medium. The tube was then centrifuged at 800 xg for 20 minutes at 15°C–20°C (without brakes). The plasma and platelet layers were discarded. The monocytes at the interface were transferred to another sterile centrifuge tube (approximately 8 ml), and at least 3 volumes (approximately 25 ml) of 1-fold PBS were added and mixed. The tube was centrifuged at 500 xg for 10 minutes at 20°C, and the supernatant was discarded. The cells were washed twice with 1-fold PBS, and finally resuspended in a culture medium suitable for cell number determination.

[0126] (ii) Isolation of Pan T cells

[0127] After determining the cell count, centrifuge at 300 xg for 10 minutes. Resuspend the resulting cell pellet in buffer, add CD3 magnetic beads, and incubate at 4-8°C for 15 minutes. Wash the cells with 1-2 mL buffer, centrifuge at 300 xg for 10 minutes, resuspend the cells in buffer, and then add them to the column under the magnetic field of a Separator QuadroMACS™. Collect the unlabeled cells that have flowed through the column and wash the column three times with 3 mL buffer. Remove the column from the separator and place it in an appropriate collection tube. Add 5 mL of 1x PBS to the column and immediately press the plunger attached to the column firmly to flush out the portion containing magnetically labeled cells. Collect the eluent and determine the T cell count.

[0128] (iii) T cell activation

[0129] Add purified T cells to Dynabeads® human T-activator CD3 / CD28 magnetic beads (beads:cells = 2:1). Change the culture medium every two days.

[0130] (iv) Viral transduction

[0131] Primary T cells were seeded at a concentration of 1.8 × 10⁶ cells per well in 6-well culture plates, and lentivirus carrying CAR-encoded nucleic acid (MOI=1; MOI: multiplicity of infection) was added. The plates were then centrifuged at 800 xg for 90 minutes and incubated overnight at 37°C. Next, 2 mL of culture medium containing IL-2 (125 U / mL) was added, and GFP(Fab) expression was detected by flow cytometry on days 4, 7, and 10.

[0132] (v) CAR-T cell expansion

[0133] Adjust the cell density to 5×10⁵ / mL with culture medium. After changing the culture medium on day 4, culture the cells in a 30 mL bioreactor (initial total cell count 9×10⁶, 120 rpm). On day 7, change the culture medium and culture the cells in a 100 mL bioreactor (initial total cell count 3×10⁷, stirring speed 90 rpm). Harvest CAR-T cells on day 10.

[0134] The CAR-T cells prepared as described above are named "Globo H / PD-12B6 scFv-Fc CAR-T cells" and "B7-H3 / PD-12B6 scFv-Fc CAR-T cells" respectively.

[0135] In addition, T cells were transfected with Globo H CAR virus, Globo H / Pembro scFv-Fc CAR virus, Globo H / Nivo scFv-Fc CAR virus and B7-H3 CAR virus, respectively. The T cells transfected with these viruses were named "Globo H CAR-T cells", "Globo H / Pembro scFv-Fc CAR-T cells", "Globo H / Nivo scFv-Fc CAR-T cells" and "B7-H3 CAR-T cells", respectively, as positive controls in this study.

[0136] Cytotoxicity analysis

[0137] Target cells were seeded at a concentration of 2 × 10⁴ cells per well in 96-well culture dishes (triple replicate). Subsequently, CAR-T cells (effective cells) were added at different effector-to-target (E:T) ratios, including 1:1, 0.5:1, and 0.25:1 NCI-N87 / PD-L1. Cells were cultured at 37°C for 24 hours. After 24 hours at 37°C, cells were washed twice with RPMI 1640 medium, and cell viability was analyzed using a CCK-8 cell counting kit to assess the cytotoxic ability of CAR-T cells.

[0138] Animal Experiments

[0139] High-expressing PD-L1 NCI-N87 cells (N87 / PD-L1) for transplantation were harvested during the logarithmic growth phase and resuspended in PBS containing 50% Matrigel® basement membrane matrix to a final concentration of 3 × 10⁷ cells / mL. N87 / PD-L1 tumor cells (3 × 10⁶ cells, 0.1 mL) were subcutaneously injected into the right anterior axillary region of mice to induce tumor growth. On day 10 post-inoculation, when the mean tumor volume (MTV) reached approximately 117 mm³, the tumor-bearing mice were randomly divided into 7 groups of 4 mice each, and injected with either CAR-T cells or control solution on the same day. CAR-T cell suspensions of Globo H, Globo H / PD-1 Nivo scFv-Fc, or Globo H / PD-12B6 scFv-Fc (6 × 10⁵ CAR-T cells per cell, injection volume 0.1 mL) were administered via single intravenous injection. Tumor volume was measured three times weekly using a digital caliper, and calculated using the formula TV = (w² × l) / 2 (in mm³), where w is the tumor width and l is the tumor length (mm). Tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1 – (T / C)] × 100%, where T and C represent the MTV of the treatment group and control group, respectively.

[0140] Cal-27 cells for transplantation were harvested during the logarithmic growth phase and resuspended in PBS containing 50% Matrigel® basement membrane matrix to a final concentration of 1×10⁷ cells / mL. Cal-27 tumor cells (1×10⁶ cells, 0.1 mL) were subcutaneously injected into the right anterior axillary region of mice to induce tumor growth. On day 10 post-inoculation, when the MTV reached approximately 141 mm³, the tumor-bearing mice were randomly divided into three groups of four mice each, and injected with either CAR-T cells or control solution on the same day. A single intravenous injection of B7-H3 or B7-H3 / PD-12B6 scFv-Fc CAR-T cell suspension (6×10⁵ CAR-T cells per mouse, 0.1 mL injection volume) was administered. Tumor volume was measured three times a week and recorded using a digital caliper. The tumor volume (TV) was calculated using the formula TV = (w² × l) / 2 (in mm³), where w is the tumor width and l is the tumor length (mm). The tumor growth inhibition rate (TGI) was calculated using the following formula: %TGI = [1 – (T / C)] × 100%, where T and C represent the MTV of the treatment group and the control group, respectively.

[0141] Example 1: ELISA determination of the binding affinity of chimeric anti-human PD-1 2B6 antibody to PD-1

[0142] The antibodies disclosed herein were evaluated for their specific binding ability to PD-1 by ELISA. In short, recombinant PD-1 protein was immobilized at a concentration of 0.1 µg per well on a 96-well ELISA plate. A serially diluted chimeric anti-human PD-1 2B6 antibody was added to the immobilized PD-1 protein, followed by detection with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody. Colorimetric analysis was performed using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, and optical density (OD) was measured at 405 nm to analyze binding activity.

[0143] As shown in Figure 3, the chimeric anti-human PD-1 2B6 antibody exhibits specific and high-affinity binding to PD-1, with a dissociation constant (KD) of 8.09 × 10⁻¹⁰ M, indicating that the antibody has a close interaction with its target.

[0144] Example 2: Chimeric anti-human PD-1 2B6 antibody exhibits dose-dependent blocking ability of PD-1 / PD-L1 interaction in bioluminescent cell assays.

[0145] To evaluate the ability of the antibody revealed in this study to block the PD-1 / PD-L1 interaction, a bioluminescent cytology assay was performed using the Promega PD-1 / PD-L1 Blocking Bioassay Kit (Promega, Madison, Wisconsin, USA). The assay included two genetically engineered cell lines: PD-1 effector cells (Jurkat T cells, expressing human PD-1 and carrying an NFAT-RE luciferase reporter gene driven by the NFAT response element) and PD-L1 aAPC / CHO-K1 cells (CHO-K1 cells, expressing human PD-L1 and carrying an engineered cell surface protein that can activate the homologous TCR in a non-antigen-dependent manner).

[0146] When these two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. Adding an antibody that blocks the PD-1 / PD-L1 interaction (such as the chimeric anti-human PD-1 2B6 antibody disclosed herein) releases this inhibitory signal, activating the TCR and dose-dependently enhancing the NFAT-RE-mediated luminescence signal. This bioluminescent signal was detected and quantified using a Bio-Glo™ luciferase detection system and a photometer (GloMax® Discover System, Promega, Madison, Wisconsin, USA).

[0147] As shown in Figure 4, the chimeric anti-human PD-1 2B6 antibody effectively and specifically blocks the PD-1 / PD-L1 interaction, with an EC50 of 3.88 × 10⁻⁹ M (approximately 3.88 nM). Other antibodies disclosed in this specification have also shown similar inhibitory activity (data not shown). These results confirm that the antibodies disclosed herein can effectively relieve PD-1 / PD-L1-induced immunosuppression, demonstrating their potential therapeutic use in treating immunosuppressive or exhaustion-driven diseases such as cancer.

[0148] Example 3: In vivo antitumor effect of chimeric anti-PD-1 2B6 antibody in MC38-hPD-L1 allogeneic tumor mouse model

[0149] An allogeneic mouse model was established according to the methods described in "Materials and Methods" to evaluate the anticancer effect of the chimeric anti-human PD-1 2B6 antibody. Briefly, B-hPD-1 / hPD-L1 mice were subcutaneously inoculated with MC38-hPD-L1 tumor cells (5 × 10⁵ cells, suspended in 0.1 mL PBS) and injected into the right anterior abdomen to establish tumors. When the average tumor volume reached 75 ± 25 mm³, the tumor-bearing mice were randomly assigned to seven study groups (G1: 6 mice; G2-G4: 8 mice each). The treatment group received intraperitoneal injections twice a week for a total of six times at a dose of 5 mg / kg. The test drugs included mouse IgG (control group), atezolizumab (Ate.), Keytruda (Key.), and the chimeric anti-PD-1 2B6 antibody. Tumor volume and body weight were measured twice a week. The study was terminated on day 28 (day 7 after the last dose), and the tumor was removed, weighed, and photographed. The results are shown in Figure 5.

[0150] The results showed that, compared with the control group, the 2B6 antibody significantly inhibited tumor growth, and its efficacy was comparable to or even better than that of commercially available anti-PD-1 / PD-L1 antibodies, demonstrating its potential as a novel immunotherapy candidate.

[0151] Example 4: Binding affinity of humanized 2B6 antibody to PD-1

[0152] The binding affinity of the antibodies listed in Tables 3 and 4 to PD-1 disclosed herein was determined according to the ELISA method described in the "Materials and Methods" section of this specification.

[0153] Compared to the parental mouse strain 2B6(MM), which has a binding affinity of approximately 4.95 × 10⁻¹⁰ M, the humanized antibodies 2B6 HdB1 Hd and 2B6 HuB1 Hd have binding affinities of 3.68 × 10⁻¹⁰ M and 4.03 × 10⁻¹⁰ M for PD-1, respectively. Compared to Hd(VH), both HdB1 and HuB1(VH) contain three beneficial mutations in their heavy chain backbone regions and exhibit higher binding affinity for PD-1 (Table 5). These residues representing the framework region indirectly promote the binding of PD-1.

[0154] Table 5: This article reveals the binding affinity of antibodies. Antibody name MM Hd-Hd HdB1 Hd HuB1 Hd K D (M) 4.95×10 -10 1.79 × 10 -7 3.68×10 -10 4.03×10 -10 The KD values ​​of the 2B6 HuB1-scFv-Fc, Pembro-scFv-Fc, and Nivo-scFv-Fc antibodies were 2.58 × 10⁻⁹, 2.79 × 10⁻⁹, and 6.68 × 10⁻⁹ M, respectively (see Table 6). The binding affinity of 2B6 HuB1-scFv-Fc was significantly better than that of Nivo-scFv-Fc and comparable to that of Pembro-scFv-Fc.

[0156] Table 6: Binding affinity of the antibodies of the present invention Antibody name 2B6 HuB1-scFv-Fc Pembro-scFv-Fc Nivo-ScFv-Fc K D (M) 2.58×10 -9 2.79 × 10 -9 6.68×10 -9

[0157] Example 5: The manifestation of the CAR structure disclosed herein

[0158] To assess the expressive power of the CAR structure disclosed herein, plastids encoding the CAR molecule were transfected into 293T cells. After culturing, the protein was collected from the supernatant, purified, and quantified using OD260 / 280 absorbance. Furthermore, the binding affinity of this purified protein to PD-1 was determined using ELISA. The results are summarized in Table 7.

[0159] Table 7: Performance results of specific CAR structures Antibody name Pembro-scFv-Fc Nivo-ScFv-Fc 2B6 HuB1-scFv-F K D (M) 4.62×10 -9 5.21 × 10 -9 1.75×10 -9 Yield (μg) 47.6 61.7 124

[0160] The data in Table 7 show that Globo H / PD-12B6 scFv-Fc CAR can be efficiently expressed in 293T cells, and the binding affinity of 2B6 HuB1-scFv-Fc to PD-1 is significantly better than that of Nivo-scFv-Fc and Pembro-scFv-Fc.

[0161] Example 6: In vitro antitumor effect of CAR-T cells disclosed in this paper

[0162] The cytotoxic activity of Globo H / PD-12B6 scFv-Fc CAR-T cells disclosed herein was determined by co-culturing Globo H / PD-12B6 scFv-Fc CAR-T cells and cancer cells at different effector cell to target cell ratios (E:T ratio). The specific methods are detailed in "Materials and Methods". Figure 6 shows that Globo H / PD-12B6 scFv-Fc CAR-T cells exhibited cytotoxicity against cancer cells in a dose-dependent manner.

[0163] Example 7: Anti-tumor effect of CAR-T cells disclosed in this paper

[0164] This example uses the N87 / PD-L1 gastric cancer model and the Cal-27 tongue squamous cell carcinoma model to evaluate the therapeutic effect of CAR-T cells on tumors as disclosed herein. According to "Materials and Methods", a single dose of CAR-T cells (6 × 10⁵ cells) was injected into mice carrying N87 or Cal-27 tumors, and tumor volume was monitored every two to three days.

[0165] Compared with the control group, the CAR-T cells disclosed in this study significantly inhibited tumor growth in the N87 / PD-L1 tumor model, with a tumor growth inhibition (TGI) rate of approximately 73% (Figure 7A). In particular, the anti-tumor effect of the CAR-T cells disclosed in this study was significantly better than that of the control groups, including Globo H CAR-T cells (GH CAR-T) and Globo H / Nivo scFv-Fc CAR-T cells (GH / Nivo CAR-T) (Figures 7A–7D).

[0166] Compared with the control group, the CAR-T cells disclosed in this study significantly inhibited tumor growth in the Cal-27 tumor model, with a TGI of approximately 47.6% (Figure 8). In particular, the anti-tumor effect of B7H3 / PD-1 2B6HuB1 CAR-T cells was significantly better than that of the control group, including groups that were given only the vector or B7H3 CAR-T cells (Figure 8).

[0167] In summary, this specification provides a novel anti-PD-1 antibody 2B6 and CAR-T cells comprising the antibody. According to the embodiments disclosed herein, the CAR-T cells (i.e., Globo H / PD-1 2B6 scFv-Fc CAR-T cells) exhibit binding affinity for PD-1 and cytotoxic activity against cancer cells. These CAR-T cells can produce and secrete anti-PD-1 scFv-Fc, thereby reducing immunosuppression in the tumor microenvironment and enhancing the anti-tumor response of CAR-T cells in solid tumors.

[0168] Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those skilled in the art to which the present invention pertains can make various modifications and alterations without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims attached to the patent application. [Simplified Explanation of the Diagram]

[0021] To make the above and other objects, features, advantages and embodiments of this disclosure more apparent and understandable, the accompanying drawings are described below:

[0022] Figure 1 is a schematic diagram illustrating a nucleic acid construct encoding CAR and the recombinant antibody of the present invention according to an embodiment of the present disclosure;

[0023] Figure 2 is a schematic diagram of a specific nucleic acid construct according to an embodiment of the present disclosure;

[0024] Figure 3 is a line graph illustrating the binding of chimeric anti-human PD-1 2B6 antibody to PD-1 by an enzyme-linked immunosorbent assay (ELISA) according to another embodiment of the present disclosure;

[0025] Figure 4 is a diagram illustrating PD-1 / PD-L1 blockade induced by chimeric anti-human PD-1 2B6 antibody according to another embodiment of the present disclosure;

[0026] Figure 5 is an in vivo efficacy diagram of chimeric anti-human PD-1 2B6 antibody in a mouse MC38 colon cancer model, illustrated according to another embodiment of the present disclosure;

[0027] Figure 6 is an illustration of the anti-tumor effect of a specific CAR-T cell according to Example 3 of this disclosure, where GH: Globo H CAR-T cell, GH / Nivo: Globo H CAR / Nivo scFv-Fc CAR-T cell, and GH / PD-1 2B6-HuB1: Globo H / PD-12B6 scFv-Fc CAR-T cell;

[0028] Figure 7 is an effect diagram of specific CAR-T cell therapy for cancer illustrated in Example 4 of this disclosure, where GH CAR-T: Globo H CAR-T cells; GH / Nivo CAR-T: Globo H CAR / Nivolumab scFv-Fc CAR-T cells; GH / PD-1 2B6-HuB1 CAR-T: Globo H / PD-12B6 scFv-Fc CAR-T cells; and

[0029] Figure 8 is a diagram illustrating the effect of specific B7H3 CAR-T cells and B7H3 / PD-1 2B6-HuB1 CAR-T cells in treating cancer, according to Embodiment 5 of this disclosure. [Biomaterial Storage]

[0170] None

Claims

1. A recombinant antibody comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain includes a first heavy chain complementarity determining region. The VL domain comprises a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2), and a third light chain CDR (CDR-L3). CDR-H1, CDR-H2, and CDR-H3 respectively contain the amino acid sequences "GFTFSSYTMS" (sequence number: 1), "TISGGGANIYYPDSVKG" (sequence number: 2), and "PYYAIDF" (sequence number: 3); and CDR-L1, CDR-L2, and CDR-L3 respectively contain the amino acid sequences "KASQDVGSAVA" (sequence number: 4), "WASTRHT" (sequence number: 5), and "QQYSTYTWT" (sequence number: 6). The recombinant antibody can bind to programmed death 1 (PD-1).

2. The recombinant antibody as claimed in claim 1, wherein the VH domain and VL domain contain amino acid sequences that are at least 85% identical to those of sequence numbers 7 and 8, respectively.

3. The recombinant antibody as claimed in claim 2, wherein the VH domain contains an amino acid sequence of sequence number 7, 12, 13 or 14; and the VL domain contains an amino acid sequence of sequence number 8 or 15.

4. The recombinant antibody as described in claim 1, wherein the recombinant antibody is secreted by immune cells or stem cells.

5. The recombinant antibody as described in claim 4, wherein the immune cell is a T cell.

6. A single cell configured to express a chimeric antigen receptor (CAR) and a recombinant antibody as described in claim 1, wherein the single cell is transfected with a nucleic acid comprising, from its 5' end to its 3' end: a first coding sequence encoding a first single-stranded variable region fragment (scFv) specific to a first antigen; a second coding sequence encoding a hinge and transmembrane (HTM) domain of a first protein; a third coding sequence encoding a co-stimulatory molecule; a fourth coding sequence encoding a cytoplasmic domain of a second protein; an internal ribosomal entry site (IRES) or a linker sequence encoding a 2A peptide; and a fifth coding sequence encoding the recombinant antibody as described in claim 1; wherein, The first to fourth coding sequences jointly encode the CAR, which is located on the cell membrane of the isolated cell; And the recombinant antibody as described in claim 1, which is secreted into the isolated cell after expression.

7. The isolated cell as described in claim 6, wherein the antigen is a tumor-associated antigen (TAA); the first and second proteins are cluster of differentiation 8 (CD8) and CD3 zeta chain (CD3ζ), respectively; and the co-stimulatory molecule is 4-1BB.

8. The isolated cell as claimed in claim 7, wherein the VH and VL domains of the recombinant antibody as claimed in claim 1 contain at least 85% identical amino acid sequences to those in sequence numbers 7 and 8, respectively.

9. The single cell as claimed in claim 8, wherein the VH domain contains an amino acid sequence of sequence number 7, 12, 13 or 14; and the VL domain contains an amino acid sequence of sequence number 8 or 15.

10. The isolated cell as claimed in claim 7, wherein the HTM domain of the CD8 contains an amino acid sequence of sequence number 9; the 4-1BB co-stimulatory molecule contains an amino acid sequence of sequence number 10; and the cytoplasmic domain of the CD3ζ contains an amino acid sequence of sequence number 11.

11. The isolated cell as claimed in claim 6, wherein the recombinant antibody as claimed in claim 1 further comprises an immunoglobulin crystallizable region (Fc segment) located at the C-terminus of the recombinant antibody.

12. The isolated cell as described in claim 11, wherein the immunoglobulin is immunoglobulin G (IgG).

13. A single isolated cell as described in claim 6, wherein the single isolated cell is an immune cell or a stem cell.

14. Use of a single cell as described in claim 6 in the preparation of a medicament for treating cancer.

15. The use as described in claim 14, wherein the cancer is stomach cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or squamous cell carcinoma of the head and neck.

16. The use as described in claim 14, wherein the isolated cell as described in claim 6 is a T cell, a natural killer (NK) cell, a macrophage, or a stem cell.

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