TW202535920A - Chimeric antigen receptor against programmed death ligand 1 (pd-l1) and application thereof - Google Patents

Abstract

The present invention relates to a chimeric antigen receptor (CAR) against programmed death ligand 1 (PD-L1) and application thereof. In particular, the CAR comprises an antigen-binding domain which binds to PD-L1; and the anti-PD-L1 CAR-T cells thus produced are useful in CAR cell therapy.

Description

Programmed death-ligand 1 (PD-L1) chimeric antigen receptor and its applications

This invention relates to a chimeric antigen receptor (CAR) targeting programmed death-ligand 1 (PD-L1) and its application. In particular, the CAR includes an antigen-binding domain that binds to PD-L1; and the resulting anti-PD-L1 CAR cells can be used in CAR cell therapy.

Programmed death-ligand 1 (PD-L1) plays a crucial role in regulating the immune response, both under normal physiological conditions and in cancer. In normal cells, PD-L1 promotes immune homeostasis by preventing T cell overactivation and protecting against autoimmunity. However, in cancer, overexpression of PD-L1 on tumor cells activates the PD-1 pathway on T cells, leading to impaired immune responses and promoting tumor immune escape. This dual function of PD-L1 reinforces its importance as a target for cancer immunotherapy. ^{^1,2}

The interaction between PD-L1 and PD-1 is complexly influenced by the glycosylation state of PD-L1. Specifically, deglycosylation of PD-L1 at N35 significantly alters its binding affinity to PD-1, affecting the immunosuppressive activity of PD-L1 in cancer.^{^3,4} In addition to N35, PD-L1 possesses additional glycosylation sites at N192, N200, and N219, each playing a crucial role in the stability and function of PD-L1.^⁵ These glycosylation sites not only enhance the structural integrity of PD-L1 but are also essential for its long-term interaction with immune cells, contributing to its immune checkpoint function.

Studies have shown that normal tissues and primitive immune cells exhibit very low levels of PD-L1 and glycosylated PD-L1 (gPD-L1), highlighting the potential for developing immunotherapies that minimize collateral damage to normal tissues. Specific targeting of gPD-L1 ^can significantly improve the safety of these therapies, thereby avoiding targeting normal cells that exhibit low levels of these molecules.

Interestingly, FDA-approved anti-PD-L1 antibodies such as atezolizumab (Tecentriq), durvalumab (Imfinzi), and avelumab (Bavencio) exhibit different affinities for glycosylated and deglycosylated forms of PD-L1. This difference in binding affinity, particularly the reduced affinity for the deglycosylated form of PD-L1, is a key consideration in the development and clinical application of these antibodies, affecting their efficacy and specificity in targeting cancer cells.^⁴

Chimeric antigen receptor T-cell (CAR-T) therapy represents a breakthrough in cancer treatment, involving the reengineering of a patient's T cells to target and eradicate cancer cells. A notable example is a clinical trial involving atezolizumab (an anti-PD-L1 antibody)-based CAR-T therapy, which was terminated early after recruiting only one patient, at least for safety reasons. This innovative approach still faces significant challenges.

This invention provides a chimeric antigen receptor (CAR) targeting PD-L1, cells expressing the CAR, and CAR cell therapy using the CAR. In particular, the generated CAR cells exhibit enhanced specific cytotoxicity against PD-L1-expressing cells and provide excellent effects in cancer immunotherapy, including significant reduction in tumor growth and durable antitumor efficacy.

In particular, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a PD-L1 binding domain, comprising (a) a heavy chain variable region ( _VH ) comprising the heavy chain complementarity-determining region 1 (HC CDR1) of SEQ ID NO:1, the heavy chain complementarity-determining region 2 (HC CDR2) of SEQ ID NO:2, and the heavy chain complementarity-determining region 3 (HC CDR3) of SEQ ID NO:3; and (b) a light chain variable region ( _VL ) comprising the light chain complementarity-determining region 1 (LC CDR1) of SEQ ID NO:4, the light chain complementarity-determining region 2 (LC CDR2) of SEQ ID NO:5, and the light chain complementarity-determining region 3 (LC CDR3) of SEQ ID NO:6.

In some specific embodiments, the CAR further includes hinge domains, transmembrane domains, and intracellular signal transduction domains.

In some specific embodiments, the CAR includes the PD-L1 binding domain, which includes _VH and _VL from the N-end to the C-end, or _VL and _VH .

In some specific embodiments, the _VH and the _VL are connected by a connector.

In some specific embodiments, the intracellular signaling domain includes at least one selected from the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD27 signaling domain, the ICOS signaling domain, the CD3ζ signaling domain, and any combination thereof.

In some embodiments, the CAR contains the amino acid sequence of SEQ ID NO: 14 or 15. In some embodiments, the CAR contains the amino acid sequence of SEQ ID NO: 20 or 21.

This invention also provides a nucleic acid molecule containing a nucleotide sequence encoding a CAR as described herein. In some instances, the nucleic acid molecule is a vector.

This invention also provides a cell comprising a nucleic acid molecule containing an nucleotide sequence encoding a CAR as described herein. These cells are engineered to express a CAR on their surface and can be used in CAR cell therapy. A pharmaceutical composition comprising cells for expressing the CAR described herein is also provided.

On the other hand, the present invention also provides a method for treating an individual suffering from a disease, condition, or symptom associated with elevated tumor antigen expression, comprising administering to the individual an effective amount of genetically modified cells expressing a CAR as described herein. The present invention also provides the use of genetically modified cells expressing a CAR as described herein to manufacture a medicament for treating an individual suffering from a disease, condition, or symptom associated with elevated tumor antigen expression.

In some specific implementations, the tumor antigen is PD-L1.

In some specific implementations, the individual had cancer.

In some specific embodiments, the cancer is selected from a group consisting of breast cancer, rectal cancer, head and neck cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, kidney cancer, glioblastoma, and leukemia.

Details of one or more specific embodiments of the present invention are set forth in the description below. Other features or advantages of the present invention will become apparent from the detailed description of the following embodiments and from the appended claims.

The following description is intended only to illustrate various embodiments of the invention. Therefore, the specific embodiments or modifications discussed herein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various changes or equivalents can be made without departing from the scope of the invention.

To provide a clear and easy understanding of the present invention, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

I. General definition

As used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to “an element” includes a plural of that element and equivalents known to those skilled in the art.

The words "comprise" or "comprsing" are generally used to mean "include" or "including," which means that one or more features, components, or parts are permitted to exist. The words "comprise" or "comprsing" cover the terms "compose" or "consisting of."

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked by a single peptide bond. The term "protein" typically refers to a relatively large polypeptide. The term "peptide" typically refers to a relatively short polypeptide (e.g., containing up to 100, 90, 70, 50, 30, 20, or 10 amino acid residues).

As used herein, the terms “approximately” or “about” refer to the degree of acceptable deviation that a person of ordinary skill in the art would understand, and this can vary to some extent depending on the context. Specifically, “approximately” or “about” can refer to a value that is within ±10%, ±5%, or ±3% of the referenced value.

As used in this article, the term "substantially identical" means that two sequences have 80% or higher, better 85% or higher, better 90% or higher, and even better 95% or higher homology.

As used herein, the term "antibody" (which may be used interchangeably in the plural form "antibodies") refers to an immunoglobulin molecule that has the ability to specifically bind to a particular target antigen molecule. As used herein, the term "antibody" includes not only the complete (i.e., full-length) antibody molecule, but also antigen-binding fragments that retain their antigen-binding ability, such as Fab, Fab', F(ab') ₂ , and Fv. Such fragments are well known in the art and are frequently used in vitro and in vivo. The term "antibody" also includes chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site with desired specificity, including amino acid sequence variations, glycosylation variations, and covalently modified antibodies.

A complete or intact antibody comprises two heavy chains and two light chains. Each heavy chain contains a variable region ( _VH ) and first, second, and third constant regions ( _CH1 , _CH2 , and _CH3 ); each light chain contains a variable region ( _VL ) and a constant region ( _CL ). This antibody has a "Y" shape, with the backbone of the Y consisting of the second and third constant regions of the two heavy chains linked together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain, which binds to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for binding to the antigen. The variable regions in these two chains are generally responsible for antigen binding. Each chain contains three highly variable regions called complementary determinant regions (CDRs): the heavy (H) chain CDRs, including HC CDR1, HC CDR2, and HC CDR3, and the light (L) chain CDRs, including LC CDR1, LC CDR2, and LC CDR3. These three CDRs are surrounded by framework regions (FR1, FR2, FR3, and FR4), which are more conserved than the CDRs and form a backbone to support the highly variable regions. The constant regions of the heavy and light chains are not responsible for antigen binding but participate in various effector functions. Immunoglobulins can be classified into different classes based on the antibody amino acid sequences of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. The heavy chain domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and m, respectively.

As used herein, terms such as "antigen-binding fragment" or "antigen-binding domain" refer to the portion or region of a complete antibody molecule that is responsible for binding to an antigen. An antigen-binding fragment can bind to the same antigen that the original antibody binds to. Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which may be monovalent fragments consisting of a _VH - _CH1 chain and a _VL - _CL chain; (ii) F(ab')2 fragments, which may be divalent fragments consisting of two Fab fragments linked by disulfide bonds in hinge regions; (iii) Fv fragments, which consist of _VH and _VL domains of an antibody molecule linked by non-covalent interactions; (iv) monochain Fv (scFv), which may be a single polypeptide chain consisting of _VH and _VL domains linked by peptide linkers; (v) (scFv) ₂ , which may include two _VH domains linked by peptide linkers and two _VL domains linked to the two _VH domains by disulfide bonds.

As used herein, the term "chimeric antibody" refers to an antibody containing polypeptides from different sources, such as different species. In some embodiments, in a chimeric antibody, the variable regions of both the light and heavy chains can mimic the variable regions of antibodies derived from the same mammalian species (e.g., non-human mammals, such as mice, rabbits, and rats), while the constant region can be homologous to sequences in antibodies derived from another mammal, such as humans.

As used in this article, the term "humanized antibody" refers to an antibody that includes a framework region derived from a human antibody and one or more CDRs derived from a non-human (usually mouse or rat) immunoglobulin.

As used herein, the term "human antibody" refers to an antibody in which the substantially complete sequence of the light and heavy chain sequences (including complementary determinant regions (CDRs)) is derived from a human gene. In some cases, the human antibody may include one or more amino acid residues not encoded by human germline immunoglobulin sequences, for example, by mutation in one or more CDRs or one or more FRs to, for example, reduce potential immunogenicity, increase affinity, and eliminate cysteine that may lead to undesirable folding.

As used herein, terms such as "specifically bind to" or "specifically bind" refer to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of its target antigen. "Specifically binding" to an antibody to a target antigen or epitope is a well-known term in the art, and the methods for determining such specific binding are also well-known in the art. An antibody is "specifically bound" to a target antigen if it has a greater affinity/stronger binding force, is easier to bind to, and/or lasts longer than it binds to other substances. In other words, by interpreting this definition, it can be understood that, for example, an antibody specifically binding to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Therefore, "specific binding" or "preferred binding" does not necessarily require (although it may include) exclusive binding. In general, the affinity of a binding can be defined by the dissociation constant ( _KD ). Typically, when used against an antibody, specific binding can refer to an antibody that specifically binds to (identifies) its target, having a _KD value less than about ^10⁻⁷ M, for example, about ^10⁻⁸ M or lower, for example, ^10⁻⁹ M or lower, about ^10⁻¹⁰ M or lower, about ^10⁻¹¹ M or lower, about ^10⁻¹² M or lower, or even lower, and binding to the specific target with an affinity corresponding to _KD , _which is at least ten times lower than its binding affinity to nonspecific antigens (such as BSA or casein), for example, at least 100 times lower, for example, at least 1,000 times lower, or at least 10,000 times lower.

As used herein, terms such as "nucleic acid" or "polynucleotide" can refer to polymers composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs, including those containing non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. It should be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". The term "cDNA" refers to DNA that is complementary to or identical to mRNA, in single-stranded or double-stranded form.

As used herein, the term "complementary" refers to the topological compatibility or matching of the interaction surfaces of two polynucleotides. A first polynucleotide is complementary to a second polynucleotide when the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Therefore, a polynucleotide with the sequence 5′-GATAT-3′ is complementary to a polynucleotide with the sequence 5′-ATATC-3′.

As used herein, the term "encode" refers to the inherent characteristic of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a given sequence of RNA transcripts (i.e., rRNA, tRNA, and mRNA) or a given amino acid sequence, and the resulting biological characteristics. Therefore, if mRNA produced by a gene is transcribed and translated into a protein in a cell or other biological system, then the gene encodes that protein. Those skilled in the art will understand that, due to the degeneracy of genetic codes, several different polynucleotides and nucleic acids can encode the same polypeptide. It should also be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotide described herein, reflecting the codon usage of any particular host organism to express the polypeptide. Therefore, unless otherwise stated, "nucleotide sequences encoding amino acid sequences" encompasses all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

As used herein, the term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having a sequence that is not naturally linked together. Recombinant nucleic acids can exist in the form of a vector. A "vector" may contain a given nucleotide sequence of interest and a regulatory sequence. A vector can be used to express a given nucleotide sequence (expression vector) or to maintain the given nucleotide sequence for replication, manipulation, or transfer between different locations (e.g., between different organisms). For the foregoing purposes, the vector can be introduced into a suitable host cell. A "recombinant cell" is a host cell in which recombinant nucleic acid has been introduced. A "transformed cell" means a cell in which DNA molecules encoding proteins of interest have been introduced using recombinant DNA technology.

Vectors can be of various types, including plasmids, myxosomes, free genomes, F myxosomes, artificial chromosomes, bacteriophages, viral vectors, etc. Typically, in a vector, a given nucleotide sequence is operatively linked to a regulatory sequence, such that when the vector is introduced into a host cell, the given nucleotide sequence is expressed in the host cell under the control of the regulatory sequence. Regulatory sequences may include, for example, but not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoters, simian virus 40 (SV40) early promoters, T7 promoters, and alcohol oxidase gene ( AOX1 ) promoters), start codons, replication origins, enhancers, secretion signal sequences (e.g., α-mating factor signals), termination codons, and other control sequences (e.g., Shine-Dalgarno sequences and termination sequences). Preferably, the vector may further include a marker sequence (e.g., an antibiotic resistance marker sequence) for subsequent screening/selection procedures. For protein production purposes, a given sequence of interest may be linked to another nucleotide sequence in addition to the aforementioned regulatory sequence within the vector, thereby generating a fusion polypeptide and facilitating subsequent purification processes. The fusion polypeptide includes a tag for purification purposes, such as a His-tag.

As used herein, the term "treatment" refers to the application or administration of one or more active agents to an individual suffering from a disease, its symptoms or condition, or the progression of the disease, with the aim of treating, curing, alleviating, reducing, altering, remedying, improving, enhancing, or affecting the disease, its symptoms or condition, the disability caused by the disease, or the progression or tendency of the disease.

II. anti- PD-L1 Antibodies

This invention is at least partially based on an anti-PD-L1 antibody for the preparation of chimeric antigen receptors (CARs). The anti-PD-L1 antibody used herein has been found to specifically target certain glycosylation sites of PD-L1. The anti-PD-L1 antibody used herein can be used to develop novel CAR constructs aimed at improving the efficacy of CAR-T therapy.

Exemplary anti-PD-L1 antibodies are described in US 11,660,352 B2, the disclosures of which are incorporated herein by reference for the purposes or subjects of this citation. Specific examples of anti-PD-L1 antibodies include heavy chain variable regions (V_{_H}) having complementary determining regions (HC_CDR1, HC_CDR2, and HC_CDR3) and light chain variable regions (V_{_L}) having complementary determining regions (LC_CDR1, LC_CDR2, and LC_CDR3), as shown in Table 1 below.

Table 1. Amino acid sequence of anti-PD-L1 antibody mTT-01 Anti-PD-L1 antibody, mTT-01 amino acid sequence Heavy Chain CDR1 NYVMS (SEQ ID NO: 1) Heavy Chain CDR2 TISSGGRYIYYTDSVKG (SEQ ID NO: 2) Heavy Chain CDR3 DGSTLYYFDY (SEQ ID NO: 3) V _H domain EVMLVESGGALVKPGGSLKLSCAASGFSLS NYVMS WVRQTPEKRLEWVA TISSGGRYIYYTDSVKG RFTIS RDNARNTLYLQMSSLRSEDTAMYYCAR DGSTLYY FDY WGQGTTLTVSS (SEQ ID NO: 7) Lightweight CDR1 SASSSVDYMY (SEQ ID NO: 4) Lightweight CDR2 DTSNLAS (SEQ ID NO: 5) Lightweight CDR3 QQWSSSPPIT (SEQ ID NO: 6) V _L domain QTVLTQSPAIMSASPGEKVTMTC SASSSVDYMY WY QQKPGSSPRLLIY DTSNLAS GVPVRFSGSGSGTSY SLTISRMEAEDAATYYC QQWSSSPPIT FGTGTKVE LK (SEQ ID NO: 8)

Table 2. DNA sequence of anti-PD-L1 antibody mTT-01 Anti-PD-L1 antibody, mTT-01 V _H domain Gaagtgatgctggtggagtctgggggagccttagtgaagcctggagggtccctgaaactctcctgtgcagcttctggattcagtttgagtaactatgtcatgtcttgggttcgccagactccagagaagaggctggagtgggtcgcaaccattagtagtggtggtaggtatatctact atacagacagtgtgaagggtcgattcaccatctccagggacaatgccaggaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattattgtgcaagagacggtagtaccttgtactactttgactattggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 9) V _L domain caaactgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtagattacatgtactggtaccagcagaagccaggatcctccccagactcctgatttatgacacatccaacctgg cttctggagtccctgttcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagtagttccccacccatcacgttcggtactgggaccaaggtggagctgaaa (SEQ ID NO: 10)

In some specific embodiments, the anti-PD-L1 antibody is a functional variant of mTT-01, characterized by comprising (a) V _H , which comprises HC CDR1 of SEQ ID NO: 1, HC CDR2 of SEQ ID NO: 2, and HC CDR3 of SEQ ID NO: 3; and (b) V _L , which comprises LC CDR1 of SEQ ID NO: 4, LC CDR2 of SEQ ID NO: 5, and HC CDR3 of SEQ ID NO: 6. In some specific embodiments, the anti-PD-L1 antibody may comprise V _H , which comprises an amino acid sequence of SEQ ID NO: 7 or substantially the same therein, and V _L , which comprises an amino acid sequence of SEQ ID NO: 8 or substantially the same therein. Specifically, the anti-PD-L1 antibody of the present invention comprises _VH , which contains an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identity with SEQ ID NO: 7, and _VL , which contains an amino acid sequence having at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%) identity with SEQ ID NO: 8. The anti-PD-L1 antibody of the present invention also includes any recombinant (engineered)-derived antibody encoded by a polynucleotide sequence encoding the relevant _VH or _VL amino acid sequence as described herein.

The term "substantially identical" can refer to a variant whose relevant amino acid sequence (e.g., in FR, CDR, _VH , or _VL ) is not substantially different from that of the reference antibody, resulting in the variant having substantially similar binding activity (e.g., affinity, specificity, or both) and biological activity relative to the reference antibody. This variant may include minor amino acid changes. It is understood that a polypeptide may have a limited number of alterations or modifications that may be made in a portion of the polypeptide unrelated to its activity or function, and still produce a variant with an acceptable amount of equivalent or similar biological activity or function. In some examples, the change in the amino acid residue is a conserved amino acid substitution, which means replacing one amino acid residue with another amino acid residue with a similar chemical structure, and having little or no effect on the function, activity, or other biological properties of the polypeptide. Typically, the FR region can undergo relatively more substitutions compared to the CDR region, as long as they do not adversely affect the binding function and biological activity of the antibody (e.g., a reduction in binding affinity of more than 50% compared to the original antibody). In some embodiments, the sequence identity between the reference antibody and the variant can be approximately 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%, or higher. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those described in *Molecular Cloning: A Laboratory Manual*, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conserved substitutions of amino acids include substitutions between amino acids in the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

The antibodies described herein may be animal antibodies (e.g., mouse-derived antibodies), chimeric antibodies (e.g., mouse-human chimeric antibodies), humanized antibodies, or human antibodies. The antigen-binding fragments of the antibodies described herein include Fab fragments, F(ab′)2 fragments, Fv fragments, single-chain Fv (scFv), and (scFv) ₂ . Antibodies or their antigen-binding fragments may be prepared using methods known in the art.

III. Preparation of antibodies or their antigen-binding fragments

Many conventional methods in this field can be used to obtain antibodies or their antigen-binding fragments.

In some specific embodiments, the antibodies provided herein can be prepared using conventional hybridoma techniques. Generally, the target antigen can be conjugated to a carrier protein such as keyhole hemocyanin (KLH) as needed, and/or mixed with an adjuvant such as complete Freund's adjuvant, to induce an immune response in the host animal to produce antibodies that bind to the antigen. Lymphocytes secreting monoclonal antibodies are harvested and fused with myeloma cells to generate hybridomas. Hybridoma colonies formed in this manner are then screened to identify and select hybridoma colonies that secrete the desired monoclonal antibody.

In some specific embodiments, the antibodies provided herein can be prepared via recombination techniques. Relatedly, isolated nucleic acids encoding the revealed amino acid sequences are also provided, as well as vectors containing these nucleic acids and host cells transformed or transfected by these nucleic acids.

For example, nucleic acids containing nucleotide sequences encoding the variable regions of the heavy and light chains of such antibodies can be selected and colonized into expression vectors (e.g., bacterial vectors such as E. coli vectors, yeast vectors, viral vectors, or mammalian vectors) using conventional techniques, and any of these vectors can be introduced into suitable cells (e.g., bacterial cells, yeast cells, plant cells, or mammalian cells) to express the antibody. Examples of nucleotide sequences encoding the variable regions of the heavy and light chains of the antibodies described herein are shown in Table 2. Examples of mammalian host cell lines include human embryonic kidney cell line (293 cells), young hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells). Recombinant vectors used to express the antibodies described herein typically contain nucleic acids encoding the amino acid sequence of the antibody, operatively linked to a promoter, whether adsorbed or inducible. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the nucleic acid encoding the antibody. The vector may, as needed, contain selection markers for prokaryotes and eukaryotes. In some instances, both the heavy chain and light chain coding sequences are included in the same expression vector. In other instances, each of the heavy chain and light chain of the antibody is selected and generated separately into its respective vector, and then allowed to stand under suitable conditions for antibody assembly.

Recombinant vectors used to express the antibodies described herein typically contain a nucleic acid encoding the amino acid sequence of the antibody, operatively linked to a promoter, whether assembled or inducible. The recombinant antibody can be generated in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, insects, and mammalian cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the nucleic acid encoding the antibody. The vector may, as needed, contain selection markers for prokaryotic and eukaryotic systems. The resulting antibody protein can be further isolated or purified to obtain a substantially homogeneous preparation for further assays and applications. Appropriate purification procedures may include, for example, separation on immunoaffinity columns or ion exchange columns, ethanol precipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), ammonium sulfate precipitation, and gel filtration.

When a full-length antibody is desired, the coding sequences of any of the _VH and _VL chains described herein can be linked to the coding sequence of the immunoglobulin Fc region, and the resulting genes encoding the heavy and light chains of the full-length antibody can be expressed and assembled in suitable host cells, such as plant cells, mammalian cells, yeast cells, or insect cells.

Antigen-binding fragments can be prepared using conventional methods. For example, the F(ab') ₂ fragment can be generated by digesting a full-length antibody molecule with pepsin, and the Fab fragment can be generated by reducing the disulfide bonds of the F(ab') ₂ fragment. Alternatively, such fragments can also be prepared using recombinant techniques by expressing heavy and light chain fragments in suitable host cells and assembling them to form the desired antigen-binding fragment, whether in vivo or in vitro. Single-chain antibodies can be prepared using recombinant techniques by linking the nucleotide sequences encoding the variable region of the heavy chain and the variable region of the light chain. Preferably, a flexible linker is added between the two variable regions.

IV. Chimeric antigen receptor ( CARs )

Chimeric antigen receptors (CARs) are artificial immune cell receptors that allow T cells to recognize specific antigens on target cells (such as tumor cells). Generally, CARs are fusion peptides containing an antigen-binding extracellular domain, a transmembrane domain, and an intracellular signaling domain that recognize target antigens. T cells expressing CAR peptides are called CAR T cells, which can specifically target certain antigens of interest.

In some embodiments, the CAR may include a signal peptide at its N-terminus. The signal peptide includes a peptide sequence that guides the delivery and localization of the peptide within and/or on the cell surface. In one embodiment, the signal peptide includes a signal peptide derived from human CD8a (MALPVTALLLPLALLLHAARP) (SEQ ID NO: 11). In another embodiment, the signal peptide includes a signal peptide derived from human CD8b (MRPRLWLLLAAQLTVLHGNSV) (SEQ ID NO: 12). Other examples include signal peptides derived from human CD45 and interleukin-2 (IL-2). Functional equivalents including these are, for example, CD8a signal peptides, CD8b signal peptides, CD45 signal peptides, and IL-2 signal peptides derived from homologous proteins of other species. The signaling peptide can be cleaved during or after translocation to produce free signaling peptides and mature proteins.

The antigen-binding extracellular domain is a region of the CAR polypeptide exposed to the extracellular fluid when the CAR is expressed on the cell surface. Typically, the antigen-binding extracellular domain is a single-chain variable fragment (scFv) derived from a monoclonal antibody, but it can also be based on other forms containing antibody-like antigen binding sites. The scFv may include an antibody heavy-chain variable region ( _VH ) and an antibody light-chain variable region ( _VL ) having a _VH - _VL or _VL - _VH orientation. In some embodiments, the antigen-binding extracellular domain is an scFv derived from an anti-PD-L1 antibody such as mTT-01 described herein. In some embodiments, _VH and _VL may be linked to each other via peptide linkers. The peptide linker can be 5-25 amino acid residues, 25-100 amino acid residues, or 50-200 amino acid residues long. In some embodiments, the peptide linker is a Gly-Ser linker. In a specific embodiment, the Gly-Ser linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). In some embodiments, the antigen-binding extracellular domain includes mTT-01 V _H , the linker, and mTT-01 V _L from the N-terminus to the C-terminus. In one specific embodiment, the antigen-binding extracellular domain contains the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the antigen-binding extracellular domain includes mTT-01 V _L , a linker, and mTT-01 V _H from the N-terminus to the C-terminus. In one specific embodiment, the antigen-binding extracellular domain contains an amino acid sequence as shown in SEQ ID NO: 15.

Table 3: Amino acid sequences of the antigen-binding extracellular domain based on mTT-01 mTT-01 V _H + Connector + mTT-01 V _L EVMLVESGGALVKPGGSLKLSCAASGFSLS NYVMS WVRQTPEKRLEWVA TISSGGRYIYYTDSVKG RFTISRDNARNTLYLQMSSLRSEDTAMYYCAR DGSTLYYFDY WGQGTTLTVSS GGGGSGGGGSGGGGS QTVLTQSPAIMSASPGEKVTMTC SASSSVDYMY WYQQKPGSSPRLLIY DTSNLA SGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSSPPIT FGTGTKVELK (SEQ ID NO: 14) mTT-01 V _L + Connector + mTT-01 V _H QTVLTQSPAIMSASPGEKVTMTC SASSSVDYMY WYQQKPGSSPRLLIY DTSNLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSSPPIT FGTGTKVELK GGGGSGGGGSGGGGS EVMLVESGGALVKPGGSLKLSCAASGFSLS NYVMS WVRQTPEKRLEWVA TISSGGRYIYYTDSVKG RFTISRDNARNTLYLQMSSLRSEDTAMYYCAR DGSTLYYFDY WGQGTTLTVSS (SEQ ID NO: 15)

The CAR peptides described herein may contain a transmembrane domain, which is typically an α-helix containing several hydrophobic residues spanning the cell membrane. This transmembrane domain provides stability to the CAR peptide containing it. In some embodiments, the transmembrane domain may be a CD28 transmembrane domain, a CD8 transmembrane domain, or a chimera of CD8 and CD28 transmembrane domains. In some embodiments, the transmembrane domain is a CD8a transmembrane domain containing the following sequence: IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 16).

The CAR peptide described herein may contain a flexure domain located between a transmembrane domain and an antigen-binding domain. In some embodiments, the flexure domain may contain up to 300 amino acids, such as 5 to 20 amino acids, 15 to 50 amino acids, 20 to 100 amino acids, or 30 to 200 amino acids. The flexure domain may provide flexibility to the CAR or prevent steric hindrance to the CAR. In some embodiments, the flexure domain is a CD8 flexure domain. In a preferred embodiment, the CD8 flexure domain is human. In some embodiments, the flexure domain is a CD28 flexure domain. In a preferred embodiment, the CD28 flexure domain is human. In some specific embodiments, the hinge field is a CD8a hinge field containing the following sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 17).

The intracellular signaling domain of the CAR peptide can activate at least one normal effector function of immune cells modified to express the CAR peptide. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. The intracellular signaling domain of the CAR peptide can be part of a protein that transmits effector function signals and directs the cell to perform specific functions. Specifically, the intracellular signaling domain is derived from the intracellular signaling domain of a natural receptor. Examples of such natural receptors include the zeta (ζ) chain of the T cell receptor or any homolog thereof (e.g., δ, γ, or ε). For example, CD3ζ (CD3-zeta) is the cytoplasmic signaling domain of the CD3 complex of the T cell receptor (TCR). It contains three immune receptor tyrosine-based activation motifs (ITAMs) that activate downstream signaling pathways. In some specific embodiments, the intracellular signaling domain includes a CD3-ζ signaling domain containing the following sequence: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 18). CD3-ζ can provide primary T cell activation signals, but it is not a fully competent activation signal and therefore may require additional co-stimulatory signals. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective lymphocyte response to antigens. Examples of co-stimulatory molecules include, but are not limited to, CD27, CD28, 4-1BB (CD137), OX40, CD30, lymphocyte function-associated antigen-1 (LFA-1), and CD2. In some cases, the co-stimulatory domains of CD28 and/or 4-1BB can be used in conjunction with CD3ζ-mediated primary signaling to transmit complete proliferation/survival signals. In some embodiments, the CAR peptides disclosed herein comprise a CD28 co-stimulatory molecule. In some embodiments, the CAR peptides disclosed herein comprise a 4-1BB co-stimulatory molecule. In some embodiments, the co-stimulatory molecule comprises a 4-1BB co-stimulatory molecule containing the sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 19). In some embodiments, the CAR peptides disclosed herein comprise a CD3ζ signaling domain and a CD28 co-stimulatory domain. In some specific embodiments, the CAR peptide disclosed herein comprises a CD3ζ signaling domain and a 4-1BB co-stimulatory domain. In other embodiments, the CAR comprises a CD3ζ signaling domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

In one specific embodiment, the CAR peptide disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 20 (TT0001: _VH -linker- _VL chain + Ala-Ala-Ala + CD8a hinge chain + CD8a transmembrane domain + 4-1BB + CD3-zeta). In another specific embodiment, the CAR peptide disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 21 (TT0002: _VL -linker- _VH chain + Ala-Ala-Ala + CD8a hinge chain + CD8a transmembrane domain + 4-1BB + CD3-zeta).

Table 4: Amino acid sequences of the mTT-01-based CAR peptides revealed in this paper. TT0001: V _H - Connector - V _L Chain + Ala-Ala-Ala + CD8a Hinge Chain + CD8a Transmembrane Domain + 4-1BB + CD3-zeta EVMLVESGGALVKPGGSLKLSCAASGFSLS NYVMS WVRQTPEKRLEWVA TISSGGRYIYYTDSVKG RFTISRDNARNTLYLQMSSLRSEDTAMYYCAR DGSTLYYFDY WGQGTTLTVSS GGGGSGGGGSGGGGS QTVLTQSPAIMSASPGEKVTMTC SASSSVDYMY WYQQKPGSSPRLLIY DTSNLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSSPPIT FGTGTKVELKAAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 20) TT0002: V _L - Connector - V _H Chain + Ala-Ala-Ala + CD8a Hinge Chain + CD8a Transmembrane Domain + 4-1BB + CD3-zeta QTVLTQSPAIMSASPGEKVTMTC SASSSVDYMY WYQQKPGSSPRLLIY DTSNLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSSPPIT FGTGTKVELK GGGGSGGGGSGGGGS EVMLVESGGALVKPGGSLKLSCAASGFSLS NYVMS WVRQTPEKRLEWVA TISSGGRYIYYTDSVKG RFTISRDNARNTLYLQMSSLRSEDTAMYYCAR DGSTLYYFDY WGQGTTLTVSSAAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21)

V. Nucleic acid (vector) and CAR Expression cells

Nucleic acid encoding the CAR described herein may be provided. This nucleic acid sequence may be, for example, a DNA, RNA, or cDNA sequence. The nucleic acid encoding the CAR may be inserted into a vector. This vector may be a plasmid or a viral vector. This vector is capable of transfecting or transducing T cells. For example, viral vectors such as retroviral vectors (e.g., oncogenic retroviral vectors, lentiviral vectors, and pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Estange-Barr virus (EBV) vectors, and herpes simplex virus (HSV) vectors may be used.

Nucleic acid encoding a CAR can be introduced into cells. In some embodiments, the CAR-encoding nucleic acid can be programmed to insert into a genomic site of interest in the host T cell. In some embodiments, the target genomic site can be located at a safe harbor locus. Specifically, the CAR-encoding nucleic acid can insert into the TRAC gene to disrupt the TRAC gene in the genetically modified T cell and express the CAR peptide. Disruption of TRAC leads to loss of endogenous TCR function. Viral vectors encoding the CAR peptide described herein, such as AAV or LV vectors, can be incubated with T cells for an appropriate time to allow the viral vector to enter the T cells. After transduction, the T cells can be cultured in a suitable cell culture medium for an appropriate period of time to allow for recovery. Genetically modified T cells can be proliferated in vitro under appropriate conditions to produce the desired genetically modified T cell population.

There are no particular restrictions on the cells used in the genetic modification process to express CAR. These cells can be any eukaryotic cell capable of expressing CAR on its surface, such as immune cells. Specifically, they can be immune effector cells, such as T cells. These T cells can include helper T cells (TH cells), cytotoxic T cells (CTLs), memory T cells, and regulatory T cells (Treg cells). Cells can be derived from samples isolated from patients, related or unrelated hematopoietic transplant donors, or completely unrelated donors; from umbilical cord blood; differentiated from embryonic cell lines or induced progenitor cell lines; or derived from self-converted cell lines. In some embodiments, CAR-expressing cells can be derived from peripheral blood mononuclear cells (PBMCs), which can be obtained from the patient's own peripheral blood or from hematopoietic stem cells from a donor peripheral blood transplant. The CAR-expressing cells thus generated can be further amplified in vitro under appropriate conditions to produce a CAR-expressing cell population to the required quantity, such as clinically relevant specifications. CAR-expressing cells generated as described herein can be harvested for therapeutic use.

VI. pharmaceutical composition

This invention also relates to a pharmaceutical composition comprising the CAR-expressing cells described herein. The CAR-expressing cells can be formulated with a pharmaceutically acceptable carrier for delivery purposes.

As used herein, "medically acceptable" means that the carrier is compatible with and preferably stabilizes the active ingredient in the composition, and is safe for the recipient individual. The carrier may be a diluent, loading agent, excipient, or matrix of the active ingredient. Typically, compositions containing CAR-expressing cells as the active ingredient, as described herein, may be in solution form, such as an aqueous solution, like physiological saline. Suitable excipients include lactose, sucrose, dextrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. As needed, the composition may contain more pharmaceutically acceptable adjuvants to bring it closer to physiological conditions, such as pH adjusters and buffers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and similar substances. The composition of the invention can be delivered via physiologically acceptable routes, typically by intravenous infusion.

V. treatment

The genetically modified CAR-expressing cell populations described herein can be administered to an individual for therapeutic purposes. Specifically, this invention provides a method for treating an individual suffering from a disease, condition, or symptom associated with elevated tumor antigen expression, comprising administering to the individual an effective amount of genetically modified cells expressing the CAR as described herein. This invention also provides the use of genetically modified cells expressing the CAR as described herein to manufacture a drug for treating an individual suffering from a disease, condition, or symptom associated with elevated tumor antigen expression.

As used herein, the term "effective amount" refers to the amount of active ingredient that imparts the desired biological effect in an individual to be treated. This effective amount can vary depending on various factors, such as the route and frequency of administration, the body weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage in each case based on the disclosures herein, established methods, and their own experience. In some instances, an effective amount of the active ingredient is used to provide an antitumor effect, such as reducing tumor size compared to no administration of the active ingredient. In some embodiments, the effective amount of genetically modified cell population may contain ^10⁵ to ^10⁷ cells, such as 1 x ^10⁵ cells, 2 x 10⁵ cells, 3 x 10⁵ cells, ⁴ x 10⁵ cells, ⁵ x 10⁵ cells, 6 x 10⁵ cells, ⁷ x 10⁵ cells, 8 x 10⁵ cells, 9 x 10⁵ cells, 1 x 10⁶ cells, 2 x 10⁶ cells, 3 x 10⁶ cells, ⁴ x 10⁶ cells, ⁵ x 10⁶ cells, ⁶ x 10⁶ cells, ⁷ x ^10⁶ cells, ⁸ x 10⁶ cells, 9 x 10⁶ ...1 x 10⁶ cells, 1 x 10⁶ cells, 1 x 10⁶ cells, ^{1 x 10⁶} cells, 1 x 10⁶ cells, ^{1 x} 10⁶ cells, 1 x 10⁶ cells, 1 x 10⁶ cells, ¹ x 10⁶ cells, 1 x ^10⁶ cells, 1 x 10⁶ cells, ^{1 x 10⁶} cells, 1 x 10⁶ cells, ^{1 x} 10⁶ cells, ¹ x 10⁶ cells, 1 x 10 ⁶ cells, 1 ^{x 10⁷} cells, 2 x 10⁷ cells, ^{3 x} 10⁷ cells, 4 x 10⁷ cells, 5 ^{x 10⁷} cells, 6 ^{x 10⁷} cells, ⁷ x 10⁷ cells, 8 x ^10⁷ cells, 9 x ^10⁷ cells, or multiples thereof.

Individuals to be treated with the methods described herein may be mammals, preferably humans. Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice, and rats. Human individuals requiring treatment may be human patients who have, are at risk of, or are suspected of having the target disease/condition. Individuals suspected of having any of these target diseases/conditions may exhibit one or more symptoms of that disease/condition. Individuals at risk of the disease/condition may be individuals with one or more risk factors for that disease/condition.

In some specific implementations, it has been determined that the individual has a relatively high level of tumor antigen compared to the reference level.

In some specific embodiments, the method may include measuring tumor antigens in a tumor sample from a patient and comparing the level of tumor antigens in the sample to a reference level. In some specific embodiments, based on this comparison, patients with a measured enhanced level of tumor antigen may be selected. Specifically, for example, the enhanced level may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more higher than the reference level. The reference level of tumor antigens as described herein may refer to the level measured in a control sample (e.g., any non-cancer tissue, cell, or biological sample from an individual or a normal population). The measurement can be performed using conventional detection and statistical methods.

In some specific implementations, the tumor antigen is PD-L1.

In some specific embodiments, the individual has cancer. Non-limiting examples of cancers for which the genetically modified population therapy described herein may be used include, but are not limited to, breast cancer, colon cancer, head and neck cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, kidney cancer, glioblastoma, and leukemia.

Administration may include the transplantation of genetically modified CAR-expressing cells, by means or routes that deliver a desired amount of genetically modified CAR-expressing cells to a desired location, such as a tumor site, to produce the desired effect. For example, in some cases, an effective amount of genetically modified CAR-expressing cells may be administered via systemic administration routes, such as injection and infusion. Injection includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracardiac, intraperitoneal, and subcutaneous administration. In some specific embodiments, the route is intravenous.

This invention is further illustrated by the following examples, which are provided for illustrative purposes and not for limitation. Based on this disclosure, those skilled in the art should understand that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of this invention, and the same or similar results can still be obtained.

Example

1. Materials and Methods

1.1 anti- -PD-L1 monoclonal antibodies

The anti-PD-L1 monoclonal antibody mTT-01 was prepared as described in US 11,660,352 B2 and has the _VH domain as shown in SEQ ID NO: 7 and the _VL domain as shown in SEQ ID NO: 8, as provided in Table 1. Chimeric atezolizumab (anti-hPD-L1-mIgG1), purchased from InvivoGen (anti-hPD-L1-mIgG1 InvivoFit, catalog number: hpdl1-mab9-1), is a recombinant monoclonal antibody characterized by a constant region of mouse IgG1 isotype and a variable region of atezolizumab.

1.2 Immunohistochemical assays for human tissues

For immunohistochemical analysis, the following modified procedure is used. Slides containing formalin-fixed paraffin-embedded (FFPE) tissue or SCMH-derived tissue microarrays (TMA) are initially prepared by incubation at 60-65°C (for FFPE tissue) or 70°C (for TMA) for 30 minutes. Dewaxing is accomplished by two consecutive immersions in Surgipath xylene (3803665, Leica) for 10 minutes each. The slides were then rehydrated using a series of graded Surgipath reagents in 100% alcohol (3803686, Leica), with concentrations decreasing from 100%, 95%, 80% to 70%, each step lasting 5 minutes, followed by rinsing with tap water.

Antigen repair was performed using a citrate-based antigen repair solution suitable for the mTT-01 antigen, pH 6 (S2369, DAKO), in a pressure cooker for 30 minutes. After cooling to room temperature, the slide was washed for 5 minutes in Tris-buffered saline solution containing Tween 20 (TBST; TBT999, Scytek). Endogenous peroxidase activity was inhibited by standing in a hydrogen peroxide blocking solution (TA-060-HP, Thermo Fisher Scientific) for 10 minutes, followed by washing twice in TBST for 3 minutes each time. Nonspecific binding was blocked for 30 minutes using 5% normal goat serum (005-000-001, Jackson ImmunoResearch Laboratories, Inc.), followed by standing with protein blocking solution (TA-060-PBQ, Thermo Fisher Scientific) for 10 minutes.

The primary antibody and 200-fold diluted mTT-01 antibody were incubated overnight at 4°C in the dark. After incubation, the slides were washed three times in TBST for 3 minutes each time. Scale-up of primary antibody binding was performed using a primary antibody amplification Quanto (TL-060-QPB) for 10 minutes, followed by three TBST washes. Detection was performed using an HRP Polymer Quanto (TL-060-QPH) for 10 minutes, with an additional three TBST washes.

Antigen-antibody complex visualization was performed for 5 minutes using DAB receptor kits (TA-060-QHSX and TA-002-QHCX, Thermo Fisher Scientific). The slides were then rinsed in tap water for 5 minutes, counterstained with Surgipath Hematoxylin Gill II (3801522, Leica) for 2 minutes, and rinsed again in tap water for 5 minutes. The slides were then dehydrated by soaking twice in 100% alcohol for 5 minutes each time, followed by rinsing twice in xylene for 5 minutes each time. Finally, the slides were mounted using Surgipath Micromount Mounting Medium (3801731, Leica).

1.3 CAR Structure and CAR T- Cell production

The CAR was constructed and expressed based on the anti-PD-L1 monoclonal antibody mTT-01. Specifically, the gPD-L1-specific second-generation CAR was designed to contain an scFv obtained from mTT-01, linked in the order of _VH to _VL (pTT0001) or _VL to _VH (pTT0002), followed by an hCD8 hindchain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. This scFv sequence was obtained from the mTT-01 monoclonal antibody. The structure of the CAR construct is shown in Figure 1. The CAR construct was integrated into a lentiviral vector using In-Fusion selection (Takara Bio, Shiga-ken, Japan). Primary human CD3+ T cells were isolated from PBMCs of healthy donors using CD3 MicroBeads (Miltenyi Biotec, North Rhine-Westphalia, Germany) and stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Stem Cell Technologies, Vancouver, Canada) and 100 IU/ml Proleukin (Novartis, Basel, Swiss). Three days later, the activated CD3+ T cells were transduced with RetroNectin (Takara Bio, Shiga-ken, Japan). The lentivirus-containing supernatant was removed two days post-infection. The CAR T-cell culture medium was replaced with fresh Proleukin-containing medium every 2-3 days. CAR expression on T cells was measured by flow cytometry seven days post-transduction. On day 14, in vitro cytotoxicity of CAR T-cells was measured and a xenograft mouse model was established.

1.4 Cell culture

Breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT549, BT474, and MCF-7), human non-small cell lung cancer (NSCLC) A549 cells, and 293T cells were purchased from ATCC. Jurkat-Lucia™ NFAT cells were purchased from InvivoGen (InvivoGen, CA, USA). MDA-MB-231 PD-L1 cells were created by ^{overexpressing} human PD-L1 using a previously described lentiviral system, along with MDA-MB-231 PD-L1 ^KO cells. BT474 and Jurkat-Lucia™ NFAT cells were cultured in RPMI 1640 (Gibco, Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum (Cytiva, MA, USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD). Other cell lines were maintained in DMEM/F12 (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. All cell lines were cultured in a humidified incubator at 37°C and 5% _CO2 .

1.5 Photoenzyme assay

TT0001 and TT0002 Jurkat NFAT reporter fluorescent enzyme (Luc) cells were generated by transducing Jurkat-Lucia™ NFAT cells with TT0001 and TT0002 lentiviruses. Stable expression of CAR on the surface of TT0001 and TT0002 Jurkat NFAT-Luc cells was confirmed by flow cytometry. MDA-MB-231, BT549, A549, and 293T (1 x ^10⁵ cells/well) were co-incubated with TT0001 and TT0002 Jurkat NFAT-Luc cells at an effector-to-target ratio (E:T) of 1:1 for two days. QUANTI-Luc™ (InvivoGen, CA, USA) was used to monitor luminescence released by TT0001 and TT0002 Jurkat NFAT-Luc cells after activation by PD-L1-expressing cells. Light emission (RLU) was measured using a GloMax96 microdisc luminometer (Promaga, WI, USA) and standardized to an unstimulated control group.

1.6 In vitro cytotoxicity assay

The cytotoxic activity of CAR-T cells was measured using the Cell Counting Manipulation-8 (CCK-8) assay (Dojindo, MD, USA). MDA-MB-231, MDA-231 PD-L1, and MDA-MB-231 PD-L1 ^KO cells (3 × ^10⁴ cells/well) were co-cultured with CAR T cells for 24 hours at E:T ratios of 10:1, 3:1, 1:1, and 0.3:1. MCF-7, BT549, M10, BT474, ZR75-1, and MDA-MB-361 cells (1.5 × ^10⁴ cells/well) were co-cultured with CAR T cells at an E:T ratio of 3:1 for 24 hours. The CAR T-cells were then removed from the target cells by washing with PBS. Tumor cell viability was assessed using CCK-8, and lysis activity was calculated using the following formula: % lysis = 1 − (percentage of CCK-8 in the CAR T-cell treated wells) / (percentage of CCK-8 in the untreated wells) × 100%.

1.7 Multi-Plex Immunoassay

CAR T cells were co-incubated with MDA-MB-231, MDA-231 PD-L1, or MDA-MB-231 PD-L1 ^KO at different E:T ratios for 48 hours. The supernatant was collected and centrifuged at 300 ×g for 10 minutes to remove cell debris. The release levels of human TNF-α, IL-6, IFN-γ, IL-10, and IL-2 were measured using a Multi-Plex immunoassay (MPI) at the Inflammation Core Facility (Institute of Biomedical Sciences, Academia Sinica, Taiwan, supported by the AS-CFII-108-118 Project). Antibody-conjugated magnetic beads were incubated with cytokine-containing samples, washed, detected with biotinylated antibodies, and then labeled with streptavidin-phycoerythrin (PE). The fluorescence content of the microbeads was detected using a Bio- ^Plex® 200 system (Bio-Rad, CA, USA), and the concentration of cytokines was standardized using standards provided by the manufacturer. All samples in this assay were protected from light and performed at room temperature.

1.8 Fluorescent activated cell sorting ( FACS Combined measurement method

To evaluate gPD-L1 expression in cancer cell lines, cells were incubated at 4°C for 30 min with 1 μg of mTT-01 monoclonal antibody (Topmunnity Therapeutics, Taiwan) in FACS buffer (2% FBS in PBS). Cells were then washed with FACS buffer and incubated at 4°C for 30 min with 0.25 μg of Alexa ^Fluor® 647 goat anti-human IgG secondary antibody in FACS buffer. Viable cells were distinguished by 7-aminoactinomycin D (7-AAD) staining prior to analysis. Transduction efficiency and CAR T-cell subset ¹¹ were measured using flow cytometry. In short, CAR T-cells were collected and blocked for 15 minutes at 4°C with Human TruStain FcX (Biolegend, CA, USA) in FACS buffer, followed by staining with anti-c-myc antibody (Roche, Basel, Switzerland) at 4°C for 30 minutes. After washing, the cells were incubated at 4°C for 30 minutes with Alexa ^Fluor® 488 goat anti-mouse IgG secondary antibody (Thermo Fisher, MA, USA) in FACS buffer. Then, the cells were probed at 4°C for 30 minutes with monoclonal antibodies targeting human CD3, CD4, CD8, CD45RA, CD45RO, and CD62L (Biolegend, CA, USA). Viable cells were measured using the eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher, MA, USA). Stained cells were analyzed using the Attune NxT Cytometer (Thermo Fisher, MA, USA), and the data were tissue-organized using FlowJo V10 software. To determine the memory function of CAR T-cells, blood, spleen, and bone marrow were collected from CAR T-cell-treated mice after tumor disappearance. Staining procedures and antibody profiles were determined based on transduction efficiency conditions and CAR T-cell subsets.

1.9 Xenotransplantation mouse model

All animal experimental procedures were reviewed and approved by the IACUC. Six-week-old female NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ (NSG) mice were purchased from the Animal Core Facility (Institute of Biomedical Sciences, Academia Sinica, Taiwan) and housed there. To establish a breast cancer tumor model, mice were subcutaneously inoculated with 5 × ^10⁶ MDA-MB-231 or BT549 cells (triple-negative breast cancer), pre-mixed with Matrigel Matrix (Corning, NY, USA) at a 1:1 ratio. When the tumor size exceeded 100 ^mm³ , the mice received two doses of CAR T-cells (a total of 2 × ^10⁷ cells in the MDA-MB-231 model and 1 × ^10⁷ cells in the BT549 model). To evaluate the memory phenotype of CAR T-cells, mice were stimulated again with 1 x ^10⁷ MDA-MB-231 or BT549 cells 12 days after tumor disappearance. The size of the tumor was calculated using the following formula: V = (length × ^width² ) / 2.

1.10 Immunohistochemistry and immunofluorescence assays for mouse tissues

Mice underwent intracardiac perfusion prior to tumor tissue extraction to assess CAR T-cell infiltration. Tumor tissue was processed as previously described, with some modifications.^{^{11 12}} Formalin-fixed paraffin-embedded (FFPE) tumor sections were dewaxed with xylene, rehydrated in fractionated ethanol, and rinsed with distilled water. Antigen repair was performed at 95°C for 30 min using 1X citrate buffer (C9999, Sigma). After cooling to room temperature, the tumor sections were washed three times with PBST (PBS + 0.05% Tween 20) and blocked at room temperature for 30 minutes with PBS solution containing 2% donkey serum (containing 1% BSA and 0.5% triton). Then, the sections were incubated overnight at 4°C with primary antibodies against hPD-L1 (1:1000) (ab205921, Abcam, Cambridge, UK), hCD3 (1:100) (MCA1477, Bio-Rad, CA, USA), hCD8a (1:50) (ab17147, Abcam, Cambridge, UK) and human granzyme B (1:70) (ab4059, Abcam, Cambridge, UK). For immunohistochemical assays, tissues were washed three times with 1X PBST and incubated at room temperature for 30 minutes with ^ImmPRESS® horse anti-rabbit, mouse, and rat IgG polymer kits (MP-5401, MP-5404, MP-5402, Vector Laboratories, Burlingame, CA, USA). Sections were then detected using ImmPACT® ^Vector® Red (SK-5105, Vector Laboratories). Final slides were counterstained with hematoxylin. Images were scanned using a PANNORAMIC 250 Flash III (3DHISTECH, Budapest, ^Öv u. 3., Hungary). For immunofluorescence assay, tissues were washed three times and incubated at room temperature for 1 hour with the secondary antibodies Alexa ^Fluor® 488 goat anti-rat IgG (1:500), Alexa ^Fluor® 568 anti-mouse IgG (1:1000), and Alexa ^Fluor® 647 donkey anti-rabbit (1:1000). Cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) in the dark for 10 minutes. Images were captured using an LSM 700 platform confocal laser scanning microscope (Zeiss, Oberkochen, Germany).

1.11 Biological distribution

To detect the distribution of CAR T-cells, seven female NSG mice were inoculated with MDA-MB-231 cells (1 × ^10⁷ cells) before intravenous injection of CAR T-cells (1 × ^10⁷ cells). Blood, lung, spleen, liver, kidney, mesenteric lymph nodes, brain, and tumor tissue were collected from the mice at 4 hours post-inoculation, and on days 1, 2, 3, 7, 14, and 21. Genomic DNA (gDNA) was extracted from these tissues using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of gDNA was measured using a Nanodrop2000 (Thermo Fisher, MA, USA) and adjusted to the appropriate concentration range. Copy number of the TT0002 gene was quantified using qPCR with the CFX connect real-time PCR detection system (Bio-Rad, France). Primers for TT0002 and mouse β-actin are listed in Table 5 (TT0002-4 and mus β-actin).

Table 5. Introduction to TT0002 and mouse β-actin (TT0002-4 and mus β-actin) Introduction Sequence (5' to 3') TT0002-4 F CTG GGA CCT CTT ACT CTC (SEQ ID NO: 22) TT0002-4 R CCA GTA CCG AAC GTG ATG (SEQ ID NO: 23) mus b- actin F TGT CAC TCT TCT CTT AGG TAT GGA (SEQ ID NO: 24) mus b- actin R GGT CTT TAC GGA TGT CAA CG (SEQ ID NO: 25)

The qPCR procedure consists of 40 cycles: 3 minutes at 95°C, followed by 10 seconds at 95°C and 30 seconds at 60°C. The specificity of each primer was confirmed by the dissociation curves after the reaction.

1.12 Statistical analysis

Student's t-test was used to analyze the two independent groups, and one-way ANOVA was used for multiple group analyses. Data are expressed as mean ± SD (standard deviation), and a p-value ≤ 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism v8.0 (GraphPad Software, San Diego, CA).

2. result

2.1 Immunohistochemical assay

Comparative analysis of the binding properties of chimeric atezolizumab and mTT-01 in human tissues highlights the enhanced specificity of mTT-01, which is attributed to its interaction with different N-glycosylation sites on PD-L1, compared with chimeric atezolizumab targets (Figures 2 to 5).

The binding properties of mTT-01 to chimeric atezolizumab (anti-hPD-L1-mIgG1 InvivoFit, catalog number: hpdl1-mab9-1, InvivoGen) were compared using human placenta, tonsils, and breast cancer tissue. Chimeric atezolizumab (anti-hPD-L1-mIgG1) is characterized by a constant region of the mouse IgG1 isotype and a variable region of atezolizumab. Positive membrane staining was observed in both mTT-01 and chimeric atezolizumab-stained placental tissue at the same working concentration (20 μg/ml) (Figure 2, arrows). However, the staining intensity in mTT-01-stained tissue was lower (Figure 2, left panel).

In tonsil tissue, mTT-01 and chimeric atezolizumab showed similar staining patterns. However, the staining intensity of mTT-01-stained tissue was lower (Fig. 3, left).

In breast cancer tissue, both mTT-01 and chimeric atezolizumab showed membrane staining patterns in tumor cells. However, the cytoplasmic staining intensity was higher in the chimeric atezolizumab-stained tissue (Figure 4). Furthermore, background staining of the stromal and nucleus was observed in the chimeric atezolizumab-stained tissue (Table 6).

Table 6. Comparison of binding characteristics of mTT-01 and chimeric atezolizumab in human placenta, tonsils, and breast cancer tissues. (20 μg/ml) mTT-01 Chimeric atezolizumab Breast cancer tissue Tumor cells membrane staining +++ ++ Cytoplasmic staining ++ +++ Immune cells - + substrate - ++ nuclear - ++ Normal tissue tonsils + ++ placenta + ++

The binding properties of mTT-01 and chimeric atezolizumab were further investigated using the SCMH breast cancer TMA (BRCA-23). A total of 43 breast cancer cores and 44 cancer-adjacent normal tissue cores were evaluated. Detailed qualitative and quantitative analyses for each core are available in the Topmunnity database. No patient demographic data were available for this TMA. Antibody binding properties were determined using TPS. A sample was considered antibody-positive if any intensity of membrane staining was observed in ≥50% of viable tumor cells with TPS. The binding properties of the antibody to normal catheters were determined using the same criteria, with any intensity of membrane staining observed in ≥50% of viable catheters defined as positive catheter staining.

Overall, positive membrane mTT-01 staining was found in 34 out of 43 breast cancer cores (79%, Fig. 5). On the other hand, positive membrane chimeric-atezolizumab staining was found in 20 out of 43 breast cancer cores (46.5%). All (20) breast cancer cores that showed chimeric-atezolizumab positive staining also showed mTT-01 positive staining.

Of the 43 breast cancer cores, 26 were identified as DCIS cores, and the other 17 were invasive cancer tissues. 22 of the 26 DCIS cores (84.6%) showed positive membrane mTT-01 staining, while 17 of the 26 DCIS cores (65.39%) showed positive membrane chimeric atezolizumab staining. The main difference was in the positive membrane staining of the invasive cancer cores, where positive membrane mTT-01 staining was found in 70.6% of the cores, while only 17.6% of the cores were chimeric-atezolizumab positive.

Conversely, in cancer-adjacent normal tissue, 3 out of 44 cores (3.6%) showed positive mTT-01 staining on the normal duct, while 10 out of 44 cores (22.7%) showed positive chimeric atezolizumab staining. All (3) adjacent normal cores showing mTT-01 positive staining also showed chimeric atezolizumab positive staining.

2.2 Jurkat Effective in cells CAR Performance

The results, as shown in Figure 6, demonstrate the successful integration and expression of the chimeric antigen receptor (CAR) in Jurkat NFAT-Luc cells (a T lymphocyte-derived immortalized cell line). These cells were engineered to include the NFAT (activated T nuclear factor)-induced Lucia reporter, which expresses luminescence upon activation via the NFAT pathway. This is crucial for evaluating the function of the CAR construct.

In the experiment, TT0001 and TT0002 CAR constructs were transduced into Jurkat NFAT-Luc cells using lentiviral vectors. Subsequent analysis showed detectable myc expression on the cell surface, a marker of CAR presence. In Figure 6, the left panel represents normal Jurkat NFAT-Luc cells, establishing a baseline for myc expression. The middle and right panels show the myc distribution in TT0001 and TT0002 CAR-Jurkat cells, respectively, showing that 81.9% of TT0001 and 78.6% of TT0002 CAR-Jurkat cells expressed myc, confirming effective CAR integration and expression.

Further bioluminescence intensities of these CAR-modified Jurkat cells are shown in Figure 7, where CAR-Jurkat cells were co-cultured with various cell lines, including MDA-MB-231, BT549, A549, and HEK293T, to evaluate their functional responses. The bioluminescence intensities of CAR-Jurkat cells showed a positive correlation between NFAT pathway activation and PD-L1 expression on the surface of co-cultured cells. This observation confirms that CAR-Jurkat cells can appropriately activate the NFAT pathway when co-cultured with target cancer cells, demonstrating the functional efficacy of the CAR signaling peptide.

2.3 CD3+ T In cells CAR Construction of the structure

This section of the patent application details the successful establishment of CAR constructs in first-generation human CD3+ T cells, as shown in Figures 8 and 9. The process involves infecting these T cells with a lentiviral vector carrying the TT0001 and TT0002 CAR constructs.

For TT0001 CAR-T, as shown in Figure 8, primary CD3+ T cells were transduced with a lentiviral vector containing the TT0001 CAR construct. Following infection, surface expression of myc (a marker of CAR expression) was analyzed using fluorescence-activated cell sorting (FACS). FACS data showed that a large proportion of T cells (over 78%) expressed myc on their surface, with a mean fluorescence intensity (MFI) of approximately 3000. This level of expression is significantly higher than the normal range for myc expression in untransduced CD3+ T cells, where the MFI was measured at 33.7. This substantial increase in MFI indicates successful expression of the TT0001 CAR construct in CD3+ T cells.

Similarly, Figure 9 focuses on the establishment of the TT0002 CAR-T. This procedure is similar to that of TT0001, in which primary CD3+ T cells are transduced with a lentiviral vector encoding the TT0002 CAR construct. Subsequent FACS analysis showed that over 79% of the transduced T cells expressed myc on their surface, with an MFI of approximately 3000, which was significantly higher than the baseline expression of myc in normal CD3+ T cells.

These results collectively confirm the successful transduction and expression of the TT0001 and TT0002 CAR constructs in CD3+ T cells. This is a key step in the development of CAR-T cell therapy because it demonstrates the ability to reprogram progenitor T cells to express specific CAR constructs, potentially guiding them to target antigens for therapeutic applications in cancer treatment.

2.4 by CAR-T Cytotoxicity and efficacy of treatment for breast cancer cell lines PD-L1 Performance Analysis

In a broad evaluation of CAR-T cells against various breast cancer cell lines, important correlations were established between the cytotoxicity of CAR-T cells and the PD-L1 expression levels of these cells, as shown in Figures 10 to 12. The upper panel of Figure 10 shows that TT0001 and TT0002 CAR-T cells exhibited cytotoxic activity against MDA-MB-231 cells at different E:T ratios. Furthermore, the lower panel of Figure 10 shows that TT0002 CAR-T cells exhibited varying degrees of cytotoxicity across different cell lines, with moderate activity observed in the MDA-MB-361 cell line, negligible activity in the BT474 cell line, and high cytotoxicity observed in other cell lines.

As shown in Figure 11, further elucidation of PD-L1 expression in the MDA-MB-231 cell line provides a deeper understanding of the variability of this response. The results indicate that the cytotoxic efficacy of TT0002 CAR-T cells is not consistent but depends on the level of PD-L1 expression in the target cells. Both the MDA-MB-231 PD-L1 overexpressing strain and the conventionally high PD-L1 expressing MDA-MB-231 strain showed significant sensitivity to treatment. In contrast, the PD-L1 knockout variant exhibited a lack of PD-L1 expression, providing insights into the mechanism of action of TT0002 CAR-T cells.

Figure 12, a comparative analysis of TT0001 and TT0002 CAR-T cells, further reinforces these observations. Notably, the cytotoxicity of these two CAR-T cell types was positively correlated with PD-L1 expression in the target cells, highlighting the crucial role of PD-L1 in regulating the response to CAR-T cell therapy.

These findings highlight the potential of CAR-T cells in treating breast cancer subtypes with different PD-L1 expressions, providing valuable insights for future treatment strategies.

2.5 Multi-Plex Analysis confirms the production of cytokines

As detailed in Figure 13, the results from the Multi-Plex assay demonstrate the efficacy of TT0002 CAR-T cells in the in vitro environment. This assay was strategically used to evaluate the cytokine release profile of these modified T cells when co-cultured with the MDA-MB-231 cell line with different PD-L1 expression levels (Figure 11). The key feature of this assay is the measurement of key cytokines, which are an integral part of the immune response and can indicate the activity of CAR-T cells.

In the upper left panel of Figure 13, IL-2 cytokine measurements show that the highest secretion occurs in co-cultures with an E:T ratio of 3:1, particularly in cell lines expressing PD-L1. This pattern illustrates that the production of IL-2 (a key cytokine for T cell activation and proliferation) increases in the presence of PD-L1-expressing target cells, thus validating the responsiveness of CAR-T cells to their intended targets.

This assay also measures TNF-α and IFN-γ, as shown in the upper middle and upper right panels of Figure 13. The increased levels of these cytokines under similar co-culture conditions further confirm the activation and functional response of CAR-T cells. The production of TNF-α and IFN-γ is crucial for an effective immune response against tumor cells, implying that CAR-T cells have the ability to recognize and respond to cancer cells displaying PD-L1.

Furthermore, this assay analyzed the levels of IL-10 and IL-6, as shown in the lower left of Figure 13. The observed cytokine profile provides additional evidence supporting the in vitro functional activity of CAR-T cells. The differences in cytokine secretion based on PD-L1 expression levels on target cells highlight the specificity and potential efficacy of TT0002 CAR-T cells.

As illustrated in Figure 13, these results thus validate the efficacy of TT0002 CAR-T cells in in vitro experiments. They demonstrate the cells' ability to generate a strong immune response when encountering target cells with different PD-L1 expressions, a key indicator of their potential effectiveness in CAR-T cell therapy.

2.6 TT0002 CAR-T Enhanced efficacy was demonstrated in xenograft mouse models.

At this crucial stage of validating CAR-T cell therapy, in vivo efficacy studies provide indispensable evidence. Figures 14 and 15 show the results of this study, comparing the efficacy of TT0001 and TT0002 CAR-T cells in a xenograft mouse model using NSG mice injected with MDA-MB-231 cells. This model is crucial for evaluating the real-world potential of these CAR-T cell therapies in targeting cancer.

In the experimental setup depicted in Figure 14, TT0001 CAR-T cells were administered when tumor size exceeded 100 ^mm³ , followed by a follow-up dose one week later. Conversely, TT0002 CAR-T cells were introduced into another group of mice with more advanced tumor growth, exceeding 200 mm³, followed by an additional dose three days later. This approach aimed to compare the efficacy of the two CAR-T cell types under different tumor loads.

Figure 15 reveals a stark contrast in tumor suppression between the two CAR-T therapies. Despite early intervention, TT0001 CAR-T cells failed to significantly affect tumor size. On the other hand, TT0002 CAR-T cells demonstrated a significant ability to control tumor growth, maintaining the tumor size at approximately 300 mm³ until day 52. This result not only highlights the superior efficacy of TT0002 CAR-T cells but also illustrates their potential effectiveness in later stages of tumor development.

The difference in outcomes between TT0001 and TT0002 CAR-T therapy underscores the importance of CAR design and screening in developing effective cancer immunotherapies. The significant tumor-suppressive effect of TT0002 CAR-T cells observed in this xenograft model supports its clinical application in cancer treatment and may provide patients with a more effective treatment option.

2.7 Demonstrated in xenograft mouse models TT0002 CAR-T Internal effects

In a crucial step in validating the in vivo efficacy of CAR-T therapy, our study focused on evaluating the effect of TT0002 CAR-T cells on tumor growth in a xenograft mouse model. The results, shown in Figures 16 and 17, provide compelling evidence of the potent tumor-suppressive capacity of TT0002 CAR-T cells.

In the experiment detailed in Figure 16, we observed a significant reduction in tumor growth following administration of TT0002 CAR-T cells. This study involved reducing the initial dose of tumor cells and then introducing TT0002 CAR-T cells when the tumors reached a measurable size. Notably, within approximately 30 days of TT0002 CAR-T cell treatment, a large number of treated mice exhibited undetectable tumor growth, a stark contrast to the PBS-treated control group. This significant reduction in tumor size highlights the potential of TT0002 CAR-T cells to effectively target and reduce tumor quality in living organisms.

To further reinforce these findings, Figure 17 details a dose-dependent study of the tumor-suppressive effect of TT0002 CAR-T cells. This study demonstrates that higher doses of TT0002 CAR-T cells lead to a more significant reduction in tumor size, and in some cases, even complete tumor disappearance. This dose-dependent response provides valuable insights into the therapeutic window and efficacy of TT0002 CAR-T cells, suggesting their suitability for customized cancer treatment regimens.

The conclusion is that the in vivo experiments, as shown in Figures 16 and 17, clearly demonstrate the dose-responsive tumor-suppressive efficacy of TT0002 CAR-T cells in xenograft mouse models. These findings are crucial for illustrating the practical application and potential of TT0002 CAR-T therapy in cancer treatment. The significant reduction in tumor size observed under different dose conditions provides important data for the further development and optimization of TT0002 CAR-T cells as a targeted oncology therapy. This evidence strengthens the potential of TT0002 CAR-T cells to make a significant contribution to the advancement of effective cancer immunotherapy.

2.8 TT0002 CAR-T Cell lifespan in restimulated xenograft mouse models

The persistence and sustained efficacy of TT0002 CAR-T cells were evaluated in an extended in vivo study using a restimulated xenograft mouse model. This study is significant for demonstrating the long-term therapeutic potential of CAR-T cells.

Initially, the study tracked the progress of two mice previously treated with TT0002 CAR-T cells, then re-injected these mice with MDA-MB-231 cells to evaluate tumor recurrence. In contrast to the control group, these mice showed no tumor growth throughout the extended observation period up to day 90. As shown in Figure 18, this result highlights the sustained antitumor activity of TT0002 CAR-T cells, demonstrating their efficacy over a considerable period after initial administration.

Subsequent analyses of blood, spleen, and bone marrow tissue from these mice provided evidence of the distribution and persistence of CAR-T cells in vivo. Flow cytometry results, shown in Figure 19, revealed the detectable presence of CAR-T cells, particularly in the bone marrow, indicating a potential reservoir or location for prolonging CAR-T cell activity.

The study was further extended to day 86 to observe the long-term effects of CAR-T cell therapy. A significant reduction in tumor growth was observed, as shown in Figure 20. This demonstrates that although the concentration of active CAR-T cells decreased over time, the residual cell population still effectively inhibited tumor growth.

These findings collectively highlight the potential of TT0002 CAR-T cells for long-term cancer treatment.

2.9 Confirmed by immunohistochemistry TT0002 CAR-T Cell penetration and activation in xenograft tumors

This section presents the results of immunohistochemical and immunofluorescence analyses to confirm the infiltration and activation of TT0002 CAR-T cells in xenograft tumor tissue, as visually supported by Figures 21 and 22.

Figure 21 shows the detection of CAR-T cells in tumor tissue of mice treated with TT0002 CAR-T cells using immunohistochemistry. Notably, in the PBS-injected control group, PD-L1 was widely expressed in the tissue sections, but human CD3-expressing cells were significantly absent. In stark contrast, the tissue sections of the TT0002 CAR-T treatment group showed a clear presence of CAR-T cells expressing human CD3, particularly around the central blood vessels. This CAR-T cell ring was surrounded by tissue with high PD-L1 expression, indicating that the CAR-T cell line targeted infiltration into the tumor region.

Figure 22 uses immunofluorescence staining to further verify the presence and activity of CAR-T cells in xenograft tumor tissue. The control group showed extensive PD-L1 expression but no significant presence of human CD3 or signs of T cell-mediated tumor cell cytotoxicity. In contrast, in the TT0002 CAR-T treatment group, human CD3 signaling was prominent and co-localized with PD-L1 signaling. Furthermore, significant presence of granzyme B signaling was detected, indicating that CAR-T cells have cytotoxic effects against target tumor cells.

The findings shown in Figures 21 and 22 provide strong evidence for the successful infiltration and activation of TT0002 CAR-T cells in the tumor microenvironment. The ability of these cells to specifically target and exert cytotoxic effects on tumor cells while infiltrating tumor tissue highlights the potential efficacy of TT0002 CAR-T therapy in cancer treatment.

2.10 CAR-T Biological distribution analysis of cells

The results of the biodistribution study of TT0002 CAR-T cells shown in Figure 23 provide important insights into the distribution and persistence of these cells in an in vivo mouse model. This analysis is essential for understanding the migration and localization of CAR-T cells after administration.

After injecting TT0002 CAR-T cells into mice with established tumors, the tumor growth curve (Figure 23, left panel) showed a significant reduction in tumor size after 35 days, consistent with that at 21 days post-injection. This reduction indicates the therapeutic effect of CAR-T cells on tumor regression.

CAR copy number in DNA samples was analyzed, as shown in the right panel of Figure 23, to determine the distribution of CAR-T cells in mice. Notably, a large number of CAR-T cells were observed in the lungs during the initial 72 hours. However, by day seven post-injection, the major localization of CAR-T cells shifted to the tumor site. This migration pattern suggests an active homing mechanism in which CAR-T cells migrate to and accumulate in tumor tissue, possibly in response to tumor-specific antigens.

The biological distribution data presented in Figure 23 highlight the targeting effect of TT0002 CAR-T cells and their ability to specifically localize tumors. The migration from the initial lung distribution to the tumor tissue underscores the cells' ability to actively seek out and influence tumor sites, which is a key factor in its therapeutic efficacy.

3. Conclusion

This study presents comprehensive data and analysis highlighting the efficacy, specificity, and long-term potential of chimeric antigen receptor T (CAR-T) cells in cancer treatment. These findings provide strong evidence supporting the innovative approach and effectiveness of CAR-T therapy, particularly for cancers expressing programmed death-ligand 1 (PD-L1).

Target recognition and cytotoxic efficacy: Immunohistochemical assays ultimately confirmed the specific binding and targeting of CAR-T cells to PD-L1-expressing tumor cells prepared in this study. These results indicate that anti-PD-L1 CAR-T cells have higher sensitivity and specificity, making them an effective treatment option for PD-L1-expressing cancers.

In vitro and in vivo validation: The successful performance of the CAR construct in Jurkat cells and primary human CD3+ T cells, and the subsequent demonstration of cytotoxicity in various breast cancer cell lines, validated the in vitro efficacy of anti-PD-L1 CAR-T cells. Furthermore, in vivo studies in xenograft mouse models further demonstrated the significant tumor-suppressive capacity of anti-PD-L1 CAR-T cells, even in late-stage tumor growth.

Lifespan and Durable Efficacy: Restimulation experiments highlight the durability and sustained antitumor efficacy of anti-PD-L1 CAR-T cells. These experimental results are significant, demonstrating that anti-PD-L1 CAR-T cells can provide long-term therapeutic effects, a key factor in durable cancer remission.

Biodistribution and targeting: Biodistribution studies confirm the targeted migration and localization of anti-PD-L1 CAR-T cells to the tumor site, highlighting their ability to actively hom and specifically exert therapeutic effects at the tumor site.

In conclusion, the comprehensive data presented in this paper confirms the potential of the CAR-T therapy of this invention as a highly effective, specific, and sustainable treatment modality in the field of cancer immunotherapy. The specificity of targeting PD-L1-expressing cells, combined with the proven efficacy profile, positions the CAR-T therapy using PD-L1-targeting CAR-T cells described in this paper as a promising candidate for future clinical applications in oncology, offering new hope to PD-L1-expressing patients battling cancer. References 1. PardollDM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer . 2012;12(4):252-264. doi:10.1038/nrc3239 2. TwomeyJD, ZhangB. Cancer Immunotherapy Update: FDA-Approved Checkpoint Inhibitors and Companion Diagnostics. AAPS J . 2021;23(2):39. doi:10.1208/s12248-021-00574-0 3. LiC-W, LimS-O, ChungEM, et al. Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1. Cancer Cell . 2018;33(2):187-201.e10. doi:https://doi.org/10.1016/j.ccell.2018.01.009 4. BenickyJ, SandaM, Brnakova KennedyZ, et al. PD-L1 Glycosylation and Its Impact on Binding to Clinical Antibodies. J Proteome Res . 2021;20(1):485-497. doi:10.1021/acs.jproteome.0c00521 5. LiC-W, LimS-O, XiaW, et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat Commun . 2016;7(1):12632. doi:10.1038/ncomms12632 6. NeelapuSS, TummalaS, KebriaeiP, et al. Chimeric antigen receptor T-cell therapy — assessment and management of toxicities. Nat Rev Clin Oncol . 2018;15(1):47-62. doi:10.1038/nrclinonc.2017.148 7. Shimabukuro-VornhagenA, BöllB, SchellonowskiP, et al. Critical care management of chimeric antigen receptor T-cell therapy recipients. CA Cancer J Clin . 2022;72(1):78-93. doi:https://doi.org/10.3322/caac.21702 8. LiuH, MaY, YangC, et al. Severe delayed pulmonary toxicity following PD-L1–specific CAR-T cell therapy for non-small cell lung cancer. Clin Transl Immunol . 2020;9(10):e1154. doi:https://doi.org/10.1002/cti2.1154 9. BajorM, Graczyk-JarzynkaA, MarhelavaK, et al. PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells. J Immunother Cancer . 2022;10(1):e002500. doi:10.1136/jitc-2021-002500 10. YangY, LiC-W, ChanL-C, et al. Exosomal PD-L1 harbors active defense function to suppress T cell killing of breast cancer cells and promote tumor growth. Cell Res . 2018;28(8):862-864. doi:10.1038/s41422-018-0060-4 11. HuangH-C, LaiY-J, LiaoC-C, et al. Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro. eBioMedicine . 2021;74. doi:10.1016/j.ebiom.2021.103712 12. HuangH-C, LiaoC-C, WangS-H, et al. Hyperglycosylated spike of SARS-CoV-2 gamma variant induces breast cancer metastasis. Am J Cancer Res . 2021;11(10):4994-5005.

without

The foregoing overview and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, preferred embodiments are shown in the accompanying drawings. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

In the following diagram:

Figure 1 shows the structure of the CAR in the viral vector backbone plasmid. pTT0001 contains the human CD8 signaling peptide, myc, the _VH of mTT-01, the GS linker, the _VL of mTT-01, the human CD8a transmembrane domain, 4-1BB, and human CD3ζ; pTT0002 contains the human CD8 signaling peptide, myc, the _VL of mTT-01, the GS linker, the _VH of mTT-01, the human CD8a transmembrane domain, 4-1BB, and human CD3ζ. Here, myc serves as a post-experimental marker for the CAR protein. Alanine-alanine-alanine is added before the human CD8a transmembrane domain as the NotI restriction endonuclease cleavage site.

Figure 2 shows a comparison of immunohistochemical staining of monoclonal antibody mTT-01 and chimeric atezolizumab in human placental tissue. The left panel shows the staining results of mTT-01; the right panel shows the staining results of chimeric atezolizumab. Therefore, the placenta, as fetal tissue, exhibits a high level of PD-L1 to protect against attack by maternal T cells. The arrows indicate that both antibodies clearly stained the cell membrane.

Figure 3 shows a comparative immunohistochemical staining of monoclonal antibody mTT-01 and chimeric atezolizumab in human tonsil tissue. The left panel represents the staining results of mTT-01; the right panel represents the staining results of chimeric atezolizumab. The tonsils are human organ tissues with high PD-L1 expression. The two antibodies showed similar staining distributions, but the staining of mTT-01 was significantly weaker than that of chimeric atezolizumab.

Figure 4 shows the comparative immunohistochemical staining of monoclonal antibody mTT-01 and chimeric atezolizumab in human breast cancer tumors. The left panel shows the staining results of mTT-01; the right panel shows the staining results of chimeric atezolizumab. Both antibodies showed similar staining distributions, with chimeric atezolizumab showing deeper staining in the cytoplasm, while mTT-01 showed deeper staining on the tumor cell membrane. Furthermore, chimeric atezolizumab stained both the matrix and nucleus of breast tissue.

Figure 5 shows a comparison of immunohistochemical staining of monoclonal antibody mTT-01 and chimeric atezolizumab in human tumors and adjacent normal tissues. Given the importance of CAR technology for immune cell recognition of cell surface biomarkers, in 43 tumor samples with positive cell membrane staining, mTT-01 identified 34 samples, a positive detection rate of 79%; while chimeric atezolizumab identified 20 samples, a positive detection rate of 46.5%. In 44 tumor-adjacent normal tissue samples, mTT-01 showed cell membrane staining in only 3 samples, with a recognition rate of 6.8% for adjacent normal tissue; chimeric atezolizumab showed staining in 10 samples, with a recognition rate of 22.7% for adjacent normal tissue. This indicates that mTT-01 has better specificity for cell membrane staining than chimeric atezolizumab, which may lead to lower toxicity to normal tissue.

Figure 6 shows Jurkat NFAT-Luc cells, an immortalized T lymphocyte-derived cell line that integrates the NFAT (nuclear factor for activated T cells)-induced Lucia reporter, resulting in the expression of luciferase upon activation of the NFAT pathway. After transducing Jurkat NFAT-Luc cells with lentiviral vectors containing TT0001 and TT0002 CARs, myc expression was detectable on the surface of Jurkat cells. The left image shows normal Jurkat NFAT-Luc cells, used to determine the normal range of myc expression, with 0.22% of cells exceeding the marked threshold. The middle image shows the distribution of myc in TT0001 CAR-Jurkat cells using the same threshold, showing that 81.9% of cells expressed myc. The right image shows the distribution of myc in TT0002 CAR-Jurkat cells using the same threshold, showing that 78.6% of cells expressed myc.

Figure 7 shows the co-culture of CAR-Jurkat cells with four cell types: MDA-MB-231, a breast cancer cell line with high PD-L1 expression; BT549, a breast cancer cell line with intermediate PD-L1 expression; A549, a lung cancer cell line with low PD-L1 expression; and HEK293T, a human embryonic kidney cell line that does not express PD-L1. When Jurkat cells are induced to activate NFAT, they produce photoenzyme, which can be detected by bioluminescence. Within two days, the normal Jurkat group showed extremely low bioluminescence, while TT0001 CAR-Jurkat and TT0002 CAR-Jurkat showed a positive correlation between bioluminescence intensity and PD-L1 expression on the surface of co-cultured cells. This confirms that CAR-Jurkat can correctly activate the NFAT pathway when co-cultured with target cancer cells, demonstrating the correct function of the CAR signaling peptide.

Figure 8 shows the establishment of the CAR construct in CD3+ T cells, TT0001 CAR-T. After infecting primary CD3+ T cells with a lentiviral vector, the surface expression of myc was detected using FACS. The upper center image shows normal CD3+ T cells, used to determine the normal range of myc expression, with cell distributions exceeding the 0.74% threshold marked. In this embodiment, three different lentiviral infection concentrations were used, with the same threshold used to separate populations. Over 78% of T cells expressed myc, with an MFI of approximately 3000, significantly higher than the MFI of 33.7 for normal CD3+ T cells, indicating successful expression of TT0001 in CAR-T cells.

Figure 9 shows the establishment of the CAR construct in CD3+ T cells, TT0002 CAR-T. After infecting primary CD3+ T cells with a lentiviral vector, the surface expression of myc was detected using FACS. The upper center image shows normal CD3+ T cells, used to determine the normal range of myc expression, with cell distributions exceeding the 0.74% threshold marked. In this embodiment, three different lentiviral infection concentrations were used, with the same threshold used to separate populations. Over 79% of T cells expressed myc, with an MFI of approximately 3000, significantly higher than the MFI of 33.7 for normal CD3+ T cells, indicating successful expression of TT0002 in CAR-T cells.

Figure 10 shows the cytotoxicity analysis of CAR-T-treated breast cancer cell lines. The upper figure shows the cytotoxicity of TT0001 and TT0002 CAR-T cells against MDA-MB-231 breast cancer cells, measured using the CCK-8 assay after 24 hours of co-culture. The results indicate that both CAR-T variants exhibited similar cytotoxic capabilities with no significant difference, and their efficacy increased with the E:T ratio. At an E:T ratio of 1:3, approximately 20% cytotoxicity was observed; at 1:1, cytotoxicity increased to approximately 60%; and at ratios higher than 3:1, cytotoxicity reached over 80%. In contrast, the control group's CD3+ T cells only exhibited significant cytotoxicity at an E:T ratio of 10:1. The figure below shows the cytotoxicity of TT0002 CAR-T in various breast cancer cell lines. TT0002 CAR-T cells exhibited varying degrees of cytotoxicity in multiple breast cancer cell lines. Notably, moderate cytotoxicity was observed in the MDA-MB-361 cell line. The remaining cell lines showed high levels of cytotoxicity to TT0002 CAR-T.

Figure 11 shows the MDA-MB-231 cell lines with different PD-L1 expression levels. The MDA-MB-231 PD-L1 cell line overexpressed PD-L1; the MDA-MB-231 cell line expressed high levels of PD-L1; and the MDA-MB-231 PD-L1 ^KO cell line did not express PD-L1.

Figure 12 shows the different cytotoxic effects of TT0002 CAR-T cells against MDA-MB-231-derived cell lines with varying PD-L1 expression. The upper figure shows that TT0002 CAR-T cells exhibited varying degrees of cytotoxicity against the MDA-MB-231 cell line series, depending on the PD-L1 expression level. A positive correlation was found between the degree of cytotoxicity and the PD-L1 expression level and the number of effective cells. The lower figure shows that CD3+ T cells did not exhibit significant cytotoxicity.

Figure 13 shows the Multi-Plex assay, used to analyze the levels of cytokines released into the culture medium after 48 hours of co-culturing TT0002 CAR-T cells with MDA-MB-231 cell lines showing different PD-L1 expression levels. The effector cells were TT0002 CAR-T cells, and the target cells were the MDA-MB-231 cell line. The upper left panel shows the results for human IL-2; regardless of the E:T ratio, IL-2 was not detected in the culture medium of the MDA-MB-231 PD-L1 ^KO co-culture. For the other two cell lines, the highest concentration of IL-2 in the medium was detected at an E:T ratio of 3:1, decreasing at lower ratios. The MDA-MB-231 PD-L1 cell line exhibited higher IL-2 activity. Although IL-2 was detected at the highest E:T ratio of 10:1, its concentration was lower than in the 3:1 group, due to consumption by TT0002 CAR-T. The MDA-MB-231 PD-L1 cell line had a slightly lower IL-2 concentration than the MDA-MB-231 cell line. This indicates that IL-2 is produced by the interaction between TT0002 CAR-T cells and cells expressing PD-L1 on their surface, with higher expression associated with an increase in T cell count and generally positively correlated with PD-L1 expression. The upper-middle figure shows the results for human TNF-α; regardless of the E:T ratio, TNF-α was not detected in the medium of the MDA-MB-231 PD-L1 ^KO co-culture. For the other two cell lines, the highest concentration of TNF-α was detected in the medium at an E:T ratio of 3:1, decreasing at lower ratios, with the MDA-MB-231 PD-L1 cell line exhibiting higher TNF-α expression. Although TNF-α was detected at the highest E:T ratio of 10:1, its concentration was lower than that in the 3:1 group. This indicates that TNF-α is produced by the interaction between TT0002 CAR-T cells and cells expressing PD-L1 on their surface, with higher expression associated with an increase in T cell count and generally positively correlated with PD-L1 expression. The upper right figure shows the results for human IFN-γ; the highest concentration of IFN-γ was detected in the culture medium at an E:T ratio of 3:1, and the concentration decreased at lower ratios. Within each E:T group, there was no significant difference between MDA-MB-231 PD-L1 and MDA-MB-231, while a small amount of IFN-γ was detected in MDA-MB-231 PD-L1 ^KO . The highest E:T ratio of 10:1 showed IFN-γ concentrations close to but lower than those in the 3:1 group. This indicates that IFN-γ is produced by the interaction between TT0002 CAR-T cells and MDA-MB-231 co-cultured cells, and its higher expression is associated with an increase in the number of T cells. Furthermore, the high expression of PD-L1 on the cell surface significantly stimulates the production of IFN-γ. The lower left figure shows the results for human IL-10, where the highest concentration of IL-10 in the culture medium was detected at an E:T ratio of 10:1, decreasing as the ratio decreased. Within each E:T group, the MDA-MB-231 PD-L1 cell line exhibited higher IL-10 concentrations at high ratios compared to the other two cell lines, with the MDA-MB-231 PD-L1 ^KO showing the lowest detectable amount of IL-10. This indicates that IL-10 is produced through the interaction between TT0002 CAR-T cells and MDA-MB-231 co-cultured cells, with higher expression correlated with an increase in T cell count. Furthermore, at high E:T ratios, high expression of PD-L1 on the cell surface significantly stimulates IL-10 production. The lower half of the figure shows the results for human IL-6. From the groups not co-cultured with TT0002CAR-T cells, it can be seen that both MDA-MB-231 PD-L1 and MDA-MB-231 cells showed high IL-6 expression, indicating that the 231 cell line itself produces IL-6. Except for the MDA-MB-231 PD-L1 ^KO group, which only expressed IL-6 after co-culture, the other two groups showed a decrease in IL-6 with increasing T cell count.

Figure 14 shows the in vivo efficacy of TT0001 CAR-T and TT0002 CAR-T. NSG mice were subcutaneously injected with 1× ^10⁷ MDA-MB-231 cells as a tumor model, with day 0 being the day of injection. On day 15, when the tumor ^exceeded 100 mm³, 1× ^10⁷ TT0001 CAR-T cells were intravenously injected. The two-dose regimen received a second intravenous injection of 1× ^10⁷ TT0001 CAR-T cells one week later; the control group received PBS on day 15. In the TT0002 CAR-T group, 1 × ^10⁷ TT0002 CAR-T cells were intravenously injected on day 21 ^when the tumor exceeded 200 mm³, followed by a second CAR-T injection three days later. The control group received PBS on day 15. Mouse weight and tumor size were measured every three days. The experiment ended with mouse sacrifice.

Figure 15 shows the therapeutic effects of TT0001 CAR-T and TT0002 CAR-T in xenograft mouse models. Data show that injection of TT0001 CAR-T did not show any tumor inhibition effect, even in small tumors; however, with TT0002 CAR-T, the tumor size was suppressed to 300 ^mm3 until day 52.

Figure 16 shows the long-term therapeutic efficacy of TT0002 CAR-T in a xenograft mouse model. Data showed that TT0002 CAR-T prolonged tumor growth. The MDA-MB-231 injection dose was reduced to 5 × ^10⁶ units, and again, when the tumor exceeded 100 ^mm³ , 1 × ^10⁷ TT0002 CAR-T units were injected. Three days later, a second dose of 1 × ^10⁷ TT0002 CAR-T units was injected. The control group received PBS at the same time point. Body weight and tumor size were measured every two to three days until the mice died or were sacrificed. The figure below shows that the TT0002 CAR-T injection group significantly inhibited tumor growth, with tumors undetectable at approximately day 30. On day 24, one CAR-T-injected mouse was sacrificed and one died, while the remaining mice survived until day 44. At this point, the control group was sacrificed, leaving two mice for subsequent restimulation experiments.

Figure 17 shows the dose-dependent tumor suppression effect of TT0002 CAR-T. The upper left figure shows the experimental design, in which 3 × ^10⁶ MDA-MB-231-Luc cells were subcutaneously injected on day 0, followed by an intravenous injection of one dose of T cells (1 × ^10⁷ CD3+ T cells, 1 × ^10⁷ TT0002 CAR-T cells, or 1 × ^10⁶ TT0002 CAR-T cells) on day 14. The lower left figure shows the different tumor-suppressing effects of different doses of CAR-T; CD3+ T cells did not inhibit the tumor; 1× ^10⁶ TT0002 CAR-T cells maintained tumor size until approximately day 28 when inhibition ceased; 1× ^10⁷ TT0002 CAR-T cells inhibited the tumor until day 42 when it almost disappeared. The right figure shows the growth of MDA-MB-231-Luc tumors in mice, where the group using both 1× ^10⁷ CD3+ T cells and 1× ^10⁶ TT0002 CAR-T cells showed tumor metastasis at day 50, while only the group using 1× ^10⁷ TT0002 CAR-T cells showed tumor shrinkage or disappearance.

Figure 18 shows the sustained efficacy of TT0002 CAR-T in a restimulated xenograft mouse model. The experiment continued as described in Figure 16, with two surviving mice restimulated by injection of 1 × ^10⁷ MDA-MB-231 tumor cells, and sacrificed on day 90. Body weight and tumor size at the injection site were measured every two to three days. The figure below shows that, compared to the control group which received concurrent tumor injections, the mice injected with TT0002 CAR-T did not develop tumors, confirming that CAR-T persisted in mice for at least 44 days and retained its tumor-suppressive efficacy.

Figure 19 shows the distribution of CAR-T cells in various tissues. This assay used FACS analysis of blood, spleen, and bone marrow tissues extracted from restimulated mice, with survival and death signals on the vertical axis and human CD3 on the horizontal axis. Cells falling into the Q3 region were identified as TT0002 CAR-T cells using the threshold drawn from the TT0002 CAR-T cells cultured in the upper left figure, to observe whether CAR-T cells were maintained in the tissues. As indicated by the arrows, very small amounts of CAR-T cells (<1%) were detected in the blood and spleen, while a larger amount (3.18%) was detected only in the bone marrow.

Figure 20 shows the sustained efficacy of TT0002 CAR-T in a restimulated xenograft mouse model. This experiment extended the tumor restimulation experiment to day 86 by administering MDA-MB-231 cells. While tumor growth was continuously suppressed, it was not completely halted. This indicates the presence of CAR-T cells in the mice on day 86, although their number was significantly reduced compared to day 44.

Figure 21 shows the penetration and activation of TT0002 CAR-T cells in xenograft tumors. Immunohistochemistry was used in this experiment to confirm the presence of CAR-T cells in mouse xenograft tumor tissues. The top image is from tumor sections of control mice injected with PBS, while the bottom image is from tumor sections of mice injected with TT-0002 CAR-T cells. In the control group, there was significant and widespread PD-L1 expression in the tissues (top right image, red area), but no cells expressing human CD3 were present (top middle image, red area). In contrast, the TT0002 CAR-T group showed a ring of CAR-T cells expressing human CD3 around the central blood vessels in the tissue (lower middle image, central red area), with an outer ring of PD-L1 highly expressed tumor tissue (lower right image, red area). The upper right portion of the slice showed the coexistence of PD-L1 and human CD3. This confirms that CAR-T cells are indeed attracted to the tumor.

Figure 22 shows the presence and activity of TT0002 CAR-T cells in xenograft tumors. This experiment used immunofluorescence staining to verify the presence of CAR-T cells in mouse xenograft tumor tissues. The top row of images is from tumor sections of mice injected with PBS (control group), and the bottom row of images is from tumor sections of mice injected with TT-0002 CAR-T. The first column on the left shows nuclear staining of the two groups of tissue sections; the second column on the left shows human CD3 staining, representing CAR-T; the middle column shows human PD-L1 staining, representing tumor cells; the second column on the right shows granzyme B staining released by T cells, indicating the effect of CAR-T on target tumor cells; the far right column shows the superposition of pseudo-colors of all staining. In the control group, extensive PD-L1 expression was observed (top center image), but no human CD3 expression was observed (top left second image), and almost no signal of T cell killing tumor cells was seen (top right second image). In contrast, the TT0002 CAR-T group showed colocalization of hCD3 signal (bottom left second image) and hPD-L1 signal (bottom center image), and a large amount of granzyme B signal was present (bottom right second image), confirming that CAR-T cells can infiltrate into tumor tissue and kill tumor cells.

Figure 23 shows the biodistribution and migration of TT0002 CAR-T in a xenograft mouse model. The biodistribution experiment of TT0002 CAR-T confirmed that it was mainly distributed in the lungs and tumors of mice during CAR-T treatment. The upper figure shows the tumor growth curve for each mouse before sacrifice; the tumors disappeared after day 35 (21 days after CAR-T injection). The lower figure shows the CAR copy number in DNA samples collected at different time points, approximating the number of CAR-T cells in the samples. It was observed that within 72 hours after CAR-T injection, CAR-T cells were mainly distributed in the lungs, while after 7 days they were mainly distributed in the tumors.

without

TW202535920A_113109555_SEQL.xml

Claims (18)

A chimeric antigen receptor (CAR) comprising a PD-L1 binding domain, comprising (a) a heavy chain variable region ( _VH ) comprising the heavy chain complementarity-determining region 1 (HC CDR1) of SEQ ID NO:1, the heavy chain complementarity-determining region 2 (HC CDR2) of SEQ ID NO:2, and the heavy chain complementarity-determining region 3 (HC CDR3) of SEQ ID NO:3; and (b) a light chain variable region ( _VL ) comprising the light chain complementarity-determining region 1 (LC CDR1) of SEQ ID NO:4, the light chain complementarity-determining region 2 (LC CDR2) of SEQ ID NO:5, and the light chain complementarity-determining region 3 (LC CDR3) of SEQ ID NO:6. The CAR of Request 1 further includes hinge domain, transmembrane domain and intracellular signal transduction domain. The CAR of claim 1, wherein the CAR includes the PD-L1 binding domain, which includes the V _H and the V _L from the N-end to the C-end, or the V _L and the V _H. For example, in Request 3, CAR, the V _H and the V _L are connected by a connector. As in claim 2, the intracellular signaling domain includes at least one selected from the CD137 (4-1BB) signaling domain, the CD28 signaling domain, the CD27 signaling domain, the ICOS signaling domain, the CD3ζ signaling domain, and any combination thereof. The CAR of claim 2, wherein the CAR contains the amino acid sequence of SEQ ID NO: 14 or 15; or, the CAR contains the amino acid sequence of SEQ ID NO: 20 or 21. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR as described in any of requests 1 to 6. For example, the nucleic acid molecule in request item 6 is a vector. A cell that contains nucleic acid molecules of request 7 or 8. A pharmaceutical composition comprising cells as described in claim 9. A method for treating an individual suffering from a disease, condition, or symptom associated with elevated tumor antigen expression, comprising administering to the individual an effective amount of genetically modified cells that express any of the CARs as claimed in claims 1 to 6. The method of claim 11, wherein the tumor antigen is PD-L1. The method described in request 11 or 12, where the individual has cancer. The method of claim 13, wherein the cancer is selected from a group consisting of breast cancer, rectal cancer, head and neck cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, kidney cancer, glioblastoma and leukemia. The use of a cell comprising a nucleic acid molecule as described in claim 7 or 8 and exhibiting a CAR as described in any of claims 1 to 6, for the manufacture of a drug for the treatment of an individual suffering from a disease, condition or symptom associated with elevated expression of a tumor antigen. For the purposes described in claim 15, the tumor antigen is PD-L1. For the purposes of requests 15 or 16, where the individual suffers from cancer. For the purposes of claim 17, the cancer is selected from a group consisting of breast cancer, rectal cancer, head and neck cancer, thyroid cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, kidney cancer, glioblastoma and leukemia.