WO2024012495A1

WO2024012495A1 - Cell expressing chimeric antigen receptor (car) targeting cd5 and use thereof

Info

Publication number: WO2024012495A1
Application number: PCT/CN2023/107024
Authority: WO
Inventors: 胡广; 张佳元; 姚小敏; 刘相元; 杨娟; 谭涛超; 赵雅; 谢萌
Original assignee: 上海驯鹿生物技术有限公司
Priority date: 2022-07-12
Filing date: 2023-07-12
Publication date: 2024-01-18
Also published as: CN117431217A

Abstract

A chimeric antigen receptor (CAR) specifically binding to a CD5 protein, comprising a CD5 binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. Further provided is an engineered immune effector cell (such as a T cell) comprising the CAR. Further provided is a use of the CAR and the engineered immune effector cell in the treatment of diseases or symptoms related to expression of CD5.

Description

Cells expressing chimeric antigen receptor (CAR) targeting CD5 and their applications

Technical field

This article relates to the field of biomedicine, specifically cells (such as T cells) expressing chimeric antigen receptors targeting CD5 and their applications.

Background technique

In recent years, chimeric antigen receptor T cell (CAR-T) technology has made breakthrough progress, especially in targeting B cell malignant tumors, and has achieved great success, represented by anti-CD19 CAR-T. So far, , three products, Kymriah, Yescarta and Tecartus, have been approved by the US FDA for marketing. Clinical trials have confirmed that CAR-T cells specifically targeting CD19 molecules can effectively treat B-cell malignancies, including relapsed/refractory B-ALL, CLL and B-cell ^lymphoma1-11 . According to literature reports, CD19 CAR-T has an effective rate of up to 90% for the treatment of relapsed/refractory ALL, and an effective rate of greater than 50% for CLL and some B-cell lymphomas. Although CAR-T therapy has achieved great success in the treatment of B-cell malignancies, its research and application in T-cell malignancies are very limited.

T-cell malignancies include acute T-lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL). T-ALL is a blood disease caused by abnormal proliferation of T lymphocytes. It is aggressive and progresses rapidly. T-cell lymphoma is a malignant tumor of T cells that can develop in lymphoid tissues (such as lymph nodes and spleen) or outside lymphoid tissues (such as gastrointestinal tract, liver, nasal cavity, skin, etc.), accounting for approximately 10% of non-Hodge patients. Golden lymphoma accounts for 10% to 15%, and the proportion is even higher in my country. CD5 is constitutively expressed on lymphocyte precursors, mature T cells, and a subset of mature B cells (Bl cells) ^12,13 . CD5 is highly expressed in approximately 85% of T-ALL and approximately 75% of peripheral T-cell lymphomas. In addition, CD5 is often expressed in mantle cell lymphoma, chronic lymphocytic leukemia (B-CLL), and hairy cell leukemia (HCL). At present, T-cell malignant tumors have a high recurrence rate and poor prognosis after radiotherapy and chemotherapy. They are hematological malignancies that are difficult to cure clinically. There is an urgent need to develop cell therapy drugs for this disease. In normal cells, the expression of CD5 is limited to mature T cells and some subtypes of B cells. The biological properties of CD5 antigen allow CD5 CAR T cells to generate effective antibodies against T-ALL and T lymphoma cells in vitro and in vivo. tumor activity. Therefore, CD5 can be used as a safe and reliable target for T cell tumors.

Mamonkin M and other researchers developed a second-generation CD5 CAR, using the scFv of mouse monoclonal antibody H65. Clinical experimental research results show that the CD5 CAR-T cells are effective in r/r CD5+T-ALL and T cells that have been treated with multiple lines in the past. It is safe and clinically effective in patients with cellular non-Hodgkin's lymphoma (T-NHL), and does not lead to complete T cell ^{depletion14,15} . More importantly, the elimination of malignant T cells through CD5 CAR-T cells may allow patients who were previously unsuitable for transplantation to receive HSCT (hematopoietic stem cell transplantation). However, according to the existing clinical follow-up data, it is known that the mouse-derived scFv CAR-T can cause the recurrence of CD5+ malignant diseases after infusion. The limited duration of CAR-T in patients may be related to the production of anti-mouse antibodies, while the fully human source scFv may be able to solve the problem of CD5 CAR-T's short persistence time in patients. The development of fully human CD5 antibodies is of great significance for the development of the next generation of CAR-T products that last longer in the body and have better long-term efficacy.

Contents of the invention

In one aspect, provided herein are immune effector cells including:

1) Chimeric Antigen Receptor (CAR) and/or its encoding nucleic acid sequence; and

2) Suicide genes and/or protein products encoded by suicide genes,

The suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene.

In some embodiments, the HSV-TK is HSV-TK mut2; preferably, the HSV-TK mut2 includes the sequence shown in SEQ ID NO: 71 or a functional variant thereof.

In some embodiments, the CAR includes a CD5 binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, the CD5 binding domain comprising one or more antibodies that specifically bind CD5 Or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), said HCDR1, The amino acid sequences of HCDR2 and HCDR3 are selected from any one of the following combinations:

(1) HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39, and HCDR3 of the sequence shown in SEQ ID NO: 40;

(2) HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42, and HCDR3 of the sequence shown in SEQ ID NO: 43;

(3) HCDR1 of the sequence shown in SEQ ID NO: 64, HCDR2 of the sequence shown in SEQ ID NO: 65, and HCDR3 of the sequence shown in SEQ ID NO: 66; and

(4) HCDR1 of the sequence shown in SEQ ID NO: 67, HCDR2 of the sequence shown in SEQ ID NO: 68, and HCDR3 of the sequence shown in SEQ ID NO: 69.

In some embodiments, the CD5-binding domain includes at least two antibodies or fragments thereof that specifically bind CD5, and the HCDR1, HCDR2, and HCDR3 contained in the antibodies or fragments thereof are independently selected from any one of the following combinations:

In some embodiments, the CD5 binding domain includes a first antibody or antigen-binding fragment thereof that specifically binds CD5 and a second antibody or antigen-binding fragment thereof, the first antibody or antigen-binding fragment thereof and the third The HCDR1, HCDR2, and HCDR3 included in the secondary antibody or its antigen-binding fragment are independently selected from any one of the following combinations:

(1) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42 and SEQ ID NO: HCDR3 of the sequence shown in 43;

(2) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment includes HCDR1 of the sequence shown in SEQ ID NO: 64, HCDR2 of the sequence shown in SEQ ID NO: 65, and HCDR3 of the sequence shown in SEQ ID NO: 66; and

(3) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 67, HCDR2 of the sequence shown in SEQ ID NO: 68 and HCDR3 of the sequence shown in SEQ ID NO: 69.

In some embodiments, the at least two antibodies or antigen-binding fragments thereof that specifically bind CD5 are connected in series.

In some embodiments, the antibody is a single domain antibody, preferably a fully human single domain antibody.

In some embodiments, the CD5 binding domain includes at least two single domain antibodies, and the single domain antibodies are connected through a linker fragment; preferably, the linker fragment includes the sequence shown in SEQ ID NO: 25.

In some embodiments, the CD5 binding domain includes the sequence set forth in SEQ ID NO: 33, 35, 37, 47, 57, 59, 61 or 63 or a functional variant thereof.

In some embodiments, the transmembrane domain includes a polypeptide from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; preferably, the transmembrane domain includes the sequence shown in SEQ ID NO: 6 or a functional variant thereof.

In some embodiments, the costimulatory domain includes a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS; preferably, the costimulatory domain includes SEQ ID NO: 8 sequence or functional variant thereof.

In some embodiments, the intracellular signaling domain comprises a signaling domain from CD3ζ; preferably, the intracellular signaling domain comprises the sequence shown in SEQ ID NO: 10 or a functional variant thereof.

In some embodiments, the CAR further comprises a hinge region connecting the CD5 binding domain and the transmembrane domain; preferably, the hinge region comprises the sequence shown in SEQ ID NO: 4 or Its functional variant.

In some embodiments, the CAR includes a CD8α signal peptide; preferably, the signal peptide includes the sequence shown in SEQ ID NO: 2 or a functional variant thereof.

In some embodiments, the nucleic acid sequence encoding the CAR and the suicide gene are located in the same nucleic acid molecule.

In some embodiments, the nucleic acid sequence encoding the CAR and the suicide gene are located in the same expression vector that is introduced into the immune effector cell.

In some embodiments, the expression vector is a lentiviral expression vector, such as a pLVx vector or a pCDH vector.

In some embodiments, a spliced peptide coding sequence is included between the nucleic acid sequence encoding the CAR and the suicide gene.

In some embodiments, the suicide gene is located in the 5' direction or the 3' direction of the nucleic acid sequence encoding the CAR.

In some embodiments, the cleavage peptide includes an amino acid sequence from a T2A peptide; preferably, the cleavage peptide includes Including the sequence shown in SEQ ID NO: 12 or its functional variant.

In some embodiments, the immune effector cells do not express CD5.

In some embodiments, the immune effector cells do not express TRAC genes and/or TRBC genes.

In some embodiments, the immune effector cells are selected from T lymphocytes and natural killer (NK) cells.

On the other hand, this article provides an isolated nucleic acid molecule, which includes the nucleic acid sequence encoding the above-mentioned CAR and the suicide gene.

In some embodiments, the coding nucleic acid sequence includes the sequence set forth in SEQ ID NO: 32, 34, 36, 46, 56, 58, 60 or 62.

In some embodiments, the cleavage peptide includes the amino acid sequence from the T2A peptide; preferably, the cleavage peptide includes the sequence shown in SEQ ID NO: 12 or a functional variant thereof.

In another aspect, provided herein are expression vectors comprising the nucleic acid molecules described above.

In some embodiments, the vector is selected from plasmids, retroviral vectors, and lentiviral vectors, such as pLVx vectors or pCDH vectors.

On the other hand, this article provides a method for preparing immune effector cells, which includes:

1) Knock out (1) CD5 gene and/or (2) TRAC gene and/or TRBC gene of the immune effector cells; and

2) Introducing the nucleic acid molecule of any one of claims 22-24 or the expression vector of claims 25 or 26 into immune effector cells.

In some embodiments, CRISPR/Cas9 technology is used to knock out the CD5 gene; preferably, the target sequence of the sgRNA used includes the sequence shown in SEQ ID NO: 70.

In another aspect, provided herein are pharmaceutical compositions comprising:

1) The immune effector cell of any one of claims 1-21, the nucleic acid molecule of any one of claims 22-24, or the expression vector of claims 25 or 26; and

2) Pharmaceutically acceptable adjuvants.

On the other hand, this article provides the use of the above-mentioned immune effector cells, nucleic acid molecules or expression vectors in the preparation of medicaments, wherein the medicaments are used to treat diseases or conditions related to the expression of CD5.

In another aspect, provided herein is a method of treating a disease or disorder associated with the expression of CD5, comprising administering a therapeutically effective amount of the above immune effector cells, nucleic acid molecules, expression vectors or pharmaceutical compositions to a subject in need thereof .

In some embodiments, administration of ganciclovir GCV to a subject in need thereof is further included to kill the immune effector cells.

In some embodiments, the disease or disorder associated with expression of CD5 is cancer or malignancy.

In some embodiments, the disease or condition associated with expression of CD5 is T lymphoblastic lymphoma or mantle cell lymphoma.

Description of drawings

Figure 1 shows a schematic diagram of the principle of anti-CD5VHs competitive binding to FACS in this experiment (Figure 1A) and the detection results of CD5 epitope binding of CD5 KO CD5 CAR-T cells using flow cytometry (Figure 1B).

Figure 2 shows the CAR structure used in this experiment (Figure 2A) and the detection of CD5 and EGFRt expression of CAR-T cells after transfection (Figure 2B).

Figure 3 shows the surface expression of CD5 in T-ALL and T-lymphoma cell lines.

Figure 4 shows the results of CD107a degranulation after co-incubation of different target cells and CAR-T cells.

Figure 5 shows the killing results of CAR-T cells on various target cells.

Figure 6 shows the results of basal apoptosis levels of CAR-T/T cells.

Figure 7 shows the proliferation of CAR-T cells after repeated stimulation of CD5 CAR-T cells by CD5-positive target cells.

Figure 8 shows the treatment results of CAR-T/T cells and PBS on mouse T cell tumor model.

Figure 9 shows the results of the binding study between CT125A cells and CD5 antigen in Example 4.

Figure 10 shows the results of degranulation activity of CT125A under stimulation of positive target cells in Example 5 (**, p<0.01; *, p<0.05).

Figure 11 shows the killing results of CT125A on different types of positive target cells in Example 6.

Figure 12 shows the results of the CT125A cytokine release study in Example 7.

Figure 13 shows the results of the study on cetuximab-mediated natural killer cell clearance of CT125A cells in vitro.

Figure 14 shows the survival rate curves of each group of animals during the experiment in Example 9.

Figure 15 shows the body weight change trend of each group of animals in Example 9.

Figure 16 shows the intensity change trend of the average tumor fluorescence signal of each group of animals in Example 9.

Figure 17 shows the changing trend of peripheral blood IL-2 of each group of animals in Example 9.

Figure 18 shows the changing trend of peripheral blood IL-4 of each group of animals in Example 9.

Figure 19 shows the changing trend of peripheral blood IL-6 of each group of animals in Example 9.

Figure 20 shows the changing trend of peripheral blood IL-10 of each group of animals in Example 9.

Figure 21 shows the change trend of peripheral blood TNF-α of animals in each group in Example 9.

Figure 22 shows the change trend of peripheral blood IFN-γ of each group of animals in Example 9.

Figure 23 shows the body weight change graph (g) of each group of animals at each time point in Example 10.

Figure 24 shows the tumor growth curves of animals in each group at each time point in Example 10.

Figure 25 shows the results of the binding study between the RD125 61-42-rFc single domain antibody rabbit Fc fusion protein and CD5-positive cells in Example 13.

Figure 26 shows the preparation of CD5 HSV-TK CAR-T with different molecular structures and changes in activity rate and cell volume. (A) Schematic diagram of the structures of four different CAR molecular vectors of CD5 HSV-TK. The core elements of the CAR molecules in the four vector structures are the same, except for the position of the HSV-TK switch and the vector backbone molecules. 2946 and 2947 are the same lentivirus backbone and carry ampicillin resistance, while 2948 and 2949 are the same lentivirus backbone. Viral backbone, carrying kanamycin resistance. (B). Brief process for preparation of CAR-T cells. (C). Statistical chart of changes in viability and cell volume of 2946 and 2947 CAR-T cells during the preparation process. D2 is before virus conversion, and D3 is before medium change. (D). Statistical chart of changes in viability and cell volume of 2948 and 2949 CAR-T cells during the preparation process. D2 is before virus conversion, and D3 is before medium change.

Figure 27 shows the proportion of CAR-positive cells during the preparation of CD5 HSV-TK CAR-T with different molecular structures. (A) The proportion of CAR-positive cells during the preparation of 2946 and 2947 CAR-T changes with the number of culture days (after transfection). (B) The proportion of CAR-positive cells during the preparation of 2948 and 2949 CAR-T changes with the number of culture days (after transfection). (C) CD5 knockout efficiency during CAR-T preparation.

Figure 28 shows the CAR-positive cell sorting results. (A) The results of sorting CAR-positive cells of 2946 and 2947 CAR-T. input refers to cells before sorting, pos refers to cells bound to the column after sorting, and neg refers to cells that are not bound to the column and flow down. (B) CAR purity detection 4 days after CAR-positive cell sorting and detection of antigen residues bound to CAR-positive cells during sorting.

Figure 29 shows the results of the in vitro tumor killing function test of CD5 CAR-T. (A) The tumor killing ability of CD5 KO T cells; (B) The tumor killing ability of CD5 CAR-T cells; (C) The tumor killing ability of anti-human CD5 CAR-T cells carrying HSV-TK suicide gene constructed using pLVx vector Functional testing.

Figure 30 shows the results of the in vitro tumor killing function test of CD5 CAR-T. (A) The tumor killing ability of CD5 KO T cells; (B) The tumor killing ability of CD5 CAR-T cells; (C) The tumor killing ability of anti-human CD5 CAR-T cells carrying HSV-TK suicide gene constructed using pCDH vector Functional testing.

Figure 31 shows the inhibition results of GCV drugs on HSV-TK positive cells. (A) Changes in the total number of CD5 CAR-T cells treated with GCV at different concentrations. (B) Changes in total cell volume of CD5 CAR-T (HSV-TK) cells constructed with pLVx vector when treated with different concentrations of GCV. (C) Changes in the number of CAR-positive cells in CD5 CAR-T cells treated with different concentrations of GCV. 6D. Changes in the number of CAR-positive cells in CD5 CAR-T (HSV-TK) cells constructed with pLVx vector when treated with different concentrations of GCV.

Figure 32 shows the inhibition results of GCV drugs on HSV-TK positive cells. (A) Changes in the total number of CD5 CAR-T cells in the presence or absence of 1ug/ml GCV. (B) Changes in the total cell volume of CD5 CAR-T (HSV-TK) cells constructed with pLVx vector in the presence or absence of 1ug/ml GCV. (C) Changes in the number of CAR-positive cells in CD5 CAR-T cells in the presence or absence of 1ug/ml GCV. (D) Changes in the number of CAR-positive cells in CD5 CAR-T (HSV-TK) cells constructed with pLVx vector in the presence or absence of 1ug/ml GCV.

Figure 33 shows the results of the in vivo tumor killing function test of CD5 CAR-T in animal experiments: the blood luciferase readings of mice in the CAR-T group G3, G4, G5 group and the control group.

Figure 34 shows the clearance of CD5 CAR-T in mice by GCV as measured by flow cytometry. CAR-T group G3 group has an obvious CAR-T cell population, that is, CD3+CAR+ cell population. The CAR-T group treated with GCV for 7 days, that is, the G4 group, and the CAR-T group treated with GCV for 14 days, that is, the G5 group, had almost no detectable CAR-T cells, that is, the CD3+CAR+ cell population.

Figure 35 shows the clearance of CD5 CAR-T in mice by GCV detected by real-time quantitative PCR. At the two time points of Day14/21, that is, 7/14 days after GCV administration, the CAR-T copy number (VCN) of the G3 group in peripheral blood, spleen and lungs was significantly higher than that of the GCV administration group.

Figure 36 shows the tumor recurrence in mice after stopping drug treatment with GCV. The G4 group stopped GCV treatment on Day 14, and the G5 group stopped GCV treatment on Day 21. That is, 14 days after stopping GCV administration, the luciferase readings in the peripheral blood of some mice were higher, indicating that anti-human antibodies carrying the HSV-TK suicide gene After CD5 CAR-T cells are cleared by GCV, swollen The tumor cells grow again.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification. The CAR described herein can specifically bind to CD5, the CAR-T cells prepared using the CAR can stably express the CAR, and the CAR-T cells prepared using the CAR have a high CAR positive rate. In addition, the CAR can promote the release of cytokines and can be used to treat diseases or conditions associated with the expression of CD5.

Unless expressly stated to the contrary, the practice of this application will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill in the art. These methods are described in, e.g., Sambrook et al., Molecule Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecule Cloning: A Laboratory Manual (2nd ed., 1989); Maniatis et al., Molecule Cloning: A Laboratory Manual (3rd ed., 2001); Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in MolecμLar Biology (John Wiley and Sons, updated July 2008); Short Protocols in MolecμLar Biology: A Compendium of Methods from Current Protocols in MolecμLar Biology, Green ePub .Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol.I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecule Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q.E.Coligan, A.M.Kruisbeek, D.H.MargμLies, E.M.Shevach and W.Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.

Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this application, the following terms are defined below.

In this article, the term "Chimeric Antigen Receptor" (Chimeric Antigen Receptor, CAR) is a fusion protein including the variable region of an antibody and a T cell signaling molecule. It allows T cells to recognize specific antigens in a non-MHC-restricted manner and exert a killing effect. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which can include tumor-associated antigen (TAA) or tumor-specific antigen (TSA) binding regions, transmembrane domains, costimulatory domains and intracellular signals The structural domain may further include a hinge region between the tumor-associated antigen binding region and the transmembrane domain. In this context, the CAR may be a genetically engineered chimeric protein capable of redirecting the cytotoxicity of immune effector cells to T cells, which specifically combines an antibody-based antigen (e.g., CD5) with a T cell receptor activating cell. The inner domains are grouped together. T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens. For descriptions of CAR and CAR T cells, see, for example, Sadelain M, Brentjens R, Rivi`ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M ,Jensen MC, Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012;24(5):633-639;Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014; 257(1):107-126; and WO2013154760, WO2016014789.

In this article, the term "CD5" is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T cell receptor signaling and promotes the survival of normal and malignant lymphocytes. CD5 is one of the characteristic surface markers of malignant T-cell tumors. 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma express CD5. In this article, the CD5 can be human CD5, and its GenBank accession number is NM_014207.4. CD5 proteins may also include fragments of CD5, such as the extracellular domain and fragments thereof. In order to prevent CD5-targeted CAR-T cells from attacking themselves, the CD5 gene of T cells can be knocked out before preparing CAR-T cells.

"TRAC gene" and "TRBC gene" herein refer to the gene encoding the constant region of the T cell receptor α chain and the gene encoding the constant region of the T cell receptor β chain, respectively. The alpha and beta chains constitute the T cell receptor (TCR), which recognizes antigens and mediates immune responses. Knockout of "TRAC gene" and/or "TRBC gene" results in cells unable to express TCR molecules. Knocking out "TRAC gene" and/or "TRBC gene" makes T cells unable to express functional TCR, which can avoid graft-versus-host disease.

In this article, the term "CD5-binding domain" generally refers to the extracellular domain of the CD5 CAR that can specifically bind to the antigen. For example, the CD5 extracellular binding domain may comprise an anti-CD5 antibody or antigen-binding fragment thereof that specifically binds to a CD5 polypeptide expressed on human cells. As used herein, the terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" are used interchangeably . The CD5 binding domain can be of natural, synthetic, semi-synthetic or recombinant origin.

As used herein, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, an antibody may include a classic immunoglobulin consisting of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule that includes an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (eg, sdAb), single chain antibodies (eg, scFv). As used herein, the "antigen-binding fragment" of an antibody refers to a fragment of an antibody that can bind to the corresponding antigen, such as Fab, Fab', and (Fab') ₂ fragments. The person skilled in the art knows how to obtain these antigen-binding fragments. For example, a classic antibody molecule can be digested with papain to obtain a Fab fragment, and pepsin to obtain F(ab') ₂ , which is formed by treatment with a reducing agent to break the disulfide bond between the hinge regions of F(ab') ₂ Fab' fragment. The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. VH regions can be further distinguished into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable region of the heavy chain contains the domain that interacts with the antigen. The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

"Single chain fragment variable (scFv)" is composed of an antibody heavy chain variable region and a light chain variable region. It is composed of short peptides connected into a peptide chain. Through correct folding, the variable regions from the heavy chain and light chain form Fv segments through non-covalent interactions, so scFv can better retain its affinity activity for antigens.

"Single domain antibody (sdAb)", also known as "VHH antibody", refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region but no light chain. From a structural point of view, single domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids. Subsequently, researchers discovered more single-domain antibodies with antigen-binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, and when used in the human body, they can easily reach tissues that are difficult for ordinary antibody molecules to reach. or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; they are more stable and can withstand changes in temperature and pH, as well as the effects of denaturants and proteases.

When referring to an antibody or antigen-binding fragment thereof, "targeting" or "specific binding" refers to the effect of one molecule (e.g., an antibody or antigen-binding fragment thereof) on another molecule (e.g., an antibody or antigen-binding fragment thereof) relative to other molecules co-present in the environment. Such as tumor cell surface antigens) have higher binding affinity. "Targeting" or "specific binding" does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.

In this article, the term "transmembrane domain" usually refers to the domain of CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays the role of transmitting signals. In the present invention, the transmembrane domain may be a CD8α transmembrane domain.

As used herein, the term "costimulatory domain" generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for an effective lymphocyte response to an antigen. The costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domains of OX40 and 4-1BB.

As used herein, the term "hinge region" generally refers to the connecting region between the antigen-binding region and the immune cell Fc receptor (FcR) binding region. In the present invention, the hinge region may be a CD8α hinge region.

As used herein, the term "intracellular signaling domain" generally refers to the component of the CAR that is located in intracellular signaling, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: it can Selected from CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.

In this article, the term "CD8α signal peptide" (Signal peptide) generally refers to a short (5-30 amino acid length) peptide chain that guides the transfer of newly synthesized proteins to the secretory pathway.

In this article, the term "marker detection signal" generally refers to a gene, protein or other molecule with a known function or sequence that can function as a specific marker and emit a signal that can be detected. The label detection signal can be a fluorescent protein, such as GFP, RFP, YFP, etc. The label detection signal may be EGFRt. The terms "EGFRt" or "tEGFR" are used interchangeably herein and refer to the gene encoding a truncated human epidermal growth factor receptor polypeptide that lacks the distal membrane EGF binding domain and cytoplasmic signaling tail but retains Extracellular epitope recognized by anti-EGFR antibodies. EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker with the function of genetically modifying cells. In this article, it can be used as a marker molecule for CAR-T cells and can also be used to eliminate CAR-T cells in the body when necessary. EGFR antibodies (e.g., Cetuximab The cetuximab mediated ADCC pathway eliminates CAR-T cells in the body (see US8802374B2), which is used as a safety switch during clinical translation.

In this article, the term "CSF2RA signal peptide", that is, colony stimulating factor 2 receptor subunit alpha signal peptide, is a peptide chain that can guide the expression of newly synthesized proteins on the surface of CAR-T cells.

In this article, "EGFR antibody" refers to an antibody-dependent cell-mediated cytotoxicity that causes immune cells to attack CAR-T cells with a truncated epidermal growth factor receptor (EGFRt). Antibodies that assist in clearing CAR-T cells. The EGFR antibody can be used when patients experience severe adverse reactions after infusion of CAR-T or other situations where CAR-T cells need to be cleared. It can assist in clearing CAR-T cells and reduce symptoms related to CAR-T treatment. The EGFR antibodies include, but are not limited to, cetuximab, panitumumab, nexituzumab, and nimotuzumab.

Another way to eliminate CAR-T cells (or NK cells) in subjects is to use suicide genes. The term "suicide gene" refers to a gene that, after being expressed in a host cell, can lead to the death of the host cell expressing the gene (including apoptosis, loss of activity, etc.). The expression of the suicide gene may be inducible, or the suicidal effect of the suicide gene may depend on additionally provided small molecule drugs. In one embodiment, the suicide gene used is the herpes simplex virus thymidine kinase (HSV-TK) gene, which can be used as a control CAR-T by providing the HSV-TK substrate ganciclovir (GCV). Cell number switch. In one embodiment, the suicide gene used is inducible Caspase-9 (iC9), which dimerizes and causes cell apoptosis by providing the small molecule drug AP1903. The use of tEGFR or these suicide genes allows clinicians to discontinue treatment when significant side effects are observed in subjects or when the cancer has been cleared. For "tEGFR" and "suicide genes" intended for molecular switches, their coding sequences can be introduced into immune effector cells (such as T cells) independently of the CAR coding sequence. Preferably, they can be introduced into immune effector cells (such as T cells) on the same expression vector as the CAR coding sequence to facilitate operation.

"Self-cleaving peptides" herein refer to short peptides that can achieve the function of cleaving proteins through ribosome skipping rather than proteolytic hydrolysis, and may include T2A, F2A, P2A, etc.

The term "functional variant" of a protein or polypeptide sequence means herein that by 1 or more, for example 1-30, or 1-20 or 1-10, for example 1 or 2 or 3 or 4 or 5 Amino acid substitutions, deletions, and/or insertions are sequences that have amino acid changes compared to the parent. Functional variants essentially retain the biological properties of the protein or polypeptide sequence prior to the alteration. In one aspect, variants of any protein or polypeptide sequence described herein are encompassed herein. In certain embodiments, functional variants of a protein or polypeptide sequence retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the pre-alteration parent. Functional variants described herein may be functional variations of a signal peptide, an antibody, a hinge region, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, a cleavage peptide, a CSF2RA signal peptide, EGFRt or HSV-TK. body. When referring to functional variants of an antibody, it also includes antibody variable regions (such as VH or VL), antibody constant regions (such as CH or CL), heavy chain CDR regions (HCDR1, HCDR2 or HCDR3), light chain CDR regions (LCDR1, LCDR2 or LCDR3), etc. Amino acid substitutions, deletions and/or insertions may occur in the heavy chain CDR region or the light chain CDR region, or the heavy chain FR region or the light chain FR region, or the heavy chain constant region or the light chain constant region, wherein the variant substantially maintains Change the biological properties of the previous antibody molecule. For an antibody, its biological activity includes, for example, antigen-binding ability. In certain embodiments, functional variants of antibodies Amino acid changes are included that do not cause the antibody variant to lose binding to the antigen, but optionally may confer properties such as increased antigen affinity and different effector functions. It can be understood that the heavy chain variable region or light chain variable region of the antibody, or each CDR region, can be changed individually or in combination. In certain embodiments, the amino acid changes in one or more or all three heavy chain CDRs are no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, the amino acid change may be an amino acid substitution, for example, it may be a conservative substitution. In certain embodiments, a functional variant has at least 80%, 85%, 90% or 95% or 99% or greater amino acid identity to the parent. Similarly, a "functional variant" of a nucleic acid molecule is used herein to refer to a nucleic acid molecule that encodes the same amino acid sequence as the parent nucleic acid molecule.

As used herein, "sequence identity" generally refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis within a comparison window. "Percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in both sequences. ) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) Number of positions to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to generate percent sequence identity. Optimal alignment for determining percent sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment within the full-length sequences being compared or within the sequence region of interest.

As used herein, the term "isolated" generally refers to the described object being in a state of separation from components of its natural environment. In certain embodiments, isolating the antibody can include purifying the antibody to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography ( For example, ion exchange or reversed phase HPLC) determination. A review of methods for evaluating antibody purity can be found in Flatman, S. et al., J. Chrom. B 848 (2007) 79-87. Similarly, "isolated nucleic acid molecules" refer to nucleic acid molecules that have been extracted or purified, and also include artificially synthesized nucleic acid molecules.

As used herein, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially synthesized. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. As used herein, nucleic acids encoding the antibodies or antigen-binding fragments thereof may be prepared by a variety of methods known in the art, including, but not limited to, using restriction fragment manipulation or using overlapping of synthetic oligonucleotides. For extension PCR, please refer to Sambrook et al., MolecμLar Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al. Current Protocols in MolecμLar Biology, Greene Publishing and Wiley-Interscience, New York N.Y.,1993.

As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for the purpose of transferring an inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and vectors primarily used for the transcription of DNA or RNA. and/or translational expression vectors. The vector may be a polynucleotide capable of transcribing and translating part of the sequence into a polypeptide when introduced into a suitable host cell, that is, an expression vector. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector. In this application, one or more of the nucleic acid molecules may be included in the vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain expression control elements that allow correct expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequences are tunable elements. The specific structure of the expression control sequence can vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, GA cap sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid. The vectors described herein may be selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors. The plasmids, retroviral vectors and lentiviral vectors described herein may contain CAR coding sequences.

In this article, the term "plasmid" usually refers to a DNA molecule outside the chromosome or nucleoid in bacteria, yeast and other organisms, which exists in the cytoplasm and has the ability to replicate autonomously, allowing it to maintain a constant copy number in progeny cells. , and express the genetic information carried. Plasmids are used as carriers of genes in genetic engineering research.

In this article, the term "retroviral vector" usually refers to a type of RNA virus whose genetic information is stored on ribonucleic acid. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains the gene for the protein that makes up the center and structure of the virus; pol, which contains the gene for reverse transcriptase; and env, which contains the gene that makes up the outer shell of the virus. Through retroviral transfection, the retroviral vector can integrate its own genome and the foreign genes it carries into the host cell genome randomly and stably. For example, CAR molecules can be integrated into the host cell.

As used herein, the term "lentiviral vector" generally refers to a diploid RNA viral vector belonging to the class of retroviruses. Lentiviral vectors are based on the genome of lentivirus, in which multiple sequence structures related to viral activity are removed to make them biologically safe, and then the sequence of the target gene required for the experiment is introduced into this genome skeleton. and vectors prepared from expression constructs. Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc., It can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, "Lentiviral vectors and their research progress, Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC). Transfection through lentiviral vectors , Retroviral vectors can integrate their own genome and the foreign genes they carry into the host cell genome randomly and stably. For example, CAR molecules can be integrated into the host cell.

As used herein, the term "transposon" generally refers to a discrete DNA segment containing a transposase gene, flanked by terminal inverted repeats (TIRs) containing transposase binding sites. Transposase binds to TIR and moves the transposon to a new site. The transposon described herein is a two-component system consisting of a plasmid carrying a CAR (transposon) and another plasmid carrying a transposase. The transposon can be introduced into target cells through electrotransduction or other methods. For example, first, the two components are electroporated into peripheral blood mononuclear cells (PBMC), and the expressed transposase acts on the terminal inverted repeats (TIR) on both sides of the CAR. , cleaves the CAR (transposon) and subsequently integrates into target cells (e.g. T cell) on the TA dinucleotide sequence in the genome. After transposition and stable genome integration are completed, the CAR protein can be expressed on the surface of the target cell (see Cheng Zhang, Jun Liu, Jiang F Zhong, et al. Engineering CAR-T cells. Biomarker Research. 2017, 5:22).

"Knockout" or "gene knockout" as used herein refers to changing the nucleotide sequence of a certain gene in a cell, whether the change is a nucleotide insertion, deletion or substitution, as long as the gene being knocked out does not produce any significant changes in the cell. Functional gene products (such as RNA or protein) are sufficient. Ideally, a gene is knocked out such that the cell or cell population completely does not form the gene product of the gene or a functional gene product. Understandably, if the amount of the gene product is significantly reduced, or the activity of the gene product is significantly reduced, it can also be considered to have achieved "gene knockout." In some cases, it may be necessary to knock out two or more genes in the cell. In some embodiments, gene knockouts can be performed sequentially, that is, knockout of one gene is followed by knockout of the next gene. In other embodiments, two or more genes can be knocked out simultaneously. For example, when using CRISPR technology to knock out multiple genes in a cell, Cas9 and multiple sgRNAs targeting each gene can be introduced into the cell at the same time.

In this article, the term "gene editing" generally refers to technologies for site-directed modification of the genome, which may include techniques based on zinc finger nucleases (ZFNs), transcription activator like effector nucleases, TALENs), regularly repeated short palindromic sequence clusters (clustered regμLarly interspaced short palindromic repeats/CRISPR-associated protein (Cas9), CRISPR/Cas9) and other technologies. It enables efficient targeted modification of the genome by adding, removing or altering genetic material at specific locations in the genome. Gene editing described herein may include introducing CAR molecules into the genome of recipient cells through gene editing technology (such as CRISPR-Cas9).

"CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology" is an RNA-guided DNA editing technology for target genes through Cas nuclease. The CRISPR gene editing system used in this technology includes Cas nuclease and guide RNA (single-guide RNA, sgRNA), and optionally ssDNA as a repair template. Part of the sequence of sgRNA can bind to Cas nuclease, and another part of the sequence (crRNA) can be complementary to part of the sequence of the target gene. With the recognition of sgRNA, Cas nuclease can form a single-stranded or double-stranded nick at a specific site of the target gene. Cells usually repair broken strands in DNA in two ways: homologous recombination repair (homology-directed repair, HDR) and non-homologous end joining (NHEJ). . When CRISPR technology is used, for example, to knock out genes in cells, it is usually only necessary to consider destroying the normal coding function of the gene, such as causing frameshift mutations or gene fragment deletions, so that products with normal functions cannot be produced (such as protein). Usually, after introducing Cas nuclease (such as Cas9) and sgRNA into the cells, cells that do not express the product of the gene to be knocked out can be screened out. "CRISPR gene editing system" in this article refers to the combination of Cas nuclease and sgRNA, which is used to edit sgRNA-targeted genes after introduction into cells.

When referring to sgRNA, the term "target sequence" refers to a nucleotide segment in the target gene or gene to be knocked out that is complementary to a partial sequence of the sgRNA (crRNA, approximately 20 bases). With the help of the sequence in the sgRNA that is complementary to the target sequence, proteins such as Cas9 can introduce nucleotide sequence changes in the target gene at a relatively certain position to achieve the effect of gene knockout. Accordingly, in this article, "sgRNA targeting a specified sequence" means that the target sequence of the sgRNA is the specified sequence.

In this article, the term "immune effector cells" generally refers to immune cells that participate in clearing foreign antigens and performing effector functions in immune responses. For example, plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.

As used herein, the term "cancer" generally refers to or describes a physiological condition in mammals that is typically characterized by dysregulated cell proliferation or survival. As used herein, hyperproliferative diseases referred to as cancer include, but are not limited to, solid tumors such as those occurring in the breast, respiratory tract, brain, reproductive organs, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid glands cancers, and their distant metastases. Such diseases also include lymphomas, sarcomas, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory cancers include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic keratinomas, cerebellar and cerebral astrocytomas, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Male genital tumors include, but are not limited to, prostate and testicular cancer. Female genital tumors include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine tumors. Gastrointestinal tumors include, but are not limited to, anus, colon, colorectum, esophagus, gallbladder, stomach, pancreas, rectum, small intestine, and salivary gland cancers. Urinary tract tumors include, but are not limited to, cancers of the bladder, penis, kidney, renal pelvis, ureters, and urethra. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatoma with or without fibrolamellar variation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi’s sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, cancers of the larynx/hypopharynx/nasopharynx/oropharynx, and cancers of the lips and oral cavity. Lymphoma includes, but is not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia.

"Treatment" refers to the treatment of a subject to obtain a beneficial or desired clinical result. "Treatment" as used herein encompasses a variety of treatments, including administration of any possible drug to the subject, surgery, radiation, etc. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g. metastasis, e.g. metastasize to the lungs or lymph nodes), prevent or delay disease recurrence, delay or slow down disease progression, improve disease conditions, inhibit disease or disease progression, block its development and remission (whether partial or complete remission). The methods provided herein encompass any one or more of these aspects of treatment. In accordance with the above, "treatment" does not require complete removal of all symptoms of a condition or disease or complete alleviation.

The term "therapeutically effective amount" refers to an amount of active compound sufficient to elicit the biological or medical response desired by the clinician in a subject. The "therapeutically effective dose" of the fusion protein of the present invention can be determined by those skilled in the art based on the route of administration, the subject's weight, age, condition and other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.

The term "pharmaceutically acceptable carrier" as used with reference to pharmaceutical compositions refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be administered safely and are suitable for use by humans and/or Administration to the animal without undue adverse side effects while being suitable for maintaining the viability of the drug or active agent therein. Depending on the route of administration, it can be administered Use various carriers well known in the art, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers solution, emulsifier, isotonic saline, and/or pyrogen-free water, etc. The pharmaceutical compositions provided herein can be made into clinically acceptable dosage forms such as powders and injections. The pharmaceutical composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, etc. route of administration.

"Pharmaceutical kit" refers to a pharmaceutical combination including at least two active ingredients. Unlike pharmaceutical compositions, pharmaceutical kits contain at least one active ingredient that is kept separate from other active ingredients.

"Subject" refers to an animal, such as a mammal, including (but not limited to) humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, goats, mammals Laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets. The subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects. In some examples, a subject refers to an individual in need of treatment of a disease or condition. In some examples, a subject receiving treatment can be a patient who has a condition associated with the treatment, or is at risk of developing the condition. In other examples, the subject is a healthy individual or an individual suffering from a disease other than that of concern. In certain examples, the subject is a human, such as a human patient. The term is often used interchangeably with "patient," "subject," "subject," etc.

The term "and/or" should be understood to mean either or both of the options.

As used herein, the term "comprises" or "includes" means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step. When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.

As used herein, the term "about" generally refers to a range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3 above or below the specified value. %, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

The present invention uses fully human monoclonal antibodies containing only the heavy chain variable region. First of all, the small molecular weight of fully human single-domain antibodies makes the structure of CAR more simplified, has lower immunogenicity than humanized mouse antibodies, and has better potential in antibody drugs or CAR-T applications. Secondly, the smaller size makes fully human single domain antibodies more likely to access narrower or partially hidden epitopes, giving them a steric advantage over larger scFvs. In addition, the smaller size increases the viral titer of its gene therapy vector and makes it easier to express it on the surface of T cells. Moreover, in the application of bispecific CAR, compared with the design of two scFvs, the use of two single domain antibodies sdAb that can recognize two antigens can simplify the structure of the bispecific CAR and improve its expression efficiency and structural stability. . After obtaining an antibody clone that specifically binds to the cell surface CD5 antigen and CD5 recombinant protein, we constructed it into an IgG expression vector with a human Fc segment, expressed the protein in CHOS cells, and performed a flow competition experiment, and obtained Fully human single domain clones that bind different epitopes of the CD5 antigen. Compared with a single single-domain clone, single-domain tandem CAR may have the function of enhancing the efficacy of CAR-T and may reduce the risk of ineffective treatment and relapse of antigen mutation. Fully human single domain clones, tandem fully human single domain clones that bind different epitopes of the CD5 antigen, and control clone H65 were constructed on the second-generation CAR structure, and then lentivirus packaging was performed and transfected into T cells. In the CAR-T At the cellular level, it was verified that the tandem fully human single-domain CD5 antibody clone FHVH3VH1 is more functional than the control clone H65 from the perspectives of target cell activation and killing, and target cell stimulation and proliferation.

In addition, the literature reports that in most constructed animal animal models of tumors, re-emerging tumor cells retain CD5 expression, indicating that tumor recurrence is not due to the loss of antigen and that the failure to eradicate all xenografts is due to the presence of CD5 CAR-T cells in Poor persistence in mice. We use CRISPR/Cas9 technology to knock out the CD5 antigen on the surface of T cells to minimize the self-activation and suicide of CD5 CAR-T and ensure its sustainability and effectiveness in clinical verification. In addition, the use of molecular switches enhances the clinical safety of CAR-T cells.

In the development process, through phage-level antibody screening/specific identification, specific antibody clones are quickly and efficiently screened, and then directly connected to CAR-T functional testing to select the best candidate antibodies and optimize the development of CAR-T. The antibody screening process improves research and development efficiency while ensuring research quality.

chimeric antigen receptor

Herein, the CAR may comprise an extracellular domain, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain that specifically binds CD5. Herein, the extracellular domain of the CAR may comprise a single domain antibody (VHH) of the invention, two or more single domain antibodies in series (2×VHH). For example, the single domain antibody can be linked to a transmembrane domain through a hinge region, such as the CD8 alpha hinge. Herein, the CAR can be used to transduce immune effector cells (eg, T cells) and be expressed on the cell surface. Therefore, this article can also provide T cells expressing the chimeric antigen receptor, and the use of the T cells and/or the CAR for preparing drugs for treating CD5-related diseases.

As used herein, the chimeric antigen receptor (CAR) may comprise a CD5 binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain. Herein, the CD5 binding domain may comprise an antibody or fragment thereof that specifically binds to CD5, and the antibody may comprise a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2) and a heavy chain complementarity determining region 2 (HCDR2). Determining region 3 (HCDR3), the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 38-43, 64-69. As used herein, the antibody may comprise a heavy chain variable region, the amino acid sequence of which is as shown in SEQ ID NO: 33, 35, 37, 47, 57, 59, 61 or 63.

As used herein, the antibody may be a single domain antibody. In certain embodiments, the antibody may comprise the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, 47, 57, 59, 61 or 63, or a functional variant thereof. For example, the single domain antibody can include FHVH1sdAb, the sequence of which is shown in SEQ ID NO: 33; the single domain antibody can include FHVH3sdAb, the sequence of which is shown in SEQ ID NO: 35; the single domain antibody can include FHVH3VH1sdAb , the sequence of which is shown in SEQ ID NO: 37; the single domain antibody may include FHVH1VH3 sdAb, the sequence of which is shown in SEQ ID NO: 47; the single domain antibody may include FHVH2 sdAb, the sequence of which is shown in SEQ ID NO: 57 shown; the single domain antibody may include FHVH4 sdAb, whose sequence is shown in SEQ ID NO: 59; the single domain antibody may include FHVH2VH1sdAb, whose sequence is shown in SEQ ID NO: 61; the single domain antibody may include FHVH4VH1sdAb , whose sequence is shown in SEQ ID NO: 63.

For example, the single domain antibody described herein can be FHVH1sdAb, the sequence of which is shown in SEQ ID NO: 33. The amino acid sequences of HCDR1-3 of the single domain antibody FHVH1 are respectively as SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID As shown in NO: 40; the single domain antibody may include FHVH3 sdAb, the sequence of which is shown in SEQ ID NO: 35, the single domain antibody FHVH3 The amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively; the single domain antibody may include FHVH1VH3 sdAb, the sequence of which is shown in SEQ ID NO: 37. The amino acid sequences of HCDR1-3 of domain antibody FHVH1VH3 are shown in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively. The single domain antibody may include FHVH3VH1sdAb, the sequence of which is shown in SEQ ID NO: 37. The amino acid sequences of HCDR1-3 of the single domain antibody FHVH3VH1 are respectively as SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40. , SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

The single domain antibody described herein can be FHVH2 sdAb, and its sequence is as shown in SEQ ID NO: 57. The amino acid sequences of HCDR1-3 of the single domain antibody FHVH2 are as SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: As shown in 66; the single domain antibody can include FHVH4 sdAb, whose sequence is shown in SEQ ID NO: 59, and the amino acid sequences of HCDR1-3 of the single domain antibody FHVH4 are respectively as SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69; the single domain antibody may include FHVH2VH1sdAb, the sequence of which is shown in SEQ ID NO: 61. The amino acid sequences of HCDR1-3 of the single domain antibody FHVH2VH1 are respectively as SEQ ID NO: 64 and SEQ ID NO: 65. SEQ ID NO: 66, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are shown. The single domain antibody may include FHVH4VH1 sdAb, the sequence of which is shown in SEQ ID NO: 63. The amino acid sequences of HCDR1-3 of the single domain antibody FHVH4VH1 are respectively as SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69. , SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 are shown.

The CAR described herein may comprise a transmembrane domain that may comprise a polypeptide from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. As used herein, the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO: 6 or a functional variant thereof. For example, the transmembrane domain of the CAR described herein may include CD8α, the sequence of which is shown in SEQ ID NO: 6.

As used herein, the costimulatory domain may comprise a polypeptide from a protein selected from the group consisting of CD28, 4-1BB, OX40 and ICOS. As used herein, the costimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 8 or a functional variant thereof.

The CARs described herein may include an intracellular signaling domain, which may include a signaling domain from CD3ζ. As used herein, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 10 or a functional variant thereof.

The CAR described herein can include a hinge region that connects the antibody and the transmembrane domain. As used herein, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 4 or a functional variant thereof.

The CAR described herein may include a signal peptide, which may, for example, be located at the N-terminus of an extracellular domain that specifically binds CD5. The signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof. For example, the signal peptide can be a CD8α signal peptide, the sequence of which is shown in SEQ ID NO: 2.

In this context, the CAR may also be linked to a cleaved peptide. As used herein, the cleaved peptide may comprise an amino acid sequence derived from a T2A peptide. As used herein, the cleavage peptide may comprise the amino acid sequence shown in SEQ ID NO: 12 or a functional variant thereof. For example, the cleavage peptide can be T2A, the sequence of which is shown in SEQ ID NO: 12.

In this article, the CAR can also be connected to an EGFRt fragment, which can be used for signal detection or used as a molecular switch for CAR-T cells.

As used herein, the CAR may comprise the amino acid sequence shown in SEQ ID NO: 27, 29, 31, 45, 49, 51, 53 or 55 or a functional variant thereof. For example, the CAR can be selected from FHVH1 CAR, the sequence of which is shown in SEQ ID NO: 27. For another example, the CAR can be selected from FHVH3 CAR, whose sequence is shown in SEQ ID NO: 29; the CAR can be selected from FHVH3VH1 CAR, whose sequence is shown in SEQ ID NO: 31. The CAR can be selected from FHVH1VH3 CAR, the sequence of which is shown in SEQ ID NO: 45. The CAR can be selected from FHVH2 CAR, whose sequence is shown in SEQ ID NO: 49; the CAR can be selected from FHVH4 CAR, whose sequence is shown in SEQ ID NO: 51; the CAR can be selected from FHVH2VH1 CAR, whose sequence is shown in SEQ ID NO: 51. The sequence is as shown in SEQ ID NO: 53; the CAR can be selected from FHVH4VH1 CAR, the sequence of which is as shown in SEQ ID NO: 55.

In certain embodiments, the CAR described herein may include a CD5 binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain in order from the N-terminus. Wherein, the CAR may include a CD5 binding domain, and the CD5 binding domain sequences are as shown in SEQ ID NO: 33 and 35. Wherein, the CD5 binding domain may include HCDR1-3, the sequences of which are shown in SEQ ID NO: 38-40; and the CD5 binding domain may include another set of HCDR1-3, whose sequences are shown in SEQ ID NO :41-43 shown. The CAR may include a FHVH3VH1 CAR or a CAR described herein having the same two sets of HCDR1-3. The CD5 binding domain includes a tandem heavy chain variable region, the sequence of which is as shown in SEQ ID NO: 37 or 47, wherein the heavy chain variable region may include HCDR1-3, whose sequence is as shown in SEQ ID NO :38-40, and the CD5 binding domain may include another set of HCDR1-3, the sequences of which are shown in SEQ ID NO:41-43.

In certain embodiments, the CAR described herein may include a CD5 binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain in order from the N-terminus. Wherein, the CAR may include a CD5 binding domain, and the CD5 binding domain sequences are as shown in SEQ ID NO: 57 and 59. Wherein, the CD5 binding domain may include HCDR1-3, the sequences of which are shown in SEQ ID NO: 64-66; and the CD5 binding domain may include another set of HCDR1-3, whose sequences are shown in SEQ ID NO :67-69 shown. The CD5 binding domain includes a tandem heavy chain variable region, the sequence of which is as shown in SEQ ID NO: 61 or 63, wherein the heavy chain variable region may include HCDR1-3, whose sequence is as shown in SEQ ID NO :64-66, 38-40, and the CD5 binding domain may include another set of HCDR1-3, the sequences of which are shown in SEQ ID NO: 67-69, 38-40.

The heavy chain variable regions may also include a connecting peptide, the sequence of which is shown in SEQ ID NO: 25. For example, the CAR may include a FHVH3VH1 CAR, a FHVH1VH3 CAR, a FHVH2VH1 CAR, a FHVH4VH1 CAR, or a CAR described herein with the same linker peptide thereto. The transmembrane domain may include a transmembrane domain derived from CD8α, and its sequence may be as shown in SEQ ID NO: 6. For example, the CAR may include a FHVH3VH1 CAR, a FHVH1VH3 CAR, a FHVH2VH1 CAR, a FHVH4VH1 CAR, or a CAR described herein having the same transmembrane domain therein. The costimulatory domain may comprise a costimulatory structure from 4-1BB, the sequence of which may be as shown in SEQ ID NO: 8. For example, the CAR may include FHVH3VH1 CAR, FHVH1VH3 CAR, FHVH2VH1 CAR, FHVH4VH1 CAR or a CAR described herein having the same costimulatory domain. The intracellular signaling domain may comprise a signaling domain from CD3ζ, the sequence of which is shown in SEQ ID NO: 10. For example, the CAR may include a FHVH3VH1 CAR, a FHVH1VH3 CAR, a FHVH2VH1 CAR, a FHVH4VH1 CAR, or a CAR described herein that has the same intracellular signaling domain. The CAR may also include a hinge region, which may be located at the C-terminus of the CD5-binding domain and at the N-terminus of the transmembrane domain, and its sequence may be, for example, as shown in SEQ ID NO: 4. For example, the CAR may include a FHVH3VH1 CAR, a FHVH1VH3 CAR, a FHVH2VH1 CAR, a FHVH4VH1 CAR, or a CAR described herein that has the same hinge region therein. The CAR can also be connected to a signal peptide, which can be located at the N-terminus of the CAR, and its sequence can be as shown in SEQ ID NO: 2.

The CAR can also be linked to a cleaved peptide, such as T2A. The cleavage peptide can be located at the C-terminus of the intracellular signal transduction domain, and its sequence can be as shown in SEQ ID NO: 12. The CAR can also be connected to the CSF2RA signal peptide, which can be located before EGFRt, and its sequence can be, for example, as shown in SEQ ID NO: 14. The CAR can also be connected to a label detection signal, which can be located at the C-terminus of the CAR (or, the cleaved peptide). The label detection signal can be selected from the following group: GFP, RFP, YFP or EGFRt. The sequence of EGFRt can be, for example, as shown in SEQ ID NO: 16.

For example, the CAR described herein can be a FHVH3VH1 CAR, whose amino acid sequences of VHH (FHVH1) HCDR1-3 are shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 respectively; the amino acid sequences of VHH (FHVH1) The amino acid sequence is shown in SEQ ID NO: 33; the amino acid sequences of VHH (FHVH3) HCDR1-3 are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively; the amino acid sequence of VHH (FHVH3) As shown in SEQ ID NO: 35, the sequence of the connecting peptide between VHH (FHVH3) and VHH (FHVH1) is as shown in SEQ ID NO: 25; its hinge region is as shown in SEQ ID NO: 4; its transmembrane domain As shown in SEQ ID NO: 6; its costimulatory domain is the 4-1BB costimulatory domain, as shown in SEQ ID NO: 8; its CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 10; so The FHVH3VH1 CAR also includes a cleaved peptide as shown in SEQ ID NO: 12, a CSF2RA signal peptide as shown in SEQ ID NO: 14, and an EGFRt as shown in SEQ ID NO: 16; as shown in SEQ ID NO: 2 The CD8α signal peptide is shown.

CD8α For example, the CAR described herein can be a FHVH1 CAR, and the amino acid sequences of its VHH (FHVH1) HCDR1-3 are shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 respectively; VHH (FHVH1) Its amino acid sequence is shown in SEQ ID NO: 33; its hinge region is shown in SEQ ID NO: 4; its transmembrane domain is shown in SEQ ID NO: 6; its costimulatory domain is the 4-1BB costimulatory structure domain, as shown in SEQ ID NO: 8; its CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 10; the FHVH1 CAR also includes a cleavage peptide as shown in SEQ ID NO: 12, as shown in SEQ ID The CSF2RA signal peptide shown in NO:14, and the EGFRt shown in SEQ ID NO:16; the CD8α signal peptide shown in SEQ ID NO:2.

For example, the CAR described herein can be a FHVH3 CAR, the amino acid sequences of VHH (FHVH3) HCDR1-3 are shown in SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively; the amino acid sequences of VHH (FHVH3) The amino acid sequence is shown in SEQ ID NO: 35; its hinge region is shown in SEQ ID NO: 4; its transmembrane domain is shown in SEQ ID NO: 6; its costimulatory domain is the 4-1BB costimulatory domain , as shown in SEQ ID NO: 8; its CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 10; the FHVH1 CAR also includes a spliced protein as shown in SEQ ID NO: 12 Cleaved peptides, such as the CSF2RA signal peptide shown in SEQ ID NO: 14, and the EGFRt shown in SEQ ID NO: 16; the CD8α signal peptide shown in SEQ ID NO: 2.

The CAR provided herein can be connected to a molecular switch (such as tEGFR or HSV-TK) through a cleaved peptide, or the CAR is considered to also include a molecular switch part. Those skilled in the art can understand that due to the effect of the cleavage peptide, the fusion protein of the CAR and the molecular switch usually does not exist in the cells expressing the CAR. The above description is for simplicity only. However, for nucleic acid molecules, the coding sequence of CAR can be combined with the coding sequence of the spliced peptide and molecular switch in an expression cassette and transcribed by the same promoter.

Nucleic acids, vectors, cells, preparation methods and compositions

In another aspect, provided herein is an isolated nucleic acid molecule encoding a CAR described herein. The isolated nucleic acid molecule encoding a CAR described herein may comprise SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17-24, 26, 28, 30, 32, 34, The nucleic acid sequence shown in 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62 or a functional variant thereof. Nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In another aspect, provided herein is a vector that may comprise the nucleic acid molecule. Herein, the vector may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. The lentiviral vectors described herein may comprise a CAR. For example, the lentiviral vectors described herein may comprise SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17-24, 26, 28, 30, 32, 34, 36, 44, The nucleic acid sequence shown in 46, 48, 50, 52, 54, 56, 58, 60, or 62 or a functional variant thereof. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain expression control elements that allow correct expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequences are tunable elements. The specific structure of the expression control sequence can vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, GA cap sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid. One or more nucleic acid molecules described herein can be operably linked to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector, including the vector sdAb plasmid and/or CAR plasmid.

On the other hand, this article provides an immune effector cell, which may include the CAR described herein, the nucleic acid molecule, or the vector. Herein, the immune effector cells may be mammalian cells. Herein, the immune effector cells may be selected from T lymphocytes and natural killer (NK) cells.

On the other hand, this article provides a method for preparing immune effector cells, which includes knocking out the immune effector cells. CD5 gene, and introduce the CAR expression vector described herein into immune effector cells. This article also provides a method for preparing immune effector cells, which includes knocking out the TRAC and/or TRBC genes of the immune effector cells, and introducing the CAR expression vector described herein into the immune effector cells. This article also provides a method for preparing immune effector cells, which includes knocking out the CD5 gene and TRAC and/or TRBC genes of the immune effector cells, and introducing the CAR expression vector described herein into the immune effector cells.

For example, a CAR expression vector described herein can be introduced into the immune effector cells, such as T lymphocytes or natural killer (NK) cells. In certain embodiments, each or each cell may comprise one or more expression vectors described herein. In certain embodiments, each or each cell may comprise multiple (eg, 2 or more) or multiple (eg, 2 or more) expression vectors described herein. Herein, the expression vector can be introduced into immune effector cells by methods known in the art. For example, immune effector cells can be transfected through retroviral vectors, and the viral genome with CAR molecules can be integrated into the host genome to ensure long-term and stable expression of the target gene. As another example, transposons are used to introduce into target cells through plasmids carrying CAR (transposons) and plasmids carrying transposase. As another example, CAR molecules can be added to the genome through gene editing (such as CRISPR/Cas9). In this article, the vector carrying the CAR molecule described herein can be introduced into the cells by methods known in the art, such as electroporation, lipofectamine 3000, Invitrogen, etc.

On the other hand, this article provides a pharmaceutical composition, which may include the immune effector cells and a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and/or non-ionic surfaces Active agents, etc. As used herein, the pharmaceutical composition may be formulated for oral administration, intravenous administration (eg, intravenous injection, I.V.), intramuscular administration (eg, intramuscular injection, I.M.), in situ at the tumor site Administration is by inhalation, rectally, vaginally, transdermally or via a subcutaneous depot.

pharmaceutical use

On the other hand, this article provides the use of the CAR, the nucleic acid molecule, the vector or the immune effector cell for preparing a medicine, wherein the medicine is used to treat diseases related to the expression of CD5 or illness. Herein, the disease or disorder associated with the expression of CD5 may be cancer or malignant tumor. In certain embodiments, the cancer or malignancy can be selected from malignant T cell tumors or malignant B cell tumors. Wherein, the malignant T cell tumor can be selected from T cell acute lymphoblastic leukemia (T-ALL), T cell lymphoma (TCL) (such as peripheral T cell lymphoma, cutaneous T cell lymphoma (CTCL), T cell non- Hodgkin lymphoma (T-NHL)); the malignant B-cell tumor can be selected from chronic lymphocytic leukemia (B-CLL) (such as hairy cell leukemia (HCL)), mantle cell lymphoma (B-MCL) , Diffuse large B lymphoma (DLBCL).

On the other hand, this article provides the CAR, the nucleic acid molecule, the vector or the immune effector cell, which treats diseases or conditions related to the expression of CD5.

On the other hand, this article provides a method of treating a disease or condition related to the expression of CD5, comprising administering the CAR, the nucleic acid molecule, the vector, or the immune effector cell to a patient.

Without intending to be bound by any theory, the following examples are merely to illustrate the working methods of the chimeric antigen receptors, vectors, cells, and compositions herein, and are not intended to limit the scope of the invention herein.

Research overview

We use fully human phage for antibody screening and directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it eliminates the difficult step of humanizing mouse antibodies, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and are better for CAR-T development. potential. After obtaining antibody clones that specifically bind to cell surface CD5 antigen and CD5 recombinant protein, we conducted in-depth research and constructed these clones and control clone H65 onto the second-generation CAR structure, then packaged lentivirus and transfected T Cells, at the CAR-T cell level, fully human CD5 antibody clones and candidate CAR-T molecules with stronger functions than the control clone H65 were screened from the perspectives of target cell activation and killing, and target cell stimulation and proliferation.

In addition, the literature reports that in most constructed animal animal models of tumors, re-emerging tumor cells retain CD5 expression, suggesting that tumor recurrence is not due to loss of antigen and that failure to eradicate all xenografts is due to the presence of CD5 CAR-T cells in Poor persistence in mice ¹⁴ . Experimental results show that CD5 knockout CAR-T cells can expand normally and maintain the killing function of CD5-positive target cells, while minimizing the self-activation and suicide of CD5 CAR-T, ensuring its clinical validation. sustainability and effectiveness. In addition, we also verified the effectiveness of the molecular switch (tEGFR or HSV-TK) in vitro and in vivo.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will appreciate, the content herein enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions herein are illustrative only and not restrictive.

Example

Example 1. CAR-T cell antigen binding detection and epitope competition experiment

Experimental purpose and principle:

As shown in Figure 1A, in order to detect whether there are clones that bind to different epitopes of the CD5 antigen in the fully human single domain clones obtained, the clones to be tested were constructed into IgG vectors with human Fc tags and expressed in CHOS cells. If the cloned IgG antibody to be tested and the CD5 KO T cells expressing the CAR to be tested bind to different epitopes of the CD5 antigen, the cloned IgG antibody to be tested and the CD5 KO T cells expressing the CAR to be tested can bind to the same CD5 antigen at the same time, and then combine APC-human Fc antibody shows APC positive in flow cytometry test, otherwise, it shows APC negative.

Brief experimental steps for cell antigen binding detection:

1) Take 1×10^6 each of CD5 KO CAR-T and MOCK T transfected with clones H65, FHVH1, and FHVH3, place at 600g, and centrifuge for 5 minutes at room temperature. Wash twice with PBS, mix with 0.2 μg of biotinylated CD5 antigen, and incubate at 4°C in the dark for 30 minutes.

2) Wash twice with PBS, add 0.2μL/test APC-streptavidin and 5μL PE-EGFR antibody, wash twice with PBS, resuspend with 100μL PBS, and detect by flow cytometer.

Brief experimental steps for epitope competition experiments:

1) Take 1×10^6 each of CD5 KO CAR-T and MOCK T clones H65, FHVH1, and FHVH3, 600g, room temperature, and centrifuge for 5 minutes. Wash twice with PBS and set aside;

2) Premix 0.5μg each of H65 antibody, FHVH3 antibody or FHVH1 antibody with human Fc tag and 0.2μg CD5 antigen, then add them to CD5 KO T cells expressing the CAR to be tested, and incubate in the dark at 4°C for 30 minutes.

3) Wash twice with PBS.

4) Add 2 μL of APC-human Fc and 5 μL of PE-EGFR antibody to each well, and incubate for 30 minutes at 4°C in the dark. After washing with PBS, resuspend in 100 μL PBS and detect by flow cytometry.

Main materials and reagents:

APC anti-human IgG,Fcγfragment specific,Jackson ImmunoResearch,Cat.109-136-170;

PE anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352904;

Experimental results:

As shown in the schematic diagram in Figure 1A, after combining the FHVH3-IgG antibody with hFc and the CD5 antigen, it can combine with the CD5 KO CAR-T cloned FHVH1 and then combine with the APC-human Fc antibody. CD5 KO CAR-T of H65, FHVH1, and FHVH3 can effectively bind to CD5 antigen, while MOCK T does not bind to CD5 antigen (upper part of Figure 1B). H65-hFc and FHVH3-hFc antibodies do not affect the binding of FHVH1 CAR-T cells to CD5 antigen, indicating that FHVH1 binds to different CD5 epitopes from H65 and FHVH3, while FHVH3 and H65 recognize overlapping CD5 epitopes (Figure 1B bottom part). After demonstrating that FHVH1 and FHVH3 bind to different CD5 epitopes, we speculate that the tandem use of FHVH1 and FHVH3 can improve efficacy and reduce the risk of tumor escape caused by antigenic mutations. Therefore, concatenating FHVH1 and FHVH3 in different sequences can be used as two candidate clones for anti-CD5 dual-epitope antibodies and anti-CD5 dual-epitope CARs.

Example 2. Preparation and detection of CD5 KO CAR-T cells

Experimental purpose and principle:

This application uses lentiviral transfection to make the knockout T cells express CAR. For the CAR-T preparation process, please refer to the patent (Zhou J, Liu J, Hu G, et al. Chimeric antigen receptor (car) binding to bcma, and uses thereof:U.S.Patent Application 16/650,580[P].2020-8-6.). Lentiviral vectors are based on the genome of lentivirus, in which multiple sequence structures related to viral activity are removed to make them biologically safe, and then the sequence of the target gene required for the experiment is introduced into this genome skeleton. and vectors prepared from expression constructs. Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc., It can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, "Lentiviral vectors and their research progress, Chinese Journal of Lung Cancer 17.12(2014):870–876.PMC). Transfection through lentiviral vectors , which can integrate CAR molecules into host cells.

T cells were knocked out of CD5 and transfected with a single single domain clone CAR lentivirus, tandem fully human single domain clone CAR lentivirus Virus (structure shown in Figure 2A), and mouse control H65 CAR lentivirus, CD5, EGFRt expression and CD5 antigen expression were detected after 5 to 7 days (Figure 2B). The CAR structure contains CD8α signal peptide, sdAb, CD8α hinge region, CD8α transmembrane region, 4-1BB costimulatory molecule and CD3ζ and uses T2A to connect a truncated EFGR molecule (EGFRt). EGFRt can be used as a safety switch during clinical translation. , and because EGFRt is co-expressed with CAR molecules, it can be used as an indirect detection indicator of the distribution of CAR molecules on the surface of T cells without affecting the structure and function of CAR.

The sequences of exemplary CAR molecules and their antigen recognition portions mentioned in this article are shown in Table 1 below.

Table 1. Exemplary CAR molecules and their antigen recognition portions

Experimental purpose and principle:

Mature T cells all express CD5 antigen on their surface. However, the CD5 CAR-T developed by Mamonkin M and other researchers without CD5 knockout has been reported to have a certain degree of ^suicide14 and has a limited duration in the patient's body, which greatly limits the patient's ability to Disease remission time and application of this CAR-T product. In order to solve this problem, this application uses CRISPR/Cas9 technology to knock out the CD5 antigen on the surface of T cells to minimize the self-activation and suicide of CD5 CAR-T and ensure its sustainability and effectiveness.

The brief experimental steps of the preparation process of CD5 KO T cells are as follows:

1) Prepare the sgRNA/Cas9RNP mixture: Calculate the required amount of sgRNA and Cas9 protein based on the cell volume (1×10^6T cells require 30 μg Cas9 protein and 20 μg sgRNA), gently mix the sgRNA and Cas9 protein and incubate at room temperature for 15 minute.

2) Gently mix the incubated sgRNA/Cas9RNP mixture and T cells and incubate at room temperature for 10 minutes.

3) Use the recommended voltage and electroporation time of the electroporation instrument to knock out the CD5 antigen of T cells.

4) Place the CD5 KO T cells in freshly rewarmed T cell culture medium, return them to a 37°C 5% carbon dioxide incubator, and perform lentiviral transfection after 24 hours. After the cell status has recovered, the cells will be tested on the 5th to 7th day. Knockout efficiency and CAR transfection efficiency. The brief experimental steps for CAR-T/T cell CD5 antigen expression detection and EGFRt expression detection are as follows:

1) Take 1×10 ⁶ CAR-T/T cells from each well, add PBS and wash once, centrifuge at 300g for 5 minutes, and discard the supernatant.

2) Resuspend the cell pellet in 100 μL PBS, add 5 μL APC-CD5 antibody and 5 μL PE-EGFR antibody respectively, and incubate at 4°C in the dark for 15 minutes.

3) Wash twice with PBS and centrifuge at 300g for 5 minutes.

4) Resuspend in 200μL PBS and run on flow cytometer for detection.

Main materials and reagents:

Chemical synthesis of EasyEdit sgRNA, Nanjing GenScript Biotechnology Co., Ltd.;

sgRNA:gctgtagaactccaccacgc (SEQ ID NO: 70);

^TrueCutTM Cas9 Protein v2,thermo,Cat.No.A36498;

APC Mouse Anti-Human CD5 antibody,Clone UCHT2,BD Pharmingen,Cat.No.555355;

PE anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352904;

Fetal bovine serum (FBS), Gibco, Cat. No. 10099141.

Experimental results:

The CAR-T studied in this application targets the CD5 target. If the CAR-T cells function well, the CAR-T cells after transfection with lentivirus can kill CAR-T/T cells that still express the CD5 antigen. In this experiment, CAR-T cells were able to eliminate cells without CD5 knockout. In order to reduce the suicide of CD5 CAR-T cells in subsequent experiments, CD5 was knocked out through CRISPR/Cas9 technology.

As shown in Figure 2B, the abscissa is the expression of EGFRt (that is, the expression of CAR indirectly represented), and the ordinate is the expression of CD5. Compared with MOCK T (non-transfected T cells), the CD5 positive expression rate of T cells after CD5 knockout is only 16.1%, indicating that the efficiency of CD5 knockout through CRISPR/Cas9 technology can reach more than 80%. Helps reduce the suicide of CD5 CAR-T cells in subsequent experiments. Among them, T cells that have not been knocked out and still express CD5 antigen are gradually cleared by CD5KO CAR-T cells during the culture process, so the CD5 antigen in CD5KO CAR-T cells is basically undetectable, proving that CD5KO CAR-T The cells clear CD5+ cells well.

Example 3. In vivo and in vitro functional verification of CAR-T cells

In vitro functional verification of CAR-T cells

Experimental purpose and principle:

Since fully human single-domain clones and tandem clones that bind CD5 antigen may not have good activation functions after being constructed into a CAR structure, their function on CAR-T cells needs to be further confirmed and the CAR molecules with the best activity need to be screened. To this end, we prepared lentiviral vectors of these CAR molecular clones and transduced T cells into CAR-T cells. Then, the in vitro biological efficacy of CAR-T cells was evaluated through CD107a degranulation assay (CD107a degranulation assay) and in vitro cell killing assay (in vitro cytotoxicity assay). Through the functional verification of these CAR-T levels, CAR molecules with ideal effectiveness and safety are finally screened for downstream CAR-T product development.

CD107a degranulation experiment

CD107a is a marker of intracellular microvesicles. When granzyme-loaded microvesicles fuse with the cell membrane, CD107a on the cell membrane will increase. When its recycling is blocked with monesin (purchased from BioLegend) , which can quantitatively reflect the intensity of microvesicle release. When CAR-T is stimulated by the target antigen on the target cell, it will cause the release of granzymes, and the activation of T cells can be judged by flow cytometric detection of the increase in CD107a.

Brief experimental steps for CD107a degranulation:

1) Verify the CD5 expression of target cells: Take 1×10 ⁶ target cells JURKAT, CCRF-CEM, CCRF-CD5 KO, MOLT-4, SUP-T1, K562, RAJI per well, add PBS and wash once, centrifuge at 300g for 5 minutes and discard the supernatant. Resuspend the cell pellet in 100 μL PBS, add 5 μL APC-CD5 antibody, and incubate at 4°C in the dark for 15 minutes. Wash twice with PBS and centrifuge at 300g for 5 minutes. After resuspending in 100 μL PBS, detect by flow cytometry.

2) Centrifuge the CAR-T cells and target cells to be tested at 300g for 5 minutes at room temperature, discard the supernatant, and resuspend them in 1640 medium + 10% FBS to 4x10 ⁶ cells/mL;

3) In the 96-well plate, add 100 μL of CAR-T cells to be tested and 100 μL of target cells respectively, and mix well;

4) Add 5 μL PE/Cy7 mouse anti-human CD107a antibody and 0.2 μl monensin to each well of cells, and then place them in a cell culture incubator (37°C, 5% CO ₂ ) and incubate for 4 hours;

5) After the incubation is completed, centrifuge at 600g for 5 minutes at 4°C, discard the supernatant, and wash the cells twice with 200 μL PBS;

6) Resuspend the cells in 100μL PBS, add 5μL APC anti-human EGFR and 5μL BV421anti-human CD8α antibody respectively, mix and incubate on ice in the dark for 20 minutes;

7) After the incubation is completed, wash the cells three times with 200 μL PBS; resuspend them with 100 μL PBS and detect them with a flow cytometer. Main samples and reagents:

Target cells JURKAT, CCRF-CEM, CCRF-CD5 KO, MOLT-4, SUP-T1, K562, RAJI;

APC Mouse Anti-Human CD5 antibody,Clone UCHT2,BD Pharmingen,Cat.No.555355;

Fetal bovine serum, Gibco, Cat.No.10099141;

Monensin,BioLegend,Cat.No.420701;

PE/Cy7 mouse anti-human CD107a,BD Pharmingen,Cat.No.561348;

BV421anti-human CD8α,Biolegend,Cat.No.301036;

APC anti-human EGFR, BioLegend, Cat. No. 352906.

Experimental results:

As shown in Figure 3, by detecting target cells CCRF-CEM, JURKAT, MOLT-4, SUP-T1, CCRF-CD5 KO, The average fluorescence intensity of K562, RAJI APC Anti-Human CD5 is used to determine the CD5 antigen expression intensity of each target cell. JURKAT and CCRF-CEM are cell lines with high expression of CD5 antigen. MOLT-4 and SUP-T1 are cell lines with medium expression of CD5 antigen. CCRF-CD5 KO, K562, and RAJI are negative cell lines. CCRF-CD5 KO is performed by knocking out CD5 from the CCRF-CEM cell line using CRISPR/Cas9 technology (the method is the same as the CD5 knockout of T cells). The knocked-out cells are then subjected to extreme dilution and then spread on a monoclonal plate. The monoclonal cells are then expanded. When the number reaches a detectable level, 2*10^5 CCRF-CD5 KO monoclonal cells are taken for flow cytometric identification to confirm that their CD5 expression is negative, and then the monoclonal cells can be used in experiments.

CAR-T cells were obtained through lentiviral transduction, and the CAR-T cells were cultured in vitro for 9 days before performing a CD107a degranulation experiment. The CAR-T cells to be tested were incubated with target cells, monensin, and CD107a antibodies for 4 hours. The cell densities of both CAR-T cells and target cells were 4 × 10 ⁵ cells/mL. Then, the samples were labeled with CD8 antibodies and EGFR antibodies, and flow cytometric detection was performed. Analyze in Flowjo software, select the live cell gate (P1) in the scatter plot, and remove cell debris; cells in the P1 gate are analyzed to select a single scattered cell gate (P2); then, CD8-positive cells are further selected in the P2 gate of cells (P3); finally, in the P3 gate, analyze the proportion of CD107a-positive cells among cells stained positive for EGFR antibody (i.e., CAR-positive cells).

The analysis results are shown in Figure 4. The results show that except for FHVH1VH3, all CAR-T cells can be specifically activated by CD5-positive target cells but not by CD5-negative target cells, and have good specificity. CAR-T cells of FHVH1, FHVH3, and FHVH3VH1 have stronger CD107a degranulation function than control CAR-T cells (H65 CAR-T cells), while FHVH1VH3 CAR-T cells themselves have non-specific activation.

In vitro cell killing assay

Experimental purpose and principle: The in vitro cell killing experiment uses CCRF-CEM, JURKAT, MOLT4 and SUP-T1 as CD5-positive target cells, and CCRF-CD5 KO, K562 and RAJI cells as CD5-negative target cells to conduct antigen-specific CD5 CAR-T cells. Evaluation of sexual lethality. Among them, the above cells were transduced with lentivirus to obtain target cells that stably express firefly luciferase. Therefore, the luciferase activity in the sample can reflect the number of target cells. CAR-T cells and target cells are co-incubated and cultured. When target cells are killed by CAR-T cells, luciferase will be released and quickly inactivated (the half-life of firefly luciferase is about 0.5 hours). If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells expand and luciferase continues to be expressed. Therefore, the killing of target cells by CAR-T can be detected through luciferase activity.

Brief experimental steps for in vitro cell killing:

1) Centrifuge the above cells at 300g for 5 minutes at room temperature. After discarding the supernatant, resuspend in 1640+10% FBS medium to 2x10 ⁵ cells/mL; add 100 μL of target cells to each well of the 96-well plate. ;

2) According to the CAR positive rate and efficacy-to-target ratio of the CAR-T sample to be tested, add the corresponding CAR-T cells to each well of the 96-well plate, and mix with the target cells; then place it in 37°C 5% CO _2. Incubate in the incubator for 24 hours;

3) Use the luciferase detection kit to detect the luciferase activity in each well of the sample.

Main samples and reagents:

Target cells CCRF-CEM, JURKAT, MOLT4, SUP-T1, CCRF-CD5 KO, K562 and RAJI;

Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

Experimental results:

The CAR-T cell sample and a fixed number of target cells (2x10 ⁴ cells) were mixed according to different effective-to-target ratios (E:T) and incubated together for 24 hours, and then the luciferase activity (RLU) in the sample was detected. Since luciferase activity can reflect the number of target cells in the sample, the killing/inhibitory ability of CAR-T cells on target cells can be obtained through changes in luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells are killed.

As shown in Figure 5, the ordinate in the figure is the ratio of target cells killed, and the abscissa is the ratio of effector cells to target cells (Effector: target cells ratio, E:T). All CAR-T cell samples were more effective in killing positive target cells than control H65 CAR-T cells, and there was no obvious killing when co-incubated with negative target cells. Therefore, CAR-T samples of FHVH1, FHVH3, FHVH1VH3, and FHVH3VH1 can specifically kill CD5-positive target cells, and have no non-specific killing of CD5-negative target cells. At a lower efficiency target ratio, especially with moderate expression of CD5 When the target cells MOLT-4 and SUP-T1 were co-incubated, the killing ability of FHVH1VH3 and FHVH3VH1 on the target cells was stronger than that of FHVH1 and FHVH3.

CAR-T/T cell apoptosis detection

1) Take 1*10^6 each of CAR-T cells, CD5 KO T and MOCK T cells that have been cultured for 10-12 days, 600g, at room temperature, centrifuge for 5 minutes, and wash twice with PBS.

2) Add 100μL binding buffer to each well to resuspend, add 10μL annexin V (FITC) and 5μL PI (PE), incubate at room temperature for 15 minutes, then add 200μL binding buffer, and run on the flow cytometer.

Main samples and reagents:

H65, FHVH1, FHVH3, FHVH1VH3, FHVH3VH1 CAR-T cells, CD5 KO T, MOCK T cells

cell;

FITC Annexin V Apoptosis Detection Kit with PI, Biolegend, Cat.No.640914.

Experimental results:

As shown in Figure 6, there was no statistical difference in the apoptosis levels of H65, FHVH1, FHVH3, FHVH1VH3, FHVH3VH1 CAR-T cells, CD5 KO T, and MOCK T cells (3 independent repeated experiments), indicating that after CD5 knockout, CD5 CAR-T cells did not show obvious self-activation or apoptosis caused by "suicide" after being cultured for 10 to 12 days, indicating that they may not lose function due to self-activation and "suicide" and can stably exist and exert their functions in the patient's body. Tumor-killing effect.

Repeated stimulation proliferation experiment

Experimental purpose and principle:

Mitomycin (Mitomycin C)-treated target cells (CCRF-CEM) were mixed with different groups of CD5 KO CAR-T cells for multiple stimulations, and then the CAR-T cells and target cells were co-incubated and cultured to determine the different The ability of CAR-T to proliferate after being continuously stimulated by target cells for multiple times.

Brief experimental steps for repeated stimulation experiments:

1) Take 7×10 ⁶ CCRF-CEM cells, 300g, room temperature, and centrifuge for 5 minutes;

2) Adjust the density of complete culture medium to 0.2×10 ⁶ cells/mL, add 5 μL Mitomycin C stock solution (1 μg/μL), mix well, and incubate at 37°C and 5% CO ₂ for 24 hours before use.

3) Take 300g of CCRF-CEM-Mitomycin C cells treated with Mitomycin C for 24 hours, centrifuge and change the medium, use Wash 6 times with PBS, resuspend the CCRF-CEM-Mitomycin C cells in CTS medium, count and adjust the density to 6×10 ⁶ cells/mL, and set aside for use.

4) Take 3×10 ⁵ CD5KO CAR-T cells respectively and transfer them into a 24-well plate. Add 50 μL of CCRF-CEM-Mitomycin cells to each well to make the effect-to-target ratio E:T=1:1. Add CTS complete culture medium to the final culture volume of 500 μL, mix well, incubate at 37°C, 5% _CO2 for 72 hours and count, treat target cells (CCRF-CEM) with Mitomycin again, and repeat the steps of repeated stimulation in 1-4 above. and draw an amplification curve.

Main samples and reagents:

FHVH1, FHVH3, FHVH3VH1, H65 CAR-T cells, CCRF-CEM cell lines;

Mitomycin C,STEMCELL Technologies,Cat.No.73274;

CTS ^TM OpTmizer ^TM T Cell Expansion SFM, Gibco, Cat. No. A1048501;

Fetal bovine serum (FBS), Gibco, Cat. No. 10099141.

Experimental results:

As shown in Figure 7, after repeated stimulation of 4 groups of CAR-T cell samples, the proliferation ability is: FHVH3VH1 CAR-T>FHVH3CAR-T>H65 CAR-T>FHVH1 CAR-T. After the target cells are stimulated 5 times, the proliferation ability of FHVH3VH1, FHVH3 and H65 CAR-T cells can still be effectively expanded. The proliferation ability of CAR-T cells after being stimulated by target cells is closely related to the patient's long-term prognosis. Therefore, FHVH3VH1 CAR-T and FHVH3 CAR-T can be considered to have the potential to proliferate long-term in the body and eliminate tumor cells.

In vivo functional verification of CAR-T cells in mouse tumor models

Experimental purpose and principle:

Because the fully human single-domain clone and tandem clone that binds to the CD5 antigen were constructed into the CAR structure, it was proven in in vitro functional verification that it has good functions of activating and killing tumor cells, and the fully human single-domain tandem clone FHVH3VH1 is effective against CD5+ target cells. The killing ability and target cell expansion ability after stimulation are enhanced compared with a single single domain clone. To this end, we performed in vivo biological efficacy assessment of CAR-T cells. The human acute T-cell leukemia cell line CCRF-CEM-ffLuc was used to establish a tumor model and conduct functional verification of CAR-T in mice to prove the effectiveness and safety of CAR-T.

Brief experimental steps:

1) Six-week-old female NCG mice were injected with 1 × 10 ⁶ CCRF-CEM-ffLuc cells through the tail vein on day 0 for tumor inoculation.

2) Inject 2×10 ⁶ and 1×10 ⁶ CD5KO CAR+T, CD5KO T cells or PBS via tail vein injection on days 4 and 7 respectively (n=5).

3) Use bioluminescence imaging weekly to assess tumor burden and monitor mouse survival curves.

Main samples and reagents:

FHVH1, FHVH3, FHVH3VH1 and H65 CAR-T cells, and human acute T-cell leukemia cell line CCRF-CEM-ffLuc cell line;

NCG severe immunodeficiency mice, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

Experimental results:

As shown in Figure 8, among the four groups of CAR-T cell samples, FHVH1, FHVH3 and FHVH3VH1 CAR-T can eliminate tumor cells in tumor-bearing mice, while the same dose of H65 CAR-T only has weak inhibition in mice. Tumor effect, FHVH1 CAR-T cells, FHVH3 CAR-T cells and FHVH3VH1 CAR-T cells have better anti-tumor effects than H65 CAR-T cells in vivo, while FHVH3VH1 CAR-T cells have stronger anti-tumor effects than FHVH1 and FHVH3 CAR- T cells. As shown in Figure 8B, as of day 23, H65, FHVH1, FHVH3 and FHVH3VH1 CAR-T cells can effectively prolong the survival time of tumor-bearing mice, with statistical differences compared to CD5KO T cells or PBS treatment group (P< 0.0001,n=5).

In order to further study the function of FHVH3VH1 CAR-T (hereinafter referred to as CT125A) cells, the following studies were conducted.

Example 4 Study on the Binding of CT125A Cells to Target Antigen CD5

Research purpose: To examine the binding ability of CT125A cells to the target antigen CD5.

Research method: After incubating CT125A cells (2×10 ⁵ cells/well) with different concentrations of fluorescent dye-labeled human CD5 protein, FCM (Flow cytometry, flow cytometry) method was used to detect the fluorescence-labeled CAR ⁺ The relationship between the percentage of positive cells and the concentration of CD5 protein was fitted by Graphpad Prism software to calculate the affinity EC50 constant. This experiment was completed by Shanghai Reindeer Biotechnology Co., Ltd., and a total of three independent repeated experiments were conducted.

Research results: In 3 batches of validation batches, the apparent affinity EC50 of CT125A and human CD5 antigen was 2.07±1.03nM. The results are shown in Table 2 and Figure 9.

Table 2 Summary of affinity test results between CT125A and human CD5 antigen

Research conclusion: CT125A cells have stable, good and specific binding ability to human CD5 antigen.

Example 5 Study on the in vitro degranulation activity of CT125A

Research purpose: To evaluate the degranulation activity of CT125A under specific stimulation of positive target cells.

Research plan: Use flow cytometry to detect the transport of CD107a molecules on the membrane surface of 3 batches of CAR-T cells. CAR-T cells contain high concentrations of cytotoxic particles in the form of vesicles in their cytoplasm. When CAR-T cells are co-incubated with target cells, the toxic particles will reach the serosal surface and fuse with the cell membrane, causing the release of the contents of the particles, ultimately leading to the death of the target cells. As degranulation occurs, CD107a molecules are transported to the cell membrane surface and can bind to CD107a antibodies. The FCM method is used to quantitatively analyze CD107a expression and evaluate the degranulation function of CT125A. This experiment was completed by Shanghai Reindeer Biotechnology Co., Ltd., and a total of three independent repeated experiments were conducted.

Research results: After incubation of CT125A with four strains of CD5-positive target cells, the degranulation reaction increased significantly. The positive rates of CD107a were 33.22% ± 3.18% (CCRF-CEM-Luc) and 35.43 ± 7.33nM (SUP-T1-Luc) respectively. ; 29.71±10.01nM (JVM-2-Luc-CD5); 36.38±8.56nM (MEC-1-CD5-Luc); without adding target cell group, the positive rate of CD107a is only 3.07%±3.34% . The data are detailed in Table 3 and Figure 10.

Table 3 Degranulation activity of CT125A under stimulation by different types of positive target cells

Research conclusion: After co-incubation of CT125A cells with the four positive target cell strains measured, the positive rate of CD107a was significantly increased compared with CT125A cells themselves. This result shows that CT125A cells have stable, specific and good degranulation activity.

Example 6 CT125A in vitro killing study

Research purpose: To evaluate the ability of CT125 injection to kill positive target cells in vitro.

Research method: CAR-T cells and target cells were co-incubated and cultured according to different effective target ratios (from 10:1 to 0.5:1, 2-fold dilution). When target cells are killed by CAR-T cells, luciferase is released and quickly inactivated. If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells expand and luciferase continues to be expressed. Therefore, the killing of target cells by CAR-T can be detected through luciferase activity.

Research results: Compared with the MOCK-T group, CT125A had a significant killing effect on the four positive target cells measured among the three effective target ratios of high, medium and low, with a dose-effect relationship. : For CCRF-CEM-Luc cells, the killing rates when the effect-to-target ratios are 2:1 and 1:1 are 96.91% and 95.58% respectively; for SUP-T1-Luc cells, the effect-to-target ratios are 2:1 and 1:1. The killing rates at 1 hour were 91.04% and 56.94% respectively; for JVM-2-Luc-CD5 cells, the killing rates at the effector-target ratios were 10:1 and 5:1 were 98.51% and 96.40% respectively; for MEC-1 -CD5-Luc cells, the killing rates were 98.25% and 97.71% when the effect-to-target ratio was 2:1 and 1:1 respectively. See Table 4 and Figure 11 for specific data.

Table 4 Killing of different types of positive target cells by CT125A

Research conclusion: CT125A cells have stable, specific and good killing activity against the four positive target cell strains tested in vitro.

Example 7 Study on the expression of gamma interferon in CT125A injection

Research purpose: To study the expression level of Interferon-γ (IFN-γ) secreted by CT125A injection after incubation with target cells to understand the killing of tumor cells by this injection.

Research method: The CBA (Cytometric Bead Array, flow microsphere chip technology) method was used to detect the secretion level of IFN-γ expression in the supernatant of different types of CD5-positive target cells co-incubated with CT125 injection.

Research results: Compared with the CAR-T cell only group: the release of IFN-γ was slightly increased under the stimulation of positive target cells CCRF-CEM-Luc and SUP-T1-Luc; the release of IFN-γ was slightly increased under the stimulation of positive target cells JVM-2- Under the stimulation of Luc-CD5 and MEC-1-CD5-Luc, the release amount of IFN-γ increased significantly; while under the stimulation of negative target cells CCRF-CEM-CD5 KO, the release amount of IFN-γ did not change significantly. The specific experimental results are shown in Figure 12.

Research conclusion: The average amount of IFN-γ released by CT125A showed an increasing trend when stimulated by the four positive target cells tested, but there was no statistically significant difference. There was no significant change in the release of IFN-γ under the stimulation of one strain of negative target cells measured.

Example 8 Study on Cetuximab-Mediated Clearance of CT125A Cells by Natural Killer Cells in Vitro

Research purpose: To investigate whether EGFRt, a molecular switch on the surface of CT125A cells, can mediate the clearance of CT125A cells by natural killer cells under the action of centuximab.

Research methods: Mainly examine the survival ratio of CAR ⁺ cells under the action of NK with different concentrations of cetuximab.

Research results: The killing data of NK derived from three test subjects showed that cetuximab can effectively kill CT125A. The three tested efficacy-target ratios were all effective and similar. The killing effect showed a dose-dependent relationship with the antibody. The EC50 was between 0.01-0.03nM and the maximum killing rate was 30%-70%. The specific experimental results are shown in Figure 13.

Research conclusion: Cetuximab can mediate the killing and clearance of CT125A by NK cells in vitro, proving that the molecular switch mechanism of EGFRt-CART is effective. It is consistent with the reported mechanisms of action of cetuximab: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement-mediated cytotoxicity (CDC). Some test subjects showed It shows that CT125A appears to cause suicide when the concentration of cetuximab is high, which may be related to the presence of a small amount of CAR-NK and CAR-NKT in the CT125A preparation (some NKT will express a small amount of CD16).

Example 9 Study on the efficacy of CT125A in immunodeficient mice bearing SUP-T1-Luci transplanted tumors

Research purpose: In this experiment, the test product CT125A injection was intravenously administered to NOG mice (NOD-Cg.Prkdc ^SCID IL-2rg ^tm1sug /JicCrl mice) transplanted with human T lymphoblastoma cells (SUP-T1-Luci). Purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.) to evaluate its inhibitory effect on tumor cell proliferation.

Research method: 65 female NOG mice were inoculated into the tail vein under sterile conditions with 0.2 mL of human T lymphoblastoma cell (SUP-T1-Luci) suspension (cell density 5.33×10 ⁶ cells/mL cell viability 100.00%) , 50 animals with appropriate tumor signals and similar weights were selected on the 4th day after vaccination, and randomly divided into 5 groups: cell protection solution group (10 animals), Mock-T group (10 animals, 9.87×10 ⁶ T cells/animal) , the test product low-dose group (10 animals, 0.3×10 ⁶ CAR-T cells/animal), the test product medium-dose group (10 animals, 1.0×10 ⁶ CAR-T cells/animal) and the test product high-dose group Group (10 animals, 3.0×10 ⁶ CAR-T cells/animal). All animals were administered a single injection into the tail vein, with the day of administration as D1, the second day after administration as D2, and so on.

After the first administration, general clinical observations were carried out twice a day. Before grouping, each animal was weighed once on D3, D7, D11, D14, D18, D21 and D23. Before grouping, all animals were weighed on D7, D14, D21 and D23. The animal live imager captures chemiluminescent signals. Lymphocyte subsets (CD45 ⁺ , CD45 ⁺ CD3 ⁺ and CD45 ⁺ CD3 ⁺ CD5 ⁺ ) and peripheral blood cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ).

Research results: 1) General clinical observation and survival rate

During the experiment, the cells in the cell protection solution group began to die on D17. The clinical symptoms before death included hunched back and/or weakness of the hind limbs. The remaining 3 animals were alive on D23. The Mock-T group began to die on D13. The clinical manifestations before death included hunched back, weakness of hind limbs, head tilt, and listlessness. The remaining 3 animals were alive on D23. The low, medium and high dose groups of the test product began to die on D21, D15 and D18 respectively, with 8, 9 and 8 animals remaining alive on D23 respectively. The remaining animals were observed and euthanized on D23. The specific results are shown in Figure 14.

2) Weight

After administration, each group of test products showed a steady upward trend. The body weight of the animals in the cell protection solution group and the Mock-T group showed a trend of first increasing and then decreasing. Compared with the cell protection solution group, there was no significant difference in the body weight of the animals in the Mock-T group during the experiment. The low, medium and high dose groups of the test product were significantly higher than the cell protection solution group from D18 onwards (P≤0.05). The results are shown in Figure 15.

3) Tumor cell proliferation (tumor cell bioluminescence intensity):

After administration, the tumor signal intensity in the cell protection solution group and Mock-T group continued to increase. The tumor signal intensity in the low, medium and high dose groups of the test product continued to decrease. Compared with the cell protection solution group, there was no significant difference in tumor signal intensity in the Mock-T group during the experiment. The tumor signal intensity in each group of the test product decreased significantly from D7 (P≤0.05), and the weakening of the tumor signal intensity decreased with the increase in the dosage of the test product, and there was an obvious dose-dependent relationship. The results are shown in Figure 16.

4) Flow cytometry

During the experiment, the CD45 ⁺ lymphocyte ratio in peripheral blood of the low-dose group of the test product basically showed a stable trend, reaching 0.4±0.3% on D23. Cell protection solution group, Mock-T group, test article medium and high dose group fluctuated slightly from D2 to D7, to D23 time rises. The ratio of peripheral blood CD45 ⁺ lymphocytes in each group of the test product showed a significant dose-dependent increase during the experiment.

During the experiment, the ratio of peripheral blood CD45 ⁺ CD3 ⁺ lymphocytes in the cell protection solution group decreased in a fluctuating manner. The Mock-T group showed a stable trend. The low, medium and high dose groups of the test product all showed a downward trend first and then an upward trend.

During the experiment, the ratio of peripheral blood CD45 ⁺ CD3 ⁺ CD5 ⁺ lymphocytes in the cell protection solution group showed an overall upward trend. The Mock-T group showed a fluctuating upward trend. The low, medium and high dose groups of the test product all showed an upward trend first and then a downward trend.

5) Cytokine detection

The peripheral blood IL-2 content in the cell protection solution group basically showed a stable trend, rising slightly to 0.36±0.09pg/mL on D23. The changes in peripheral blood IL-2 content in the Mock-T group were basically stable. The low, medium and high dose groups of the test product all showed a downward trend overall. The results are shown in Figure 17.

During the experiment, the IL-4 content in peripheral blood of the cell protection solution group increased from 0.00±0.00pg/mL on D2 to 58.55±9.42pg/mL on D23. The IL-4 content in peripheral blood of the remaining groups remained basically unchanged and remained stable near the baseline of 0.00±0.00pg/mL. The peripheral blood IL-6 content of animals in all groups was basically stable around the baseline of 0.00±0.00pg/mL. The results are shown in Figure 18-19.

During the experiment, the IL-10 content in peripheral blood of the cell protection solution group and the Mock-T group fluctuated slightly and was basically stable near the baseline. The high dose group of the test product increased slightly from 0.35±6.29pg/mL on D2 to 6.29±3.38pg/mL on D28. The peripheral blood IL-10 content of the remaining groups was basically stable near the baseline, with no obvious change trend. The results are shown in Figure 20.

During the experiment, the TNF-α content in peripheral blood of the cell protection solution group increased slightly to 0.29±0.26pg/mL on D23. The Mock-T group first increased and then decreased. The levels in the low, medium and high dose groups of the test product increased to D5 and then decreased. They all dropped to 0.00±0.00pg/mL at D7, and then slightly increased again at D23, showing fluctuating changes. The TNF-α content in the peripheral blood of each group of the test product showed a significant dose-dependent increase on D2 to D5 and D23, suggesting that the changes in the TNF-α content in the peripheral blood of each group of the test product may be dose-dependent. The results are shown in Figure 21.

During the experiment, the IFN-γ content in peripheral blood of the cell protection solution group was basically stable at 0.00±0.00pg/mL. The Mock-T group showed a continuously increasing trend. In the low-dose group of the test product, the level increased at D5 and then dropped to D23. The test product in the medium and high dose group increased to D5, then decreased to D7, and then increased to D23. The IFN-γ content in peripheral blood of each group of the test product at each time point showed a significant dose-dependent increase, suggesting that the changes in peripheral blood IFN-γ content of each group of the test product may be dose-dependent. The results are shown in Figure 22.

Conclusion: This experiment successfully established a NOG mouse vein transplantation tumor model of human T lymphoblastoma cells (SUP-T1-Luci). Under the conditions of this experiment, the test product CT125A injection was administered at a dose of 0.3×10 ⁶ , 1.0×10 ⁶ and 3.0×10 ⁶ CAR-T cells/cell in a single intravenous injection against human T lymphoblastoma SUP. -T1-Luci cell NOG mouse intravenous transplanted tumors have inhibitory and clearing effects on the growth, and the tumor inhibitory and clearing effects increase with the increase in the dosage of the test product, and there is an obvious dose-dependent relationship.

Example 10 CT125A systemic transplantation tumor model of human mantle cell lymphoma JVM-2-Luc-CD5 cell line NPG mouse drug efficacy experiments

Research purpose: To study the anti-tumor effect of CD5-targeted CAR-T cell injection CT125A on systemic xenograft tumors of CD5-positive human mantle cell lymphoma JVM-2-Luc-CD5 cell NPG mice.

Research methods:

1) Cell culture

The human mantle cell lymphoma JVM-2-Luc-CD5 cell suspension used in this experiment was homemade by the inventor. Commercially available JVM-2 cells were used to overexpress Luc to construct the JVM-2-Luc cell line, and then the CD5 Expressed in the JVM-2-Luc cell line, JVM-2-Luc-CD5 cells were obtained. The cells were collected and counted, resuspended in PBS, and inoculated into the tail veins of 50 NPG mice. Each mouse was inoculated with 3×10 ⁶ JVM-2-Luc-CD5 cells, resuspended in PBS, and inoculated into the mice via tail vein injection with an inoculation volume of 200 μL.

2) Animal grouping and dosing schedule

On the 4th day after vaccination, the mice will be randomly divided into 3 groups using E-workbook according to body weight and tumor size (fluorescence intensity), with 10 mice in each group. The grouping information and dosing schedule are shown in Table 5. CAR-T cells were injected on the day of grouping, and the day of grouping and administration was defined as Day 0.

Table 5. Grouping and dosing schedule

Note: 1. N represents the number of animals in each group; 2. Dosing volume: The dosing volume is adjusted to 200 μL/animal. 3.CT125A cells As mentioned above, CS10 is a commercially available cell cryopreservation solution and used as a blank control.

3) Drug preparation method

Drug preparation method: Take out 4 tubes of CT125A from the cryogenic storage place and quickly place them in a 37°C water bath. Shake them gently until the cells are completely thawed, and then aseptically remove the cells in a biosafety cabinet. Transfer CT125A cells to a suitable sterile container, mix by inverting upside down, and then take an appropriate amount of cells to count the cell density and viability. The density of CT125A viable cells is 2.58×10 ⁷ /mL (viability rate 85.52%), and the density of CAR+ (41.59%) viable cells is 1.07×10 ⁷ /mL, a total of 4mL, then add CS10 to dilute to the required density and adjust the volume to 4.28mL. Use the same method to adjust the Mock T cell density to be consistent with the CT125A total T cell density. The prepared cell suspension was placed on wet (crushed) ice and transported to the animal room for animal administration, and administration was completed within 2 hours after recovery.

4)Evaluation method

Cage-side observation: Observe the appearance and behavior of each mouse every day, from the beginning of grouping to the end of the experiment. All abnormal appearance and behavioral activities are recorded in the clinical observation form of Pengli Biological Laboratory.

In vivo imaging: before grouping (Day0), Day 5, Day 10, Day 15 and Day 18 after grouping, a total of 4 times. Before imaging, mice were maintained anesthetized with 3-4% isoflurane.

Animal weight: After grouping, measure and record the weight of mice twice a week until the end of the experiment.

Clinical observation of animal life: Observations are made once a day during the experiment, and the observation content includes but is not limited to the animal's mental state, diet, etc. All abnormal appearance and behavioral activities are recorded in the clinical observation form of Pengli Biological Laboratory and reported back in time. Give to the client. Observe whether there are allergic reactions, ascites, etc. after administration. If there are abnormalities or deaths, they need to be recorded and reported.

Sample collection: On Day 7 after grouping, collect approximately 100 μL of whole blood from each mouse and store it at -80°C; at the end of the Day 18 experiment, collect approximately 300 μL of whole blood from each mouse and store it at -80°C.

5)Euthanasia

During the experiment, if any one or more of the following conditions occur, the animal should be euthanized:

1) Health status: severe weight loss and body score (score based on SOP PL-ONC023) less than 2.

2) Impaired animal functions: The nodules interfere with normal animal functions (such as eating, drinking, or walking around).

3) Other disease symptoms.

Animals showing the above symptoms will be recorded as "abnormal".

At the end of the in vivo experiment on Day 18, all mice were asphyxiated with _CO2 and then killed by cervical dislocation. No samples will be collected from animals that have died before the end of the in vivo experiments.

6) Statistical analysis

Results will be presented as mean ± S.E.M. The body weight and tumor fluorescence intensity of mice in each group were graphed and statistically analyzed using the statistical software GraphPad Prism8 for Windows (Serial Number: GPS-1766552-EDSH-0204F). After the tumor fluorescence intensity is Log transformed (if there is zero value data, take 1 and perform Log transformation), use Two-way ANOVA (mixed model) and Turkey test to compare whether there is a significant difference in the fluorescence signal values between each group, P <0.05 is considered a significant difference.

Research result:

1) Animal weight and status

During the experiment, some animals in the CS10 control group and CT125A administration group experienced more than 15% weight loss and animal death. Among them, 2 mice out of 10 mice in the CS10 group died, and 1 mouse out of the CT125A administration group. The mouse died; on Day 18, the average weight loss rate of the CS10 control group was 8.96%, and the average weight loss rate of the CT125A administration group was 8.64%, indicating that the model itself would cause moderate weight loss in the animals, while CT125A had no effect on the animal weight. , no obvious toxicity was produced. The changes in body weight of animals in each group at each time point are shown in Figure 23, and the statistical data of body weight in each group at each time point are shown in Table 6.

Table 6. Body weight statistics of each group at each time point (g)

2) Tumor growth

Four days after inoculation of the human mantle cell lymphoma JVM-2-Luc-CD5 cell line, administration was carried out in groups. The day of administration is regarded as Day0. During the experiment, fluorescence imaging was performed on Day5, Day10, Day15 and Day18, and statistical analysis was performed on the tumor growth in each group of animals. The tumor growth and data statistics of each group of animals at each time point are shown in Figure 24 and Table 7.

During the experiment, the tumor fluorescence intensity of mice in the vehicle CS10 group (G1) first decreased and then increased; on Day0, the tumor fluorescence intensity was 8.77×10 ⁷ ±0.88×10 ^{7 p/s; on Day5, the tumor fluorescence intensity was 8.77×10 7 ±0.88×10 7} p/s. The tumor fluorescence intensity dropped to 1.19*×10 ⁷ ±0.17×*10 ⁷ p/s; after that, the tumor fluorescence intensity maintained an upward trend; by Day 18, the fluorescence intensity of the control group animals was 6.65×10 ⁷ ±2.57×10 ⁷ p/s ; The results show that this study successfully established a human mantle cell lymphoma JVM-2-Luc-CD5 cell systemic transplantation tumor model in NPG mice.

The animals in the G2 group were given a single dose of Mock T cells at a dose of 4.81×10 ⁶ T cells/mouse on Day 0 and the tumor fluorescence intensity was measured regularly. , the data showed that the change trend of tumor fluorescence intensity of animals in Mock T group (G2) was similar to that of the control group. By Day 18, the tumor fluorescence intensity was 1.36×10 ⁸ ±2.44×10 ⁷ p/s; the tumor fluorescence intensity at each time point was the same as that of the control group. There was no significant difference compared with the solvent control group CS10 during the same period.

After animals in the G3 group were given a single dose of CT125A cells at a dose of 2×10 ⁶ CAR ⁺ cells/mouse on Day 0, the tumor fluorescence intensity was measured regularly. The data shows that the tumor fluorescence intensity of animals in the CT125A administration group (G3) at each time point after administration decreased significantly compared with the day of grouping. The tumor fluorescence intensity on Day0 was 8.77×10 ⁷ ±9.08×10 ⁶ p/s. On Day 18, the tumor fluorescence intensity was 1.15×10 ⁷ ±1.15×10 ⁷ p/s. The tumor fluorescence intensity of animals in the CT125A administration group at each time point was significantly lower than that of the CS10 control group or Mock T group during the same period (P<0.01, see Table 7). And at the end of the experiment, 8 of the 9 surviving mice in the CT125A administration group had tumor fluorescence intensity of zero, while none of the surviving animals in the CS10 control group and Mock T group had tumor fluorescence intensity of zero.

Table 7. Tumor growth in each group at each time point (×10 ⁷ , Bioluminescence intensity, p/s)

in conclusion

In summary, under the conditions of this experiment, CT125A injection was administered as a single intravenous injection at a dose of 2×10 ⁶ CAR-T cells/mouse to treat human mantle cell lymphoma JVM-2-Luc-CD5 cell NPG mice. It has strong inhibitory and clearing effects on the growth of systemic transplanted tumors and is well tolerated by animals.

Example 11 Human membrane protein array research

Research purpose: To investigate the non-specific cross-reaction of CT125A tandem single domain antibody rabbit FC fusion protein RD125 61-42 rFc (composed by fusing the above-mentioned FHVH3VH1 and rFc) with human membrane proteins.

Research method: Using the membrane protein array platform (Membrane Proteome Array, MPA) of the American company Integral Molecular, which consists of about 6000 different human membrane protein clones, the CT125A single chain antibody rabbit FC fusion protein RD125 61-42 rFc and the expression of human Cell binding of membrane protein clones, screening out positive binding cells, and secondary confirmation of the results.

Research results: In the screening test, at a concentration of 5 μg/mL, RD125 61-42 rFc had a strong signal with cells expressing human CD5 clones and weak binding to a small number of human membrane protein cells. In the secondary concentration gradient confirmation test, it was confirmed that RD125 61-42 rFc has a dose-dependent strong binding to human CD5 membrane-expressing cells and a weak binding to CAMK1G calmodulin kinase membrane-expressing cells; at different concentrations, it binds to human CD5 membrane-expressing cells. The fluorescence value bound to CD5 membrane-expressing cells is about 40-100 times higher than the fluorescence value bound to CAMK1G membrane-expressing cells.

Research conclusion: Human membrane protein array detection results show that the CT125A single chain antibody rabbit FC fusion protein RD125 61-42 rFc only has weak non-specific binding to CAMK1G calmodulin kinase, indicating that the CT125A single domain antibody has good specificity.

Example 12. Determination of affinity of anti-CD5 sdAbs

Experimental purpose and principle:

The affinity between CD5sdAbs and antigens may have an important impact on the killing effect and duration of CAR-T in patients. In order to determine this important property, we used ForteBio's Octet molecular interaction technology to measure it. The biofilm interference technology used by the Octet system is a label-free technology that provides high-throughput biomolecule interaction information in real time. The instrument emits white light to the sensor surface and collects the reflected light. The reflection spectra of different frequencies are affected by the thickness of the light film layer of the biosensor. The reflected light of some frequencies forms constructive interference (blue), while others are affected by destructive interference. Interference (red). These interferences are detected by the spectrometer and form an interference spectrum, which is displayed as the phase shift intensity (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the shift of the interference spectrum in real time, and this shift directly reflects the thickness of the biofilm on the sensor surface, from which information on the interaction of biomolecules can be obtained. High-quality data can be used to measure the kinetic parameters of biomolecular interactions (Kon, Kdis and KD), providing important information for the research and development process.

Brief experimental steps:

1) Dilute anti-CD5 IgG (consisting of fusion of the VHH sequence of CD5 and human IgG4Fc) to 20μg/mL with loading buffer (1×PBS, pH 7.4, 0.01% BSA and 0.02% Tween 20), and incubate in the biosensor Load sample, about 0.8nM.

2) After a 60s equilibrium phase, the binding kinetics of CD5 antigen (Acro, CD5-H52H5) were monitored at various antigen concentrations (100 to 1.563nM). Proceed to 160 s for association and 300 s for dissociation at each concentration.

3) Wash 3 times with 10mM Glycine-HCl, pH 1.5 to regenerate the chip.

4) Analyze binding constants by using a 1:1 binding site model (Biacore X-100 evaluation software).

Experimental results:

Affinity refers to the strength with which a single molecule binds to its ligand, and is usually measured and reported through the equilibrium dissociation constant (KD), which can be used to evaluate and rank the strength of the interaction between two molecules. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactant. The smaller the KD value, the greater the affinity of the antibody for its target. As shown in Table 8, H65, FHVH1, FHVH3, FHVH3VH1, FHVH4 and FHVH2 can all bind to the CD5 antigen, and the affinity of FHVH3VH1 is slightly higher than that of H65, FHVH1, FHVH3, FHVH4 and FHVH2.

Table 8 Anti-CD5 IgG affinity determination

Example 13. Binding study of tandem single domain antibodies to CD5 ⁺ target cells

Research purpose: To examine the binding ability of CD5 single domain antibodies to CD5 ⁺ target cells.

Research methods: After incubating different concentrations of CD5 tandem single domain antibody rabbit Fc fusion protein 61-42-rFc (composed by fusing FHVH3VH1 with rFc) with CD5 ⁺ target cells, the cells were washed twice with PBS and then fluorescent dye-labeled Rabbit Fc antibody labels positive cells and detects them using FCM (Flow cytometry) method. By analyzing the relationship between the percentage of fluorescently labeled positive CD5 ⁺ target cells and different concentrations of CD5 tandem single domain antibodies, Graphpad Prism software Perform a fit to calculate the EC50 constant. This experiment was carried out three independent repetitions.

Research results: CD5 tandem single domain antibody rabbit FC fusion protein 61-42 rFc has high affinity with four strains of CD5 positive cells, Kd are: 2.99±0.35nM (CCRF-CEM-Luc); 4.02±0.92 nM (SUP-T1-Luc); 0.64±0.07nM (JVM-2-Luc-CD5); 1.14±0.16nM (MEC-1-CD5-Luc). The specific results are shown in Table 9 and Figure 25.

Table 9 The affinity between 61-42-rFc single domain antibody and various CD5 positive cells is summarized in the table.

Research conclusion: The CD5 tandem single domain antibody rabbit Fc fusion protein 61-42-rFc has stable, good and specific binding ability to the four strains of CD5-positive cells measured, with EC50 values ranging from 1 to 5nM.

Example 14: CD5-targeting autologous CAR-T cells carrying HSV-TK suicide gene

Methods and Materials

1. Sorting and activation of CD4 ⁺ T and CD8 ⁺ T cells

Resuscitate frozen healthy donor (specific information confidential) PBMC, a total of 1.0×10 ⁸ cells per tube, quickly thaw and resuspend in 8 ml of preheated Rinsing buffer, take a small amount of cell suspension for cell counting. Centrifuge the PBMC suspension at 300g (↑8↓8) for 10 minutes. After centrifugation, discard the supernatant and add 20ul/10 ⁷ anti-CD4 and anti-CD8 magnetic beads respectively. Mix well and place in a 4°C refrigerator for 20 minutes. During this period, flick the tube wall several times every 10 minutes to avoid cell precipitation. . After the incubation, add Rinsing buffer, rinse once, centrifuge (400g 10min↑8↓8), and resuspend the cells in 500μl Rinsing buffer. At the same time, place the LS sorting column on the Miltenyi magnetic sorting stand. After rinsing once with 2ml Rinsing buffer, add 500μl of cell suspension. After the cell suspension is exhausted, add 2ml of Rinsing buffer twice. on the LS column. Use 5 mL of Rinsing buffer to wash out the target cells from the LS column and collect them. After making appropriate dilutions, count the target cells. Take about 1×10 ⁵ cells to determine the purity of the sorted T cells by flow cytometry. Then centrifuge the cell suspension at 300g for 10 minutes, adjust the cell density to 1×10 ⁶ /ml with fresh T cell culture medium, and add the T cell activator TransAct at a concentration of 10ul/10 ⁶ cells for activation, 4 mL per well. Seed into a 12-well plate and culture in a 37°C, _CO2 incubator.

2. T cells were electroporated after 48 hours of activation.

Cells were collected in a centrifuge tube and centrifuged (300g for 15 min up 8 down 8); after completion, discard the supernatant, resuspend the cells together with an appropriate amount of compound electrolyte, and count the cells; prepare a corresponding amount of RNP (Cas9) based on the cell count results. Complex of protein and sgRNA), incubate at 37° for more than 10 minutes; at the same time, centrifuge the cells again. After the end, resuspend the cells with the corresponding amount of electroporation buffer, add the incubated RNP, mix gently, and then add the Lonza electroporation instrument. In the electroporation cup, select the program EH-115 for electroporation of activated T cells, electroporate, then immediately add a small amount of warmed T cell culture medium, put it in the incubator to recover for more than 15 minutes, and then remove the cell suspension from the electroporation cup. Transfer it to a suitable culture flask and add T cell culture medium to make the culture density 2M/ml.

3. Lentiviral transduction of CAR

Five hours after cell electroporation, lentiviral transduction of CAR was performed. Conduct viability testing and cell counting on the cell suspension, add the corresponding amount of lentivirus according to the cell counting results, with an MOI of 3, then add 1% DMSO transfer agent, mix gently, and continue culturing in a 37°C incubator. . After 24 hours, the medium was changed to remove the virus, and fresh medium was used to continue culturing T culture cells at a density of 1M/ml.

4.FACS (flow cytometry) detection

Take about 2×10^5 cell suspension in a 1.5ml centrifuge tube, centrifuge at 300g for 5 minutes, wash once with PBS+2% fetal calf serum buffer, completely discard the supernatant, and resuspend the cells in 100μl buffer. Then add 1 μl of the corresponding antibody, mix, and incubate in the dark at 4°C for 30 minutes. Add 100 ul of buffer, wash once, resuspend with 100 ul of buffer containing DAPI or 7AAD, and run on the machine for detection.

5. CAR-positive cell sorting

After counting the cells, collect the cells and centrifuge at room temperature (300g for 15 minutes, rise 8 and drop 8). After the end, discard the supernatant, resuspend the cells in Rinsing Buffer (80ul/10^7cells), and add CD5-PE antigen protein (3ul/10^6cells) ), incubate at four degrees in the dark for 50 minutes; after incubation, add 10ml of Rinsing Buffer to resuspend the cells, centrifuge at room temperature, discard the supernatant after completion, resuspend the cells in Rinsing Buffer (80ul/10^7cells), anti-PE-beads (1.5ul/10^6cells), incubate at 4 degrees in the dark for 30 minutes; after incubation, add 10ml of Rinsing Buffer to resuspend the cells, centrifuge at room temperature, and rinse the LS column with 3ml of buffer; after centrifugation, discard the supernatant and use Resuspend the cells in Rinsing Buffer (1*10^8/ml), pass through the column three times (1ml/column), then wash the column three times with 3ml of buffer, and finally use 5ml of Rinsing Buffer to blow out the positive cells on the column. Count, take a small amount of cells for FACS to detect the purity of CAR positive sorting, centrifuge the positive cells and culture them in a culture bottle with an appropriate amount of T cell complete culture medium.

Main reagents

Main consumables

14.1 Preparation of CD5 HSV-TK CAR-T cells

For the CD5 CAR-T switched by HSV-TK, the inventor first designed four structural molecules (Figure 26A): Among them, the core components of the CAR molecule (extracellular signal peptide-binder-transmembrane structure-intracellular costimulatory molecule) are all For the CD5 CAR-T that is the same as the tEGFR switch from the company where the inventor belongs, the inventor placed the HSV-TK switch in front and behind the CAR molecule and used two different lentiviral backbones.

Next, the inventors prepared CAR-T molecules of these four structures and evaluated which CAR-T structure was the best by comparing their activity rates, amplification speeds, CAR conversion efficiency, tumor killing functions, etc. . The preparation process of CAR-T is shown in Figure 26B: Generally, on Day 0, anti-CD4 and anti-CD8 microbeads are used to sort CD4- and CD8-positive T cells from cryopreserved recovery or fresh PBMC, and then TransAct The activator was activated for 48 hours. On day 2, electroporation was performed to knock out the CD5 protein. 4 hours after electroporation, lentiviral transduction of CAR was performed. On day 3, the medium was changed to remove the virus. On day 5, the proportion of CAR-positive cells was detected by FACS. On day 6, CD5-PE protein and Anti- PE magnetic beads will sort out the CAR-positive cells and continue to culture them. On days 8 and 10, the sorted cells will be tested for CD5-PE protein residue and purity of CAR-positive cells. When there is no residual CD5-PE protein and the number of cells is sufficient, can be frozen at any time.

The viability of the four CAR-T cells during the preparation process and the expansion rate of the total cells are shown in Figures 26C and 26D: the viability of the cells can be recovered quickly after transfection, and can generally reach 90% on day 5. There is no significant difference between the four CAR-Ts; the expansion speed of total cells on day 7, the speed of 2948 and 2949 is slightly higher than that of 2946 and 2947, there is no difference between 2948 and 2949, while between 2946 and 2947, 2946 expanded faster than 2947 after day7.

The results of the CAR conversion efficiency of the four CAR-Ts and the changes in the proportion of CAR-positive cells with the number of culture days are shown in Figure 27. From the perspective of 3 days after conversion, under the same MOI, the CAR conversion efficiency of 2948 and 2949 About twice as high as 2946 and 2947, the proportion of CAR-positive cells of the four CAR-Ts will increase with the increase of culture days, and the knockout efficiency of CD5 is above 96%.

To sum up, among the four CAR-T structural molecules, 2948 may be the best choice.

The inventor used 2946 and 2947 CAR-T cells to conduct a test of CAR-positive cell sorting, and the results are shown in Figure 28. After sorting, the positive cells are basically all CAR-positive, but there are still a lot of CAR-positive cells among the negative cells that have not been sorted. From a proportional point of view, the sorting efficiency of CAR-positive cells is very low; from the perspective of positive cells after sorting Judging from the MFI (mean fluorescence intensity) of the CAR-positive cells in the cells and negative cells, using antigen to sort CAR-positive cells can only sort out cells with very high CAR expression. It is speculated that this may be due to the interaction between the antigen protein and the CAR molecule. The binding force is relatively weak (3A).

Four days after sorting, the sorted CAR-positive cells were directly tested by flow cytometry without staining, and it was found that there was basically no antigen. protein-positive cells, and the cells were re-stained with the antigen protein and tested, and it was found that basically all cells can be bound by the antigen protein. This shows that the antigen protein bound during the previous sorting has been basically degraded, and the cells after sorting The purity of CAR-positive cells remained unchanged after 4 days of culture. This experiment shows that cells can be cryopreserved 4 days after CAR-positive cell sorting.

14.2 Testing of tumor killing function of anti-human CD5 CAR-T cells carrying HSV-TK suicide gene

Experiment on killing tumor cells in vitro using anti-human CD5 CAR-T (HSV-TK) cells constructed using pLVx vector

The anti-human CD5 CAR-T (HSV-TK) cells constructed using the pLVx vector can be preliminarily identified in vitro for their tumor-killing function. Generally, CD5 CAR-T (HSV-TK) and tumor target cells (stably expressing luciferase protein) are mixed at different effect-to-target ratios and co-cultured for 24 hours, and the expression level of luciferase protein in the cells after co-culture is detected. The survival of tumor cells is then used to determine the tumor-killing function of CD5 CAR-T (HSV-TK).

Figure 29 shows the tumor killing performance of CD5 CAR-T (HSV-TK) constructed and prepared using pLVx vector. Figure 29A shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5-knocked-out T cells. CD5-knocked-out T cells did not kill CCRF and CD5 KO CCRF when the effective-to-target ratio was 2-0.125. Figure 29B shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5 CAR-T cells. CD5 CAR-T cells have an obvious killing effect on CCRF cells. When the effective-target ratio is 2-0.25, the killing efficiency is close to 100%; CD5 CAR-T cells have no killing effect on CD5 KO CCRF when the effective-target ratio is 2-0.125. Figure 29C shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5 CAR-T (HSV-TK) cells constructed and prepared using pLVx vector. CD5 CAR-T (HSV-TK) cells have obvious effects on CCRF cells. The killing effect. When the effective-target ratio is 2-0.5, the killing efficiency is close to 100%; while for CD5 KO CCRF, there is no killing when the effective-target ratio is 2-0.125.

Experiment on killing tumor cells in vitro using anti-human CD5 CAR-T (HSV-TK) cells constructed using pCDH vector

The anti-human CD5 CAR-T (HSV-TK) cells constructed using the pCDH vector can be preliminarily identified in vitro for their tumor-killing function. Generally, CD5 CAR-T (HSV-TK) and tumor target cells (stably expressing luciferase protein) are mixed at different effect-to-target ratios and co-cultured for 24 hours, and the expression level of luciferase protein in the cells after co-culture is detected. The survival of tumor cells can be used to determine the tumor-killing function of CD5 CAR-T (HSV-TK).

Figure 30 shows the tumor killing performance of CD5 CAR-T (HSV-TK) constructed and prepared using pCDH vector. Figure 30A shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5-knocked-out T cells. CD5-knocked-out T cells did not kill CCRF and CD5 KO CCRF when the effect-to-target ratio was 2-0.125. Figure 30B shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5 CAR-T cells. CD5 CAR-T cells have an obvious killing effect on CCRF cells. When the effective-target ratio is 2-0.25, the killing efficiency is close to 100%; CD5 CAR-T cells have no killing effect on CD5 KO CCRF when the effective-target ratio is 2-0.125. Figure 30C shows the killing of target cells CCRF and negative control cells CD5 KO CCRF cells by CD5 CAR-T (HSV-TK) cells constructed and prepared using pCDH vector. CD5 CAR-T (HSV-TK) cells have obvious effects on CCRF cells. The killing effect. When the effective-target ratio is 2-0.5, the killing efficiency is close to 100%; while there is no killing effect on CD5 KO CCRF when the effective-target ratio is 2-0.125.

14.3 Inhibition of CAR-T by HSV-TK combined with ganciclovir (GCV)

HSV-TK can efficiently bind to GCV and monophosphorylate it, and then intracellular kinases diphosphorylate and triphosphorylate it. The structure of triphosphorylated GCV is very similar to intracellular nucleosides, so it will Competitively binds to DNA polymerase or breaks the ratio of the four nucleosides in cells to inhibit DNA synthesis, leading to cell death.

The inventor used different concentrations of GCV drugs to treat CD5 CAR-T (HSV-TK) (pLVx) cells and CD5 CAR-T cells respectively, and obtained the total number of cells and CAR positivity through cell counting and CAR positivity rate flow cytometry every 3 days. Cell change data. The results are shown in Figure 31. For CD5 CAR-T cells that do not contain HSV-TK structures, GCV concentrations of 0.3ug/ml, 1ug/ml, and 3ug/ml in the culture system have no effect on the expansion of the cells (Figure 31A) , and had no effect on the number of CAR-positive cells (Figure 31C); GCV did not affect the growth of CD5 CAR-T cells.

For CD5 CAR-T (HSV-TK) cells, GCV concentrations of 0.3ug/ml, 1ug/ml, and 3ug/ml in the culture system can effectively inhibit the expansion of CD5 CAR-T (HSV-TK) cells (Figure 31B) , the number of CAR-positive cells was significantly inhibited by GCV (Figure 31D); GCV can inhibit the growth of CD5 CAR-T (HSV-TK) cells at a lower concentration (0.3ug/ml).

In order to clarify whether the number of CD5 CAR-T cells carrying the HSV-TK structure will rebound after GCV removal, the inventors used CD5 CAR-T (HSV-TK) (pLVx) cells and CD5 CAR-T cell culture systems for the first 5 days. Add 1ug/ml GCV drug every day, start removing GCV on the 6th day, and add 1ug/ml GCV again on the 13th day. The change data of the total number of cells and CAR-positive cells were obtained through cell counting and CAR-positive rate flow cytometry every 3-4 days. The results are shown in Figure 32. For CD5 CAR-T cells that do not contain HSV-TK structures, the presence or absence of 1ug/ml GCV in the culture system has no effect on the expansion of the cells (Figure 32A). The CAR-positive cells of the cells There was no effect on the number of CD5 CAR-T cells (Figure 32C); GCV did not affect the growth of CD5 CAR-T cells. Consistent with previous experimental conclusions.

For CD5 CAR-T (HSV-TK) cells, 1ug/ml GCV in the culture system can effectively inhibit the expansion of CD5 CAR-T (HSV-TK) cells (Figure 32B). The number of CAR-positive cells in the cells was significantly increased by GCV. Inhibition (Figure 32D); when GCV was withdrawn on day 6, an increase in the number of CD5 CAR-T (HSV-TK) cells and CAR-positive cells was seen on day 9. When 1ug/ml GCV was added to the culture system again on the 13th day, the number of CD5 CAR-T (HSV-TK) cells and the number of CAR-positive cells decreased again. This experiment once again confirmed that CD5 CAR-T carrying HSV-TK can be effectively inhibited by GCV. When the GCV drug is withdrawn, the CD5 CAR-T (HSV-TK) cells that have not been cleared can gradually recover. These cells should It will still be suppressed when GCV is encountered again.

14.4: The tumor killing function of anti-human CD5 CAR-T cells carrying HSV-TK suicide gene in mice, and the confirmation of the killing effect of GCV on anti-human CD5 CAR-T cells carrying HSV-TK suicide gene in vivo

In order to confirm whether the conclusions in Sections 14.2 and 14.3 of this Example are applicable in the in vivo environment, that is, to confirm the tumor killing function of anti-human CD5 CAR-T cells carrying the HSV-TK suicide gene in the in vivo environment, and to confirm the effect of GCV on carrying the HSV-TK suicide gene in the in vivo environment. The inventor conducted this part of the animal experiment on the killing effect of HSV-TK suicide gene against human CD5 CAR-T cells.

NPG mice were injected with 1*10 ⁶ SUP_T1-luciferase tumor cells through the tail vein, and CAR-T cells were injected 4 days later. Blood was collected from the test mice regularly to detect the level of SUP_T1-luciferase, and survival and weight changes were recorded to obtain Know the clearance of tumor cells by CAR-T. At the same time, GCV administration was also carried out in two other CAR-T experimental groups. After treatment, 1 to 2 weeks after administration, the CAR-T levels in different tissues of the mice were detected to monitor the killing effect of GCV. At the same time, blood was collected from the mice to detect the levels of SUP_T1-luciferase to monitor the tumor removal after CAR-T. relapse.

The animal grouping settings are as follows:

Brief experimental steps:

1) On Day 0, inoculate 1*10 ⁶ SUP_T1-luciferase tumor cells into each experimental mouse;

2) Four days later, that is, on Day 4, 3*10 ⁶ CAR-T cells were injected into each mouse in the CAR-T experimental group. There were 12 mice in each experimental group. In addition, a CD5 KO T control group and a PBS control group were set up. The control group had 6 test mice in each group;

3) Seven days after the tumor cells were inoculated, that is, Day 7, GCV administration was started to the two GCV experimental groups, and peripheral blood was collected before administration;

4) 14 days after inoculation of tumor cells, that is, Day 14, GCV administration was stopped in the first GCV experimental group, and peripheral blood was collected from each mouse;

5) 21 days after the tumor cells were inoculated, that is, Day 21, GCV administration was stopped in the second GCV experimental group, and peripheral blood was collected from each mouse. At the same time, 3 mice in each group were killed and tissues were collected;

6) During the 21-50 days after inoculation of tumor cells, that is, from Day 21 to Day 50, peripheral blood and tissues of the remaining mice were collected every week; on Day 25, spleen samples were taken from the mice, and flow cytometry was used to detect the content of the spleen. CAR-T cells;

7) Every time blood is taken after Day 25, the luciferase reading value in the fresh blood is also measured on the day of blood collection to detect the level of tumor cells. After all mouse tissue samples are collected, the genome is extracted and the CAR-T cell vector copy number (VCN) is detected.

Experimental results:

1) As shown in Figure 33, 21 days after the injection of CAR-T cells, that is, on Day 25, fresh blood samples were taken from the mice in the control group and the CAR-T experimental group and the luciferase readings were measured. The luciferase readings of the three CAR-T groups were The reading values were all significantly lower than those in the control group, indicating that the levels of tumor cells in each CAR-T cell group were very low, and the anti-human CD5 CAR-T cells carrying the HSV-TK suicide gene had normal tumor killing function in the in vivo environment.

2) As shown in Figure 34, after the GCV administration treatment, on Day 25, fresh spleen samples were taken from the mice in the control group and the CAR-T experimental group and the cells were separated for flow cytometry detection. Experiments show that the CAR-T group without GCV treatment, that is, the G3 group, can detect the CAR-T cell population, that is, the CD3+CAR+ cell population. In the CAR-T group treated with GCV for 7 days, that is, the G4 group, and in the CAR-T group treated with GCV for 14 days, that is, the G5 group, almost no CAR-T cells were detected, indicating that GCV administration can significantly eliminate HSV-TK-carrying cells. Suicide gene anti-human CD5 CAR-T cells.

3) As shown in Figure 35, after the GCV administration treatment, in addition to flow cytometry detection, the inventor also used real-time quantitative PCR counting to detect the number of CAR-T vector copies in different tissue samples of mice. That is VCN. The detection experiment of peripheral blood samples showed that 7 days after GCV administration of mice in the CAR-T cell group, that is, on Day 14, the VCN of the CAR-T group without GCV treatment, that is, the G3 group, was significantly higher than that of the GCV administration group. Groups, namely G4 and G5 groups, indicate that GCV administration can more completely eliminate anti-human CD5 CAR-T cells carrying HSV-TK suicide genes in peripheral blood. Detection experiments on spleen and lung tissue samples showed that 14 days after GCV administration to the mice in the CAR-T cell group, that is, on Day 21, the VCN of the CAR-T group without GCV treatment, that is, the G3 group, was significantly higher than that of the GCV-treated mice. The drug treatment groups, namely the G4 and G5 groups, indicate that GCV administration can more completely eliminate anti-human CD5 CAR-T cells carrying HSV-TK suicide genes in the spleen and lungs.

4) The CAR-T group was treated with GCV for 7/14 days and then stopped. The anti-human CD5 CAR-T cells carrying the HSV-TK suicide gene had been cleared by GCV, and there was recurrence of residual tumor cells in the mice. As shown in Figure 36, 14 days after stopping GCV administration, some mice had tumor recurrence, indicating that GCV treatment can eliminate anti-human CD5 CAR-T cells carrying the HSV-TK suicide gene and lose the ability of CAR-T cells. After inhibition, tumor cells relapse.

Accordingly, this article provides at least the following technical solutions:

Scheme 1: Immune effector cells, which include:

2) Suicide genes and/or protein products encoded by suicide genes,

Wherein the CAR includes a CD5 binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and the CD5 binding domain includes one or more antibodies or antigen-binding fragments thereof that specifically bind to CD5 ,

The suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene.

Scheme 2: The immune effector cell as described in Scheme 1, wherein the HSV-TK is HSV-TK mut2; preferably, the HSV-TK mut2 includes the sequence shown in SEQ ID NO: 71 or a functional variant thereof.

Option 3: The immune effector cell as described in Option 1 or 2, wherein:

The antibody or antigen-binding fragment thereof includes heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3). The amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected. Any combination from:

(2) HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42, and SEQ ID NO: HCDR3 of the sequence shown in 43;

Scheme 4: The immune effector cell according to any one of schemes 1-3, wherein the CD5 binding domain includes at least two antibodies or fragments thereof that specifically bind CD5, and the antibodies or fragments thereof comprise HCDR1, HCDR2 and HCDR3 are independently selected from any one of the following combinations:

Embodiment 5: The immune effector cell as described in any one of Embodiments 1-4, wherein the CD5-binding domain includes a first antibody or an antigen-binding fragment thereof that specifically binds CD5 and a second antibody or an antigen-binding fragment thereof, so The HCDR1, HCDR2, and HCDR3 included in the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment are independently selected from any one of the following combinations:

(1) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42 and HCDR3 of the sequence shown in SEQ ID NO: 43;

(3) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains at least two of the HCDR1 of the sequence shown in SEQ ID NO: 67, the HCDR2 of the sequence shown in SEQ ID NO: 68, and the HCDR3 of the sequence shown in SEQ ID NO: 69 that specifically bind to CD5. Antibodies or their antigen-binding fragments are connected in series.

Embodiment 6: The immune effector cell according to any one of Embodiments 1-5, wherein the antibody is a single domain antibody.

Embodiment 7: The immune effector cell according to any one of Embodiments 1-6, wherein the CD5 binding domain includes at least two single domain antibodies, and the single domain antibodies are connected through a linker fragment; preferably, the linker fragment includes the sequence shown in SEQ ID NO: 25.

Scheme 8: The immune effector cell according to any one of Schemes 1-7, wherein the CD5 binding domain includes the sequence shown in SEQ ID NO: 33, 35, 37, 47, 57, 59, 61 or 63 or its sequence Functional variant.

Embodiment 9: The immune effector cell of any one of Embodiments 1-8, wherein the transmembrane domain includes a polypeptide from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; Preferably, the transmembrane domain includes the sequence shown in SEQ ID NO: 6 or functional variants thereof.

Embodiment 10: The immune effector cell according to any one of Embodiments 1-9, wherein the costimulatory domain includes a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS; Preferably, The costimulatory domain includes the sequence shown in SEQ ID NO: 8 or a functional variant thereof.

Scheme 11: The immune effector cell according to any one of Schemes 1-10, wherein the intracellular signaling domain comprises a signaling domain from CD3ζ; preferably, the intracellular signaling domain comprises SEQ The sequence shown in ID NO: 10 or its functional variant.

Scheme 12: The immune effector cell as described in any one of Schemes 1-11, wherein the CAR further comprises a hinge region, the hinge region connects the CD5 binding domain and the transmembrane domain; Preferably, The hinge region includes the sequence shown in SEQ ID NO: 4 or a functional variant thereof.

Option 13: The immune effector cell as described in any one of Schemes 1-12, wherein the CAR includes a CD8α signal peptide; preferably, the signal peptide includes the sequence shown in SEQ ID NO: 2 or a functional variant thereof .

Embodiment 14: The immune effector cell according to any one of Embodiments 1-13, wherein the nucleic acid sequence encoding the CAR and the suicide gene are located in the same nucleic acid molecule.

Embodiment 15: The immune effector cell according to any one of Embodiments 1-14, wherein the coding nucleic acid sequence of the CAR and the suicide gene are located in the same expression vector introduced into the immune effector cell.

Embodiment 16: The immune effector cell according to any one of Embodiments 1-15, wherein the expression vector is a lentiviral expression vector, such as pLVx vector or pCDH vector.

Scheme 17: The immune effector cell according to any one of Schemes 1-16, wherein a spliced peptide coding sequence is included between the nucleic acid sequence encoding the CAR and the suicide gene.

Scheme 18: The immune effector cell according to any one of Schemes 1-17, the suicide gene is located in the 5' direction or the 3' direction of the nucleic acid sequence encoding the CAR.

Option 19: The immune effector cell as described in any one of Schemes 1-18, wherein the cleavage peptide includes the amino acid sequence from the T2A peptide; preferably, the cleavage peptide includes the sequence shown in SEQ ID NO: 12 or functional variants thereof.

Embodiment 20: The immune effector cell according to any one of Embodiments 1-19, wherein the immune effector cell does not express CD5.

Embodiment 21: The immune effector cell according to any one of Embodiments 1-20, wherein the immune effector cell does not express TRAC gene and/or TRBC gene.

Embodiment 22: The immune effector cell according to any one of Embodiments 1-21, wherein the immune effector cell is selected from T lymphocytes B cells and natural killer (NK) cells.

Scheme 23: Isolated nucleic acid molecule, which includes the nucleic acid sequence encoding the CAR and the suicide gene described in any one of Schemes 1-22.

Scheme 24: The nucleic acid molecule as described in Scheme 23, wherein the coding nucleic acid sequence includes the sequence shown in SEQ ID NO: 32, 34, 36, 46, 56, 58, 60 or 62.

Option 25: The nucleic acid molecule as described in Scheme 23 or 24, wherein the cleavage peptide includes the amino acid sequence from the T2A peptide; preferably, the cleavage peptide includes the sequence shown in SEQ ID NO: 12 or a functional variation thereof. body.

Scheme 26: Expression vector, which includes the nucleic acid molecule described in any one of Schemes 23-25.

Embodiment 27: The expression vector according to Embodiment 26, wherein the vector is selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors, such as pLVx vector or pCDH vector.

Scheme 28: Method for preparing immune effector cells, which includes:

2) Introducing the nucleic acid molecule described in any one of Schemes 22-24 or the expression vector described in Scheme 25 or 26 into immune effector cells.

Scheme 29: The method as described in Scheme 28, wherein CRISPR/Cas9 technology is used to knock out the CD5 gene; preferably, the target sequence of the sgRNA used includes the sequence shown in SEQ ID NO: 70.

Option 30: Pharmaceutical composition, comprising:

1) The immune effector cell described in any one of Schemes 1-22, the nucleic acid molecule described in any one of Schemes 23-25, or the expression vector described in Scheme 26 or 27; and

2) Pharmaceutically acceptable adjuvants.

Scheme 31: Use of the immune effector cells described in any one of Schemes 1-22, the nucleic acid molecule described in any one of Schemes 23-25, or the expression vector described in Scheme 26 or 27 in the preparation of medicines, wherein said Medications are used to treat diseases or conditions associated with the expression of CD5.

Option 32: A method for treating diseases or conditions related to the expression of CD5, which includes a therapeutically effective amount of the immune effector cells described in any one of Schemes 1-22, and the nucleic acid molecules described in any one of Schemes 23-25. , the expression vector described in Scheme 26 or 27, or the pharmaceutical composition described in Scheme 30 is administered to a subject in need.

Embodiment 33: The method of Embodiment 32, further comprising administering ganciclovir GCV to a subject in need to kill the immune effector cells.

Embodiment 34: The use of embodiment 31 or the method of embodiment 32 or 33, wherein the disease or disorder associated with the expression of CD5 is cancer or malignant tumor.

Embodiment 35: The use of embodiment 31 or the method of embodiment 32 or 33, wherein the disease or disorder associated with the expression of CD5 is T lymphoblastic lymphoma or mantle cell lymphoma.

references:

1.Castella,M.,et al.,Development of a Novel Anti-CD19 Chimeric Antigen Receptor:A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.MolecμLar Therapy-Methods & Clinical Development,2019.12:p.134-144 .

2.Castella, M., et al., Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial.Frontiers in immunology, 2020.11:p .482.

3. Ortíz-Maldonado, V., et al., CART19-BE-01: A MμLticenter Trial of ARI-0001 Cell Therapy in Patients with CD19(+)Relapsed/Refractory Malignancies. Mol Ther, 2020.

4.Gill, S., et al., CD19 CAR-T cells combined with ibrutinib to induce complete remission in CLL.Journal of Clinical Oncology, 2017.35(15_suppl):p.7509-7509.

5. Neelapu, S.S., et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. New England Journal of Medicine, 2017.377(26):p.2531-2544.

6. Maude, S.L., et al., CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood, 2015.125(26):p.4017.

7.Jacobson,C.A.,CD19 Chimeric Antigen Receptor Therapy for Refractory Aggressive B-Cell Lymphoma.Journal of Clinical Oncology, 2018.37(4):p.328-335.

8.Kochenderfer,J.,et al.,Anti-CD19 chimeric antigen receptor T cells preceded by low-dose chemotherapy to induce remissions of advanced lymphoma. Journal of Clinical Oncology, 2016.34(18_suppl):p.LBA3010-LBA3010.

9.Grupp, S.A., et al., Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia. New England Journal of Medicine, 2013.368(16):p.1509-1518.

10.Hirayama, A.V., et al., The response to lymphodepletion impacts PFS in aggressive non-Hodgkin lymphoma patients treated with CD19 CAR-T cells. Blood, 2019: p.blood-2018-11-887067.

11.Davila, M.L., et al., Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci Transl Med, 2014.6(224):p.224ra25.

12. Jones, N.H., et al., Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1. Nature, 1986.323(6086): p.346-349.

13. Huang, H.J., et al., MolecμLar cloning of Ly-1, a membrane glycoprotein of mouse T lymphocytes and a subset of B cells: molecμLar homology to its human counterpart Leu-1/T1(CD5). Proceedings of the National Academy of Sciences of the United States of America,1987.84(1):p.204-208.

14.Mamonkin,M.,et al.,A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.Blood,2015.126(8):p.983-992.

15.Hill,L.C.,et al.,Safety and Anti-Tumor Activity of CD5 CAR T-Cells in Patients with Relapsed/Refractory T-Cell Malignancies.Blood,2019.134(Supplement_1):p.199-199.

The specific sequences of the amino acid and nucleotide sequences mentioned in the examples of this article and elsewhere are as follows:

Claims

Immune effector cells, which include:

1) Chimeric Antigen Receptor (CAR) and/or its encoding nucleic acid sequence; and

2) Suicide genes and/or protein products encoded by suicide genes,

Wherein the CAR includes a CD5 binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, and the CD5 binding domain includes one or more antibodies or antigen-binding fragments thereof that specifically bind to CD5 ,

The suicide gene is the herpes simplex virus thymidine kinase (HSV-TK) gene.
The immune effector cell of claim 1, wherein the HSV-TK is HSV-TK mut2; preferably, the HSV-TK mut2 includes the sequence shown in SEQ ID NO: 71 or a functional variant thereof.
The immune effector cell of claim 1, wherein:

The antibody or antigen-binding fragment thereof includes heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3). The amino acid sequences of HCDR1, HCDR2 and HCDR3 are selected. Any combination from:

(1) HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39, and HCDR3 of the sequence shown in SEQ ID NO: 40;

(2) HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42, and HCDR3 of the sequence shown in SEQ ID NO: 43;

(3) HCDR1 of the sequence shown in SEQ ID NO: 64, HCDR2 of the sequence shown in SEQ ID NO: 65, and HCDR3 of the sequence shown in SEQ ID NO: 66; and

(4) HCDR1 of the sequence shown in SEQ ID NO: 67, HCDR2 of the sequence shown in SEQ ID NO: 68, and HCDR3 of the sequence shown in SEQ ID NO: 69;

Preferably, the CD5-binding domain includes at least two antibodies or fragments thereof that specifically bind CD5, and the HCDR1, HCDR2, and HCDR3 contained in the antibodies or fragments thereof are independently selected from any one of the following combinations:

(1) HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39, and HCDR3 of the sequence shown in SEQ ID NO: 40;

(2) HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42, and HCDR3 of the sequence shown in SEQ ID NO: 43;

(3) HCDR1 of the sequence shown in SEQ ID NO: 64, HCDR2 of the sequence shown in SEQ ID NO: 65, and HCDR3 of the sequence shown in SEQ ID NO: 66; and

(4) HCDR1 of the sequence shown in SEQ ID NO: 67, HCDR2 of the sequence shown in SEQ ID NO: 68, and HCDR3 of the sequence shown in SEQ ID NO: 69;

Preferably, the CD5-binding domain includes a first antibody or an antigen-binding fragment thereof that specifically binds to CD5 and a second antibody or an antigen-binding fragment thereof, the first antibody or an antigen-binding fragment thereof and the second antibody or its antigen-binding tablet The HCDR1, HCDR2, and HCDR3 included in the segment are independently selected from any one of the following combinations:

(1) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 41, HCDR2 of the sequence shown in SEQ ID NO: 42 and HCDR3 of the sequence shown in SEQ ID NO: 43;

(2) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment includes HCDR1 of the sequence shown in SEQ ID NO: 64, HCDR2 of the sequence shown in SEQ ID NO: 65, and HCDR3 of the sequence shown in SEQ ID NO: 66; and

(3) The first antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 38, HCDR2 of the sequence shown in SEQ ID NO: 39 and HCDR3 of the sequence shown in SEQ ID NO: 40; the first The secondary antibody or its antigen-binding fragment contains HCDR1 of the sequence shown in SEQ ID NO: 67, HCDR2 of the sequence shown in SEQ ID NO: 68 and HCDR3 of the sequence shown in SEQ ID NO: 69;

Preferably, the at least two antibodies or antigen-binding fragments thereof that specifically bind CD5 are connected in series;

Preferably, the antibody is a single domain antibody;

Preferably, the CD5 binding domain includes at least two single domain antibodies, and the single domain antibodies are connected through a linker fragment; more preferably, the linker fragment includes the sequence shown in SEQ ID NO: 25;

Preferably, the CD5 binding domain includes the sequence shown in SEQ ID NO: 33, 35, 37, 47, 57, 59, 61 or 63 or a functional variant thereof;

Preferably, the transmembrane domain includes a polypeptide from a protein selected from the group consisting of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33 , CD37, CD64, CD80, CD86, CD134, CD137 and CD154; More preferably, the transmembrane domain includes the sequence shown in SEQ ID NO: 6 or a functional variant thereof;

Preferably, the costimulatory domain includes a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS; more preferably, the costimulatory domain includes the sequence shown in SEQ ID NO: 8 or its functional variants;

Preferably, the intracellular signaling domain includes a signaling domain from CD3ζ; more preferably, the intracellular signaling domain includes the sequence shown in SEQ ID NO: 10 or a functional variant thereof;

Preferably, the CAR also includes a hinge region, which connects the CD5 binding domain and the transmembrane domain; more preferably, the hinge region includes the sequence shown in SEQ ID NO: 4 or its function. sexual variants;

Preferably, the CAR includes a CD8α signal peptide; more preferably, the signal peptide includes the sequence shown in SEQ ID NO: 2 or a functional variant thereof;

Preferably, the immune effector cells are selected from T lymphocytes and natural killer (NK) cells.
The immune effector cell according to any one of claims 1 to 3, wherein the coding nucleic acid sequence of the CAR and the suicide gene are located in the same nucleic acid molecule; preferably, the coding nucleic acid sequence of the CAR and the suicide gene The gene is located in introduced into the same expression vector of the immune effector cells.
The immune effector cell according to any one of claims 1 to 4, wherein the expression vector is a lentiviral expression vector, such as pLVx vector or pCDH vector.
The immune effector cell according to any one of claims 1 to 5, wherein a cleavage peptide coding sequence is included between the nucleic acid sequence encoding the CAR and the suicide gene.
The immune effector cell according to any one of claims 1-6, wherein the immune effector cell does not express:

1)CD5; and/or

2)TRAC gene and/or TRBC gene.
An isolated nucleic acid molecule or expression vector, which includes the nucleic acid sequence encoding the CAR according to any one of claims 1-7 and a suicide gene.
A method for preparing immune effector cells, comprising:

1) Knock out (1) CD5 gene and/or (2) TRAC gene and/or TRBC gene of the immune effector cells; and

2) Introducing the nucleic acid molecule of claim 8 into immune effector cells.
A method for treating diseases or conditions related to the expression of CD5, which includes administering a therapeutically effective amount of the immune effector cell of any one of claims 1-7 or the nucleic acid molecule of any one of claim 8 to a person in need. subjects were administered the drug.