Enhancement of CAR T cell function by chimeric antigen receptor (CAR) spacer modification

Abstract

This invention provides a chimeric antigen receptor (CAR) containing an inactive and modifiable spacer that avoids off-target binding by Fc receptor (FcR)-expressing cells in CAR T cell therapy. [Solution] A chimeric antigen receptor (CAR) is provided, comprising an extracellular spacer containing at least one Ig-like C1 domain or a fragment or variant thereof of signal regulatory protein alpha (SIRP-alpha) and an antigen-binding domain, wherein the antigen-binding domain targets a tumor antigen, and the tumor antigen is selected from CD19 or HER-2.

Description

[Technical Field]

[0001] The present invention relates to a chimeric antigen receptor (CAR) comprising an inactive and modifiable spacer that avoids off-target binding by Fc receptor (FcR)-expressing cells in CAR T cell therapy. The spacer is based on the Ig-like C1 domain of the signal regulatory protein alpha. [Background technology]

[0002] Chimeric antigen receptor (CAR)-based T-cell therapy is a new treatment approach for hematological malignancies, showing remarkable results in the treatment of refractory and relapsed patients with acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and non-Hodgkin lymphoma. However, in this cutting-edge therapy, current CARs need to be refined to achieve highly efficient and tolerable cytotoxicity by preventing previously identified or potentially identified side effects. Fine-tuning CARs to avoid spacer-related interactions with off-target cells, or comparing optimal spacer modifications, has not been widely studied, and more precise insights are needed to modulate cytotoxic responses.

[0003] The structurally functional spacer between the cell membrane and the antigen-binding domain plays a crucial role in the fine-tuning of CAR-related antigen-independent or antigen-dependent signaling. Commonly used CARs consist of an immunoglobulin G (IgG) constant domain, an extracellular domain of CD8-alpha or CD28, and an extracellular component of NGFR (Casucci et al., 2018) or NKG2D (Sentman et al., 2014). In typical IgG-based CARs (IgG1-CARs), the IgG1-CH2 domain of the Fc region can interact with FcR-expressing myeloid cells, usually monocytes or macrophages, or with NK cells, potentially inducing myeloid activation and inflammation (Almaasbak et al., 2015). FcR binding to CARs can cause CAR T cell activation and destruction of FcR-expressing myeloid cells, sequesterion of CAR T cells in the lungs, activation-induced cell death (AICD), and an overall decrease in CAR T cell activity (Almaasbak et al., 2015; Hombach et al., 2010; Hudecek et al., 2015). Side effects, which are thought to be unwanted interactions with off-target cells, must be avoided in order to achieve functional therapeutic CAR T cells.

[0004] The signal-regulating protein (SIRP) family (also known as SHPS, CD172, etc.) consists of membrane proteins involved in regulating leukocyte function (van Beek et al., 2005). The extracellular domain of SIRP family members typically consists of one Ig-like V domain and two Ig-like C1 domains. SIRP-alpha (also known as SHPS-1, BIT, MFR, CD172a, p84) is a SIRP family member with a typical extracellular domain consisting of one Ig-like V domain, an Ig-like C1-1 domain, and an Ig-like C1-2 domain (van Beek et al., 2005). While the extracellular domain of SIRP-alpha is only known to bind to the target ligand CD47 via its N-terminal Ig-like V domain extracellularly (Hatherley D et al., 2009), the Ig-like C1 domain of SIRP-alpha is currently known as an inactive scaffold. [Overview of the project]

[0005] The present invention relates to a chimeric antigen receptor (CAR) comprising an extracellular spacer containing at least one Ig-like C1 domain or a fragment or variant thereof of signal regulatory protein alpha (SIRP-alpha).

[0006] In some embodiments, the Ig-like C1 domain of SIRP-alpha is selected from (i) the type 1 domain or a fragment or variant thereof relating to Sequence ID No. 1; or (ii) the type 2 domain or a fragment or variant thereof relating to Sequence ID No. 2.

[0007] In some embodiments, the extracellular spacer includes the type 1 domain and the type 2 domain of Ig-like C1 of SIRP-alpha.

[0008] In some embodiments, the extracellular spacer further comprises at least one multimerization domain, the multimerization domain or multiple multimerization domains selected from the IgG1 hinge region relating to SEQ ID NO: 4 or SEQ ID NO: 80, the IgG2 hinge region relating to SEQ ID NO: 81, the IgG3 hinge region relating to SEQ ID NO: 82, the IgG4 hinge region relating to SEQ ID NO: 83, and / or the extracellular domain of CD28 relating to SEQ ID NO: 3, and / or fragments and variants thereof. In some embodiments, the multimerization domain or multiple multimerization domains are selected from the IgG1 hinge region relating to SEQ ID NO: 4 or a fragment thereof and / or the extracellular domain of CD28 relating to SEQ ID NO: 3 or a fragment thereof. In some embodiments, the multimerization domain or multiple multimerization domains are selected from the IgG4 hinge region relating to SEQ ID NO: 83 or a fragment thereof and / or the extracellular domain of CD28 relating to SEQ ID NO: 3 or a fragment thereof.

[0009] In some embodiments, the extracellular spacer is located between the transmembrane domain and the antigen-binding domain. In some embodiments, the antigen-binding domain is a single-chain variable region (scFv).

[0010] In some embodiments, the extracellular spacer dimerizes the CAR by at least one disulfide crosslink. The extracellular CD28 contains one disulfide crosslink. The IgG hinge region contains two disulfide crosslinks. In some embodiments, the CAR is dimerized by one disulfide crosslink, two disulfide crosslinks, or three disulfide crosslinks.

[0011] The present invention also relates to a CAR comprising an extracellular spacer containing an amino acid sequence relating to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61.

[0012] In some embodiments, the CAR includes any of the following: an extracellular spacer domain, an antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and optionally a co-stimulatory domain.

[0013] In some embodiments, the antigen-binding domain of the CAR contains an antibody or a fragment thereof.

[0014] In some embodiments, the antigen-binding domain of the CAR includes a single-chain variable region fragment (scFv).

[0015] In some embodiments, the antigen-binding domain of the CAR targets a tumor antigen or cancer antigen. The tumor antigen can be selected from CD19, HER-2, BCMA, CD22, CS1, CD38, CD33, CD20, CD30, CD38, CD123, TAA, GD2, MSLN, EGFR, EBV, GPC3, MUC1, PSMA, and NY-ESO-1, as discussed by Yu et al., 2020 and Townsend et al., 2018. The tumor antigen targeted by the CAR of the present invention is preferably selected from CD19 or HER-2.

[0016] In some embodiments, the transmembrane domain of the CAR is selected from the transmembrane domain of a membrane protein. The transmembrane domain can be selected from CD28, CD8, CD8 alpha, OX40L receptor (also known as CD134), 4-1BB (also known as CD137), CD3, CD3 delta, CD3 gamma, CD3 epsilon, or CD3 zeta, or fragments thereof. In a preferred embodiment, the transmembrane domain of the CAR includes the transmembrane domain of CD28 or a fragment thereof, as described in SEQ ID NO: 23.

[0017] The intracellular signaling domain of CAR can be selected from the intracellular domains of CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD28, Fc gamma RIII, FcR cytoplasmic tail, or tyrosine kinase or fragments thereof. In a preferred embodiment, the intracellular signaling domain includes the intracellular domain of CD3 zeta or a fragment thereof, as described in SEQ ID NO: 25.

[0018] The CAR co-stimulatory domain can be selected from CD28, CD8, CD8 alpha, OX40L receptor (also known as CD134), 4-1BB (also known as CD137), KIR2DS2, ICOS, CD27, MYD88-D40, or fragments thereof or variants thereof. Preferably, the CAR co-stimulatory domain includes intracellular CD28 or a fragment thereof as described in SEQ ID NO: 24.

[0019] The present invention also relates to i. single-chain variable region fragment (scFv); ii. an IgG hinge domain; iii. a type 1 Ig-like C1 domain and / or a type 2 Ig-like C1 domain of signal regulatory protein alpha-1; iv. CD3 zeta; v. a CD28 transmembrane domain; vi. optionally, a CD28 extracellular domain and / or a CD28 intracellular domain a chimeric antigen receptor (CAR) comprising the foregoing components.

[0020] The present invention also relates to a CAR comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67.

[0021] The present invention further relates to a polynucleotide encoding any one of the CARs described above.

[0022] The present invention also relates to a vector comprising a polynucleotide encoding any one of the CARs described above.

[0023] The present invention also relates to a cell comprising any one of the CARs described above or any polynucleotide encoding the same. In some embodiments, the cell is a T cell.

[0024] The present invention further relates to a method for adjusting the length of a CAR by selecting at least two domains from the group consisting of (i) an IgG hinge domain, (ii) the type 1 Ig-like C1 domain of signal regulatory protein alpha-1, (iii) the type 2 Ig-like C1 domain of signal regulatory protein alpha-1, or (iv) a CD28 extracellular fragment for a spacer domain, whereby chimeric antigen receptors having various lengths are obtained.

[0025] In some embodiments, the extracellular spacer domain does not bind to an Fc receptor or has reduced binding affinity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] [Figure 1A] It is a schematic diagram of the kinetics of spacer-modified CAR and T cell expansion (n=3). It shows the CAR domains and designed structures in the schematic model. CAR 1S and CAR X1S are not shown in the figure. CAR 1S and CAR X1S respectively correspond to CAR 2S and CAR X2S, except that the type 2 domain of SIRP-alpha Ig-like C1 is replaced with the type 1 domain of SIRP-alpha Ig-like C1. [Figure 1B] It is a schematic diagram of the kinetics of spacer-modified CAR and T cell expansion (n=3). T cell viability was evaluated by trypan blue, and cells were counted on days 2, 3, 6, 8, and 10 before passaging using a Bio-Rad TC20 automatic cell counter. The results are shown as average values with standard deviation. [Figure 1C] It is a schematic diagram of the kinetics of spacer-modified CAR and T cell expansion (n=3). Fold expansion based on passaging was counted every 2 to 3 days, and fold expansion between subcultures was evaluated. Lines represent the average values (with SD) of different CARs. [Figure 1D]This is a schematic diagram (n=3) of the dynamics of spacer-modified CARs and T cell expansion. CAR expression on day 13 was analyzed by flow cytometry. The results are shown as individual data points and mean values ​​(line). [Figure 2A] Cellular phenotype after expansion. T cell products (n=3) were expanded for 13 days, and their phenotypes were analyzed by flow cytometry. Results are shown as points for individual data with mean values. Cellular phenotypes were determined by the following antibody combinations: T cells CD3+CD56-; NKT cells CD3+CD56+; NK cells CD3-CD56+; and other cells CD3-CD56-. [Figure 2B] Cellular phenotype after expansion. T cell products (n=3) were expanded for 13 days, and their phenotypes were analyzed by flow cytometry. Results are shown as points for individual data with mean values. CD4 and CD8-positive cell populations within the T cell population. [Figure 2C] Cellular phenotype after expansion. T cell products (n=3) were expanded for 13 days, and their phenotypes were analyzed by flow cytometry. Results are shown as individual data with mean values. CD4 and CD8-positive cell populations within the NKT cell population. [Figure 3A] Percentages of T and NKT cells with different memory phenotypes, exhaustion, and eventual differentiation. Results (measured by flow cytometry) are shown as mean values ​​with minimum and maximum values. On day 13 of expansion, cells were analyzed for memory phenotype. [Figure 3B] The proportion of T and NKT cells with different memory phenotypes, exhaustion, and ultimately divergent forms. Results (measured by flow cytometry) are shown as points for individual data with mean values. SCM-like and CM memory phenotypes were collectively classified as the "initial memory phenotype" group, and as the "effector phenotype" group, EM and Eff. [Figure 3C]Percentages of T and NKT cells with different memory phenotypes, exhausted, and ultimately differentiated. Results (measured by flow cytometry) are shown as points for individual data with mean values. Cells were analyzed as exhausted (PD-1 positive) and ultimately differentiated (CD57 positive) groups. [Figure 4A] T cell response and cytotoxicity against CD19-positive Nalm-6 cells. Mean (black horizontal line) and individual data points are shown. CAR T cells were co-cultured with Nalm-6 cells in a 1:1 E:T ratio for 18 hours. Cytokines were analyzed from the culture supernatant using a CBA array based on flow cytometry. [Figure 4B] T cell response and cytotoxicity to CD19-positive Nalm-6 cells. Mean (black horizontal line) and individual data points are shown. T cell degranulation by CD19-positive Nalm-6 cells was analyzed by staining CD107a in T cells after 4 hours of co-culture in the presence of the GolfiStop protein transport inhibitor. Results show the percentage of CD107a-expressing cells in T cells and, from that value, the percentage of CD4 and CD8-positive cells. [Figure 4C] T cell response and cytotoxicity against CD19-positive Nalm-6 cells. Luciferase activity was measured to analyze the in vitro cytotoxicity of CAR T cells against luciferase-expressing CD19+ Nalm-6 cells at various E:T ratios. Mean ± SD is shown. [Figure 5A] CAR T cell interactions with FcR-expressing THP-1 monocytes. CAR T cells were co-cultured with monocytes in a 1:1 ratio (effector cells:off-target cells). CAR T cell activation was measured by staining cell surface activation markers (CD25, CD69; flow cytometry). [Figure 5B]CAR T cell interactions with FcR-expressing THP-1 monocytes. CAR T cells were co-cultured with monocytes in a 1:1 (effector cells:off-target cells) ratio. CAR T cell activation was measured by measuring CAR T cell activation-inducing cytokines (CAR T cells: IFN-gamma and IL-2) using a flow cytometry-based CBA array. [Figure 5C] CAR T cell interaction with FcR-expressing THP-1 monocytes. CAR T cells were co-cultured with monocytes in a 1:1 ratio (effector cells:off-target cells). CAR T cell activation was measured by measuring monocyte activation-inducing cytokines (monocytes: IL-1 beta) using a flow cytometry-based CBA array. [Figure 6A] CAR expression and cytotoxicity of Jurkat T cells encoding CARs of various lengths. CAR expression was measured by flow cytometry after transduction (mock, CAR2S, and IgG CAR) or after transduction and positive selection (CAR M, CAR XM, CAR L, and CAR XL). Results are shown in contour plots. [Figure 6B] CAR expression and cytotoxicity of Jurkat T cells encoding CARs of various lengths. In vitro cytotoxicity was evaluated by measuring luciferase activation in CD19Nalm-6-Luc cells at various E:T ratios. Results are expressed as mean ± SD (n=3). [Figure 7] Cytotoxicity of CARs (Caribouflage-Associated Caribouflage) and CAR M cells, which possess HER-2 targeting domains, against HER-2-positive SKBR-3 breast cancer cells. Luciferase activity was measured by quantifying the in vitro cytotoxicity of HER-2-targeted CAR T cells against luciferase-expressing HER-2-positive SKBR-3 cells at various E:T ratios. Results are shown as mean ± SD. [Figure 8]Cytotoxicity of HER-2-targeting CAR M-expressing T cells against HER-2-positive SKBR-3 breast cancer cells. Luciferase activity was measured by quantifying the in vitro cytotoxicity of HER-2-targeting CAR T cells against luciferase-expressing HER-2-positive SKBR-3 cells at various E:T ratios. [Figure 9A] Cytotoxicity of CAR constructs containing cell expansion, CAR expression, and modified multimerization domains. Expansion of T cells derived from the same donor transduced with lentiviral vectors containing CAR constructs, CAR M, CAR XM, CAR M1, CAR XM2, CAR XM3, CAR M4, CAR 2S5, and CAR M6. Expansion folds correlated with the number of T cells at the start of the experiment. Expansion folds were measured on days 1, 3, 6, 8, and 10. [Figure 9B] Cytotoxicity of CAR constructs possessing cell expansion, CAR expression, and modified multimerization domains. Chimeric antigen receptor expression of CAR constructs. Chimeric antigen receptor expression on T cells was detected from the cell surface using antibodies. Vector copy number was measured by quantitative PCR from isolated genomic DNA. The percentage of viable cells and vector copy number is shown for CAR constructs, CAR M, CAR XM, CAR M1, CAR XM2, CAR XM3, CAR M4, CAR 2S5, and CAR M6. [Figure 9C] Cytotoxicity of CAR constructs possessing cell expansion, CAR expression, and modification multimerization domains. The cytotoxicity of CAR-effector T cells (CAR constructs, CAR M, CAR XM, CAR M1, CAR XM2, CAR XM3, CAR M4, CAR 2S5, and CAR M6) was investigated by co-culturing them with NALM-6 target cells in various ratios for 24 hours. Effector-target (E:T) ratios of 4:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, and 0.0625:1 were used. The amount of target-specific transgene (luciferase) was measured, and the percentage of killing of target cells only was determined. [Modes for carrying out the invention]

[0027] Features and embodiments of the present invention are described in this disclosure as non-limiting examples. This disclosure should not be considered as limiting to any specific compounds, compositions, methods, or uses described herein. Those skilled in the art should understand that obvious modifications and variations can be made to the present invention and its embodiments. The singular forms a, an, and the used in this application refer to one or more.

[0028] To carry out the present invention and its embodiments, those skilled in the art can use general techniques and methods of biology, molecular biology, microbiology, chemistry, biochemistry, immunology, and oncology. General techniques and methods are described in the literature, for example, in laboratory manuals and laboratory protocols. Such literature includes, for example, *Current Protocols in Cell Biology*, *Current Protocols in Immunology*, *Current Protocols in Molecular Biology*, *Current Protocols in Microbiology*, and *Molecular Cloning: A Laboratory Manual*. The technical and scientific terms used have the general meanings that are understood by those skilled in the art based on scientific literature and technical dictionaries.

[0029] Chimeric antigen receptors (CARs) refer to receptor proteins that bind to specific antigens and are involved in cell activation. CARs include an antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain, and optionally a costimulatory domain. Cells expressing CARs can bind to specific antigens and, as a result, activate themselves. CAR cells are preferably T cells, naive T cells, memory T cells, and effector T cells.

[0030] The spacer domain is the extracellular domain of a CAR. It is located between the transmembrane domain and the antigen-binding domain, connecting them. The spacer domain plays a role in fine-tuning CAR signaling.

[0031] An immunoglobulin (Ig)-based spacer domain is derived from or contains a fragment derived from the immunoglobulin Fc region. The immunoglobulin Fc region may be derived from IgG, IgM, IgA, or IgE. The IgG Fc region may be derived from IgG1, IgG2, IgG3, or IgG4. An IgG-based spacer domain contains CH2 and CH3 domains derived from the IgG Fc region. An IgG-based spacer domain having the IgG constant regions CH2 and CH3 is described, for example, in Hombach et al., 2010.

[0032] For example, members of the signal regulatory protein (SIRP) family, also known as SHPS and CD172, are membrane proteins involved in regulating leukocyte function (van Beek et al., 2005). The extracellular domain of SIRP family members typically consists of one Ig-like V domain and two Ig-like C1 domains. SIRP-alpha (also known as SHPS-1, BIT, MFR, CD172a, and p84) is a SIRP family member with a typical extracellular domain having one Ig-like V domain, an Ig-like C1 type 1 domain, and an Ig-like C1 type 2 domain (van Beek et al., 2005). The extracellular domain of SIRP-alpha is known only to bind to the target ligand CD47 at its N-terminus via its V-type Ig-like domain (Hatherley D et al., 2009), while the Ig-like C1 domain of SIRP-alpha is currently known as an inactive scaffold. The Ig-like domain typically has dimensions of approximately 4 × 2.5 × 2.5 nm. The amino acid sequence of SIRP-alpha exists in the UniProt database with accession number P78324.

[0033] Extracellular spacer domain The spacer domain of the present invention comprises at least one Ig-like domain of signal-regulating protein alpha (SIRP-alpha). Signal-regulating protein alpha is abbreviated as SIRP-alpha throughout this application. The SIRP-alpha Ig-like C1 domain is selected from a type 1 domain (SEQ ID NO: 1) and / or a type 2 domain (SEQ ID NO: 2). In one embodiment, the spacer comprises a type 1 domain of SIRP-alpha Ig-like C1. In another embodiment, the spacer comprises a type 2 domain of SIRP-alpha Ig-like C1. In yet another embodiment, the spacer comprises a type 1 domain and a type 2 domain of SIRP-alpha Ig-like C1. The spacer may contain multiple type 1 domains and / or type 2 domains of SIRP-alpha Ig-like C1.

[0034] The spacer may include a macromerization domain. The macromerization domain macromerizes the CAR monomer. In macromerization, the CAR forms a dimer, trimer, tetramer, pentamer, or macromer from the CAR monomer. Preferably, the CAR forms a dimer formed from two CAR monomers. The macromerization domain can form bonds between the monomers of the CAR. Preferably, the bonds between monomers are disulfide crosslinks. Preferably, the macromerization domain forms at least one, two, or three disulfide bonds between monomers. In some embodiments of the present invention, the macromerization domain of the spacer is selected from the group consisting of IgG1 hinge region, IgG2 hinge region, IgG3 hinge region, IgG4 hinge region, extracellular CD28 domain, or fragments or variants thereof. In some embodiments, the spacer includes a macromerization domain containing an IgG1 hinge region or a fragment thereof. In some embodiments, the spacer includes a macromerization domain containing an IgG4 hinge region or a fragment thereof. In preferred embodiments, the macromerization domain includes the amino acid sequence relating to SEQ ID NO: 4. In a preferred embodiment, the multimerization domain comprises the amino acid sequence relating to SEQ ID NO: 80 or SEQ ID NO: 83. The IgG1 hinge region or a fragment thereof binds to the SIRP-alpha-Ig-like C1 domain from one end and to the antigen-binding domain of the CAR from the other end. The IgG4 hinge region or a fragment thereof binds to the SIRP-alpha-Ig-like C1 domain from one end and to the antigen-binding domain of the CAR from the other end. Additional linker sequences may be used for combination. In another embodiment, the spacer comprises a multimerization domain comprising an extracellular CD28 domain or a fragment thereof. In a preferred embodiment, the multimerization domain comprises the amino acid sequence relating to SEQ ID NO: 3. The extracellular CD28 domain or a fragment thereof binds to the SIRP-alpha-Ig-like C1 domain from one end and to a transmembrane domain from the other end, for example, the transmembrane domain of CD28 (SEQ ID NO: 23). Additional linker sequences may be used for combination. The spacer may comprise multiple multimerization domains.In some embodiments, the spacer includes both the IgG1 hinge region and the extracellular CD28 domain. In some embodiments, the spacer includes both the IgG4 hinge region and the extracellular CD28 domain.

[0035] The spacer domain is positioned between the transmembrane domain and the antigen-binding domain, linking them together. The spacer domain plays a role in fine-tuning the antigen signaling of the CAR. In this invention, the length of the spacer can be adjusted using various domains and combinations thereof in the spacer. As a result, different spacer lengths optimize the binding of the CAR to its antigen. In some embodiments, the domains in the spacer can be selected from the type 1 domain of SIRP-alpha Ig-like C1, the type 2 domain of SIRP-alpha Ig-like C1, the extracellular CD28 domain and / or the IgG hinge region and / or fragments or variants thereof. Table 1 shows the amino acid sequences of various CAR spacers containing the selected domains resulting in spacers of different lengths (SEQ ID NOs: 10-18, 56-61).

[0036] In immunoglobulin (Ig)-based CARs, the CH2 domain interacts with the Fc receptor (FcR) on myeloid cells. Myeloid cells that express FcR include, for example, monocytes, macrophages, and NK cells. FcR binding to CARs can lead to CAR T cell activation, destruction of FcR-expressing myeloid cells, sequestration of CAR T cells in the lungs, activation-induced cell death (AICD), and an overall decrease in CAR T cell activity (Almaasbak et al., 2015; Hombach et al., 2010; Hudecek et al., 2015). Unwanted interactions with off-target cells and potential side effects must be avoided in order to obtain functional therapeutic CAR T cells.

[0037] In the present invention, the spacer domain comprises at least one Ig-like C1 domain or fragment thereof of signal regulatory protein alpha. The Ig-like C1 domain is selected from a type 1 domain and / or a type 2 domain. Preferably, the spacer comprises both a type 1 domain and a type 2 domain of Ig-like C1. The spacer domain of the present invention does not interact with FcR on myeloid cells that result in functional action. T cells having the CAR of the present invention do not result in CAR T cell activation caused by off-target binding, destruction of FcR-expressing myeloid cells, sequestration of CAR T cells in the lungs, activation-induced cell death (AICD), and an overall decrease in CAR T cell activity.

[0038] In a preferred embodiment of the present invention, the spacer domain comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, or a variant or fragment thereof. These variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 10 to 18. The amino acid sequences of the spacer domain are summarized in Table 1.

[0039] In a preferred embodiment of the present invention, the spacer domain includes SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61, or a variant or fragment thereof. These variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs. 56-61. The amino acid sequences of the spacer domain are summarized in Table 1.

[0040] The CAR spacer XS associated with Sequence ID No. 10 contains an IgG1 hinge region and a CD28 extracellular fragment.

[0041] The CAR spacer 1S associated with Sequence ID No. 11 includes an IgG1 hinge region and a type 1 domain of SIRP-alpha Ig-like C1.

[0042] The CAR spacer 2S associated with Sequence ID No. 12 includes an IgG1 hinge region and a type 2 domain of SIRP-alpha Ig-like C1.

[0043] The CAR spacer X1S associated with Sequence ID No. 13 contains an IgG1 hinge region, a type 1 domain of SIRP-alpha Ig-like C1, and a CD28 extracellular fragment.

[0044] The CAR spacer X2S associated with Sequence ID No. 14 contains an IgG1 hinge region, a type 2 domain of SIRP-alpha Ig-like C1, and a CD28 extracellular fragment.

[0045] The CAR spacer M associated with Sequence ID No. 15 includes an IgG1 hinge region, a type 1 domain of SIRP-alpha-Ig-like C1, and a type 2 domain of SIRP-alpha-Ig-like C1.

[0046] The CAR spacer XM associated with Sequence ID No. 16 includes an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular fragment.

[0047] The CAR spacer L associated with Sequence ID No. 17 includes an IgG1 hinge region, a type 2 domain of SIRP-alpha-Ig-like C1, a type 1 domain of SIRP-alpha-Ig-like C1, and a type 2 domain of SIRP-alpha-Ig-like C1.

[0048] The CAR spacer XL associated with Sequence ID No. 18 includes an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular fragment.

[0049] The CAR spacer M1 associated with sequence number 56 includes an IgG4 hinge region, a type 1 domain of SIRP-alpha-Ig-like C1, and a type 2 domain of SIRP-alpha-Ig-like C1.

[0050] The CAR spacer XM2 associated with Sequence ID No. 57 includes an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular fragment.

[0051] The CAR spacer XM3 associated with Sequence ID No. 58 includes an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, another IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular fragment.

[0052] The CAR spacer M4 associated with Sequence ID No. 59 includes an IgG4 hinge region, a type 1 domain of SIRP-alpha-Ig-like C1, an IgG4 hinge region, a type 2 domain of SIRP-alpha-Ig-like C1, and an IgG4 hinge region.

[0053] The CAR spacer 2S5 associated with Sequence ID No. 60 includes an IgG4 hinge region, a type 2 domain of SIRP-alpha-Ig-like C1, and another IgG4 hinge region.

[0054] The CAR spacer M6 related to sequence number 61 includes a type 1 domain and a type 2 domain of SIRP-alpha-Ig-like C1.

[0055] All of the above CAR spacers may include linker sequences that connect the domains to each other. All CAR spacers and their amino acid sequences are summarized in Table 1.

[0056] antigen-binding domain The antigen-binding domain of a chimeric antigen receptor recognizes an antigen. The antigen-binding domain of a CAR binds to the epitope of the antigen. The antigen-binding domain may include a protein, peptide, or mimetics thereof that binds to the antigen. In some embodiments, the antigen-binding domain is an antibody or a functional fragment thereof. An antibody refers to an immunoglobulin that specifically binds to the epitope of an antigen. An antibody may be a monoclonal antibody or a polyclonal antibody. Antibodies or functional fragments thereof include, but are not limited to, chimeric antibodies, humanized antibodies, bispecific antibodies, nanobodies, camelid antibodies, antigen-binding fragments (Fab), bivalent Fab regions (F(ab')2), single-chain antibody fragments (scAb)Fv, single-chain variable region fragments (scFv), and bivalent scFv (sc(Fv)2). In some embodiments, the antigen-binding domain includes a single-chain variable region fragment (scFv). scFv includes a variable light chain variable (VL) and a variable heavy chain (VH).

[0057] Various antigens are known to be involved in cancer. Cancer-associated antigens may be antigens expressed by cancer cells. Cancer-associated antigens may be overexpressed by cancer cells. Cancer-associated antigens may be the products of mutated genes or the products of normal genes expressed in amounts that can be targeted on cancer cells using CARs. Cancer-associated antigens may be proteins, peptides, carbohydrates, glycoproteins, glycolipids, proteoglycans, proteolipids, or combinations thereof. Several cancer-associated antigens have been reviewed by Townsend et al., 2018 and Yu et al., 2020.

[0058] In some embodiments, the antigen-binding domain of the CAR binds to a cancer-related antigen. The cancer-related antigen can be selected from, for example, known cancer-related antigens. Such antigens are reviewed in Townsend et al., 2018 and Yu et al., 2020. In some embodiments, the antigen-binding domain binds to CD19. In some embodiments, the antigen-binding domain that binds to CD19 is a single-chain variable region fragment (scFv). In some embodiments, the antigen-binding domain that binds to CD19 is an scFv containing SEQ ID NO: 22 or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 22. In some embodiments, the antigen-binding domain binds to HER-2. In some embodiments, the antigen-binding domain that binds to HER-2 is a single-chain variable region fragment (scFv). In some embodiments, the antigen-binding domain that binds to HER-2 is an scFV containing SEQ ID NO: 53 or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 53.

[0059] transmembrane domain The transmembrane domain of a CAR may be selected from or derived from any transmembrane domain of a membrane protein. The transmembrane domain of a CAR may, for example, be the transmembrane domain of CD28, CD8, CD8 alpha, OX40L receptor (also known as CD134), 4-1BB (also known as CD137), CD3, CD3 delta, CD3 gamma, CD3 epsilon, or CD3 zeta. In some embodiments, the transmembrane domain of a CAR is the transmembrane domain of CD28 or a fragment thereof or a variant thereof. In some embodiments, the transmembrane domain of a CAR includes the amino acid sequence relating to SEQ ID NO: 23.

[0060] Signal transduction domains CAR may include an intracellular signaling domain. The intracellular signaling domain may be cytoplasmic. The intracellular signaling domain of CAR mediates signals that result in effector function in cells expressing CAR. For example, the intracellular signaling domain of CAR can mediate CAR signaling to T cell activation. The intracellular signaling domain can be selected from the intracellular domains of CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD28, Fc gamma RIII, FcR cytoplasmic tail, and tyrosine kinases. In some embodiments, the intracellular signaling domain includes CD3 zeta or a fragment thereof. In some embodiments, the intracellular signaling domain includes the amino acid sequence or a fragment thereof relating to SEQ ID NO: 25.

[0061] Co-stimulatory domain CAR optionally contains one or more co-stimulatory domains. The co-stimulatory domains are cytoplasmic and can influence cell proliferation and phenotypic differentiation. The co-stimulatory domains of CAR can be selected from, for example, CD28, CD8, CD8 alpha, OX40L receptor (also known as CD134), 4-1BB (also known as CD137), KIR2DS2, ICOS, CD27, MYD88-D40 or its fragments or variants. In some embodiments, the co-stimulatory domain of CAR includes intracellular CD28 or its fragments or variants. In some embodiments, the co-stimulatory domain of CAR includes the amino acid sequence corresponding to SEQ ID NO: 24.

[0062] In some embodiments, the intracellular or cytoplasmic region of the CAR includes an intracellular signaling domain and a co-stimulatory domain. In some embodiments, the intracellular region of the CAR includes a CD3 zeta or a fragment thereof and an intracellular CD28 domain or a fragment thereof. In some embodiments, the cytoplasmic region of the CAR includes the amino acid sequence or a fragment thereof relating to SEQ ID NO: 24 and the amino acid sequence or a fragment thereof relating to SEQ ID NO: 25.

[0063] CAR A CAR comprises an antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain, and optionally a costimulatory domain. The CARs of the present invention can be selected from the amino acid sequences relating to SEQ ID NOs. 26, 27, 28, 29, 30, 31, 32, 33, 34, or 54, or their variants or fragments. These variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs. 26-34 or 54. The structures and amino acid sequences of the CARs are summarized in Table 1.

[0064] The CARs of the present invention can be selected from the amino acid sequences relating to SEQ ID NOs. 62, 63, 64, 65, 66, or 67, or their variants or fragments. These variants have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs. 62-67. The structures and amino acid sequences of the CARs are summarized in Table 1.

[0065] The CAR XS associated with Sequence ID No. 26 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region and an extracellular CD28 fragment as a spacer domain, a CD28 fragment as a transmembrane domain, an intracellular CD28 fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0066] The CAR 1S associated with Sequence ID No. 27 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region and a type 1 domain of SIRP-alpha-Ig-like C1 as a spacer domain, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0067] The CAR 2S associated with Sequence ID No. 28 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region and a type 2 domain of SIRP-alpha-Ig-like C1 as a spacer domain, a CD28 fragment as a transmembrane domain, an intracellular CD28 fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0068] The CAR X1S associated with Sequence ID No. 29 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a type 1 domain of SIRP-alpha Ig-like C1, and an extracellular domain of CD28 as spacer domains, a CD28 fragment as a transmembrane domain, an intracellular fragment of CD28 as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0069] The CAR X2S associated with Sequence ID No. 30 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha Ig-like C1 type 2 domain, and a CD28 extracellular domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0070] The CAR M associated with Sequence ID No. 31 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, and a SIRP-alpha-Ig-like C1 type 2 domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0071] The CAR XM associated with Sequence ID No. 32 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0072] The CAR L associated with Sequence ID No. 33 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, a SIRP-alpha-Ig-like C1 type 1 domain, and a SIRP-alpha-Ig-like C1 type 2 domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0073] The CAR XL associated with Sequence ID No. 34 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0074] The HER-2 CAR M associated with Sequence ID No. 54 contains an scFv that binds to HER-2 as an antigen-binding domain, an IgG1 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, and a SIRP-alpha-Ig-like C1 type 2 domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0075] CAR M1, associated with Sequence ID No. 62, contains an scFv that binds to CD19 as an antigen-binding domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, and a SIRP-alpha-Ig-like C1 type 2 domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0076] CAR XM2, associated with Sequence ID No. 63, contains an scFv that binds to CD19 as an antigen-binding domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0077] CAR XM3, associated with Sequence ID No. 64, contains an scFv that binds to CD19 as an antigen-binding domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, and a CD28 extracellular domain as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0078] CAR M4, associated with Sequence ID No. 65, contains an scFv that binds to CD19 as an antigen-binding domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 1 domain, an IgG4 hinge region, a SIRP-alpha-Ig-like C1 type 2 domain, and an IgG4 hinge region as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0079] CAR 2S5 associated with Sequence ID No. 66 contains an scFv that binds to CD19 as an antigen-binding domain, an IgG4 hinge region and a type 2 domain of SIRP-alpha-Ig-like C1 and an IgG4 hinge region as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0080] CAR M6, associated with Sequence ID No. 67, contains an scFv that binds to CD19 as an antigen-binding domain, a type 1 domain and a type 2 domain of SIRP-alpha-Ig-like C1 as spacer domains, a CD28 fragment as a transmembrane domain, a CD28 intracellular fragment as a co-stimulatory domain, and a CD3 zeta fragment as an intracellular signaling domain.

[0081] All of the above CARs may contain linker sequences that connect the domains to each other. All of these CARs and their amino acid sequences are summarized in Table 1.

[0082] The CAR of the present invention has a signal-regulating protein alpha (SIRP alpha) system scaffold to provide an inactive and modifiable universal spacer that avoids off-target binding to Fc receptor (FcR)-expressing cells in CAR T cell and other cell therapies. Off-target binding to myeloid cells via FcR leads to interference with CAR T cells, decreased cytokine production, and overall dysfunction of CAR T cells.

[0083] All novel CARs with a SIRP Alf frame are CD4 + It exhibited a small change in the CD4:CD8 ratio favorable to the population, and nevertheless possessed comparable cytotoxicity and functionality to conventional IgG-based CARs.

[0084] T cells carrying SIRP-alpha CARs did not show activation levels after co-culture with THP-1 monocytes, unlike T cells with hIgG-CH2CH3 CARs expressing high levels of the initial activation marker CD69, IL-2, and IFN-gamma, which acted as a control. In contrast to T cells with IgG CARs, monocyte activation, as measured by IL-beta production, was also avoided in SIRP-alpha CAR T cells.

[0085] Polynucleotides and vectors This invention relates to a polynucleotide encoding a chimeric antigen receptor. The polynucleotide may be DNA or RNA or modified DNA or modified RNA or a nucleic acid analog. The polynucleotide may be single-stranded or double-stranded. The polynucleotide of this invention can be isolated, purified, recombinantly produced or synthesized by any method available to those skilled in the art. The nucleosides of the polynucleotide may be chemically modified. The nucleic acid analog is a compound structurally similar to DNA and RNA. The nucleic acid analog may be, for example, peptide nucleic acid (PNA), locked nucleic acid (LNA), cross-linked nucleic acid (BNA), or morpholino. The polynucleotide may contain one or more nucleoside analogs.

[0086] It should also be understood that similar nucleic acid sequences can be encoded by alternative polynucleotide sequences. Codon optimization in the present invention was performed using homosapiens codons with estimated probabilities based on frequency analysis in endogenous receptors. In some embodiments of the present invention, the polynucleotide sequence encoding the CAR spacer can be selected from SEQ ID NOs. 35, 36, 37, 38, 39, 40, 41, 42, or 43. In some embodiments of the present invention, the polynucleotide sequence encoding the CAR spacer can be selected from SEQ ID NOs. 68, 69, 70, 71, 72, or 73. In some embodiments of the present invention, the polynucleotide sequence encoding the CAR spacer can be selected from SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 52, or 55. In some embodiments of the present invention, the polynucleotide sequence encoding the CAR spacer can be selected from SEQ ID NOs. 74, 75, 76, 77, 78, or 79.

[0087] The CAR-coding polynucleotide of the present invention may form an expression cassette. The expression cassette contains genetic information for encoding the CAR of the present invention. The expression cassette comprises a polynucleotide sequence encoding the CAR of the present invention. The expression cassette may include a coding sequence for an antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain, and optionally a costimulatory domain. In addition to the coding sequence, the expression cassette may include a sequence selected from a promoter sequence, an enhancer sequence, a translation stop sequence, and a transcription termination sequence. The expression cassette encoding the CAR of the present invention can be introduced into host cells by viral or nonviral means.

[0088] In nonviral methods, CAR-coding polynucleotides are introduced into host cells by methods based on, for example, opening the lipid membrane of the target cell by electric current and / or binding the polynucleotide to the lipid envelope. The expression cassette may be a plasmid encoding the CAR or as mRNA encoding the CAR. The expression cassette may include parts that enable integration into the host cell. Any available nonviral gene delivery method can be selected by those skilled in the art. Such methods include, for example, transfection and nucleofection methods, liposomes, cationic agents, and the use of electroporation. Nonviral methods and their uses are reviewed in Harris et al., 2020 and Riedl et al., 2018.

[0089] In the viral method, a viral vector is used to introduce the CAR-coding nucleotides of the present invention into host cells. The viral vector may be, for example, a retroviral vector, a lentiviral vector, or an adenoviral vector. The viral vector can be prepared using a plasmid containing an expression cassette comprising CAR-coding material, packaging material, and envelope-related material. Plasmids can be selected from, for example, pRRL.SIN-19, RSV-rev, pMDLg / pRRE, and pMD.G. Other expression cassette materials can be selected from chimeric 5'LTR-packaging signal-REV-response element-promoter-transgene cassettes, REV expression plasmids, matrix and expression vectors for precursor proteins for capsids and nucleocapsids, and for precursors for reverse transcriptase and integrase components, and expression vectors for envelope proteins such as VSV-G. Such plasmids are introduced into host cells, resulting in the production of self-inactivating viral particles containing the CAR expression cassette insert. Such vectors can integrate the cassette into the recipient cell genome. Those skilled in the art may use available viral-based methods to introduce the polynucleotide encoding the CAR of the present invention into host cells. Viral vectors and related methods are described, for example, in reference to Dull et al., 1998 and Levine et al., 2016.

[0090] cell The host cell of the present invention means a cell expressing the CAR of the present invention. The polynucleotide encoding the CAR of the present invention can be introduced into the host cell by a viral or nonviral method. The host cell may be a eukaryotic cell or a prokaryotic cell. A prokaryotic cell may be, for example, a bacterial cell. A eukaryotic cell may be, for example, an animal cell, a plant cell, a fungal cell, or an insect cell. The host cell may be a cultured cell line. Such a cell line may be, for example, NK92 or Jurkat T cells. The host cell can be isolated from living organisms such as animals, plants, fungi, or insects. Preferably, the host cell is isolated from a human. The host cell may be, for example, a blood cell, a nerve cell, an epithelial cell, an endothelial cell, or a hepatocyte. Preferably, the host cell is a blood cell, and more preferably a leukocyte. The host cell may be a leukocyte selected from neutrophils, eosinophils, basophils, lymphocytes, and monocytes. The host cell may be a leukocyte selected from natural killer cells (NK), T lymphocytes (T cells) and / or B lymphocytes (B cells) or plasma cells. Preferably, the host cell of the present invention is a T cell. The T cell is a helper T cell (T H ) cells, cytotoxic T(T C ) cells, regulatory T (T reg ) cells, which may be natural killer T (NKT) cells. T cells have specific cell surface molecules, such as T cell CD3, T H Cell CD4,T cCells can express CD8. Various memory phenotypes include naive T cells, stem cell memory-like T (TSCM-like) cells, central memory T (TCM) cells, stem cell memory T (TSCM) cells, effector T (Teff) cells, and effector memory T (TEM) cells. Memory phenotypes can be identified based on cell surface molecule expression, such as CD95, CD45RO, CD45RA, CD27, etc. Memory T cells and their cell surface markers are summarized in Table 2. Memory T cells can express CD4 or CD8. The host cells may consist of a single cell type or a population of different cell types, preferably a population consisting of a specific T cell type or a specific NK cell type, or a population consisting of multiple T cell types and / or multiple NK cell types. In the present invention, the host cells may be a population of different cell types, for example, peripheral blood mononuclear cells isolated from a blood sample. The host cells may be T cells isolated from peripheral blood mononuclear cells. T cells are understood as cells that express CD3 on their surface. Cells may include natural killer T (NKT) cells, various T cell phenotypes, memory T cells, helper T cells, effector T cells, and NK cells. Specifically, cells may express cell surface markers such as CD3, CD4, and / or CD8. Populations of different cell types within a cell population may vary.

[0091] The cell population may include T cells and NKT cells. Preferably, the host cell population contains more than 80%, more than 86%, or more than 90% T cells. Preferably, the host cell population contains less than 15%, less than 13%, or less than 9% NKT cells. In a preferred embodiment, the host cell population contains more than 86% T cells and less than 13% NKT cells. The T cells of the host cells may include, for example, CD4-positive and CD8-positive cells. The host cell population may also contain T cells, with less than 40% of the cells being CD57-positive and / or PD-1-positive.

[0092] The CAR of the present invention, a polynucleotide encoding a spacer-modified CAR, a vector containing a polynucleotide encoding a spacer-modified CAR, and / or cells expressing the CAR of the present invention can be used to treat diseases associated with antigens targeted by the antigen-binding domain of the CAR. When the CAR binds to an antigen, it causes cytotoxicity to target cells expressing the antigen. Cells expressing the CAR of the present invention can be used in cell therapy for cancer diseases, preferably in the treatment of refractory and relapsed patients with hematological malignancies, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and non-Hodgkin lymphoma. Target antigens of CAR-expressing cells, preferably T cells, may be, for example, CD19, HER-2, and other cancer-related target antigens selected from cancer-related antigens reviewed, for example, Townsend et al., 2018 and Yu et al., 2020. Therapeutic CAR T cells can be used in cancer immunotherapy. Therapeutic CAR T cells may be autologous or allogeneic. Autologous cells are isolated from the patient, a polynucleotide encoding the CAR is introduced into those cells via a vector, and the cells expressing the CAR are then returned to the patient. Allogeneic cells are isolated from a different individual but are genetically similar to the patient's cells.

[0093] CAR-expressing cells, preferably T cells, may be administered to a patient in a pharmaceutical composition. In addition to CAR-expressing cells, the pharmaceutical composition may also contain other pharmaceutically active agents, preservatives, and / or buffering agents. [Examples]

[0094] Example 1: Materials and Method CAR design The sequences of the FMC63 antibody clone variable regions (gene bank: immunoglobulin light chain, variable region; CAA74660.1 and immunoglobulin heavy chain, variable region; CAA74659.1) were modified to design CD19-targeted single-chain variable region fragments (scFv). The variable light and heavy chains were linked by four canonical GGGGS linkers. The hinge region derived from the IgG1-CH1-domain was used to link its spacer to the CD19-binding domain. The antigen-binding domain and the spacer between it and the cell membrane were constructed from SIRP-alpha Ig-like C1-1 and / or C1-2 domains. The primary structure of SIRP-alpha was obtained from the Uniprot database (P78324) and back-translated using homosapiens codons based on estimated frequency distribution. Several spacer structures were constructed to include additional extracellular fragments of the T cell-specific surface glycoprotein CD28. The transmembrane (TM) and intracellular (IC) sequences were derived from the T cell-specific surface glycoprotein CD28 and the intracellular T lymphocyte activation domain of the T cell receptor (TCR, CD3 zeta chain, Uniprot P20963-3, CD28, Uniprot P10747). The amino acid sequences of various CARs are summarized in Table 1.

[0095] The human Ab4D5 (Carter et al., 1992) antibody clone was used to design a HER-2 targeted single-chain variable region fragment. In the HER-2 targeted CAR construct, the other domains of the CAR were identical to those of the CD19 targeted CAR M. The HER-2 targeted CAR was otherwise manufactured in the same manner as the CD19 targeted CAR.

[0096] An IgG1-based CAR (FMC63 scFv, IgG1-CH2-CH3 spacer, CD28 transmembrane and intracellular domains, and CD3 zeta-signaling domain) was used as a positive control. The FcR-binding site-free control was a CD28-based CAR (CAR XS; FMC63 scFv, CD28-derived IgG hinge region, extracellular, transmembrane and intracellular sequences, and an intracellular sequence derived from the CD3 zeta-signaling domain). To evaluate (CAR-)T cell-specific interactions with post-transformed target cells, a negative transformation control and an empty pLV- vector (mock) were used.

[0097] T cell expansion CAR T cells were produced from peripheral blood mononuclear cells isolated from the pia mater, as previously described (Kaartinen et al., 2017). In the T cell cultures, X-VIVO medium (Lonza, Basel, Switzerland) supplemented with 5% human AB serum (Cellalab, Oviedo, Spain) and 100 U / ml IL-2 (Proleukin, Novartis, Basel, Switzerland) was used. The T cell density was 1 × 10⁶ on days 0–2. 6 The cells / ml were adjusted, and on day 3, after washing the vector, the T cell density was increased to 0.5 × 10⁶ by adding fresh culture medium. 6 The cells were adjusted to a cell density of 0.5 × 10⁶ cells / ml. On day 2, T cells were converted using third-generation lentiviral vectors (Koponen et al., 2003) or mock vectors containing sequences encoding various CAR structures. CAR T cells were cultured until day 10 and then frozen to await further cellular function analysis. To evaluate the functionality of CAR T cells, CAR T cells were thawed on day 10, adjusted to a cell density of 0.5 × 10⁶ cells / ml, and cultured until day 13 before analysis. For memory phenotyping analysis, CAR T cells were cultured until day 13 without freezing.

[0098] cell line NALM-6 (CD19+B lineage, acute lymphoblastic leukemia, ALL) cells, THP-1 (FcR+ monocytes, acute monocytic leukemia) cells, and E6.1 Jurkat T cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, USA), 100 IU / mL penicillin, and 100 μg / m³ streptomycin (Thermo Fisher Scientific). Additionally, 2 mM L-glutamine was added for Jurkat T cells. The NALM-6-luc cell line was produced as described by Dufva et al., 2019.

[0099] Flow cytometry Cells were fixed with 1% paraformamide (10 minutes, +4°C) before staining with anti-human antibodies. Fluorescence-minus 1 (FMO) and / or appropriate isotype controls were used as controls. Samples were subjected to a BD FACSAria IIu cytometer (BD Biosciences, Franklin Lakes, USA), and results were analyzed using FlowJo (version 10.5.3, BD Biosciences) software.

[0100] CAR T cell memory phenotyping After expansion, T cell subtypes, residual NK cells and NKT cells (Table 2) were stained using the following anti-human antibodies from BD Biosciences: CD3 (clone UCHT1)-fluorescein isothiocyanate (FITC), CD4 (clone SK3)-BD Horizon™ Brilliant Violet™ 510 (BV510), CD8 (RPA-T8)-BD Horizon™ Brilliant Violet™ 421 (BV421), CD56 (clone B159)-allophycocyanin (APC). Memory T cell phenotypes were identified using CD27 (clone M-T271)-peridinin-chlorophyll protein (PerCP) conjugated cyanine 5.5 (Cy5.5), CD45RA (clone HI100)-APC, CD45RO (clone UCHL1)-phycoerythrin (PE) conjugated cyanine 7 (Cy7) and CD95 (clone DX2)-PE.

[0101] T cell memory phenotypes were defined for CD4 and CD8 subpopulations using the expression markers shown in Table 2. To identify T cell maturation and exhaustion to the terminal effector phenotype, antibodies against CD57 (clone NK-1)-BD Horizon™ Brilliant Violet™ 421 (BV421) and CD279 (clone MIH4)-AF647 were used. Expression of programmed cell death protein 1 (CD279) and the T cell terminal effector induction marker CD57 were assessed in the CD95+CD27+ / -CD45RO+ / - population. CAR expression was measured using F(ab')2 fragment goat anti-human immunoglobulin (Ig)G (H+L) conjugated Alexa Fluor® 647 (Jackson ImmunoResearch, West Grove, USA).

[0102] Cytotoxicity Assay To evaluate the cytotoxic efficacy of spacer-modified CARs, cells were Luc +NALM-6 cells were co-cultured for 18 hours with various T cell:B cell ratios (effector:target ratio, E:T). At the end of the co-culture, luciferin (ONE-Glo luciferase reagent, Promega) was added, and the presence of live target cells was quantified using a CLARIOstar Plus Multi-Mode microplate reader (BMG Lab-Tek) according to the manufacturer's instructions.

[0103] Degranulation assay To measure T cell degranulation induced by target cells, cells were co-cultured with NALM-6 target cells in a 1:1 (E:T) ratio for 4 hours in the presence of lysosome-associated membrane protein 1 (CD107a) antibody (PE-conjugated, clone H4A3, BD Biosciences) and GolgiStop® protein transport inhibitor (BD Biosciences). Degranulation was measured by flow cytometry of cell surface expression of CD107a from all T cells in the co-culture. + It was evaluated as a population of T cells.

[0104] Analysis to prove CAR T cell interaction with monocytes To analyze the effects of CAR T cells binding to monocytes, T cells were co-cultured with THP-1 monocytes in a 1:1 ratio at +37°C for 18 hours. Cell surface activation markers CD25 (clone BC96, BioLegend) and CD69 (clone FN50, BD BioScience) on T cells were measured using flow cytometry, and the cell culture medium was collected for further analysis of activation-inducing cytokines (monocytes: IL-1 beta and CAR T cells: IFN-gamma and IL-2).

[0105] Cytokine assay To quantify activation-inducing cytokines from cytotoxicity assays (IFN-gamma and IL-2) and analyses demonstrating CAR T cell interactions with monocytes (IFN-gamma, IL-2, and IL-1β), cell culture media (effector:target ratio; 1:1) were analyzed using a Cytometric Bead Array (CBA Human Soluble Protein Master Buffer Kit CBA Flex Sets, BD Biosciences) with IL-2, IFN-gamma, and IL-1β, according to the manufacturer's instructions. Results were analyzed using FCAP Array Software v3.0 (BD Biosciences).

[0106] Magnetic microbead selection of antibody-bound and CAR-positive Jurkat T cells The SIRP-alpha conjugated antibody (SE12B6; Seiffert et al., 2001) was conjugated to cyanine 5 (Cy5) fluorescent dye using the LYNX Rapid Plus Cy5 antibody conjugation kit (Bio-Rad, Milteny Biotech) according to the manufacturer's instructions. Jurkat T cells were selected by single-cell isolation (anti-Cy5 / anti-Alexa Fluor 647 microbeads, Milteny Biotech) according to the manufacturer's instructions, and their expression was confirmed by flow cytometry.

[0107] Example 2: T cell expansion and CAR expression The CAR constructs, CAR XS, CAR XM, and CAR M contain an scFv moiety derived from the monoclonal antibody FMC63, extracellular spacers derived from the Ig-like C1-1 and Ig-like C1-2 domains of SIRP-alpha, an IgG hinge region and / or CD28, a transmembrane domain derived from CD28, and intracellular domains derived from CD28 and CD3 zeta (Figure 1A). CARs were transduced into T cells using a lentiviral vector (pLV) under the hPGK promoter (Koponen JK et al., 2003).

[0108] Various CAR-transduced T cells expanded 48-260 times within 13 days (Figure 1B). Although there was no significant difference in expansion rate, CAR XM-transduced cells showed a tendency towards slower proliferation. Even if differences in proliferation data were observed in the early stages, CAR M and XM appear to have a second peak characteristic of proliferation after thawing cells at day 10, in contrast to IgG1-CAR.

[0109] On day 2 of the expansion, T cells were stably transduced with CAR gene-carrying lentivirus or mock vectors. Thirteen days after cell production, the cells were analyzed for CAR expression, and measured by subtracting the CAR antibody binding result of empty vector-transduced T cells (mock 13.25±5.2). CAR was detected in 25.3%–88.8% of the cells (mean ±SD; IgG1-CAR 88.8±5.6, CAR M 45.0±22.6, CAR XM 60.6±22.6, and CAR XS 25.3±14.3) (Figure 1D).

[0110] All CARs were successfully expressed on T cells, but the expansion rates of CAR XS, CAR M, and CAR XM T cells after 6 days of culture appeared to be somewhat lower than those of IgG-CAR and mock T cells (Figure 1C).

[0111] Example 3: Characterization of T cell phenotype and maturation among various CAR-expressing cells. Thirteen days after expansion, the majority of cells (86%–90%) were T cells (CD3+ CD56-), with 9–13% being NKT cells (CD3+ CD56+), and a very small additional contribution from NK cells (CD3- CD56+), or the remainder being CD3- CD56- cells (Figure 2A). In addition to the cellular phenotype, the T cell memory phenotype was then evaluated. Previously, we reported that the concentration of IL-2 during CAR T cell expansion affects the T cell memory phenotype (Kaartinen T et al., 2017). Therefore, we used 100 U / ml of IL-2 in the culture to prevent excessive T cell differentiation. The repertoire of T cell memory phenotypes is shown in Figure 3A. To more easily differentiate the effects of the expansion process and various CARs on the memory phenotype of T cells, we grouped T cell memory subgroups into an early memory group (=Tscm, Tscm-like, and Tcm) and an effector group (=Tem and Teff) (Figure 3B). Up to day 13 of expansion, T cells with SIRP-alpha-based CARs M and CAR XM tended to preferentially differentiate into CD4+ cells (Figure 2B), and the proportion of effector T cells among them tended to be higher, in contrast to CD8 cells which showed a stronger dominance of early memory cells. Nevertheless, due to variability among different donors, no significant differences related to various CARs in T memory cell differentiation were detected. Otherwise, the memory phenotype was the same, and the exhaustion levels, as measured by PD-1 surface expression and proliferative capacity as a T cell terminal effector maturation-related marker CD57, were also the same. Thirteen days after expansion, most CD4 and CD8 cells were negative for the exhaustion markers CD57 and PD-1 (66.2–79.9%), with a small number expressing one or both of these surface markers (Figure 3C). Again, CAR did not specifically affect the expression of exhaustion markers in T cells.

[0112] Example 4: Activation and cytotoxic activity of CAR T cells CAR T cell interactions with target antigen-carrying cells induce T cell activation and target cell killing. After establishing the successful production of T cells carrying spacer-modified CAR constructs, we then analyzed the functional characteristics of CAR T cells through target-dependent activation. To analyze CAR function in T cell activation by CD19+ target cells, we measured cytokine production from overnight co-cultures using a 1:1 effector:target cell ratio (Figure 4A). All T cells carrying different CARs produced similar amounts of IL-2, with a non-significant trend (~1.3 times) towards higher IL-2 production by CAR M-carrying cells. No difference was detected in IFN-gamma production.

[0113] Next, we investigated the ability of T cells to degranulate in response to 4 hours of co-culture with CD19+ target cells by measuring the cell surface expression status of CD107a. Populations of CAR-expressing cells were directly related to the fraction of degranulating cells in response to target cells (Figure 4B), confirming the functionality of CAR-expressing cells. CAR expression levels in IgG1-CAR were higher than those of CAR M, CAR XM, or CAR XS, but similar to CD107a expression in CD4+ cells. In contrast, IgG1-CAR and CAR XS showed higher CD107a expression in CD8+ cells than CAR M and CAR XM.

[0114] Despite differences in CAR expression and CD8+ cell degranulation levels, all CAR T cell lines exhibited remarkably similar cytotoxicity rates against NALM-6 cell targets (Figure 4C). In 18-hour co-culture experiments with CD19+ target cells, all CAR T cell lines demonstrated 100% killing efficacy at a 2:1 (E:T) ratio, and similar capabilities at lower E:T ratios.

[0115] Example 5: Spacer-modified CAR T cells did not show activation in "off-target" bone marrow cells. SIRP-alpha FiCARs were designed to evade interaction with Fc receptor-expressing myeloid cells. We evaluated CAR T cell interactions with myeloid cells by co-culturing CAR T cells with THP-1 monocytes in a 1:1 (effector:off-target cell; E:OT) ratio. CAR T cell activation was determined by staining for the cell surface activation markers CD25 (indicating long-term activation) and CD69 (indicating short-term activation) (Figure 5A), and by measuring cytokines produced by T cells (Figure 5B: CAR T cells: IFN-gamma and IL-2) and monocytes (Figure 5C: monocytes; IL-1 beta) in response to CAR-related activation. All CAR T cells expressed CD25 activation markers at levels higher than or equal to CD25 activation markers, both with and without THP-1 monocytes. Furthermore, in co-culturing CAR T cells with THP-1 monocytes, Fc-region-containing CARs, IgG1-CARs, expressed high levels of the cell surface early activation marker CD69. In contrast, T cells with spacer-modified CAR constructs, namely CAR XS, CAR M, and CAR XM cells, did not express CD69 along with mock T cells. A similar pattern was observed in cytokine production; activation induced by IgG1-CAR, in addition to THP-1-induced activation inducing the cytokine IL-1 beta, induced by IgG1-CAR-induced activation inducing the cytokines IL-2 and IFN-gamma. Spacer-modified CAR T cells produced low levels of IL-2 and IFN-gamma, all comparable to mock-transformed control T cells, either with or without THP-1 monocytes. Furthermore, THP-1 monocytes co-cultured with spacer-modified T cells produced IL-1 beta at levels as low as THP-1 cells alone or together with mock-transformed control T cells. Taken together, these data indicate that co-culture of spacer-modified CAR T cells with FcR-carrying monocytes does not result in excessive activation of T cells or monocytes.

[0116] Example 6 Modification of SIRP-Alpha Spacer Length To further investigate whether the CAR skeletal structure can be modified for better binding of membrane-proximal or membrane-distal antigens on target cells, we designed CARs of various lengths for targeting CD19. By adjusting the spacer length using different Ig-like C1 domains of SIRP-alpha, we designed length-modified CARs by removing another Ig-like C1 domain from CAR M or CAR XM, or by adding an additional Ig-like C1 domain to CAR M and CAR XM.

[0117] First, to ensure high expression of CARs of various lengths, CAR-expressing Jurkat T cells were selected using single-cell microbead isolation. Next, to measure expression, CARs of various lengths were stained using biotinylated anti-human CD19 CAR detection reagent (Milteny Biotech) and a biotin antibody conjugated with APC as a secondary antibody (Milteny Biotech). Staining was performed according to the manufacturer's instructions. All transduced Jurkat T cell cultures showed high expression levels of various CARs compared to mock Jurkat T cells transduced with an empty vector exhibiting nonspecific binding to the antibody (Figure 6A: CAR 2S 90.6%, CAR L 88.1%, CAR XL 95.4%).

[0118] Furthermore, to evaluate the functionality of CARs of various lengths, the cellular efficacy of CAR-transduced Jurkat T cells against CD19-positive Nalm-6-Luc cells was examined at several E:T ratios (Figure 6B). Jurkat T cells expressing any of the various CARs (CAR 2S, CAR L, and CAR XL) all exhibited similar killing efficacy to Jurkat T cells with CAR M, CAR XM, and IgG1-based control CARs, ranging from 0–20% to 66.8–82% depending on the E:T ratio. As a positive control for killing, we used primary T cells showing superior killing efficacy (48–94.7%), and as a negative control, we used mock-transduced Jurkat T cells showing non-CAR-related killing efficacy of 0–15.4% at different E:T ratios.

[0119] Example 7: HER-2 targeting in CARs based on a SIRP alpha skeleton After demonstrating that the spacer length could be adjusted, we designed a novel CAR by replacing the CD19-targeted scFv domain in the previous CAR M structure with a HER-2-targeted scFv domain. To demonstrate the function of the HER-2-targeted CAR M, the CAR was transduced into primary T cells. After expansion, HER-2-targeted CAR T cells were co-cultured with HER-2-positive SKBR-3-eGFP-Luc breast cancer cells in various effector-target (E:T) ratios (Figure 7). In an 18-hour preliminary cytotoxicity test (n=1), T cells transduced with the HER-2-targeted CAR M showed higher killing efficacy compared to mock transduced T cells, with minimal CAR-independent cell killing by themselves.

[0120] Example 8: Cytotoxicity of T cells expressing CAR M with scFv-targeted HER-2 T cells were isolated from healthy donor pial membranes and transduced with lentiviral vectors carrying the HER-2 CAR M gene construct at various MOIs (metamorphosis of infection) of 1.25, 2.5, and 5, and expanded for 11 days. T cells expressing HER-2 CAR M with an alternative scFv targeting HER-2 (effector cells) were cultured together with firefly luciferase-expressing HER-2+ SKBR-3 breast cancer cells (target cells) at effector-target (E:T) ratios of 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8. After 24 hours, luciferin was added, and the number of viable target cells was quantified, showing high killing efficacy at all different E:T ratios compared to empty vector (mock) transduced T cells.

[0121] Example 9: Cytotoxicity of CAR constructs having cell expansion, CAR expression, and modification multimerization domains CD4+ and CD8+ T cells were purified from peripheral blood mononuclear cells using magnetic beads (Milteny Biotech). Purified CD4+ and CD8+ T cells were transduced with lentiviral vectors encoding CAR constructs (CAR M, CAR XM, CAR M1, CAR XM2, CAR XM3, CAR M4, CAR 2S5, CAR M6) and expanded in cell medium containing IL-7 and IL-15 (Milteny Biotech) at 12.5 ng / ml. Cell volume and viability were measured throughout the expansion. The effect of various CAR constructs on expansion was investigated up to day 10 (Figure 9A). The various constructs did not significantly affect cell expansion, and all constructs reached over 20-fold expansion.

[0122] Cells were examined for CAR expression using flow cytometry. CAR constructs were detected using biotin-labeled antibodies that detect specific domains present in all CAR constructs (Figure 9B). Vector copy number (VCN) was determined by isolating genomic DNA and detecting the integrated gene using transgene-specific primers (Figure 9B). In the cell population, with approximately 1 VCN, more than 50% of cells expressed the CAR transgene on the cell surface.

[0123] CAR-T cells (after thawing) were co-cultured for 24 hours with CD19+NALM-6 target cells in various ratios of effector (CAR-T) and target (cancer) cells. The cells were then lysed, and target cell-specific (transduced) gene activity was measured (Figure 9C). In the killing assay, CAR M, XM, M1, and M6 tended to show higher killing efficacy than other CAR constructs, but all constructs showed significantly increased target cell killing efficacy compared to untransduced T cells or empty vector (mock) transduced T cells.

[0124] [References] Almaasbak H et al (2015) Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model. Gene Ther 22: 391-403. Casucci M et al (2018) Extracellular NGFR spacers allow efficient tracking and enrichment of fully functional car-t cells co-expressing a suicide gene. Front Immunology 9 Carter P et al (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89:4285-4289. Dull et al (1998) A Third-Generation Lentivirus Vector with a Conditional Packaging System. J Virol 72 (11): 8463-8471. Harris E et al (2020) Optimization of electroporation and other non-viral gene delivery strategies for T cells. Biotechnol Progress, e3066. Hatherley D et al (2007) The structure of the macrophage signal regulatory protein α (SIRP-alpha) inhibitory receptor reveals a binding face reminiscent of that used by T cell receptors. J Biol Chem 282: 14567-14575. Hatherley D et al (2009) Structure of signal-regulatory protein α: A link to antigen receptor evolution. J Biol Chem 284: 26613-26619. Hombach A et al (2010) Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response. Gene Ther 17: 1206 Hudecek M et al (2015) The Nonsignaling Extracellular Spacer Domain of Chimeric Antigen Receptors Is Decisive for In Vivo Antitumor Activity. Cancer Immunol Res 3: 125-135. Kaartinen T, Luostarinen A, Maliniemi P, et al (2017) Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion. Cytotherapy 19:689-702. Koponen JK et al (2003) Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. Gene Ther 10: 459-466. Levine B et al (2017) Global Manufacturing of CAR T Cell Therapy. Molecular Therapy: Methods & Clinical Development Vol. 4: 92-101 Riedl S et al (2018) Non-Viral Transfection of Human T Lymphocytes. Processes, 6, 188. Seiffert M et al (2001) Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34+CD38- hematopoietic cells. Blood 97:2741-2749. Sentman CL et al (2014) NKG2D CARs as Cell Therapy for Cancer. Cancer J 20: 156-159. Townsend MH et al (2018) The expansion of targetable biomarkers for CAR T cell therapy. Journal of Experimental & Clinical Cancer Research 37:163 van Beek EM et al (2005) Signal Regulatory Proteins in the Immune System. J. Immunol. 175: 7781-7. Yu JX et al (2020) Cancer cell therapies: the clinical trial landscape. Nature Reviews Drug Discovery 19, 583-584.

[0125] [Table 1] TIFF2026143693000002.tif239164 TIFF2026143693000003.tif243164 TIFF2026143693000004.tif243164 TIFF2026143693000005.tif243164 TIFF2026143693000006.tif242164 TIFF2026143693000007.tif237164 TIFF2026143693000008.tif242164 TIFF2026143693000009.tif242164 TIFF2026143693000010.tif242164 TIFF2026143693000011.tif242164 TIFF2026143693000012.tif242164 TIFF2026143693000013.tif242164 TIFF2026143693000014.tif242164 TIFF2026143693000015.tif242164 TIFF2026143693000016.tif242164 TIFF2026143693000017.tif242164 TIFF2026143693000018.tif231157 TIFF2026143693000019.tif244164 TIFF2026143693000020.tif246164 TIFF2026143693000021.tif244164 TIFF2026143693000022.tif246164 TIFF2026143693000023.tif246164 TIFF2026143693000024.tif242164 TIFF2026143693000025.tif242164 TIFF2026143693000026.tif244164 TIFF2026143693000027.tif22164

[0126] Table 2

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular spacer containing at least one Ig-like C1 domain or a fragment or variant thereof of signal regulatory protein alpha (SIRP-alpha).

2. The CAR according to claim 1, wherein the Ig-like C1 domain of SIRP-alpha is selected from (i) the type 1 domain or a fragment or variant thereof relating to SEQ ID NO: 1; or (ii) the type 2 domain or a fragment or variant thereof relating to SEQ ID NO:

2.

3. The CAR according to claim 1 or 2, wherein the extracellular spacer comprises the type 1 domain and the type 2 domain of Ig-like C1 of SIRP-alpha.

4. The CAR according to any one of claims 1 to 3, wherein the extracellular spacer further comprises at least one multimerization domain.

5. The CAR according to claim 4, wherein the multimerization domain, or a plurality of multimerization domains, are selected from an IgG hinge region selected from the IgG1 hinge region related to SEQ ID NO: 4 or SEQ ID NO: 80, the IgG2 hinge region related to SEQ ID NO: 81, the IgG3 hinge region related to SEQ ID NO: 82, the IgG4 hinge region related to SEQ ID NO: 83, and / or the extracellular domain of CD28 related to SEQ ID NO: 3 and / or fragments or variants thereof.

6. The CAR according to claim 4, wherein the multimerizing domain, or a plurality of multimerizing domains, are selected from the IgG1 hinge region or a fragment thereof relating to SEQ ID NO: 4 and / or the extracellular domain of CD28 or a fragment thereof relating to SEQ ID NO:

3.

7. The CAR according to claim 4, wherein the multimerizing domain, or a plurality of multimerizing domains, are selected from the IgG4 hinge region or a fragment thereof relating to SEQ ID NO: 83 and / or the extracellular domain or a fragment thereof of CD28 relating to SEQ ID NO:

3.

8. The CAR according to any one of claims 1 to 7, wherein the extracellular spacer is located between and bound to the transmembrane domain and the antigen-binding domain.

9. The CAR according to claim 8, wherein the antigen-binding domain comprises a single-chain variable region fragment (scFv).

10. The CAR according to any one of claims 1 to 9, wherein the spacer dimerizes the CAR by at least one disulfide crosslink.

11. A CAR comprising an extracellular spacer containing an amino acid sequence relating to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO:

61.

12. (i) an extracellular spacer according to any one of claims 1 to 11, (ii) an antigen-binding domain, (iii) a transmembrane domain, (iv) an intracellular signaling domain, and (v) optionally a co-stimulatory domain, comprising a chimeric antigen receptor (CAR).

13. The CAR according to claim 12, wherein the antigen-binding domain comprises an antibody or a fragment thereof.

14. The CAR according to claim 12, wherein the antigen-binding domain comprises a single-chain variable region fragment (scFv).

15. The CAR according to any one of claims 12 to 14, wherein the antigen-binding domain targets a tumor antigen.

16. The CAR according to claim 15, wherein the tumor antigen is selected from CD19 or HER-2.

17. The CAR according to any one of claims 12 to 16, wherein the transmembrane domain includes the transmembrane domain of CD28 related to Sequence ID No.

23.

18. The CAR according to any one of claims 12 to 17, wherein the intracellular signaling domain and / or co-stimulatory domain comprises the intracellular domain or fragment of CD3 zeta according to SEQ ID NO: 25 and / or the intracellular domain or fragment of CD28 according to SEQ ID NO:

24.

19. (i) Single-chain variable region fragment (scFv); (ii) IgG hinge domain; (iii) Ig-like C1 type 1 and / or Ig-like C1 type 2 domains of signal regulatory protein alpha-1; (iv) CD3 zeta; (v) CD28 transmembrane domain; (vi) Optionally, the extracellular domain of CD28 and / or the intracellular domain of CD28 A chimeric antigen receptor (CAR) that includes this receptor.

20. A CAR comprising or consisting of the amino acid sequence relating to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO:

67.

21. A polynucleotide encoding a CAR according to any one of claims 1 to 20.

22. A vector comprising the polynucleotide according to claim 21.

23. A cell comprising a CAR according to any one of claims 1 to 20 or a polynucleotide according to claim 21.

24. The cell according to claim 23, wherein the cell is a T cell.

25. A method for adjusting the length of a chimeric antigen receptor (CAR) to produce chimeric antigen receptors of different lengths by selecting at least two domains from the group (i) IgG hinge domain, (ii) type 1 domain of Ig-like C1 of signal regulatory protein alpha-1, (iii) type 2 domain of Ig-like C1 of signal regulatory protein alpha-1, or (iv) spacer domain from an extracellular CD28 fragment.

26. The method according to claim 25, wherein the spacer domain does not bind to the Fc receptor or has a low binding affinity to the Fc receptor.

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