Parallel chimeric antigen receptors (pCAR) and adaptor chimeric antigen receptors comprising alternative signaling domains and methods of use thereof

Abstract

Provided herein are second generation chimeric antigen receptors (CARs), parallel CARs (pCARs), and adaptor CARs comprising intracellular signaling domains modified to achieve optimal signaling. Also provided herein are compositions and methods for increasing the anti-tumor efficacy and restimulation ability of CAR T cells, pCAR T cells, and adaptor CAR T cells.

Description

[0001] 1. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. The XML copy, created on April 4, 2024, is named P4861PC00 Sequence Listing and is 115,824 bytes in size. BACKGROUND

[0003] Chimeric antigen receptors (CARs), sometimes referred to as artificial T cell receptors, chimeric T cell receptors (cTCRs), or chimeric immunoreceptors, are now well known engineered receptors in the art. They are primarily used to transform immune effector cells, particularly T cells, to provide these cells with a desired engineered specificity. In the field of cancer therapy, adoptive cell therapy using CAR-T cells has been of particular interest for research. In these therapies, T cells are taken from a patient and modified so that they express a CAR specific for an antigen found in a particular form of cancer. The CAR-T cells, which can then recognize and kill cancer cells, are reintroduced to the patient.

[0004] First generation CARs provide a TCR-like signal, most commonly using the CD3 zeta (CD3 zeta) intracellular signaling domain, and thereby elicit tumor killing function. However, engagement of the CD3 zeta-chain fusion receptor can be insufficient to elicit substantial IL-2 secretion and / or T cell proliferation in the absence of a concomitant costimulatory signal. In physiologic T cell responses, optimal lymphocyte activation requires the engagement of one or more costimulatory receptors, such as CD28 or 4-1BB.

[0005] Second generation CARs have been constructed to transduce, in addition to the antigen-dependent TCR-like signal, a functional antigen-dependent costimulatory signal in human primary T cells, in addition to allowing tumor killing activity, also allowing T cell proliferation. Most commonly, second generation CARs provide costimulation using a costimulatory domain (synonymously, costimulatory signaling region) derived from CD28 or 4-1BB. The combined delivery of costimulation plus CD3 zeta signaling makes second generation CARs functionally superior to their first generation counterparts (CD3 zeta signal only). Examples of second generation CARs can be found in U.S. Patent No. 7,446,190, which is incorporated herein by reference.

[0006] More recently, so-called third generation CARs have been prepared. These CARs combine multiple costimulatory domains (synonymously, costimulatory signaling regions) in cis with a TCR-like signaling domain, such as CD28+4-1BB+CD3zeta or CD28+OX40+CD3zeta, to further enhance potency. In third generation CARs, the costimulatory domains are arranged in tandem in the CAR endodomain and are typically upstream of CD3zeta or its equivalent.

[0007] However, generally, results with these third generation CARs have been disappointing, showing only a small improvement over second generation configurations, with some third generation CARs being less effective than second generation configurations.

[0008] We recently described a new format in which an immune responsive cell, such as a T cell, is engineered to express two constructs in parallel - a second generation CAR and a chimeric costimulatory receptor (CCR). The second generation CAR comprises, from C-terminal to N-terminal (from intracellular to extracellular), the following domains: (a) a signaling region; (b) a costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen. The CCR comprises, from C-terminal to N-terminal (from intracellular to extracellular): (a) a costimulatory signaling region that is different from the costimulatory signaling region of the CAR; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with an epitope on a target antigen. The CAR and CCR can recognize the same epitope, different epitopes on the same antigen, or epitopes present on two different antigens. Unlike the CAR, the CCR lacks a TCR-like signaling region, such as CD3zeta. These parallel CAR (pCAR) engineered T cells exhibit superior activity and anti-exhaustion capacity compared to first, second, and third generation CAR-T cells. See U.S. Pre-Grant Publication 2019 / 0002521 as well as PCT Publications WO 2020 / 183158 and WO 2021 / 0383036, each of which is incorporated herein by reference in its entirety.

[0009] These properties of pCAR-T cells make them attractive candidates for treating refractory malignancies in which first, second, and third generation CAR-T cells show limited efficacy, in part due to T cell exhaustion. However, there is a need to further develop this technology to improve its therapeutic effectiveness. SUMMARY

[0010] In one aspect, described herein is a parallel chimeric antigen receptor (pCAR) comprising:

[0011] (1) a second generation chimeric antigen receptor (second generation CAR) comprising:

[0012] a) an intracellular signaling domain comprising a modified CD3 zeta polypeptide;

[0013] b) a costimulatory signaling region;

[0014] c) a transmembrane domain; and

[0015] d) a first binding element that specifically interacts with a first epitope on a first target antigen,

[0016] wherein the modified CD3 zeta polypeptide comprises an unmodified immunoreceptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and / or ITAM3; and

[0017] (2) a chimeric costimulatory receptor (CCR) comprising at least one CCR polypeptide comprising:

[0018] e) a costimulatory signaling region that is different from the costimulatory signaling region of (b);

[0019] f) a transmembrane domain; and

[0020] g) a second binding element that specifically interacts with a second epitope on a second target antigen. The present disclosure also provides:

[0021] - one or more polynucleotide constructs encoding the pCAR of the invention,

[0022] - one or more expression vectors comprising the one or more polynucleotide constructs of the invention,

[0023] - an immunoresponsive cell comprising the pCAR of the invention, the one or more polynucleotide constructs of the invention, or the one or more expression vectors of the invention,

[0024] - a pharmaceutical composition comprising the one or more polynucleotide constructs of the invention, the one or more expression vectors of the invention, or the immunoresponsive cell of the invention, and an excipient,

[0025] - the pCAR of the invention, the one or more polynucleotide constructs of the invention, the one or more expression vectors of the invention, the immunoresponsive cell of the invention, or the pharmaceutical composition of the invention, for use in therapy, and

[0026] - the pCAR of the application, the polynucleotide construct(s) of the application, the expression vector(s) of the application, the immune responsive cell of the application, or the pharmaceutical composition of the application for use in the treatment of cancer.

[0027] In another aspect, described herein is a fusion polypeptide comprising a DNAX-activating protein 10 (Dap10) polypeptide or functional variant thereof, and a modified CD3 zeta polypeptide comprising an unmodified immunoreceptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and / or ITAM3.

[0028] In another aspect, described herein is a fusion polypeptide comprising (i) a DNAX-activating protein 10 (Dap10) polypeptide or functional variant thereof, and (ii) a modified CD3 zeta polypeptide comprising an unmodified immunoreceptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and / or ITAM3. BRIEF DESCRIPTION OF DRAWINGS

[0029] These and other features, aspects, and advantages of the present application will become better understood with reference to the following description and appended claims, taken in conjunction with the accompanying drawings, where:

[0030] FIGS. 1A, IB, and 1C illustrate schematic diagrams of CAR and pCAR constructs. FIG. 1A shows schematic diagrams of TBB / H, H2, TBB / H 1XX, and H2 1XX constructs. H2 comprises, from intracellular to extracellular: a CD3 zeta signaling region, a CD28 co-stimulation and transmembrane domain, and a HMFG2 single chain antibody (scFv) domain targeting human MUC1. TBB / H is a pCAR utilizing a second generation “H2” CAR targeting MUC1. The chimeric co-stimulatory receptor (CCR) in the TBB / H pCAR has a pan-ErbB specific T1E binding domain and a 4-1BB co-stimulation domain fused to a CD8a transmembrane domain. H2 1XX is a modified version of the H2 CAR that contains mutations (Y to F) in both distal CD3 zeta immunoreceptor tyrosine activation motifs (ITAMs) to form a single functional ITAM (proximal). TBB / H 1XX is a modified version of the TBB / H pCAR that contains mutations (Y to F) in both distal CD3 zeta immunoreceptor tyrosine activation motifs (ITAMs) to form a single functional ITAM (proximal).

[0031] FIG. IB shows TBB / H-1, TBB / H-1 3, TBB / H-1 6, TBB / H-1 9 and TBB / H-1 9 YRSpCAR constructs. TBB / H-1 is a modified version of TBB / H in which the CD3ζ polypeptide is truncated to remove two distal ITAMs (ITAM2 and ITAM3). TBB / H-1 3, TBB / H-1 6 and TBB / H-1 9 are modified versions of TBB / H-1 that further lack 3, 6, or 9 amino acid residues upstream of the remaining ITAM in the truncated CD3ζ polypeptide, respectively. TBB / H-1 9 YRS is a modified version of TBB / H-1 9 that further comprises deletion of the C-terminal YRS motif of the CD28 costimulatory region.

[0032] Figure 1C shows a schematic of FcεR1γ and DAP12 pCAR constructs that are modified versions of the TBB / H pCAR in which the CD3ζ intracellular signaling domain of the TBB / H pCAR is replaced by an activation domain from FcεR1γ or DAP12, each of which comprises a single ITAM.

[0033] Figures 2A-2E show results of antigen re-stimulation assays with T cells expressing the CARs and pCARs shown schematically in Figure 1A on a monolayer of BXPC3 tumor cells. Figures 2A-2B H2 1XX CAR-T cells (open diamonds) showed anti-tumor activity in more re-stimulation cycles than H2 CAR-T cells, comparable to TBB / H pCAR-T cells. Figures 2C-2E TBB / H 1XX-T cells showed better re-stimulation capacity on a monolayer of BXPC3 tumors than TBB / H-T cells, with increased proliferative capacity.

[0034] Figures 3A-3B Figures 3A-3B show results of antigen re-stimulation assays with T cells expressing the pCARs shown schematically in Figures 1A and 1B on a monolayer of BXPC3 tumor cells. Figure 3A shows that TBBH / H 1XX-pCAR T cells had better anti-tumor efficacy than pCAR-T cells expressing truncated CD3ζ signaling domains. Figure 3B shows that pCAR-T cells expressing truncated CD3ζ had reduced proliferative capacity compared to TBB / H 1XX pCAR-T cells.

[0035] Figures 4A-4CResults of antigen re-stimulation assays with T cells expressing the CAR and pCAR indicated in FIGS. 1A and 1C on monolayers of BXPC3 tumor cells are shown. TBB / H 1XX pCAR-T cells (large inverted triangle), have superior anti-tumor activity (FIG. 4A), proliferation potential (FIG. 4B), and re-stimulation capacity (FIG. 4C) compared to pCAR-T cells expressing either DAP12 (square) or FcεR1γ (open diamond) signaling domains.

[0036] Figures 5A-5D are schematic diagrams of NKG2D_bb / H 1XX, NKG2D(trimer) / H 1XX, Pbb(trimer) / H 1XX, and 44bb(trimer) / H 1XX pCAR constructs. The second generation CAR in each pCAR construct (“H 1XX”) comprises from intracellular to extracellular: a CD3ζ signaling region (this region comprises a mutation (Y to F) in both distal CD3ζ ITAMs to form a single functional ITAM (proximal)), a CD28 costimulatory and transmembrane domain, and a HMFG2 single chain antibody (scFv) domain targeting human MUC1. FIG. 5A is a schematic diagram of the NKG2D_bb / H 1XX pCAR construct. FIG. 5B is a schematic diagram of the NKG2Dbb(trimer) / H 1XX pCAR construct. FIG. 5C is a schematic diagram of the Pbb(trimer) / H 1XX pCAR construct. FIG. 5D is a schematic diagram of the 44bb(trimer) / H 1XX pCAR construct.

[0037] FIGS. 6A-6I show the growth of BXPC3 tumor xenografts expressing firefly luciferase (ffLUC) in mice treated with CAR or pCAR-T cells as determined by bioluminescence imaging (BLI). Results shown are from mice treated with: NKG2D_bb / H 1XX pCAR-T cells (FIG. 6A), NKG2D(trimer) / H 1XX pCAR-T cells (FIG. 6B), Pbb(trimer) / H 1XX pCAR-T cells (FIG. 6C), 44bb(trimer)H 1XX pCAR-T cells (FIG. 6D), TBB / H pCAR-T cells (FIG. 6E), TBB / H 1XX pCAR-T cells (FIG. 6F), H2 1XX CAR-T cells (FIG. 6G). Mice treated with T cells transduced with CD19 specific F28z (FIG. 6H) or PBS (FIG. 6I) were included as negative controls. The inset in FIGS. 6A-6G is a schematic of the CAR or pCAR administered.

[0038] Figure 7 are survival curves for mice evaluated for tumor growth in FIGS. 6A-6I.

[0039] Figure 8 A schematic diagram of the connector CAR constructs NKG2D / Dap10-12, NKG2D / Dap10-CD3ζ and NKG2D / Dap10-CD3ζ(1XX) and the linear CAR NKG2D-CD3ζ is shown.

[0040] Figure 9A -9C G shows the transduction efficiency of the adaptor CAR and linear CAR targeting the NKG2D ligand on days 3 and 10 after gene transfer. Figure 9A The results of the determination of CD4+ / CD8+ T cell ratio (Figure 9B) and NKG2D expression (Figure 9C). Figure 9A Figure 9B shows the transduction efficiency of the indicated CARs on days 3 and 10 post-gene transfer. Data are presented as median + interquartile range. ****p < 0.0001, verified by the Mann-Whitney test. Figure 9C shows the mean fluorescence intensity of NKG2D expression in the indicated CAR T cells analyzed on day 10 post-gene transfer. Data are presented as median + interquartile range. *p < 0.001; ****p < 0.0001, verified by the Kruskal-Wallis test.

[0041] Figures 10A-10E The results of in vitro assays were presented, evaluating the cytotoxicity achieved when BXPC3 and Ren tumor cells were co-cultured with adaptor CAR T cells (NKG2D / Dap10-12; NKG2D / Dap10-CD3ζ; NKG2D / Dap10-CD3ζ (1XX)), linear CAR T cells (NKG2D-CD3ζ), or untransduced T cells (Untrans). Figure 10A ), cytokine release ( Figures 10B-10C ), number of effective tumor restimulation cycles ( Figure 10D ) and maximum T cell expansion fold ( Figure 10E ). Figure 10A The results of the MTT cytotoxicity assay are shown, in which indicated CAR T cells were co-cultured with BXPC3 or Ren tumor cells at a specified target cell to effector cell ratio for 24 hours. Data shown are mean ± SEM, p < 0.0001, by two-factor ANOVA. Figure 10BThe data show the IL-2 concentration measured in the supernatant collected after 24 hours of co-culturing tumor cells with the indicated T cell population (effective cell:target cell ratio of 1:1). Data are presented as median + interquartile range. Figure 10C The IFN-γ concentration is shown in the supernatant collected after 72 hours of co-culturing tumor cells with the indicated T cell population (effective cell:target cell ratio of 1:1). Data are presented as median + interquartile range. Figure 10D The number of twice-weekly restimulation cycles achieved on BXPC3 and Ren tumor cells by NKG2D adaptor CAR T cells (NKG2D / Dap10-12 (also known as “N / 1012”); NKG2D / Dap10-CD3ζ (also known as “N / 10Z”); NKG2D / Dap10-CD3ζ(1XX) (also known as “N / 10Z(1XX)”), CAR T cells (NKG2D-CD3ζ (also known as “NZ”)) and control T cells (NKG2D; Untrans.)) are shown. Data are presented as median + interquartile range. *p < 0.01; **p < 0.01; ****p < 0.0001, by Kruskalwallis test. Figure 10E The maximum fold increase of the indicated T cell population after iterative tumor restimulation is shown. Data are presented as median + interquartile range. *p < 0.01; ***p < 0.001; ****p < 0.0001, passed by Kruskalwallis test.

[0042] Figures 11A-11F show the growth of subcutaneous BXPC3 tumor xenografts in mice after administration of NKG2D / Dap10-12 adaptor CAR T cells (Figure 11A); NKG2D-CD3ζ linear CAR T cells (Figure 11B); NKG2D / Dap10-CD3ζ adaptor T cells (Figure 11C); NKG2D / Dap10-CD3ζ(1XX) adaptor T cells (Figure 11D); PBS (Figure 11E); or untransduced T cells (Figure 11F). BXPC3 cells (1 x 10⁻¹²) were used to treat the tumors. 5 10 million T cells were established subcutaneously in NSG mice for 17 days, followed by intravenous (iv) administration of the indicated T cell population or administration of PBS as a control. ****p < 0.0001; NS – not significant, by two-factor ANOVA (after treatment).

[0043] Figure 12 These are survival curves for mice used to assess tumor growth in Figures 11A-11F. Detailed Implementation

[0044] 5.1. Definition

[0045] As used herein, the term "variant" refers to polypeptide sequences that are naturally occurring polymorphic forms of the basic sequence as well as synthetic variants in which one or more amino acids in the basic sequence are inserted, removed, or replaced. However, the variants produce similar biological effects as the basic sequence. For example, a variant of the intracellular domain of human CD3 zeta chain will function in a similar manner as the intracellular domain of human CD3 zeta chain. An amino acid substitution can be considered "conservative" where the one amino acid is replaced with a different amino acid in the same class having broadly similar properties. Non-conservative substitutions are where the amino acid is replaced with a different type or class of amino acid.

[0046] As is well known to those skilled in the art, changing the primary structure of a peptide by conservative substitutions can not significantly alter the activity of the peptide, because the side chains of the amino acids inserted into the sequence can be able to form similar bonds and contacts as the side chains of the amino acids that are replaced. This is true even when the substitutions are in critical regions that determine the conformation of the peptide. Non-conservative substitutions are also possible, provided that these substitutions do not destroy the function of the polypeptide, as described above. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Typically, the variant will have an amino acid sequence that will be at least 70%, for example at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%, 95%, 96%, or 98% identical to the basic sequence. Identity in this context can be determined using the BLASTP computer program.

[0047] As used herein, the term "antigen" refers to any member of a specific binding pair that will bind to a binding element. The term includes a receptor on a target cell.

[0048] As used herein and with respect to a binding element to a target molecule, the terms "binds," "specifically binds to," and "selectively binds" a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen (e.g., a polypeptide target), "specifically interacts with," "selectively interacts with," "is specific for," and "is selective for" a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen (e.g., a polypeptide target) mean binding that is measurably different from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule.

[0049] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises a scFv.

[0050] As used herein, the term "parallel chimeric antigen receptor" or "pCAR" refers to a parallel chimeric antigen receptor comprising a combination of a second generation chimeric antigen receptor (CAR) and a parallel chimeric costimulatory receptor (CCR). pCARs have been described in WO2017 / 021701, which is incorporated by reference herein in its entirety.

[0051] As used herein, the term "adaptor CAR" refers to an adaptor chimeric antigen receptor comprising a combination of (i) at least one adaptor protein comprising an activating signaling domain and a costimulatory signaling region, with (ii) at least one binding domain (targeting moiety) that specifically binds a target antigen. The at least one adaptor protein (i) and the at least one targeting moiety (ii) are expressed as two or more separate polypeptides that associate through non-covalent interactions.

[0052] As used herein, the term "linear CAR" refers to a chimeric antigen receptor comprising at least one extracellular binding domain (targeting moiety) and one or more intracellular signaling domains configured in a single polypeptide to span the plasma membrane of a cell. Examples of linear CARs include, but are not limited to, second generation CARs.

[0053] 5.2. Other interpretative conventions

[0054] In the claims, the articles "a," "an," and "the" can mean one or more than one unless otherwise indicated or otherwise evident from the context. A claim or description employing the phrase "or the other comprises "or" between one, more or all members of a group of members is considered satisfied unless otherwise indicated or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group of members exists or is applied or otherwise given in a given product or process, or in which more than one member of the group of members exists or is applied or otherwise given in a given product or process. The invention includes embodiments in which more than one member or all members of the group of members exist or are applied or otherwise given in a given product or process.

[0055] It should also be noted that the term "comprising" is intended to be open-ended and allow for the inclusion of additional elements or steps without, however, requiring the inclusion of additional elements or steps. When the term "comprising" is used, the term "consisting of" is also intended to be encompassed and disclosed.

[0056] Where a range is given, endpoints are included. Furthermore, it is to be understood that the limits of any numerical range specified do not suggest that when the minimum and maximum values are used, the application is limited to only that particular range. It is also to be understood that the application is not limited to the referenced parts of the materials, instruments, devices, and / or methods described unless the context clearly indicates otherwise. In addition, it is to be understood that the numerical properties of the values expressed as ranges in the various embodiments of the application can employ any particular value or sub-range within the stated range to the tenth of the range lower limit unit, unless the context clearly indicates otherwise.

[0057] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if the cited sources are not explicitly indicated as being incorporated by reference. In the event of an inconsistency between a cited source and a statement of the current application, the statement of the current application prevails.

[0058] Section and table headings are not limiting.

[0059] 5.3. Chimeric antigen receptors (CARs)

[0060] In one aspect, a second generation chimeric antigen receptor (second generation CAR) is provided. The second generation CAR comprises (a) an intracellular signaling domain comprising a modified CD3 zeta polypeptide; (b) a costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen, wherein the modified CD3 zeta polypeptide comprises an unmodified ITAM1, and a mutation in ITAM2 and / or ITAM3.

[0061] In some embodiments, the second generation CAR is a single polypeptide.

[0062] In some embodiments, the CAR is H or H2 as shown in FIG. 1A. The H2 CAR is a second generation (2G) CAR, originally described in Wilkie et al., J. Immunol. 180:4901-9 (2008), which is incorporated by reference in its entirety herein. It comprises, from intracellular to extracellular, a CD3 zeta signaling region, a CD28 costimulation, a transmembrane and extracellular spacer domain, and a HMFG2 scFv domain targeting human MUC1.

[0063] In some embodiments, the CAR is the H2 1XX shown in Figure 1A. The H2 1XX CAR is a second-generation (2G) CAR that comprises, from intracellular to extracellular: a modified CD3ζ signaling region, CD28 co-stimulatory domains, transmembrane and extracellular septal domains, and an HMFG2 scFv domain targeting human MUC1.

[0064] In some embodiments, the CAR is an H2BB CAR. H2BB is a 2G CAR that comprises, from intracellular to extracellular, a CD3ζ signaling region, 41 BB The co-stimulatory domain, the CD8α transmembrane and extracellular septal domain, and the HMFG2 scFv domain targeting human MUC1.

[0065] 5.3.1. Intracellular signal transduction domains

[0066] The CAR construct contains a signal transduction region (i.e., a TCR-like signal transduction region). In some embodiments, the signal transduction region contains an immune receptor tyrosine activation motif (ITAM), as reviewed, for example, by Love et al., Cold Spring Harbor Perspect. Biol [Cold Spring Harbor Biological Perspective] 2010 2(6)1 a002485. In some embodiments, the signal transduction region contains an intracellular domain of the human CD3ζ chain (CD3 zeta) (as described, for example, in U.S. Patent No. 7,446,190, which is incorporated herein by reference) or a variant thereof. In a particular embodiment, the signal transduction region contains a domain of amino acid residues 52-163 spanning the full-length human CD3ζ chain (e.g., SEQ ID NO: 1 or 2). The CD3ζ chain has many known polymorphic forms (e.g., Sequence ID: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein:

[0067]

[0068] (SEQ ID NO: 1);

[0069]

[0070] (SEQ ID NO: 2).

[0071] Alternative signal transduction regions of the CD3ζ domain include, for example, FcεR1γ, CD3ε, DAP12, and multiple ITAM. See Eshhar Z et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 90:720-724 (1993); Nolan et al., Clin Cancer Res [Clinical Cancer Research] 5: 3928-3941 (1999); Zhao et al., J Immunol [Journal of Immunology] 183: 5563-5574 (2009); Topfer et al., J Immunol [Journal of Immunology] 194: 3201-3212 (2015); and James JR, Sci Signal [Science Signal] 11(531)eaan1088 (2018), the disclosures of which are incorporated herein by reference in their entirety.

[0072] In some embodiments, the signal transduction region comprises an intracellular domain of FcεR1γ or a variant thereof. In a particular embodiment, the FcεR1γ signal transduction region comprises the amino acid sequence of SEQ ID NO: 20 as shown below:

[0073] RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ

[0074] (SEQ ID NO: 20).

[0075] In some embodiments, the signal transduction region comprises the DAP12 intracellular domain or a variant thereof. In a particular embodiment, the DAP12 signal transduction region comprises the amino acid sequence of SEQ ID NO: 58.

[0076] YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0077] (SEQ ID NO: 58).

[0078] 5.3.1.1 Immune Receptor Tyrosine Activating Motif (ITAM)

[0079] The CAR construct includes an intracellular signal transduction domain containing at least one immune receptor tyrosine activation motif (ITAM). In some embodiments, the intracellular signal transduction domain is TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In specific embodiments, the intracellular signal transduction domain is FcεR1γ, DAP12, or CD3ζ. In a typical embodiment, the intracellular signal transduction domain is CD3ζ. The CD3ζ domain has three ITAMs, named ITAM1, ITAM2, and ITAM3 from proximal to distal membrane.

[0080] Primary signaling domains that regulate TCR complex activation may include ITAM. Non-limiting examples of intracellular signaling domains containing ITAM include domains derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0081] 5.3.1.2 Modified CD3ζ intracellular signal transduction domain

[0082] In some embodiments, the CD3ζ intracellular signal transduction domain is modified. In some embodiments, the CD3ζ intracellular signal transduction domain includes modification in at least one of the three ITAMs. In some embodiments, the CD3ζ intracellular signal transduction domain is truncated. In some embodiments, the CD3ζ intracellular signal transduction domain is truncated to remove one or more of the three ITAMs. In some embodiments, the CD3ζ intracellular signal transduction domain is modified by selective splicing. In some embodiments, the CD3ζ intracellular signal transduction domain is engineered to express a fusion peptide between CD3ε, CD3δ, or CD3θ and CD3ζ.

[0083] In various embodiments, at least one, at least two, or all three ITAMs are modified. In some embodiments, at least two ITAMs of CD3ζ are modified. In some embodiments, all three ITAMs of CD3ζ are modified. In some embodiments, one ITAM of CD3ζ is unmodified, and at least one of the other two ITAMs is modified.

[0084] In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM1 variant containing one or more loss-of-function mutations. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM1 variant containing two loss-of-function mutations. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM1. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM1 to a phenylalanine residue.

[0085] In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM2 variant containing one or more loss-of-function mutations. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM2 variant containing two loss-of-function mutations. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM2. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM2 to a phenylalanine residue.

[0086] In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM3 variant containing one or more loss-of-function mutations. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM3 variant containing two loss-of-function mutations. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM3. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM3 to a phenylalanine residue.

[0087] In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM2 variant and an ITAM3 variant containing one or more loss-of-function mutations. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an ITAM2 variant and an ITAM3 variant, which contain two loss-of-function mutations. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in each of ITAM2 and ITAM3. In some embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in each of ITAM2 and ITAM3 to a phenylalanine residue.

[0088] In some embodiments, the modified CD3ζ intracellular signaling domain comprises ITAM1 and one or more modifications on at least one of ITAM2 and / or ITAM3. In some embodiments, the modified CD3ζ intracellular signaling domain comprises ITAM1 and one or more loss-of-function mutations in ITAM2 and / or ITAM3. In some embodiments, the modified CD3ζ intracellular signaling domain comprises ITAM1 and two loss-of-function mutations in each of ITAM2 and ITAM3.

[0089] In some embodiments, the modified CD3ζ comprises one or more ITAMs containing two tyrosine (Y) to phenylalanine (F) mutations. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in ITAM2 and / or ITAM3. In some embodiments, the modified CD3ζ intracellular signaling domain comprises an unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in each of ITAM2 and ITAM3. In some embodiments, the modified CD3ζ intracellular signaling domain comprises tyrosine (Y) to phenylalanine (F) mutations in at least one, at least two, or all three ITAMs (e.g., ITAM1, ITAM2, and ITAM3).

[0090] In various embodiments, the modified CD3ζ polypeptide comprises at least one functional ITAM. In some embodiments, the modified CD3ζ polypeptide contains mutations in both ITAM2 and ITAM3. In some embodiments, ITAM2 and ITAM3 in the modified CD3ζ are nonfunctional. In some embodiments, each of ITAM2 and ITAM3 contains at least one Tyr-to-Phe mutation. In typical embodiments, each of ITAM2 and ITAM3 contains two Tyr-to-Phe mutations. In some embodiments, the CD3ζ intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 48 (ITAM1 is bolded and underlined). Residues mutated from tyrosine (Y) (relative to unmodified CD3ζ) to phenylalanine (F) are double-underlined.

[0091]

[0092] (SEQ ID NO: 42)

[0093]

[0094] (SEQ ID NO: 43)

[0095]

[0096] (SEQ ID NO: 48).

[0097] In some embodiments, the second-generation CAR comprises a truncated CD3ζ intracellular signaling domain. In some embodiments, CD3ζ is truncated to remove at least one of the three ITAMs (e.g., ITAM1, ITAM2, ITAM3), or a combination thereof. In some embodiments, CD3ζ is truncated to remove ITAM1. In some embodiments, CD3ζ is truncated to remove ITAM2. In some embodiments, CD3ζ is truncated to remove ITAM3. In some embodiments, CD3ζ is truncated to remove both ITAM2 and ITAM3. In some embodiments, the truncated CD3ζ intracellular signaling domain has the amino acid sequence of SEQ ID NO: 49 as shown below:

[0098]

[0099] (SEQ ID NO: 49).

[0100] In various embodiments, the modified CD3ζ polypeptide comprises a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof. In some embodiments, the modified CD3ζ polypeptide comprises a deletion of ITAM3 or a portion thereof and a deletion of ITAM2 or a portion thereof. In some embodiments, the modified CD3ζ polypeptide comprises a deletion of both ITAM2 and ITAM3. In some embodiments, the modified CD3ζ polypeptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the N-terminal 40 amino acids of SEQ ID NO: 41 or SEQ ID NO: 48. In some embodiments, the modified CD3ζ polypeptide comprises the N-terminal 40 amino acids of the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 48.

[0101] In some embodiments, the modified CD3ζ polypeptide further comprises the deletion of one or more amino acid residues at the N-terminus of ITAM1. In some embodiments, the modified CD3ζ polypeptide comprises a sequence having at least 70%, 80%, 90%, 95%, or 99% identity with SEQ ID NO: 50-53. In some embodiments, the modified CD3ζ polypeptide comprises a sequence selected from SEQ ID NO: 50-53.

[0102] Those skilled in the art will be able to introduce mutations into the nucleic acid sequence of a gene or gene product (e.g., ITAM) using standard techniques. For example, point mutations can be introduced by site-directed mutagenesis using PCR.

[0103] 5.3.2. Co-stimulation signal transduction region

[0104] In CAR, the co-stimulatory signal transduction region is appropriately located between the signal transduction region and the transmembrane structural domain, and is far from the binding element.

[0105] Suitable costimulatory signal transduction regions are well known in the art and include those of B7 / CD28 family members, such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, or PDCD6; or ILT / CD85 family proteins, such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin α, OX40, RELT, TACI, TL1A, TNF-α, or TNF. RII; or SLAM family members, such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A, or SLAM; or TIM family members, such as TIM-1, TIM-3, or TIM-4; or other co-stimulatory molecules, such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, dendritic cell-associated type C phytohemagglutinin-1 (Dectin-1), DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), CD7, NKG2X, CD83, or variants thereof.See Mondino A et al., J LeukocBiol. [Journal of Leukocyte Biology] 55:805-815 (1994); Thompson CB, Cell. [Cell] 81:979-982 (1995); Somoza C et al., Res Immunol. [Immunological Research] 146:171-176 (1995); Rhodes DA et al., Annu Rev Immunol. [Annual Review of Immunology] 34:151-172 (2016); Foell J et al., Curr Cancer Drug Targets. [Contemporary Cancer Drug Targets] 7:55-70 (2007); Greenwald RJ et al., Annu Rev Immunol. [Annual Review of Immunology] 23:515-548 (2005); Flem-Karlsen K et al., Trends Cancer. [Cancer Trends] 4:401-404 (2018); Flies DB et al., J Immunother. [Journal of Immunotherapy] 30:251-260 (2007); Gavrieli M et al., Adv Immunol. [Advances in Immunology] 92:157-185 (2006); Zhu Y et al., Nat Commun. [Nature Communications] 4:2043 (2013); Omar HA et al., Crit RevOncol Hematol. [Reviews in Oncology and Hematology] 135:21-29 (2019); Hashemi M et al., Oncotarget. [Tumor Targets] 9:24857-24868 (2018); Kang X et al., Cell Cycle. [Cell Cycle] 15:25-40 (2016); Watts TH, Annu Rev Immunol. [Annual Review of Immunology] 23:23-68 (2005); Bryceson YT et al., Immunol Rev. 214:73-91 (2006); Sharpe AH, Curr Opin Immunol. 7:389-395 (1995); Wingren AG et al., Crit RevImmunol. 15:235-253 (1995), the contents of which are incorporated herein by reference in their entirety.

[0106] The co-stimulatory signaling region can be selected based on the specific intended use of the immune response cells. In some embodiments, the co-stimulatory signaling region is selected from the co-stimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, Dap10, CD30, GITR, HVEM, DR3, and CD40, or variants thereof. In some embodiments, the co-stimulatory signaling region is selected from the co-stimulatory signaling regions of CD28, 4-1BB, CD27, OX40, Dap10, and ICOS, or variants thereof.

[0107] In certain embodiments, the co-stimulatory signaling region of CAR is the co-stimulatory signaling region of CD28. In some embodiments, the CD28 co-stimulatory signaling region contains a modification of one or more tyrosine residues in the CD28 cytoplasmic domain. In some embodiments, the CD28 co-stimulatory signaling region contains a mutation at the C-terminal tyrosine residue. In some embodiments, the CD28 co-stimulatory signaling region contains a modified YRS motif. In some embodiments, the CD28 co-stimulatory signaling region contains a mutation in the YRS motif. In some embodiments, the CD28 co-stimulatory signaling region lacks the YRS motif.

[0108] In some embodiments, the co-stimulatory signaling region of CAR is the co-stimulatory signaling region of CD28. In some embodiments, the co-stimulatory signaling region of CD28 comprises the amino acid sequence of SEQ ID NO: 4 as shown below, wherein the intracellular domain is shown in bold:

[0109]

[0110] (SEQ ID NO: 4).

[0111] In some embodiments, the co-stimulatory signal transduction region of CD28 comprises the amino acid sequence of SEQ ID NO: 25 as shown below:

[0112]

[0113] (SEQ ID NO: 25).

[0114] 5.3.3. Transmembrane domain

[0115] Suitable transmembrane domains are known in the art and include, for example, transmembrane domains of CD8α, CD28, CD4, CD3ζ, FcεR1γ, or variants thereof. Choosing CD3ζ as the transmembrane domain may result in association between the CAR and other elements of the TCR / CD3 complex. This association may recruit more ITM, but may also lead to competition between the CAR and endogenous TCR / CD3.

[0116] In some embodiments, the transmembrane domain of the CAR is selected from the transmembrane domains of CD28 and CD8α. In a particular CAR embodiment, the transmembrane domain of the CAR is the transmembrane domain of CD28.

[0117] In some embodiments, the CAR includes a portion of the extracellular domain and the transmembrane domain of CD28.

[0118] In some embodiments, a portion of the CD28 extracellular domain and the transmembrane domain comprise the amino acid sequence of SEQ ID NO: 35 as shown below, wherein the transmembrane domain is shown in bold:

[0119]

[0120] (SEQ ID NO: 35).

[0121] 5.3.4. Co-stimulatory signal transduction domains and transmembrane domains

[0122] In some embodiments where the co-stimulatory signaling region of CAR is or includes the co-stimulatory signaling region of CD28, the CD28 transmembrane domain represents a suitable, and generally preferred, choice of transmembrane domain. The full-length CD28 protein is a 220-amino acid protein of SEQ ID NO: 3, wherein the transmembrane domain is shown in bold:

[0123]

[0124] (SEQ ID NO: 3).

[0125] In some embodiments, the co-stimulatory signal transduction region comprises a CD28-based hinge region and also suitably a transmembrane domain and an intracellular domain. In some embodiments, the co-stimulatory signal transduction region comprises amino acids 114-220 of SEQ ID NO: 3, shown below as SEQ ID NO: 4, wherein the transmembrane domain is shown in bold:

[0126]

[0127] (SEQ ID NO: 4).

[0128] In some embodiments where the co-stimulatory signaling region of CAR is or includes the co-stimulatory signaling region of Dap10, the Dap10 transmembrane domain represents a suitable, and generally preferred, choice of transmembrane domain. The full-length Dap10 protein is a 93-amino acid protein of SEQ ID NO: 47, wherein the transmembrane domain is shown in bold:

[0129]

[0130] (SEQ ID NO: 47)

[0131] 5.3.5. CAR bonding element

[0132] The CAR's bonding element is bonded to at least one epitope.

[0133] In various embodiments, the binding element of the CAR specifically binds to epitopes.

[0134] In various embodiments, the binding element of the CAR binds to a first target antigen. In some embodiments, at least one antigen is associated with a disease (e.g., cancer).

[0135] Therefore, a suitable binding element can be any element that provides the CAR with the ability to recognize targets of interest. The targets targeted by the CAR disclosed herein can be any clinically interesting targets for which it is desired to guide a T-cell response.

[0136] In various embodiments, the binding element used in the CAR described herein is the antigen-binding site (ABS) of an antibody. In typical embodiments, the ABS used as the binding element is arranged in the form of scFv or a single-domain antibody derived from a camel, human, or other species.

[0137] Alternatively, the binding element of a CAR may include a ligand that binds to a surface protein of interest.

[0138] In some embodiments, the binding element associates with a leader sequence on the cell surface that promotes expression. Many leader sequences are known in the art, and these sequences include macrophage colony-stimulating factor receptor (FMS) leader sequences or CD124 leader sequences.

[0139] In some embodiments, the binding element specifically binds to tumor antigens or tumor-associated antigens, providing a means of targeting tumor cells while limiting damage to non-tumor cells or tissues.

[0140] In some embodiments, the binding element specifically binds to a first epitope selected from the following first target antigens: NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB1-4, HER2, B7-H3, Claudin 18.2, Claudin 6, Glypican 3, ALK, CD70, GD2, and prostate-specific membrane antigen (PSMA).

[0141] In some embodiments, the binding element specifically binds to epitopes on antigen MUC1.

[0142] 5.3.5.1MUC1 CAR

[0143] In a particular embodiment, the binding element specifically interacts with an epitope on the MUC1 target antigen.

[0144] In presently preferred embodiments, the CAR binding element specifically interacts with a first epitope on the MUC1 target antigen. In some embodiments, the CAR binding element comprises an antigen-binding site for an antibody specific to MUC1. In some embodiments, the CAR binding element comprises a CDR for an antibody specific to MUC1. In some embodiments, the CAR binding element comprises a V-shaped surface area for an antibody specific to MUC1. H Sequence and V L sequence.

[0145] In some embodiments, the CAR-binding element comprises an antigen-binding site of the HMFG2 antibody. In some embodiments, the CAR-binding element comprises a CDR of the HMFG2 antibody. The CDR sequence of the HMFG2 antibody was determined using tools available at www.abysis.org and is shown below as SEQ ID NO: 8-13:

[0146] V H CDR1GTFFSNY (SEQ ID NO: 8);

[0147] V H CDR2RLKSNNYA (SEQ ID NO: 9);

[0148] V H CDR3GNSFAY (SEQ ID NO: 10);

[0149] V L CDR1RSSTGAVTTSNYAN (SEQ ID NO: 11);

[0150] V L CDR2GTNNRAP (SEQ ID NO: 12);

[0151] V L CDR3ALWYSNHWV (SEQ ID NO: 13).

[0152] In some embodiments, the CAR binding element comprises the V of the HMFG2 antibody. H Domain and V L Domain. V of HMFG2 antibody H Domain sequence and V L The domain sequences are shown below as SEQ ID NO: 14-15:

[0153] EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSS

[0154] (SEQ ID NO: 14);

[0155] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE

[0156] (SEQ ID NO: 15).

[0157] In a particularly preferred embodiment, the CAR-binding element comprises an antigen-binding site for the HMFG2 antibody arranged in the form of an scFv. In some embodiments, the amino acid sequence of the scFv of the HMGF2 antibody is identical to that shown below in SEQ ID NO: 1670%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the following:

[0158] EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGS GGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE

[0159] (SEQ ID NO: 16).

[0160] In some embodiments, the nucleic acid encoding the scFv of the HMGF2 antibody is SEQ ID NO: 17 shown below:

[0161] GAGGTGCAGCTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACAT TATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCA GGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCCGTGGTCACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGT GGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGATCAGAG

[0162] (SEQ ID NO: 17).

[0163] 5.3.5.2NKG2DL CAR

[0164] In a particular embodiment, the binding element interacts specifically with an epitope on the NKG2D receptor ligand.

[0165] In some embodiments, the CAR-binding element comprises a human NKG2D receptor polypeptide. In some embodiments, the CAR-binding element comprises a fragment, portion, or variant of the human NKG2D receptor polypeptide.

[0166] The NKG2D-CD3ζ linear CAR (SEQ ID NO: 69) contains an NKG2D ligand-binding domain (SEQ ID NO: 75) fused with a CD3ζ peptide (SEQ ID NO: 1).

[0167] The protein sequence of the NKG2D-CD3ζ linear CAR is shown below as SEQ ID NO: 69. The CD3ζ sequence is in italics, while the ITM of CD3ζ is in bold; the NKG2D sequence is double-underlined.

[0168]

[0169] (SEQ ID NO: 69).

[0170] 5.4. Parallel chimeric antigen receptor (pCAR)

[0171] In one aspect, a parallel chimeric antigen receptor (pCAR) is provided. The pCAR comprises a second-generation CAR and a chimeric co-stimulatory receptor (CCR). The pCAR comprises a second-generation CAR containing (a) an intracellular signaling domain comprising a CD3ζ polypeptide; (b) a co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen, wherein the modified CD3ζ polypeptide comprises an unmodified immune receptor tyrosine activation motif 1 (ITAM1) and mutations in ITAM2 and / or ITAM3; and a CCR containing (e) a co-stimulatory signaling region different from the co-stimulatory signaling region of (b); (f) a transmembrane domain; and (g) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0172] In some embodiments, the pCAR is the TBB / H pCAR shown in Figure 1A. The TBB / H pCAR utilizes a second-generation "H2" CAR targeting MUC1 and a co-expressed chimeric co-stimulatory receptor (CCR). The CCR in the TBB / H pCAR has fusion with the CD8α transmembrane domain. T 1E combines structural domains and 4-1 BBCo-stimulatory domain. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) and is a hybrid ErbB ligand. See Wingens et al., J. Biol. Chem. 278:39114-23 (2003) and Davies et al., Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entirety. Unlike CARs, CCRs lack TCR-like signaling domains, such as CD3ζ. Compared with first-generation, second-generation, and third-generation CAR-T cells, parallel CAR (pCAR) engineered T cells exhibit superior activity and resistance to exhaustion. See PCT Publication WO 2020183158 A1, which is incorporated herein by reference in its entirety.

[0173] In some embodiments, the pCAR is the TBB / H 1XX pCAR shown in Figure 1A. Except that the CD3ζ peptide is modified to include one or more mutations in ITAM2 and ITAM3, the TBB / H 1XX pCAR is the same as the TBB / H pCAR described above, as described in 5.3.1.2 above.

[0174] In some embodiments, the pCAR is a TBB / H 1XX pCAR variant as shown in Figure 1B. In some embodiments, the TBB / H 1XX pCAR variant is TBB / H-1, which does not contain the two distal ITAMs (ITAM2 and ITAM3) of CD3ζ. In some embodiments, the TBB / H 1XX pCAR variant contains a mutation in the proximal ITAM (ITAM1) and one or more tyrosine (Y) to phenylalanine (F) in each of ITAM2 and ITAM3. In some embodiments, the TBB / H 1XX pCAR contains a mutation in the proximal ITAM (ITAM1) and two tyrosine (Y) to phenylalanine (F) in each of ITAM2 and ITAM3.

[0175] In some embodiments, the pCAR is a variant of the TBB / H-1 pCAR, and when compared to the TBB / H-1 pCAR, it lacks one or more amino acids upstream of the functional ITAM (ITAM1) of CD3ζ. In some embodiments, when compared to the TBB / H-1 pCAR, the TBB / H-1 pCAR variant lacks three amino acid residues upstream of the functional ITAM (ITAM1) of CD3ζ (TBB / H-1). 3). In some embodiments, the TBB / H-1 pCAR variant lacks six amino acid residues upstream of the functional ITAM (ITAM1) of CD3ζ compared to the TBB / H-1 pCAR. 6). In some embodiments, the TBB / H-1 pCAR variant lacks 9 amino acid residues upstream of the functional ITAM (ITAM1) of CD3ζ compared to TBB / H-1 pCAR. 9).

[0176] In some embodiments, the TBB / H-1 pCAR variant lacks 9 amino acid residues upstream of the functional ITAM (ITAM1) at CD3ζ, and further lacks the YRS motif at CD28 (i.e., TBB / H-1). 9 YRS).

[0177] In some embodiments, the pCAR is the TBB / H-FcεR1γ pCAR or the TBB / H-DAP12 pCAR shown in FIG1C. In such embodiments, the TBB / H-FcεR1γ pCAR includes an FcεR1γ substitution activation domain replacing the CD3ζ domain. In such embodiments, the TBB / H-DAP12 pCAR includes a DAP12 substitution activation domain replacing the CD3ζ domain.

[0178] 5.4.1. Chimeric Co-stimulatory Receptors (CCRs)

[0179] The chimeric costimulatory receptor (CCR) comprises, from the C-terminus to the N-terminus (or, if expressed in an immune-responding cell, from intracellular to extracellular), at: (e) at least one second costimulatory signaling region, which is distinct from the first costimulatory signaling region of the CAR; (f) at least one second transmembrane domain; and (g) at least one second binding element that specifically binds to a second epitope on a second target antigen.

[0180] In some embodiments, CCR is a single polypeptide. In some embodiments, CCR is two polypeptides. In some embodiments, CCR is three polypeptides. In some embodiments, CCR is more than three polypeptides.

[0181] In some embodiments, the second binding element specifically binds to a second epitope selected from the following second target antigens: ErbB1-4, HER2, CD19, B7-H3, GD2, αvβ6 integrin, NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), NKp30 ligands (e.g., BAG6, B7-H6), NKp44 ligands (e.g., NKp44L (MLL5), PCNA, viral hemagglutinin, nestin-1, galactoglucan-3, other proteoglycans), NKp46 ligand, MUC1, sealing protein 18.2, sealing protein 6, phosphatidylinositol proteoglycan 3, ALK, CD70, GD2, PD-L1, and PSMA or variants thereof.

[0182] The second epitope may be the same as or different from the first epitope.

[0183] In some embodiments, the pCAR comprises a CCR, which comprises two CCR peptides that dimerize upon ligand binding. In some embodiments, the pCAR comprises a CCR, which has three CCR peptides that trimerize upon ligand binding.

[0184] In some embodiments, CCR is in monomeric form. In some embodiments, CCR is in dimeric form. In some embodiments, CCR is in trimeric form.

[0185] In some embodiments, the dimerized CCR includes a second binding element that specifically binds to an epitope on the ErbB antigen. In some embodiments, the second binding element is a T1E peptide. In some embodiments, the second binding element has the amino acid sequence of SEQ ID NO: 18.

[0186] In some embodiments, the dimerized CCR includes a second binding element that specifically binds to an NKG2D ligand (e.g., MICA, MICB, ULBP1-ULBP6). In some embodiments, the second binding element is an NKG2D peptide. In some embodiments, the second binding element has the amino acid sequence of SEQ ID NO: 75. In some embodiments, the dimerized CCR has the amino acid sequence of SEQ ID NO: 77.

[0187] In some embodiments, the trimerized CCR includes a second binding element that specifically binds to an NKG2D ligand (e.g., MICA, MICB, ULBP1-ULBP6). In some embodiments, the second binding element is an NKG2D peptide. In some embodiments, the second binding element has the amino acid sequence of SEQ ID NO: 75. In some embodiments, the trimerized CCR has the amino acid sequence of SEQ ID NO: 79.

[0188] In some embodiments, the trimerized CCR includes a second binding element that specifically binds to a PD-1 ligand. In some embodiments, the second binding element is a PD-1 peptide. In some embodiments, the second binding element has the amino acid sequence of SEQ ID NO: 86. In some embodiments, the trimerized CCR has the amino acid sequence of SEQ ID NO: 81.

[0189] In some embodiments, the trimerized CCR includes a second binding element that specifically binds to an NKp44 ligand. In some embodiments, the second binding element is an NKp44 polypeptide. In some embodiments, the second binding element has the amino acid sequence of SEQ ID NO: 87. In some embodiments, the trimerized CCR has the amino acid sequence of SEQ ID NO: 83.

[0190] 5.4.1.1 Co-stimulatory signal transduction region

[0191] In CCR, the co-stimulatory signal transduction region is appropriately located near the transmembrane structural domain and far from the binding element.

[0192] Suitable costimulatory signal transduction regions are well known in the art and include those of B7 / CD28 family members, such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, or PDCD6; or ILT / CD85 family proteins, such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin α, OX40, RELT, TACI, TL1A, TNF-α, or TNF. RII; or SLAM family members, such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A, or SLAM; or TIM family members, such as TIM-1, TIM-3, or TIM-4; or other co-stimulatory molecules, such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, dendritic cell-associated type C phytohemagglutinin-1, DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), CD7, NKG2X, CD83, or variants thereof.See Mondino A et al., J Leukoc Biol. [Journal of Leukocyte Biology] 55:805-815 (1994); Thompson CB, Cell. [Cell] 81:979-982 (1995); Somoza C et al., Res Immunol. [Immunological Research] 146:171-176 (1995); Rhodes DA et al., AnnuRev Immunol. [Annual Review of Immunology] 34:151-172 (2016); Foell J et al., Curr Cancer DrugTargets. [Contemporary Cancer Drug Targets] 7:55-70 (2007); Greenwald RJ et al., Annu Rev Immunol. [Annual Review of Immunology] 23:515-548 (2005); Flem-Karlsen K et al., Trends Cancer. [Trends in Cancer] 4:401-404 (2018); Flies DB et al., J Immunother. [Journal of Immunotherapy] 30:251-260 (2007); Gavrieli M et al., Adv Immunol. [Advances in Immunology] 92:157-185 (2006); Zhu Y et al., Nat Commun. [Nature Communications] 4:2043 (2013); Omar HA et al., Crit Rev Oncol Hematol. [Reviews in Oncology and Hematology] 135:21-29 (2019); Hashemi M et al., Oncotarget. [Tumor Targets] 9:24857-24868 (2018); Kang X et al., Cell Cycle. [Cell Cycle] 15:25-40 (2016); WattsTH, Annu Rev Immunol. [Annual Review of Immunology] 23:23-68 (2005); Bryceson YT et al., Immunol Rev. [Immunol Review] 214:73-91 (2006); Sharpe AH, Curr Opin Immunol. [Current Opinion on Immunology] 7:389-395 (1995); Wingren AG et al., Crit Rev Immunol. [Immunol Review Review] 15:235-253 (1995), the contents of which are incorporated herein by reference in their entirety.

[0193] The co-stimulatory signaling region can be selected based on the specific intended use of the immune-responding cells. Specifically, the co-stimulatory signaling region of the CCR can be selected to function superimposed or synergistically with the co-stimulatory signaling region of the CAR. In some embodiments, the co-stimulatory signaling region of the CCR is selected from the co-stimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, Dap10, and CD40, or variants thereof.

[0194] In some embodiments, the co-stimulatory signal transduction region of the CCR is selected from the 4-1BB co-stimulatory domain, the CD28 co-stimulatory domain, the OX40 co-stimulatory domain, and the ICOS co-stimulatory domain. In some embodiments, the co-stimulatory signal transduction region of the CCR has an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 25, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

[0195] In some embodiments, the co-stimulatory signal transduction region of the CCR is a 4-1BB co-stimulatory domain.

[0196] In some embodiments, one co-stimulatory signal transduction region of the pCAR is the CD28 co-stimulatory signal transduction region, and the other is the 4-1BB co-stimulatory signal transduction region. In some embodiments, one co-stimulatory signal transduction region of the pCAR is the CD28 co-stimulatory signal transduction region, and the other is the CD27 co-stimulatory signal transduction region. In some embodiments, one co-stimulatory signal transduction region of the pCAR is the CD28 co-stimulatory signal transduction region, and the other is the OX40 co-stimulatory signal transduction region. In some embodiments, one co-stimulatory signal transduction region of the pCAR is the ICOS co-stimulatory signal transduction region, and the other is the 4-1BB co-stimulatory signal transduction region.

[0197] In a specific pCAR embodiment, the co-stimulatory signal transduction region of the CAR is the co-stimulatory signal transduction region of CD28, and the co-stimulatory signal transduction region of the CCR is the co-stimulatory signal transduction region of 4-1BB.

[0198] In some embodiments, the co-stimulatory signal transduction region of the CCR is the 4-1BB co-stimulatory signal transduction region. In some embodiments, the 4-1BB co-stimulatory signal transduction region comprises the amino acid sequence of SEQ ID NO: 37 as shown below:

[0199] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0200] (SEQ ID NO: 37).

[0201] 5.4.1.2 Transmembrane domain

[0202] The transmembrane domains of the CAR and CCR constructs can be the same or different. In the currently preferred embodiments, when the CAR and CCR constructs are expressed by a single vector, the transmembrane domains of the CAR and CCR are different to ensure that the constructs are separated on the cell surface. Choosing different transmembrane domains can also enhance the stability of the expression vector, as viral vectors contain homologous nucleic acid sequences that facilitate rearrangement and the deletion of sequences between homologous repeats. In embodiments where the CAR and CCR of pCAR are chosen to have the same transmembrane domain, this risk can be mitigated by modifying or “wobbling” the codons that encode the same protein sequence.

[0203] Suitable transmembrane domains are known in the art and include, for example, transmembrane domains of CD8α, CD28, CD4, CD3ζ, FcεR1γ, or variants thereof. Choosing CD3ζ as the transmembrane domain may result in association between the CAR and other elements of the TCR / CD3 complex. This association may recruit more ITM, but may also lead to competition between the CAR and endogenous TCR / CD3.

[0204] In some embodiments, the transmembrane domain of the CCR is the transmembrane domain of CD8α.

[0205] In some embodiments, the CCR includes a portion of the extracellular domain and the transmembrane domain of CD8α.

[0206] In some embodiments, a portion of the CD8a extracellular domain and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36 as shown below:

[0207] PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH

[0208] (SEQ ID NO: 36)

[0209] 5.4.1.3 CCR bonding element

[0210] The binding elements of the CAR construct and CCR construct of pCAR are respectively bound to the first epitope and the second epitope.

[0211] In a typical embodiment, the bonding elements of the CAR construct and the CCR construct are different from each other.

[0212] In various embodiments, the binding elements of the CAR and CCR specifically bind to a first epitope and a second epitope of the same antigen. In some embodiments of these embodiments, the binding elements of the CAR and CCR specifically bind to the same epitope, overlapping epitope, or different epitope of the same antigen. In embodiments where the first epitope and the second epitope are the same or overlapping, the binding elements on the CAR and CCR may compete for their binding.

[0213] In various embodiments, the binding elements of the pCAR CAR construct and the CCR construct bind to different antigens. In some embodiments, the antigens are different but may be associated with the same disease (e.g., the same specific cancer).

[0214] Therefore, a suitable binding element can be any element that provides the CCR with the ability to recognize targets of interest. The targets targeted by the CCR disclosed herein can be any clinically interesting targets for which it is desired to guide a T-cell response.

[0215] In various embodiments, the binding element used in the CCR of the pCAR described herein is the antigen-binding site (ABS) of an antibody. In typical embodiments, the ABS used as the binding element is arranged in the form of a single-chain antibody (scFv) or a single-domain antibody derived from a camel, human, or other species.

[0216] Alternatively, the binding element of the CCR of the pCAR disclosed herein may include a ligand that binds to a surface protein of interest.

[0217] In some embodiments, the binding element associates with a leader sequence on the cell surface that promotes expression. Many leader sequences are known in the art, and these sequences include macrophage colony-stimulating factor receptor (FMS) leader sequences or CD124 leader sequences.

[0218] In some embodiments, the binding element specifically binds to tumor antigens or tumor-associated antigens, providing a means of targeting tumor cells while limiting damage to non-tumor cells or tissues.

[0219] In some embodiments, the binding element of the CCR specifically binds to epitopes selected from the following second target antigens: NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, sealing protein 18.2, sealing protein 6, phosphatidylinositol proteoglycan 3, ALK, CD70 and PSMA, PD-L1, and NKp30 ligands (e.g., BAG6 and B7-H6).

[0220] In some embodiments, the binding element of the CCR specifically binds to epitopes on NKG2D ligands (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6). In some embodiments, the binding element of the CCR is an NKG2D peptide or a fragment or functional variant thereof.

[0221] In some embodiments, the binding element of the CCR specifically binds to an epitope on a PD-L1 ligand (e.g., PD-L1). In some embodiments, the binding element of the CCR is a PD-1 peptide or a fragment or functional variant thereof.

[0222] In some embodiments, the binding element of the CCR specifically binds to an epitope on the NKp44 ligand. In some embodiments, the binding element of the CCR is an NKp44 peptide or a fragment or functional variant thereof.

[0223] In some embodiments, the binding element of the CCR specifically interacts with an epitope on the ErbB receptor. In some embodiments, the binding element of the CCR is a T1E peptide, an antigen-binding site of HER2, or an antigen-binding site of another ErbB family member. In some embodiments, the binding element of the CCR is a T1E peptide.

[0224] In some embodiments, the binding element of the CCR is a T1E peptide that binds to both ErbB homodimers and heterodimers. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), and is a hybrid ErbB ligand. The T1E peptide is a chimeric fusion protein comprising the entire mature human EGF protein, excluding the five amino acids closest to the N-terminus (amino acids 971-975 of the pre-epidermal growth factor precursor (NP 001954.2)), which have been replaced with the seven amino acids closest to the N-terminus of the mature human TGF-α protein (amino acids 40-46 of pre-transforming growth factor α isoform 1 (NP 003227.1)). See Wingens et al., J. Biol. Chem. 278:39114-23 (2003) and Davies et al., Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entirety. The sequence of T1E is shown below as SEQ ID NO: 18:

[0225] VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

[0226] (SEQ ID NO: 18).

[0227] In some embodiments, the binding element of the CCR is ICR62, which binds to EGFR. In a particular embodiment, the binding element of the CCR comprises an antigen-binding site of an ICR62 antibody arranged in the form of an scFv. In some embodiments, the amino acid sequence of the scFv of the ICR62 antibody is identical to 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of SEQ ID NO:44 shown below.

[0228] QVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWVKQSHGKSLEWIGYFNPNSGYSTYNEKFKSKATLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVMDAWGQGASVTVSSAQTTAPSVYPLAPGSG GGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQKLGEAPKKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDFATYFCLQHNSFPTFGAGTKLELKRADAAPTVSIFPPSKS

[0229] (SEQ ID NO: 44).

[0230] In some embodiments, the binding element of the CCR specifically binds to PD-L1.

[0231] In some embodiments, the binding element specifically binds to NKp30 ligands selected from BAG6 and B7-H6.

[0232] In some embodiments, the second-generation CAR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, and the CCR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 24.

[0233] 5.4.2. Exemplary pCAR

[0234] TBB / H pCAR (SEQ ID NO: 7) comprises: (i) a CCR comprising: a T1E binding domain fused to a portion of the CD8α extracellular domain, followed by a CD8α transmembrane domain and a 4-1BB costimulatory domain (“TBB”) (SEQ ID NO: 24); and (ii) a second-generation CAR comprising: an HMFG2 scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain (“H2”) (SEQ ID NO: 21). The CCR and CAR are linked via a furin cleavage site (RRKR (SEQ ID NO: 31)), a Ser-Gly linker (SGSG (SEQ ID NO: 32)), and a T2A ribosomal skip peptide (EGRGSLLTCGDVEENPGP (SEQ ID NO: 33)).

[0235] The protein sequence of TBB / H pCAR is shown below as SEQ ID NO: 7. V of HMFG2 H and V L The sequences are underlined and bolded; the T1E peptide sequence is italicized, underlined, and bolded; the CD3ζ signal transduction region sequence is italicized; and the ITAM sequence is bolded and italicized.

[0236]

[0237] (SEQ ID NO: 7).

[0238] The protein sequence of the CCR (“TBB”) of TBB / H is shown below as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold:

[0239]

[0240] (SEQ ID NO: 24).

[0241] The protein sequence of the CAR (“H2”) of TBB / H is shown below as SEQ ID NO: 21, wherein the V of HMFG2 H and V L The sequence is underlined and bolded, while the ITM of the CD3ζ polypeptide is italicized and bolded:

[0242]

[0243] (SEQ ID NO: 21).

[0244] The TBB / H 1XX pCAR (SEQ ID NO: 23) comprises (i) a CCR containing a T1E binding domain fused to a portion of the CD8α extracellular domain, followed by a CD8α transmembrane domain and a 4-1BB co-stimulatory domain (“TBB”) (SEQ ID NO: 24); and (ii) a second-generation CAR containing an HMFG2 scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3ζ signaling region (“H2 1XX”) (SEQ ID NO: 59). The modified CD3ζ peptide (SEQ ID NO: 48) contains the wild-type immune receptor tyrosine activation motif 1 (ITAM1) and two tyrosine-to-phenylalanine mutations in each of ITAM2 and ITAM3. CCR and CAR are linked via furin cleavage site (RRKR (SEQ ID NO: 31)), Ser-Gly linker (SGSG (SEQ ID NO: 32)) and T2A ribosomal skipping peptide (EGRGSLLTCGDVEENPGP (SEQ ID NO: 33)).

[0245] The protein sequence of TBB / H 1XX pCAR is shown below as SEQ ID NO: 23. V of HMFG2 H and V L The sequence is underlined and bolded, while the ITM of the CD3ζ polypeptide is italicized and bolded. Residues mutated from tyrosine (Y) (as opposed to unmodified CD3ζ) to phenylalanine (F) are double-underlined.

[0246]

[0247] (SEQ ID NO: 23).

[0248] The protein sequence of the CCR (“TBB”) of TBB / H 1XX is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0249] The protein sequence of the CAR (“H 1XX”) of TBB / H 1XX is shown below as SEQ ID NO: 59, wherein the V of HMFG2 H and V L The sequence is underlined and bolded, and the ITAM of CD3ζ is italicized and bolded.

[0250]

[0251] (SEQ ID NO: 59)

[0252] The TBB / H-1 pCAR (SEQ ID NO: 26) comprises (i) a CCR containing a T1E-binding domain fused to a portion of the CD8α extracellular domain, followed by a CD8α transmembrane domain and a 4-1BB co-stimulatory domain (“TBB”) (SEQ ID NO: 24); and (ii) a second-generation CAR containing an HMFG2 scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3ζ signaling region (“H-1”) (SEQ ID NO: 22). The modified CD3ζ peptide contains only one immune receptor tyrosine activation motif 1 (ITAM1), wherein each of ITAM2 and ITAM3 is deleted from the CD3ζ peptide. CCR and CAR are linked via furin cleavage site (RRKR (SEQ ID NO: 31)), Ser-Gly linker (SGSG (SEQ ID NO: 32)) and T2A ribosomal skipping peptide (EGRGSLLTCGDVEENPGP (SEQ ID NO: 33)).

[0253] The protein sequence of TBB / H-1 pCAR is shown below as SEQ ID NO: 26. The V of HMFG2 H and V L The sequence is underlined and bolded, and the ITAM1 of the CD3ζ polypeptide is in italics and bolded.

[0254]

[0255] (SEQ ID NO: 26).

[0256] The protein sequence of the CCR (“TBB”) of TBB / H-1 is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0257] The protein sequence of the CAR (“H-1”) of TBB / H-1 is shown below as SEQ ID NO: 22, wherein the V of HMFG2 H and V L The sequence and the ITM1 of the CD3ζ polypeptide are in italics and bold:

[0258]

[0259] (SEQ ID NO: 22).

[0260] Various TBB / H-1 The pCAR comprises (i) a CCR comprising: a T1E binding domain fused to a portion of the CD8α extracellular domain, followed by a CD8α transmembrane domain and a 4-1BB co-stimulatory domain (“TBB”) (SEQ ID NO: 24); and (ii) a second-generation CAR comprising: an HMFG2scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3ζ signaling region (“H-1”). The region lacks each of ITAM2 and ITAM3 and has three missing from the N-terminus of ITAM1 ("H-1"). 3”), six (“H-1”) 6) or nine ("H-1") 9” amino acid residues. CCR and CAR are linked by furin protease cleavage site (RRKR (SEQ ID NO:31)), Ser-Gly linker (SGSG (SEQ ID NO:32)) and T2A ribosomal skip peptide (EGRGSLLTCGDVEENPGP (SEQ ID NO:33)).

[0261] TBB / H-1 The protein sequence of 3 pCARs is shown below as SEQ ID NO: 27. HMFG2 V H and V L The sequence is underlined and bolded, and the ITAM1 sequence of CD3ζ is italicized and bolded.

[0262]

[0263] (SEQ ID NO: 27).

[0264] TBB / H-1 The protein sequence of CCR (“TBB”) of 3 is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0265] TBB / H-1 3 of the CAR ("H-1") The protein sequence of HMFG2 (3) is shown below as SEQ ID NO: 62, wherein the V of HMFG2 is... H and V L The sequence is underlined and bolded, while ITAM1 of CD3ζ is italicized and bolded.

[0266]

[0267] (SEQ ID NO: 62).

[0268] TBB / H-1 3 of H-1 The protein sequence of 3 contains a truncated CD3ζ polypeptide with the amino acid sequence shown in SEQ ID NO: 50. The ITAM1 sequence is in italics and bold.

[0269]

[0270] (SEQ ID NO: 50)

[0271] TBB / H-1 The protein sequence of 6 pCAR is shown below as SEQ ID NO: 28. V of HMFG2 H and V L The sequence is underlined and bolded, and the ITAM1 sequence of CD3ζ is italicized and bolded.

[0272]

[0273] (SEQ ID NO: 28).

[0274] TBB / H-1 The protein sequence of CCR (“TBB”) of 6 is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0275] TBB / H-1 6 of the CAR ("H-1") The protein sequence of HMFG2 (6) is shown below as SEQ ID NO: 63, wherein the V of HMFG2 is... H and V L The sequence is underlined and bolded, while ITAM1 of CD3ζ is italicized and bolded.

[0276]

[0277] (SEQ ID NO: 63).

[0278] TBB / H-1 6 of H-1 The protein sequence of 6 contains a truncated CD3ζ polypeptide with the amino acid sequence shown in SEQ ID NO: 51. The ITAM1 sequence is in italics and bold.

[0279]

[0280] (SEQ ID NO: 51).

[0281] TBB / H-1 The protein sequence of pCAR 9 is shown below as SEQ ID NO: 29. HMFG2 V H and V L The sequences are underlined and bolded, the sequences of the CD28 co-stimulatory region are double-underlined, and the ITM1 sequence of CD3ζ is italicized and bolded.

[0282]

[0283] (SEQ ID NO: 29).

[0284] TBB / H-1 The protein sequence of CCR (“TBB”) of 9 is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0285] TBB / H-1 9 of the CAR ("H-1") The protein sequence of HMFG2 (9") is shown below as SEQ ID NO: 64, wherein the V of HMFG2 is... H and V L The sequence is underlined and bolded, while ITAM1 of CD3ζ is italicized and bolded.

[0286]

[0287] (SEQ ID NO: 64).

[0288] TBB / H-1 9 H-1 The protein sequence of number 9 contains a truncated CD3ζ polypeptide with the amino acid sequence shown in SEQ ID NO: 52. The ITAM1 sequence is in italics and bold.

[0289]

[0290] (SEQ ID NO: 52).

[0291] TBB / H-1 9 YRS pCAR is TBB / H-1 A modified version of 9, in which the YRS motif in the CD28 co-stimulatory region is missing.

[0292] TBB / H-1 The protein sequence of 9 YRS is shown below as SEQ ID NO: 30. V of HMFG2 H and V L The sequences are underlined and bolded, the sequences of the CD28 co-stimulatory region are double-underlined, and the ITM1 sequence of CD3ζ is italicized and bolded.

[0293]

[0294] (SEQ ID NO: 30).

[0295] TBB / H-1 The protein sequence of the CCR (“TBB”) of 9 YRS is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0296] TBB / H-1 9 YRS's CAR ("H-1") The protein sequence of "9 YRS" is shown below as SEQ ID NO: 65, wherein the V of HMFG2 H and V L The sequences are underlined and bolded, the sequences of the CD28 co-stimulatory region are double-underlined, and the ITM1 sequence of CD3 is italicized and bolded.

[0297]

[0298] (SEQ ID NO: 65).

[0299] TBB / H-1 9 YRS H-1 The protein sequence of 9 YRS contains a truncated CD3ζ polypeptide with the amino acid sequence shown in SEQ ID NO: 53. The ITAM1 sequence is in italics and bold.

[0300]

[0301] (SEQ ID NO: 53).

[0302] The protein sequence of TBB / H-FcεR1γ pCAR is shown below as SEQ ID NO: 54. V of HMFG2 H and V L The sequence is underlined and bolded, and the ITAM of FcεR1γ is italicized and bolded.

[0303]

[0304] (SEQ ID NO: 54).

[0305] The protein sequence of the CCR (“TBB”) of TBB / H-FcεR1γ is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0306] The protein sequence of the CAR (“FcεR1γ”) of TBB / H-FcεR1γ is shown below as SEQ ID NO: 55, wherein the V of HMFG2 H and V L The sequence is underlined and bolded, and the ITAM of FcεR1γ is italicized and bolded.

[0307]

[0308] (SEQ ID NO: 55).

[0309] The protein sequence of TBB / H-DAP12 pCAR is shown below as SEQ ID NO: 56. V of HMFG2 H and V L The sequence is underlined and bolded, and ITAM in DAP12 is italicized and bolded.

[0310]

[0311] (SEQ ID NO: 56).

[0312] The protein sequence of the CCR (“TBB”) of TBB / H-DAP12 is shown above as SEQ ID NO: 24, with the T1E peptide underlined and highlighted in bold.

[0313] The protein sequence of the CAR (“H-DAP12”) of TBB / H-DAP12 is shown below as SEQ ID NO: 57, wherein the V of HMFG2 H and V L The sequence is underlined and bolded, and ITAM in DAP12 is italicized and bolded.

[0314]

[0315] (SEQ ID NO: 57).

[0316] NKG2D_bb / H 1XX pCAR (SEQ ID NO: 76) comprises (i) two CCR peptides, each containing a CD124 leader peptide, an NKG2D extracellular domain fused to a G4S-G4D linker (amino acids 82-216), an IgG1 spacer region, followed by a CD8a transmembrane domain and a 4-1BB costimulatory domain (“NKG2D_bb”) (SEQ ID NO: 77) and (ii) a second-generation CAR containing an HMFG2 scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 costimulatory domain, and a modified CD3ζ signaling region (“H2 1XX”) (SEQ ID NO: 59). The modified CD3ζ polypeptide (SEQ ID NO: 48) contains the wild-type immune receptor tyrosine activation motif 1 (ITAM1) and two tyrosine-to-phenylalanine mutations in each of ITAM2 and ITAM3. CCR and CAR are linked via a T2A ribosomal skipping peptide.

[0317] The protein sequence of NKG2D_bb / H 1XX pCAR is shown below as SEQ ID NO: 76. The NKG2D sequence is underlined with a single underline, while the ITM of the CD3ζ polypeptide is italicized and bolded, with mutated residues underlined with double underlines.

[0318]

[0319] (SEQ ID NO: 76).

[0320] The protein sequence of the CCR (“NKG2D_bb”) of NKG2D_bb / H 1XX is shown below as SEQ ID NO: 77, wherein the NKG2D polypeptide sequence is bolded and the 4-1BB co-stimulatory signal transduction region sequence is underlined.

[0321]

[0322] (SEQ ID NO: 77).

[0323] The protein sequence of the CAR (“H 1XX”) of NKG2D_bb / H 1XX is shown above as SEQ ID NO: 59.

[0324] NKG2Dbb (trimer) / H 1XX pCAR (SEQ ID NO: 78) comprises (i) three CCR peptides, each containing a CD124 leader peptide, an NKG2D extracellular domain fused to a G4S-G4D-G4 linker (amino acids 82-216), a coiled-coil domain of Coronin 1A, a G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB costimulatory domain (“NKG2Dbb (trimer)”) (SEQ ID NO: 79) and (ii) a second-generation CAR containing an HMFG2 scFv fused to a portion of the CD28 extracellular domain targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 costimulatory domain, and a modified CD3ζ signaling region (“H2 1XX”) (SEQ ID NO: 59). The modified CD3ζ polypeptide (SEQ ID NO: 48) contains the wild-type immune receptor tyrosine activation motif 1 (ITAM1) and two tyrosine-to-phenylalanine mutations in each of ITAM2 and ITAM3. CCR and CAR are linked via a furin cleavage site (RRKR)-SGSG linker-T2A ribosomal skip peptide.

[0325] The protein sequence of NKG2Dbb (trimer) / H 1XX pCAR is shown below as SEQ ID NO: 78. The NKG2D sequence is underlined with a single underline, while the ITM of the CD3ζ polypeptide is italicized and bolded, with mutated residues underlined with double underlines.

[0326]

[0327] (SEQ ID NO: 78).

[0328] The protein sequence of the CCR (“NKG2Dbb (trimer)”) of NKG2Dbb (trimer) / H 1XX is shown below as SEQ ID NO: 79, wherein the NKG2D polypeptide sequence is bolded and the 4-1BB co-stimulatory signal transduction region sequence is underlined.

[0329]

[0330] (SEQ ID NO: 79).

[0331] The protein sequence of the CAR (“H 1XX”) of NKG2D_bb / H 1XX is shown above as SEQ ID NO: 59.

[0332] Pbb(trimer) / H 1XX pCAR (SEQ ID NO: 80) comprises (i) three CCR peptides, each containing a PD-1 peptide fused to a G4 linker, a coiled-coil domain of crown protein 1A, a G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB costimulatory domain (“Pbb(trimer)”) (SEQ ID NO: 81) and (ii) a second-generation CAR containing an HMFG2 scFv fused to a portion of the extracellular domain of CD28 targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 costimulatory domain, and a modified CD3ζ signaling domain (“H2 1XX”) (SEQ ID NO: 59). The modified CD3ζ peptide (SEQ ID NO: 48) contains the wild-type immune receptor tyrosine activation motif 1 (ITAM1) and two tyrosine-to-phenylalanine mutations in each of ITAM2 and ITAM3. CCR and CAR are linked via a furin cleavage site (RRKR)-SGSG linker-T2A ribosomal skip peptide.

[0333] The protein sequence of Pbb (trimer) / H 1XX pCAR is shown below as SEQ ID NO: 80. The PD-1 sequence is underlined with a single underline, while the ITM of the CD3ζ polypeptide is italicized and bolded, with mutated residues double-underlined.

[0334]

[0335] (SEQ ID NO: 80).

[0336] The protein sequence of the CCR (“Pbb(trimer)”) of Pbb(trimer) / H 1XX is shown below as SEQ ID NO: 81, with the PD-1 polypeptide sequence bolded and the 4-1BB co-stimulatory signal transduction region sequence underlined.

[0337]

[0338] (SEQ ID NO: 81).

[0339] The protein sequence of the CAR (“H 1XX”) of NKG2D_bb / H 1XX is shown above as SEQ ID NO: 59.

[0340] The 44bb (trimer) / H 1XX pCAR (SEQ ID NO: 82) comprises (i) three CCR peptides, each containing an NKp44 peptide fused to a G4 linker, a coiled-coil domain of crown protein 1A, a G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB costimulatory domain (“44bb (trimer)”) (SEQ ID NO: 83) and (ii) a second-generation CAR comprising an HMFG2 scFv fused to a portion of the extracellular domain of CD28 targeting human MUC1, followed by a CD28 transmembrane domain, a CD28 costimulatory domain, and a modified CD3ζ signaling domain (“H2 1XX”) (SEQ ID NO: 59). The modified CD3ζ peptide (SEQ ID NO: 48) contains the wild-type immune receptor tyrosine activation motif 1 (ITAM1) and two tyrosine-to-phenylalanine mutations in each of ITAM2 and ITAM3. CCR and CAR are linked via a furin cleavage site (RRKR)-SGSG linker-T2A ribosomal skip peptide.

[0341] The protein sequence of 44bb (trimeric) pCAR is shown below as SEQ ID NO: 82. The NKp44 sequence is underlined, while the ITM of the CD3ζ polypeptide is italicized and bolded, with mutated residues double-underlined.

[0342]

[0343] (SEQ ID NO: 82).

[0344] The protein sequence of the CCR (“44bb (trimer)”) of 44bb (trimer) / H 1XX is shown below as SEQ ID NO: 83, wherein the NKp44 polypeptide sequence is bolded and the 4-1BB costimulatory signal transduction region sequence is underlined.

[0345]

[0346] (SEQ ID NO: 83).

[0347] The protein sequence of the CAR (“H 1XX”) of 44bb (trimer) / H 1XX is shown above as SEQ ID NO: 59.

[0348] 5.4.2.1 Other Exemplary pCARs

[0349] In some embodiments, the pCAR is I62BB / H 1XX. The CCR in the I62BB / H 1XX pCAR has an ICR62 binding domain fused with the CD8α transmembrane domain and a 4-1 BB Co-stimulatory domains. The binding element for CCR is ICR62—the scFv domain that targets EGFR. See Modjtahedi et al., Cell Biophys. 22(1-3):129-146 (1993), which is incorporated herein by reference in its entirety. The binding element for CAR is the HMFG2 scFv that targets human MUC1.

[0350] The protein sequence of I62BB / H 1XX pCAR is shown below as SEQ ID NO: 45. V of HMFG2 and ICR62 H and V L The sequences are underlined and bolded, while the sequences in the CD3ζ signal transmission region are in italics, with the ITAM sequence being bolded and italicized.

[0351]

[0352] (SEQ ID NO: 45).

[0353] The protein sequence of the CCR (“I62BB”) of I62BB / H is shown below as SEQ ID NO: 46, wherein the V of ICR62 H and V L The sequence is underlined and highlighted in bold:

[0354]

[0355] (SEQ ID NO: 46).

[0356] The protein sequence of the CAR (“H 1XX”) of I62BB / H 1XX is shown above as SEQ ID NO: 59, wherein the V of HMFG2 H and V L The sequence is underlined and bolded, while the ITAM of CD3ζ is italicized and bolded.

[0357] 5.5. Connector CAR

[0358] In one aspect, an adaptor chimeric antigen receptor (adaptor CAR) is provided. The adaptor CAR comprises, from the C-terminus to the N-terminus (as in intracellular expression in immune-responsive cells, from intracellular to extracellular), (i) at least one adaptor protein comprising an activation signaling domain and a co-stimulatory signaling region; and (ii) at least one third binding element that specifically binds to a third epitope on a third antigen. At least one adaptor protein (i) and at least one third binding element (ii) are expressed as two or more separate polypeptides associated in the plasma membrane via non-covalent interactions, such association being driven by complementary charged amino acids in each polypeptide.

[0359] In some embodiments, the adaptor CAR comprises (i) an adaptor protein comprising a CD3ζ activation signal transduction domain and a Dap10 polypeptide; and (ii) an NKG2D ligand-binding domain polypeptide. In some embodiments, the CD3ζ activation signal transduction domain is modified.

[0360] In some embodiments, the modified CD3ζ activation signaling domain comprises at least one ITAM variant containing one or more loss-of-function mutations. In some embodiments, the modified CD3ζ activation signaling domain comprises an ITAM2 variant containing one or more loss-of-function mutations, and an ITAM3 variant containing one or more loss-of-function mutations. In some embodiments, the ITAM2 variant comprises two loss-of-function mutations, and the ITAM3 variant comprises two loss-of-function mutations. In some embodiments, each loss-of-function mutation comprises a tyrosine-to-phenylalanine substitution.

[0361] In some embodiments, the adaptor CAR comprises (i) an adaptor protein comprising a Dap10 co-stimulatory peptide and a Dap12 activation signal transduction domain; and (ii) an NKG2D ligand-binding domain peptide.

[0362] As described elsewhere in this document, adaptor CARs are named according to the following conventions: (ii) Targeting moiety (containing a binding element that specifically interacts with the target antigen) / (i) Co-stimulatory signaling domain-activation signaling domain, where a forward slash ( / ) indicates the adaptor (non-covalent) association between the targeting moiety (ii) and the adaptor protein (signaling unit) (i). A dash (-) in the adaptor CAR name indicates a direct fusion between the two components (e.g., between the co-stimulatory signaling domain and the activation signaling domain).

[0363] In some embodiments, the connector CAR is as follows Figure 8 The NKG2D / Dap10-12 is shown. In some embodiments, the connector CAR is as follows: Figure 8The NKG2D / Dap10-CD3ζ shown is illustrated. In some embodiments, the connector CAR is as follows: Figure 8 The NKG2D / Dap10-CD3ζ(1XX) shown.

[0364] 5.5.1. Connector Proteins

[0365] The adaptor CAR comprises one or more adaptor proteins. In some embodiments, the adaptor protein comprises an activation signal transduction domain. In some embodiments, the adaptor protein comprises a co-stimulatory signal transduction region. In some embodiments, the adaptor protein is a fusion of the activation signal transduction domain and the co-stimulatory region. In some embodiments, the adaptor protein is a modified fusion of the activation signal transduction domain and the co-stimulatory region.

[0366] In some embodiments, the activation signaling domain of the adaptor protein includes at least one ITAM. In a particular embodiment, the activation signaling domain is Dap12, CD3ζ, or a variant thereof (e.g., CD3ζ 1XX).

[0367] In some embodiments, the co-stimulatory signal transduction region is Dap10 or a variant thereof.

[0368] In some embodiments, the adaptor protein is a fusion of an activation signal transduction domain and a co-stimulatory region (which are directly bonded to each other in a continuous polypeptide chain). In some embodiments, the adaptor protein is a fusion of an activation signal transduction domain and a co-stimulatory region (which are indirectly bonded to each other through a suitable linker (e.g., a peptide linker).

[0369] Peptide linkers are commonly used in fusion peptides, and methods for selecting or designing such linkers are well known in the art. (See, for example, Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10): 13570-1369 and Wriggers W et al., 2005, Biopolymers 80:736-746). Linkers can also be used to link the fusion peptide of this disclosure to another peptide.

[0370] In some embodiments, the adaptor protein is a fusion of the Dap10 polypeptide or a functional variant thereof with the CD3ζ polypeptide or a functional variant thereof. In some embodiments, the adaptor protein is a fusion of Dap10 and CD3ζ (e.g., Dap10-CD3ζ in the NKG2D / DAP10-CD3ζ adaptor CAR). In specific embodiments, the adaptor protein is a fusion of Dap10 and CD3ζ 1XX (e.g., Dap10-CD3ζ 1XX in the NKG2D / Dap10-CD3ζ 1XX adaptor CAR).

[0371] The adaptor protein is further described in PCT / EP2020 / 076566 (published as WO 2021 / 058563), which is incorporated herein by reference in its entirety.

[0372] 5.5.2. Connector CAR coupling element

[0373] In some embodiments, the CAR-linked linker specifically binds to NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6).

[0374] 5.5.3. Exemplary Connector CAR

[0375] The NKG2D / Dap10-12 adaptor CAR (SEQ ID NO: 67) comprises a first polypeptide (i.e., the NKG2D ligand-binding domain (SEQ ID NO: 75)) and a second polypeptide (i.e., a fusion of the Dap10 co-stimulatory region (SEQ ID NO: 47) and the Dap12 activation signal transduction domain (SEQ ID NO: 85)). The first and second polypeptides are initially expressed as a single mRNA transcript, wherein the N-terminus of NKG2D is linked to the C-terminus of the DAP12 activation signal transduction domain via a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and a P2A ribosomal skipping peptide (SEQ ID NO: 34).

[0376] The protein sequence of the NKG2D / Dap10-12 adaptor CAR is shown below as SEQ ID NO: 67. The Dap10 sequence is bolded; the Dap12 sequence is underlined, with Dap 12 ITAM bolded; and the NKG2D sequence is double-underlined.

[0377]

[0378] (SEQ ID NO: 67).

[0379] The NKG2D / Dap10-12 connector CAR is described in more detail in PCT / EP2020 / 076566 (published as WO 2021 / 058563), which is incorporated herein by reference in its entirety.

[0380] The NKG2D / Dap10-CD3ζ adaptor CAR (SEQ ID NO: 72) comprises a fusion of a first polypeptide (i.e., the NKG2D ligand-binding domain (SEQ ID NO: 75)) and a second polypeptide (i.e., the Dap10 co-stimulatory polypeptide (SEQ ID NO: 47) and the CD3ζ activation signal transduction domain (SEQ ID NO: 1)). The first and second polypeptides are initially expressed as a single mRNA transcript, wherein the N-terminus of NKG2D is linked to the C-terminus of CD3ζ via a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and a P2A skipping peptide (SEQ ID NO: 34).

[0381] The protein sequence of the NKG2D / Dap10-CD3ζ adaptor CAR is shown below as SEQ ID NO: 72. The Dap10 sequence is bolded; the CD3ζ sequence is underlined with a single underline, where ITM of CD3ζ is italicized; the NKG2D sequence is underlined with a double underline.

[0382]

[0383] (SEQ ID NO: 72).

[0384] The NKG2D / Dap10-CD3ζ 1XX adaptor CAR (SEQ ID NO: 74) comprises a first polypeptide (i.e., the NKG2D ligand-binding domain (SEQ ID NO: 75)) and a second polypeptide (i.e., a fusion of the Dap10 co-stimulatory region and the modified CD3ζ activation signal transduction domain (SEQ ID NO: 88)). The first and second polypeptides are initially expressed as a single mRNA transcript, wherein the N-terminus of NKG2D is linked to the C-terminus of CD3ζ via a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and a P2A skipping peptide (SEQ ID NO: 34).

[0385] The protein sequence of the NKG2D / Dap10-CD3ζ 1XX adaptor CAR is shown below as SEQ ID NO: 74. The Dap10 sequence is bolded; the CD3ζ sequence is underlined with a single underline, while the ITM of CD3ζ is in italics, and the mutated residues of CD3ζ are underlined with double underlines; the NKG2D sequence is underlined with double underlines.

[0386]

[0387] (SEQ ID NO: 74).

[0388] 5.6. Immune Response Cells

[0389] In one aspect, immune-response cells are provided. In some embodiments, the immune-response cells express a second-generation chimeric antigen receptor (CAR) as described herein. In some embodiments, the immune-response cells express a pCAR comprising a second-generation chimeric antigen receptor (CAR) and a parallel chimeric co-stimulatory receptor (CCR).

[0390] The second-generation CAR comprises (a) an intracellular signal transduction domain containing a modified CD3ζ peptide; (b) a co-stimulatory signal transduction region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen, wherein the modified CD3ζ peptide contains immune receptor tyrosine activation motif 1 (ITAM1) and mutations in ITAM2 and / or ITAM3.

[0391] The pCAR comprises a second-generation CAR and a chimeric co-stimulatory receptor (CCR). The pCAR comprises a second-generation CAR containing (a) an intracellular signaling domain comprising a modified CD3ζ polypeptide; (b) a co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen, wherein the modified CD3ζ polypeptide comprises an immune receptor tyrosine activation motif 1 (ITAM1) and mutations in ITAM2 and / or ITAM3; and a CCR containing (e) a co-stimulatory signaling region different from the co-stimulatory signaling region of (b); (f) a transmembrane domain; and (g) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0392] 5.6.1. Cells

[0393] In some embodiments, the immune response cells are T cells.

[0394] In some embodiments, the immune response cells are αβ T cells. In a particular embodiment, the immune response cells are cytotoxic αβ T cells. In a particular embodiment, the immune response cells are αβ helper T cells. In a particular embodiment, the immune response cells are regulatory αβ T cells (Tregs).

[0395] In some embodiments, the immune response cells are γδ T cells. In a particular embodiment, the immune response cells are Vδ2 cells. + γδ T cells. In a specific embodiment, the immune response cells are Vδ2 cells. – T cells. In a specific embodiment, Vδ2 – T cells are Vδ1 + cell.

[0396] In some embodiments, the immune response cells are natural killer (NK) cells.

[0397] In some embodiments, immune response cells do not express additional exogenous proteins. In other embodiments, immune response cells are engineered to express additional exogenous proteins, such as cytokines, receptors, or derivatives thereof.

[0398] In some embodiments, the immune-response cells are cells derived from a cell line. In some embodiments, the immune-response cells are primary T cells. In some embodiments, the immune-response cells are human cells, optionally primary human T cells.

[0399] In some embodiments, the immune response cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune response cells are obtained from tumors. In some embodiments, the immune response cells obtained from tumors are tumor-infiltrating lymphocytes (TILs). In some embodiments, the TILs are αβ T cells. In other specific embodiments, the TILs are γδ T cells, and particularly Vδ2. + or Vδ2 – γδ T cells.

[0400] 5.7. Nucleic Acids and Methods for Preparing pCAR T Cells

[0401] This document also provides a combination of a first nucleic acid and a second nucleic acid, the first nucleic acid encoding a second-generation CAR as described above, and the second nucleic acid encoding a CCR as described above. As noted above, for convenience, the combination of CAR and CCR is referred to herein in the singular as pCAR, although the CAR and CCR are separate, co-expressed proteins. Based on the description of CAR and CCR above, suitable sequences of nucleic acids will be apparent to those skilled in the art. Sequences can be optimized for use in cells requiring a desired immune response. However, in some cases, as discussed above, codons can be altered to differ from the optimal choice or "wobbled" to avoid repetitive sequences. Specific instances of such nucleic acids encode the preferred embodiments described above.

[0402] To achieve transduction, the nucleic acid encoding pCAR is appropriately introduced into one or more vectors (e.g., plasmids, retroviruses, or lentiviruses). Such vectors (including plasmid vectors) or cell lines containing them constitute another aspect of the present invention.

[0403] In a typical embodiment, immune-responding cells are genetically modified, for example via retroviral or lentiviral-mediated transduction, to introduce CAR and CCR-encoding nucleic acids into the host T-cell genome, thereby allowing stable CAR and CCR expression. These can then optionally be reintroduced into the patient after amplification to provide beneficial therapeutic effects, as described below.

[0404] The first and second nucleic acids encoding the CAR and CCR can be expressed from the same vector or different vectors. This disclosure further provides kits for generating immune-responding cells, such as pCAR T cells as described herein. The kit may contain a combination of a first and a second nucleic acid, the first nucleic acid encoding a second-generation CAR as described above, and the second nucleic acid encoding a CCR as described above. In some embodiments, the kit contains a combination of one or more vectors containing a first nucleic acid encoding a second-generation CAR and a second nucleic acid encoding a CCR. In some embodiments, the kit further contains reagents for genetically modifying immune-responding cells.

[0405] In some embodiments, the method includes: (i) obtaining T cells and / or NK cells from a subject, (ii) transducing a polynucleotide or one or more vectors encoding the CAR peptide and CCR peptide disclosed herein into the T cells and / or NK cells, and (iii) culturing the T cells and / or NK cells to express the CAR and CCR.

[0406] In some embodiments, the method includes: (i) obtaining T cells and / or NK cells from a subject, (ii) transducing a polynucleotide or one or more vectors encoding the CAR and CCR disclosed herein into the T cells and / or NK cells, and (iii) culturing the T cells and / or NK cells to express the CAR, CCR, and chimeric cytokine receptor or autocrine loop.

[0407] 5.8. Pharmaceutical Compositions

[0408] This disclosure further provides pharmaceutical compositions comprising one or more polynucleotides encoding CARs and CCRs, one or more carriers encoding CARs and CCRs, or immune-responding cells expressing the pCARs described herein. The pharmaceutical compositions may further comprise pharmaceutically or physiologically acceptable diluents, carriers, and / or excipients. Physiologically acceptable diluents, carriers, and / or excipients are generally selected to suit the intended mode of administration and may include agents for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. These carriers may include aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including saline and / or buffer media.

[0409] 5.9. How to use pCAR

[0410] In some embodiments, one or more polynucleotides encoding CARs and CCRs, one or more vectors encoding CARs and CCRs, or the immunoresponsive pCAR cells disclosed herein can be used to guide a therapy for a T cell-mediated immune response against target cells. Therefore, in another aspect, methods are provided for guiding a T cell-mediated immune response against target cells in a patient in need. The methods include administering to a patient a therapeutically effective amount of one or more polynucleotide constructs, one or more vectors, or a population of immunoresponsive cells as described above, wherein the binding element is specific to the target cells. In a typical embodiment, the target cells express MUC1 and / or one or more NKG2D ligands.

[0411] In another aspect, methods for treating cancer in patients of need are provided. The methods include administering to a patient a therapeutically effective amount of one or more polynucleotide constructs, one or more vectors, or a population of immune-responding cells as described above, wherein the binding element is specific to target cells. In a typical embodiment, the target cells express MUC1. In a typical embodiment, the target cells express one or more NKG2D ligands. In various embodiments, the patient has breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal cancer, thyroid cancer, or renal cell carcinoma. In some embodiments, the patient has colon cancer, breast cancer, ovarian cancer, lung cancer, or pancreatic cancer. In some embodiments, the patient has breast cancer. In some embodiments, the patient has tumor cells expressing MUC1. In some embodiments, the patient has been identified as having tumor cells expressing MUC1. In some embodiments, the patient has tumor cells expressing one or more NKG2D ligands. In some embodiments, the patient has been identified as having tumor cells expressing one or more NKG2D ligands.

[0412] In some embodiments, the treatment method further includes the following prior steps: (i) obtaining immune response cells from a subject, (ii) transducing the immune response cells with a polynucleotide or one or more vectors encoding the CAR peptide and CCR peptide disclosed herein, and (iii) culturing the immune response cells to express the CAR and CCR.

[0413] In another aspect, the present invention provides immune-response cells, pharmaceutical compositions, polynucleotides, collections of polynucleotides, or kits for use in therapy. The present invention also provides immune-response cells, pharmaceutical compositions, polynucleotides, collections of polynucleotides, or kits for use in cancer treatment. The present invention is also provided for use in the manufacture of medicaments for treating cancer. In various embodiments, the patient has breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal cancer, thyroid cancer, or renal cell carcinoma. In some embodiments, the patient has colon cancer, breast cancer, ovarian cancer, lung cancer, or pancreatic cancer. In some embodiments, the patient has breast cancer. In some embodiments, the patient has tumor cells expressing MUC1. In some embodiments, the patient has been identified as having tumor cells expressing MUC1. In some embodiments, the patient has tumor cells expressing one or more NKG2D ligands. In some embodiments, the patient has been identified as having tumor cells expressing one or more NKG2D ligands. In some embodiments, the patient has been pretreated with chemotherapy agents. In some embodiments, administration of immune response cells to a patient results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% reduction in tumor size compared to untreated tumors. The amount of immune response cells administered to the patient should take into account the route of administration, the type of cancer being treated, the patient's weight, and / or the patient's age. Typically, approximately 1 x 10⁻⁶ cells are administered to the patient. 6 To approximately 1 x 10 11 Cells. In one embodiment, approximately 1 x 10 cells are administered to the patient. 7 To approximately 1 x 10 10 One cell, or about 1 x 10 8 To approximately 1 x 10 9 Each cell.

[0414] 6. Sequence

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442] 7. Example

[0443] 7.1. Methods used in the studies of Examples 1-4

[0444] Generation of the H2 1XX CAR construct. The H2 1XX CAR is a modified version of the second-generation H2 CAR described in Wilkie et al., J. Immunol. [Journal of Immunology] 180:4901-9 (2008) and WO 2020 / 183158, both of which are incorporated herein by reference in their entirety. Briefly, two tyrosine (Y) residues in each of the two distal immune receptor tyrosine activation motifs (ITAMs) of the CD3ζ polypeptide in the H2 CAR were mutated to phenylalanine (F) residues. The resulting H2 1XX CAR contains a CD3ζ polypeptide with functional ITAM1 and nonfunctional ITAM2 and 3 (“1XX”). The H2 1XX CAR has the amino acid sequence of SEQ ID NO: 59.

[0445] Generation of the TBB / H 1XX pCAR construct. The TBB / H 1XX pCAR is a modified version of the TBB / H pCAR described in WO 2020 / 183158, which is incorporated herein by reference in its entirety. Briefly, two tyrosine (Y) residues in each of the two distal immune receptor tyrosine activation motifs (ITAMs) of the CD3ζ polypeptide in the H2 CAR of the TBB / H pCAR were mutated to phenylalanine (F) residues. The resulting TBB / H 1XX pCAR contains a CAR having a CD3ζ polypeptide comprising a functional ITAM1 and nonfunctional ITAM2 and 3 (“1XX”). The TBB / H 1XX pCAR has the amino acid sequence of SEQ ID NO: 23. TBB / H-1—a pCAR having a truncated CD3ζ polypeptide comprising only one ITAM (ITAM1)—was generated by deleting the two distal ITAMs (ITAM2 and ITAM3). The TBB / H-1 pCAR has the amino acid sequence of SEQ ID NO: 26. To generate TBB / H-1... 3. TBB / H-1 6 and TBB / H-1 The 9 pCAR was deleted to remove amino acid residues 3, 6, and 9 upstream of the single remaining ITAM (ITAM1) in the TBB / H-1 pCAR. TBB / H-1 3. TBB / H-1 6 and TBB / H-1 The 9 pCARs each possess the amino acid sequences of SEQ ID NO: 27-29. This is for the generation of TBB / H-1. 9 YRS pCAR, for TBB / H-1 9 pCARs were further modified to remove the YRS motif at the C-terminus of the CD28 co-stimulatory signal transduction region. TBB / H-1 9 YRS pCAR has the amino acid sequence of SEQ ID NO: 30.

[0446] Generation of TBB / H pCAR alternatives. The CD3ζ domain in TBB / H pCAR was replaced by an activation domain from FcεR1γ or DAP12, respectively, to generate TBB / H-FcεR1γ pCAR or TBB / H-DAP12 pCAR. The FcεR1γ pCAR has the amino acid sequence of SEQ ID NO: 54. The DAP12 pCAR has the amino acid sequence of SEQ ID NO: 56.

[0447] Generation of the NKG2D_bb / H 1XX pCAR construct. The NKG2D_bb / H 1XX pCAR utilizes the same second-generation CAR targeting MUC1 as the TBB / H1XX pCAR described above. The CCR in the NKG2D_bb / H 1XX pCAR contains two extracellular NKG2D domains, each fused with a transmembrane domain and an intracellular 4-1BB co-stimulatory domain. To generate this construct, the corresponding sequence in TBB / H 1XX was replaced with a synthetic cDNA encoding the CCR. The resulting NKG2D_bb / H 1XX pCAR contains a MUC1-targeting CAR (which has a CD3ζ polypeptide containing functional ITAM1 and non-functional ITAM2 and 3 (“1XX”)) and a CCR that binds to an NKG2D ligand. The NKG2D_bb / H 1XX pCAR has the amino acid sequence of SEQ ID NO: 76 and the nucleic acid sequence of SEQ ID NO: 89.

[0448] Generation of the NKG2Dbb(trimer) / H 1XX pCAR construct. The NKG2Dbb(trimer) / H 1XX pCAR utilizes the same second-generation CAR targeting MUC1 as the TBB / H 1XX pCAR described above. The CCR in the NKG2Dbb(trimer) / H 1XX pCAR contains three extracellular NKG2D domains, each fused with a transmembrane domain and an intracellular 4-1BB co-stimulatory domain via a trimerization domain. To generate this construct, the corresponding sequence in TBB / H 1XX was replaced with a synthetic cDNA encoding the CCR. The resulting NKG2Dbb(trimer) / H 1XX pCAR contains a CAR having a CD3ζ polypeptide comprising functional ITAM1 and nonfunctional ITAM2 and 3 (“1XX”). The NKG2Dbb(trimer) / H 1XX pCAR has the amino acid sequence of SEQ ID NO: 78 and the nucleic acid sequence of SEQ ID NO: 90.

[0449] Generation of the Pbb(trimer) / H 1XX pCAR construct. The Pbb(trimer) / H 1XX pCAR utilizes the same second-generation CAR targeting MUC1 as the TBB / H 1XX pCAR described above. The CCR in the Pbb(trimer) / H 1XX pCAR contains three extracellular PD-1 domains, each fused with a transmembrane domain and an intracellular 4-1BB co-stimulatory domain. To generate this construct, the corresponding sequence in TBB / H 1XX was replaced with a synthetic cDNA encoding the CCR. The resulting Pbb(trimer) / H 1XX pCAR contains a CAR having a CD3ζ polypeptide comprising functional ITAM1 and nonfunctional ITAM2 and 3 (“1XX”). The Pbb(trimer) / H 1XX pCAR has the amino acid sequence of SEQ ID NO: 80 and the nucleic acid sequence of SEQ ID NO: 91.

[0450] Generation of the 44bb(trimer) / H 1XX pCAR construct. The pbb(trimer) / H 1XX pCAR utilizes the same second-generation CAR targeting MUC1 as the TBB / H 1XX pCAR described above. The CCR in the 44bb(trimer) / H 1XX pCAR contains three extracellular NKp44 domains, each fused with a transmembrane domain and an intracellular 4-1BB co-stimulatory domain. To generate this construct, the corresponding sequence in TBB / H 1XX was replaced with a synthetic cDNA encoding the CCR. The resulting 44bb(trimer) / H 1XX pCAR contains a CAR having a CD3ζ polypeptide comprising functional ITAM1 and nonfunctional ITAM2 and 3 (“1XX”). The 44bb(trimer) / H 1XX pCAR has the amino acid sequence of SEQ ID NO: 82 and the nucleic acid sequence of SEQ ID NO: 92.

[0451] T cell culture and transduction. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood samples using density gradient centrifugation with Ficoll-Paque. PBMCs were activated with 5 μg / mL phytohemagglutinin (PHA)-L and cultured in RPMI containing GlutaMax supplemented with 5% human AB serum for 24–48 hours. IL-2 (100 U / ml) was supplemented to cells 24 hours before T cell retroviral transduction of the CAR construct. CAR T cells were expanded in RPMI containing GlutaMax supplemented with 5% human AB serum and IL-2 (100 U / ml) for 10–14 days and then used for functional assays. Transduced cells were labeled according to the CAR construct they were transduced for (e.g., H2 T cells, H2 1XX T cells, TBB / H 1XX T cells).

[0452] Repeated antigen stimulation assay. The efficacy of CAR T cells was tested in the repeated antigen stimulation assay. BXPC3 tumor cells and CAR T cells were co-cultured at a 1:1 effector cell to target cell ratio for 72–96 hours. T cells were removed, resuspended in fresh culture medium, counted, and then added to fresh BXPC3 tumor monolayers. Residual tumor cell viability was measured by MTT assay after each restimulation. CAR T cells were added to fresh monolayers until the viability of the tumor cell monolayer reached 80% compared to the untreated monolayer.

[0453] In vivo tumor monitoring was performed using bioluminescence imaging. Six- to ten-week-old NSG mice were used in the in vivo experiments and assigned to experimental groups based on similar average tumor burden before treatment. BXPC3 tumor cells were transduced using an SFG retroviral vector co-expressing firefly luciferase (ffLuc) and dsTomato red fluorescent protein (RFP) and purified by flow cytometry before in vivo transplantation. Cells were inoculated intraperitoneally (ip) at doses of 1 x 10⁻⁶ cells. 5 1 x 10 BXPC3 cells were collected and the tumor was allowed to establish for 11 days. At this point, the tumor was injected via the ip route at a rate of 1 x 10⁻⁶ cells / mL. 7 CAR T cells were administered at a dose per cell, or PBS was administered as a control. A CD19-specific F28z CAR, containing an FMC63 scFv coupled to the CD28 spacer region, a transmembrane domain, an intracellular domain, followed by a CD3ζ intracellular domain, was used as an additional negative control. Tumor burden was monitored using a series of bioluminescent imaging techniques. Monitoring was performed using the IVIS Spectrum imaging platform (PerkinElmer) and Living Image software. To monitor tumor status, mice were injected intraperitoneally with D-luciferin (150 mg / kg) and imaged 20 minutes later under isoflurane anesthesia. Animals were examined daily and weighed weekly in all experiments.

[0454] In vivo tumor monitoring was performed using tumor volume measurements. Six- to ten-week-old NSG mice were used in the experiment and assigned to experimental groups based on similar average tumor burden prior to treatment. BXPC3 cells (1 x 10⁻⁶) were introduced into the mice. 5 T cells (10 million cells) were established subcutaneously in NSG mice for 17 days, followed by intravenous administration of the indicated T cell population or administration of PBS as a control. Tumor volume was monitored using calipers and calculated using the formula π / 6 x W x W x L, where W = width; L = length.

[0455] 7.2. Methods used in the study of Example 5

[0456] Cell lines and tissue cultures. BXPC3 and REN tumor cell lines were used in these studies. Tumor cell lines were grown in R10 or D10 media containing RPMI or DMEM supplemented with 10% FBS and GlutaMax. PG13 and 293VEC-RD114 cells (retroviral packaging cells) were maintained in D10. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2. Cell lines were validated by STR typing, and mycoplasma contamination was monitored periodically. Cell lines were engineered to express RFP / ffLuc when indicated.

[0457] Human study supervision. Blood samples were obtained from healthy male and female volunteers aged 18-65.

[0458] Generation of the CAR adapter instead of NKG2D.

[0459] All CARs were constructed using human codon-optimized sequences and, where appropriate, ligated digested DNA fragments through gene synthesis and cloning (Genscript, Hong Kong, and Leiden, Netherlands).

[0460] To construct SFG NKG2D / Dap10-12, a synthetic DNA encoding the following elements was constructed: a full-length human Dap10-Dap12 intracellular domain (UniProt amino acids 62-113) – RRKR (furin protease cleavage site) – [serine-glycine]2 linker – porcine chezovirus (P2A) ribosomal skip-read peptide – an NKG2D – XhoI restriction site including a stop codon. This sequence was side-joined at the 5' side by a sequence from the unique AgeI to NcoI restriction sites within SFG (the NcoI site overlaps with the start codon in the synthetic fragment, thus disrupting the site). After digestion with AgeI and XhoI, these sequences were ligated to similarly digested SFG. The NKG2D / Dap10-12 adaptor CAR has the nucleic acid sequence of SEQ ID NO: 66. The NKG2D / Dap10-12 adaptor CAR has the amino acid sequence of SEQ ID NO: 67.

[0461] To generate SFG NKG2D, the SFG NKG2D / Dap10-12 construct was digested with NcoI and XhoI (releasing the NKG2D cDNA sequence). This fragment was then ligated with SFG that had undergone similar digestion. NKG2D has the amino acid sequence of SEQ ID NO: 75.

[0462] To generate SFG NKG2D-CD3ζ, a synthetic cDNA encoding the start codon (ATG)-CD3ζ intracellular domain-full-length NKG2D-stop codon-XhoI restriction site was synthesized as described above. The sequence from the unique AgeI-NcoI restriction site within SFG was then side-joined at the 5' side, as in SFG NKG2D / Dap10-12. This fragment was then inserted into SFG after digestion with AgeI and XhoI. The NKG2D-CD3ζ CAR has the nucleic acid sequence of SEQ ID NO: 68. The NKG2D-CD3ζ CAR has the amino acid sequence of SEQ ID NO: 69.

[0463] To generate SFG NKG2D / Dap10-CD3ζ, a synthetic cDNA encoding the full-length human Dap10-CD3ζ intracellular domain-RRKR (furin protease cleavage site)-[serine-glycine]2 linker-porcine cyclophosphovirus (P2A) ribosomal skip-read peptide-full-length NKG2D-stop codon-XhoI restriction site was synthesized and side-joined at the 5' side by the unique AgeI-NcoI restriction site within SFG, as in SFG NKG2D / Dap10-CD3ζ. This cDNA was then inserted into SFG after digestion with AgeI and XhoI. The NKG2D / Dap10-CD3ζ adaptor CAR has the amino acid sequence of SEQ ID NO: 72 and the nucleic acid sequence of SEQ ID NO: 71.

[0464] The SFG NKG2D / Dap10-CD3ζ(1XX) adaptor CAR was generated by insertional mutagenesis, converting all four tyrosine residues in CD3ζ ITAM2 and 3 of SFG NKG2D / Dap10-CD3ζ to phenylalanine. CD3ζ 1XX has the amino acid sequence of SEQ ID NO: 42 (variant 2) or SEQ ID NO: 43 (variant 1). The NKG2D / Dap10-CD3ζ 1XX adaptor CAR has the nucleic acid sequence of SEQ ID NO: 73. The NKG2D / Dap10-CD3ζ 1XX adaptor CAR has the amino acid sequence of SEQ ID NO: 74.

[0465] T cell culture and transduction of the adaptor CAR. Viral vectors were prepared as described using the PG13 cell line, 293VEC-RD114 cells, or via triple transfection with 293T cells. For the latter, 1.65 x 10⁻⁶ cells were used. 6Low-passaged 293T cells were uniformly distributed in 11 mL IMDM + 10% FBS on 10 cm plates. After 8–24 h, 30 μL of GeneJuice was added to 470 μL of serum-free IMDM and gently mixed. After incubation at room temperature for 5 min, 3.125 μg of RD114 plasmid, 4.6875 μg of pEQ-Pam3 plasmid, and 4.6875 μg of the SFG vector of interest were added to the GeneJuice / medium mixture, gently mixed, and incubated at room temperature for 15 min. The transfection mixture was added dropwise to the plates and gently swirl to ensure uniform distribution. After incubation at 37°C and 5% CO2 for 48 h, the medium was removed for rapid freezing in an ethanol dry ice bath and the medium was replaced. This process was repeated after another 24 h. The frozen virus was aliquoted and stored at -80°C. Retroviral transduction of phytohemagglutinin-activated T cells was performed using RetroNectin-coated plastic dishes.

[0466] Flow cytometry analysis. All cell staining reactions were performed on ice. For intracellular antigen detection, cells were first stained with a fixable live / dead dye, followed by surface protein staining on ice for 30 min. Intracellular staining was performed by fixation with 0.01% formaldehyde followed by permeabilization with PBS + 0.5% bovine serum albumin + 0.1% saponin. Cells were then stained for intracellular proteins at 4°C for 30 min. All gating was performed using an isotype control antibody or a fluorescence minus one control. Where necessary, viability staining agents were included, and appropriate Fc blocking agents were used prior to staining to limit non-specific antibody binding. Flow cytometry was performed using a FACSCalibur flow cytometer equipped with CellQuest Pro software or a BD LSRFortessa flow cytometer equipped with BD FACSDiva software, and data were analyzed using FlowJo, LLC.

[0467] Enzyme-linked immunosorbent assay (ELISA). The supernatant collected from the co-culture of tumor cells and CAR T cells was analyzed using human IFN-γ or human IL2 ELISA, performed according to the manufacturer's instructions, with a sensitivity limit of 1 pg / mL. In the combined restimulation assay, cytokine production was set to zero for each cycle following T cell culture.

[0468] Cytotoxicity assays. Tumor cells were incubated with T cells at a specified effector cell to target cell ratio (E:T). For adherent target cells, residual tumor cell viability was quantified using an MTT assay. After removing the supernatant and residual T cells, 500 μg / mL MTT was added to D10 medium and incubated at 37°C and 5% CO2 for 40 min. Formazan crystals were resuspended in DMSO, and absorbance was measured at 560 nm. Alternatively, tumor cell viability was monitored by a luciferase assay. 150 mg / mL D-luciferin was added immediately before reading the luminescence. In each case, tumor cell viability was calculated as follows: (absorbance or luminescence value of tumor cells cultured with T cells / absorbance or luminescence value of untreated monolayer alone) x 100%. In combined restimulation assays, tumor viability was set to 100% for each cycle following T cell culture failure.

[0469] Tumor restimulation assay. Transduced T cells were co-cultured with tumor cell lines in the absence of exogenous cytokine support. The cells were then subjected to a 5 x 10⁻⁶ m² / h²⁻¹² m²⁻¹ ... 4 CAR T cells were added to an equal number of tumor cells for tumor restimulation assay. Every 3-4 days, T cells were transferred to 5 x 10⁶ cells. 4 On a fresh monolayer of tumor cells. Restimulation continued until no more T cells were recovered or tumor cell destruction was less than 70%. The supernatant was collected after 24 h for cytokine analysis, and tumor cell viability was determined by MTT or luciferase assays after 24 h or 72 h. If T cells could not be restimulated, tumor viability was set to 100%, and cytokine production was set to zero.

[0470] 7.3. Example 1: Evaluation of the antitumor activity of pCAR-T cells targeting MUC1 with modified CD3ζ signaling domains and second-generation CAR-T cells targeting MUC1.

[0471] T cells expressing CAR or pCAR (Figure 1A) were subjected to several rounds of antigen stimulation as described above. In short, untransduced (UT) T cells and T cells expressing CAR (H2; H2 1XX) or pCAR (TBB / H; TBB / H 1XX) were co-cultured with BXPC3 tumor cells in vitro at a 1:1 effector cell:target cell ratio (T cells:tumor cells) for 72 hours. The T cells were removed, resuspended in fresh culture medium, counted, and then added to a fresh BXPC3 tumor monolayer. Residual tumor cell viability was quantified by MTT assay. The results in Figures 2A-2E show the percentage of BXPC3 tumor cells surviving after restimulation (Figures 2A and 2C), the T cell count after restimulation (Figures 2B and 2D), and the restimulatory capacity of T cells on the BXPC3 tumor monolayer (Figure 2E).

[0472] The antitumor activity and restimulation ability of H2 1XX CAR T cells on tumor cells were evaluated. Figures 2A-2B As shown, compared to H2 CAR T cells, H2 1XX CAR T cells (hollow rhombus) exhibited antitumor activity over more restimulation cycles, comparable to TBB / H CAR T cells.

[0473] The antitumor activity and restimulation capacity of TBB / H 1XX CAR-T cells on tumor cells were evaluated. Figures 2C-2E The results showed that, compared to TBB / HT cells or untransduced T cells (UT), TBB / H 1XX T cells (inverted triangle) exhibited better restimulation capacity and increased proliferation on BXPC3 tumor monolayers. The data demonstrated that, in the context of pCAR, a single functional ITAM (proximal) in the CD3ζ signaling domain is sufficient to generate highly efficient antitumor activity. The data further demonstrated that, compared to T cells expressing TBB / H pCAR (with three functional ITAMs in the CD3ζ signaling domain), T cells expressing TBB / H 1XX pCAR (with only a single functional ITAM in the CD3ζ signaling domain) exhibited improved restimulation capacity and increased proliferation on BXPC3 tumor monolayers.

[0474] 7.4. Example 2: Evaluation of the antitumor activity of pCAR T cells targeting MUC1 with truncated CD3ζ signaling domains

[0475] To evaluate whether T cells expressing pCARs containing CD3ζ signaling domains including functional ITAM1 and non-functional ITAM2 and ITAM3 are as effective as T cells expressing pCARs lacking CD3ζ signaling domains in killing tumor cells, several truncated versions of the TBB / H pCAR (TBB / H-1, TBB / H-1...) were generated. 3. TBB / H-1 6. TBB / H-1 9 and TBB / H-1 9 YRS (see Method and Figure 1B).

[0476] Restimulation assays using pCAR T cells expressing the truncated CD3ζ signaling domain demonstrated that the removal of the two distal ITAMs from CD3ζ impaired the antitumor efficacy of T cells compared to TBB / H 1XX pCAR T cells (Figure 3A). T cell counts after each stimulation cycle further demonstrated that pCAR T cells expressing the truncated CD3ζ exhibited reduced proliferative capacity compared to TBB / H 1XX pCAR T cells (Figure 3B). The data demonstrate that while a single proximal ITAM within the CD3ζ signaling domain is sufficient to generate potent antitumor activity, the inclusion of two distal nonfunctional ITAMs improved both antitumor efficacy and proliferative capacity compared to T cells expressing the truncated CD3ζ signaling domain (ITAM1 only).

[0477] 7.5. Example 3: Evaluation of the antitumor activity of pCAR T cells targeting MUC1 with alternative intracellular signal transduction domains.

[0478] To assess whether pCAR T cells expressing signaling domains other than CD3ζ possess similar antitumor activity and proliferative potential to TBB / H 1XX pCAR T cells, pCARs with alternative signaling domains derived from DAP12 and FcεR1γ were generated (see Methods and Figure 1C). Figures 4A-4C As shown, compared with pCAR T cells expressing DAP12 or FcεR1γ signaling domains, TBB / H 1XX pCAR T cells (inverted triangle) exhibited superior antitumor activity (Fig. 4A), proliferation potential (Fig. 4B), and restimulation capacity (Fig. 4C). Equivalents and by reference are incorporated herein by reference.

[0479] 7.6. Example 4: Evaluation in vivo of the antitumor efficacy of pCAR T cells targeting MUC1 with alternative chimeric co-stimulatory receptors (CCRs).

[0480] To evaluate the efficacy of pCAR T cells expressing a substitute chimeric co-stimulatory receptor (CCR) targeting MUC1 in vivo against pancreatic tumors, pCARs with substitute CCRs were generated, and T cells expressing these constructs were prepared and injected into mice carrying BXPC3 tumor xenografts expressing firefly luciferase (ffLuc) (see Methods and...). Figures 5A-5D Tumor growth was then monitored using bioluminescence imaging.

[0481] In short, 11 days after tumor inoculation, 1 x 10⁻⁶ cells were administered via intraperitoneal injection. 7One pCAR T cell was injected into mice carrying a BXPC3 xenograft. Control mice were injected with PBS or a similar number of F28z (CD19-specific control) CAR T cells.

[0482] The results shown in Figures 6A-6I demonstrate that treatment with NKG2D_bb / H 1XX pCAR-T cells reduced tumor growth in almost all test mice (Figure 6A). Similar results were observed in mice treated with NKG2Dbb (trimer) / H 1XX and Pbb (trimer) / H 1XX pCAR T cells (Figures 6B and 6C, respectively). These results are comparable to those in mice treated with TBB / H 1XX pCAR T cells (Figure 6F). In contrast, mice treated with 44bb (trimer) / H 1XX (Figure 6D), TBB / H (Figure 6E) pCAR T cells, and H2 1XX CAR T cells (Figure 6G) showed similar tumor growth to the control group (Figures 6H and 6I). The data suggest that pCARs with alternative CCRs (e.g., NKG2D or PD-1 as the targeting domain) inhibit tumor growth in this model (Figures 6B and 6C). Furthermore, data show that modifying the CD3ζ activation domain of pCAR to have only a single functional ITAM (ITAM1) enhances antitumor activity (compare results in Figure 6E and Figure 6F).

[0483] Figure 7 Survival curves of mice carrying BXPC3 tumor xenografts treated with CAR or pCAR T cells indicated in Figures 6A-6I are shown. Consistent with the BLI assessment of tumor growth shown in Figures 6A-6I, mice treated with NKG2D_bb / H 1XX, NKG2Dbb (trimer) / H 1XX, and Pbb (trimer) / H 1XX pCAR T cells had longer survival times compared to mice treated with 44bb (trimer) / H 1XX, TBB / H 1XX, H 1XX, or TBBH pCAR T cells.

[0484] The results demonstrated that NKG2D_bb / H 1XX, NKG2Dbb / H 1XX, and Pbb / H1XX pCARs exhibited superior antitumor efficacy in the BXPC3 mouse model compared to pCARs containing CCRs targeting NKp44 ligands or ErbB (e.g., 44bb(trimer) / H1XX, TBB / H, TBB / H 1XX). The combination of 41BB as a co-stimulatory domain in the CCR and the CD3ζ(1XX) mutation activation domain in the CAR appears to provide a balanced activation and co-stimulatory signaling, thereby inducing potent cytotoxicity while maintaining persistence—two properties crucial for targeting solid tumors.

[0485] 7.7. Example 5: Evaluation of the role of CAR structures in targeting NKG2D ligands

[0486] To evaluate alternative activation signaling domains in adaptor CARs targeting the NKG2D ligand, three adaptor CARs were engineered. Each adaptor CAR contains two peptides: (i) an adaptor protein, a fusion of a T cell activation signaling domain and a Dap10 co-stimulatory signaling domain; and (ii) an NKG2D targeting moiety (binding element). These adaptor CARs differ in the activation signaling domain of the adaptor protein: Dap12, CD3ζ, or CD3ζ (1XX).

[0487] The antitumor activity and amplification capacity of the adaptor CAR (CD3ζ) having an activation signaling domain containing three ITAMs per CAR monomer were evaluated and compared with those of the adaptor CARs (Dap12 and CD3ζ (1XX)) having an activation signaling domain containing a single ITAM. The CD3ζ 1XX domain was generated by replacing each tyrosine (Y) residue in ITAM2 and ITAM3 of wild-type CD3ζ with a phenylalanine (F) residue, resulting in a CD3ζ domain with a single functional ITAM: ITAM1.

[0488] Furthermore, by comparing the cell surface expression of the aforementioned adaptor CAR with that of a linear 2G CAR containing an NKG2D binding element (targeting portion) and a CD3ζ activation signaling domain, the role of exogenously expressed Dap10 in improving the cell surface expression of CARs targeting the NKG2D ligand was investigated.

[0489] Schematic diagrams of the adaptor CARs (NKG2D / Dap10-12; NKG2D / Dap10-CD3ζ; NKG2D / Dap10-CD3ζ (1XX)) and linear CARs (NKG2D-CD3ζ) evaluated in these studies are shown below. Figure 8 As shown.

[0490] Highly efficient expression of each adaptor CAR (NKG2D / Dap10-12; NKG2D / Dap10-CD3ζ; NKG2D / Dap10-CD3ζ (1XX)) and linear CAR (NKG2D-CD3ζ) was achieved in transduced T cells, with progressive enrichment in transduced cells between day 3 and day 10. Figure 9A This causes transduced T cell cultures to favor CD8. + T cell subsets (Figure 9B).

[0491] T cells transduced with an adaptor CAR expressing Dap10 showed higher cell surface CAR expression compared to cells transduced with a linear CAR NKG2D-CD3ζ that associates with endogenous Dap10 (Fig. 9C). Cytotoxicity was assessed. Figure 10A ), cytokine release ( Figures 10B-10C ) and tumor restimulation ( Figure 10D In vitro assays did not demonstrate any significant difference in antitumor activity among the adaptor CARs (NKG2D / Dap10-12; NKG2D / Dap10-CD3ζ; NKG2D / Dap10-CD3ζ (1XX)). Measurements of the maximum T-cell expansion achieved during iterative restimulation on tumor cell monolayers demonstrated that both NKG2D / Dap10-12 and NKG2D / Dap10-CD3ζ (1XX) achieved maximum expansion in these assays. Figure 10E ).

[0492] Next, we compared these CARs in vivo in a subcutaneous BXPC3 xenograft model, further comparing them with untransduced T cells and PBS-treated mice. Tumors were allowed to establish for 17 days before intravenous (iv) delivery of CAR T cells. Results showed that antitumor activity was split between two groups. Significant antitumor activity was observed in mice treated with NKG2D / Dap10-12 (Fig. 11A) and NKG2D / Dap10-CD3ζ (1XX) (Fig. 11D) CAR T cells. In contrast, NKG2D-CD3ζ (Fig. 11B) and NKG2D / Dap10-CD3ζ (Fig. 11C) CAR T cells—each possessing an activation signaling domain containing three ITAMs—did not exhibit any significant antitumor activity compared to untransduced T cells (Fig. 11F). These data suggest that the superior efficacy of adaptor CAR T cells depends on a reduced ITAM dose. High cell surface CAR expression achieved through exogenous expression of Dap10 did not improve efficacy, as evidenced by the poor antitumor activity observed in mice treated with NKG2D / Dap10-CD3ζ CAR T cells (Fig. 11C).

[0493] The survival rate of BXPC3 xenograft mice treated with NKG2D / Dap10-12; NKG2D / Dap10-CD3ζ; NKG2D / Dap10-CD3ζ (1XX); or NKG2D-CD3ζ CAR T cells was as follows: Figure 12 As shown in the figure. The results showed that mice treated with NKG2D / Dap10-CD3ζ (1XX) or NKG2D / Dap10-12 T cells had significantly prolonged survival.

[0494] All references cited herein are incorporated by reference to the extent that each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is specifically and individually indicated as incorporated in its entirety by reference for all purposes. The applicant intends to incorporate this statement by reference pursuant to 37 CFR §1.57(b)(1) to refer to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identified in accordance with the requirements of 37 CFR §1.57(b)(2), even if such reference is not immediately adjacent to the specific statement of inclusion by reference. Any specific statement of inclusion by reference included in the specification (if any) does not diminish this general statement of inclusion by reference. The citation of references herein does not constitute an admission that the reference is relevant prior art, nor does it constitute any admission of the content or dates of such publications or documents.

[0495] Although the invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0496] 8. Examples

[0497] Example 1. A second-generation chimeric antigen receptor (second-generation CAR), the second-generation chimeric antigen receptor comprising:

[0498] a) An intracellular signal transduction domain comprising a modified CD3ζ polypeptide;

[0499] b) Co-stimulatory signal transduction region;

[0500] c) Transmembrane domains; and

[0501] d) A first binding element that specifically interacts with a first epitope on the first target antigen.

[0502] The modified CD3ζ polypeptide contains unmodified immune receptor tyrosine activation motif 1 (ITAM1) and one or more mutations in ITAM2 and / or ITAM3.

[0503] Example 2. A second-generation CAR as described in Example 1, wherein the modified CD3ζ polypeptide contains mutations in both ITAM2 and ITAM3.

[0504] Example 3. The second-generation CAR as described in Example 2, wherein ITAM2 and ITAM3 in the modified CD3ζ are non-functional.

[0505] Example 4. A second-generation CAR as described in any one of Examples 1-3, wherein each of the ITAM2 and ITAM3 contains at least one Tyr-to-Phe mutation.

[0506] Example 5. A second-generation CAR as described in any one of Examples 1-4, wherein each of the ITAM2 and ITAM3 contains two Tyr-to-Phe mutations.

[0507] Example 6. A second-generation CAR as described in any one of Examples 1-3, wherein the modified CD3ζ polypeptide comprises a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof.

[0508] Example 7. A second-generation CAR as described in Example 6, wherein the modified CD3ζ polypeptide further comprises the deletion of one or more amino acid residues on the N-terminal side of the ITM1.

[0509] Example 8. A second-generation CAR as described in any of the preceding examples, wherein the modified CD3ζ polypeptide comprises a sequence selected from SEQ ID NO: 48-53.

[0510] Example 9. A second-generation CAR as described in any of the preceding examples, wherein the first target antigen is selected from NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, Claudin 18.2, Claudin 6, Glypican 3, anaplastic lymphoma kinase (ALK), CD70, and prostate-specific membrane antigen (PSMA).

[0511] Example 10. A second-generation CAR as described in any of the preceding examples, wherein the first target antigen is MUC1.

[0512] Example 11. A second-generation CAR as described in any of the preceding examples, wherein the first binding element comprises a CDR of an HMFG2 antibody.

[0513] Example 12. A second-generation CAR as described in Example 11, wherein the first binding element comprises the V of the HMFG2 antibody. H Domain and V L Structural domain.

[0514] Example 13. A second-generation CAR as described in any of the preceding examples, wherein the first binding element comprises an HMFG2 single-stranded variable fragment (scFv).

[0515] Example 14. A second-generation CAR as described in any of the preceding examples, wherein the second-generation CAR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59.

[0516] Example 15. A parallel chimeric antigen receptor (pCAR), the parallel chimeric antigen receptor comprising a second-generation CAR as described in any of the preceding examples, and a chimeric co-stimulatory receptor (CCR), the chimeric co-stimulatory receptor comprising:

[0517] e) Co-stimulatory signal transduction region, which is different from the co-stimulatory signal transduction region of (b);

[0518] f) Transmembrane domains; and

[0519] g) A second binding element that specifically interacts with a second epitope on a second target antigen.

[0520] Example 16. A second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in Example 15, wherein the co-stimulatory signal transduction region is selected from the co-stimulatory signal transduction regions of B7 / CD28 family members, ILT / CD85 family members, tumor necrosis factor (TNF) superfamily members, SLAM family members and TIM family members.

[0521] Example 17. A second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in Example 15 or Example 16, wherein the co-stimulatory signal transduction region is selected from the CD28 co-stimulatory domain, CD27 co-stimulatory domain, OX40 co-stimulatory domain, ICOS co-stimulatory domain and 4-1BB co-stimulatory domain.

[0522] Example 18. A second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in any one of Examples 15-17, wherein the co-stimulatory signal transduction region of (b) is a CD28 co-stimulatory signal transduction domain.

[0523] Example 19. A second-generation CAR or pCAR as described in Example 18, wherein the CD28 co-stimulatory domain contains a mutation in the YRS motif.

[0524] Example 20. pCAR as described in any one of Examples 15-19, wherein the co-stimulatory signal transduction region of (e) is a 4-1BB co-stimulatory domain.

[0525] Example 21. A second-generation CAR as described in Example 1 or a pCAR as described in Example 15, wherein the co-stimulatory signal transduction region is a co-stimulatory molecule selected from CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, dendritic cell-associated type C phytohemagglutinin-1, DPPIV, EphB6, LAG-3, TSLPR or variants thereof.

[0526] Example 22. A second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in any one of Examples 15-21, wherein the transmembrane domain is selected from the transmembrane domains of CD8α, CD28, CD4, CD3ζ, and FcεR1γ.

[0527] Example 23. A second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in any one of Examples 15-22, wherein the transmembrane domain of (c) is a CD28 transmembrane domain.

[0528] Example 24. pCAR as described in any one of Examples 15-23, wherein the transmembrane domain of (f) is a CD8α transmembrane domain.

[0529] Example 25. pCAR as described in any one of Examples 15-24, wherein the second target antigen is selected from NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, GD2, sealing protein 18.2, sealing protein 18.6, phosphatidylinositol proteoglycan 3, anaplastic lymphoma kinase (ALK), CD47, CD70, prostate-specific membrane antigen (PSMA), PD-L1, and NKp30 ligands (e.g., BAG6 and B7-H6).

[0530] Example 26. pCAR as described in any one of Examples 15-25, wherein the second binding element specifically interacts with a second epitope on the ErbB antigen.

[0531] Example 27. pCAR as described in any one of Examples 15-26, wherein the second binding element is a T1E peptide, an antigen-binding site of ICR12, or an antigen-binding site of ICR62.

[0532] Example 28. pCAR as described in any one of Examples 15-27, wherein the second binding element is a T1E peptide.

[0533] Example 29. pCAR as described in any one of Examples 15-25, wherein the second binding element specifically binds to PD-L1.

[0534] Example 30. pCAR as described in any one of Examples 15-25, wherein the second binding element specifically binds to an NKp30 ligand selected from BAG6 and B7-H6.

[0535] Example 31. The pCAR as described in any one of Examples 15-30, wherein the second-generation CAR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, and the CCR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 24.

[0536] Example 32. One or more polynucleotide constructs encoding a second-generation CAR as described in any one of Examples 1-14 or a pCAR as described in any one of Examples 15-31.

[0537] Example 33. One or more expression vectors comprising one or more polynucleotide constructs as described in Example 32.

[0538] Example 34. An immune response cell comprising a second-generation CAR as described in any one of Examples 1-14, a pCAR as described in any one of Examples 15-31, one or more polynucleotide constructs as described in Example 32, or one or more expression vectors as described in Example 33.

[0539] Example 35. Immune response cells as described in Example 34, wherein the immune response cells are αβ T cells, γδ T cells, or natural killer (NK) cells.

[0540] Example 36. A pharmaceutical composition comprising one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, or a population of immune-response cells as described in Example 34 or Example 35, and an excipient.

[0541] Example 37. A method for guiding a T-cell-mediated immune response against target cells in a subject in need, the method comprising administering to the subject a therapeutically effective amount of one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, immune-responding cells as described in Example 34 or 35, or a pharmaceutical composition as described in Example 36.

[0542] Example 38. A method of treating cancer in a subject in need, the method comprising administering a therapeutically effective amount of one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, immune response cells as described in Example 34 or 35, or a pharmaceutical composition as described in Example 36.

[0543] Example 39. The method as described in Example 34 or 35, wherein the subject has cancer.

[0544] Example 40. The method as described in Example 39, wherein the subject has cancer selected from the group consisting of: breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal cancer, thyroid cancer, and renal cell carcinoma.

[0545] Example 41. A second-generation CAR as described in any one of Examples 1-14, a pCAR as described in any one of Examples 15-31, one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, immune response cells as described in Example 34 or 35, or a pharmaceutical composition as described in Example 37 for therapeutic purposes.

[0546] Example 42. A second-generation CAR as described in any one of Examples 1-14, a pCAR as described in any one of Examples 15-31, one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, immune response cells as described in Example 34 or 35, or a pharmaceutical composition as described in Example 37 for the treatment of cancer.

[0547] Example 43. Use of a second-generation CAR as described in any one of Examples 1-14, a pCAR as described in any one of Examples 15-31, one or more polynucleotide constructs as described in Example 32, one or more expression vectors as described in Example 33, immune response cells as described in Example 34 or 35, or a pharmaceutical composition as described in Example 37 in the preparation of a medicament for treating cancer.

Claims

1. A parallel chimeric antigen receptor (pCAR), said parallel chimeric antigen receptor comprising: (1) Second-generation chimeric antigen receptor (CAR), which includes: a) An intracellular signal transduction domain comprising a modified CD3ζ polypeptide; b) Co-stimulatory signal transduction region; c) Transmembrane domains; and d) A first binding element that specifically interacts with a first epitope on a first target antigen; The modified CD3ζ polypeptide comprises unmodified immune receptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and / or ITAM3; and (2) A chimeric co-stimulatory receptor (CCR), wherein the chimeric co-stimulatory receptor comprises at least one CCR polypeptide, the CCR polypeptide comprising: e) A co-stimulatory signal transduction region that is different from the co-stimulatory signal transduction region described in (b); f) Transmembrane domains; and g) A second binding element that specifically interacts with a second epitope on a second target antigen.

2. The pCAR of claim 1, wherein the modified CD3ζ polypeptide contains mutations in both ITAM2 and ITAM3.

3. The pCAR of claim 2, wherein ITAM2 and ITAM3 in the modified CD3ζ are non-functional.

4. The pCAR of any one of claims 1-3, wherein (i) each of the ITAM2 and the ITAM3 contains at least one Tyr-to-Phe mutation or two Tyr-to-Phe mutations, or (ii) the modified CD3ζ polypeptide contains a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof.

5. The pCAR of claim 4, wherein the modified CD3ζ polypeptide further comprises the deletion of one or more amino acid residues on the N-terminal side of the ITAMS1.

6. The pCAR as claimed in any of the preceding claims, wherein the modified CD3ζ polypeptide comprises a sequence selected from SEQ ID NO: 48-53.

7. The pCAR as claimed in any of the preceding claims, wherein the first target antigen is selected from NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, Claudin 18.2, Claudin 18.6, Glypican 3, ALK, CD70, and prostate-specific membrane antigen (PSMA).

8. The pCAR as claimed in any of the preceding claims, wherein the first binding element comprises the CDR of the HMFG2 antibody and the V of the HMFG2 antibody. H and V L Structural domain, or HMFG2 single-chain variable fragment (scFv).

9. The pCAR as claimed in any of the preceding claims, wherein the second-generation CAR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

59.

10. The pCAR as claimed in any of the preceding claims, wherein the co-stimulatory signal transduction region is selected from (i) co-stimulatory signal transduction regions of members of the B7 / CD28 family, members of the ILT / CD85 family, members of the tumor necrosis factor (TNF) superfamily, members of the SLAM family, and members of the TIM family; (ii) the CD28 co-stimulatory domain, the 4-1BB co-stimulatory domain, the OX40 co-stimulatory domain, and the ICOS co-stimulatory domain; or (iii) co-stimulatory molecules selected from CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, Dectin-1, DPPIV, EphB6, LAG-3, TSLPR, or variants thereof.

11. The pCAR of claim 10, wherein the CD28 co-stimulatory domain comprises a mutation in the YRS motif.

12. The pCAR as claimed in any of the preceding claims, wherein the co-stimulatory signal transduction region of (e) is a 4-1BB co-stimulatory domain.

13. The pCAR as claimed in any of the preceding claims, wherein the transmembrane domain is selected from the transmembrane domains of CD8α, CD28, CD4, CD3ζ, and FcεR1γ.

14. The pCAR as claimed in any of the preceding claims, wherein the transmembrane domain of (c) is a CD28 transmembrane domain, and / or the transmembrane domain of (f) is a CD8α transmembrane domain.

15. The pCAR as claimed in any of the preceding claims, wherein the second target antigen is selected from NKG2D ligands (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, GD2, Claudin 18.2, Claudin 18.6, Glypican 3, ALK, CD47, CD70, prostate-specific membrane antigen (PSMA), PD-1 ligands (e.g., PD-L1), NKp44 ligands (e.g., NKp44L (MLL5), PCNA, viral hemagglutinin, nestin-1, galactoglucan-3, other proteoglycans), and NKp30 ligands (e.g., BAG6 and B7-H6).

16. The pCAR as claimed in any of the preceding claims, wherein the second binding element specifically binds to the ErbB antigen, NKG2D ligand, PD-1 ligand, NKp44 ligand, or NKp30 ligand.

17. The pCAR as claimed in any of the preceding claims, wherein the second binding element is any one of the following: T1E peptide, NKG2D peptide, PD-1 peptide, NKp44 peptide, NKp30 peptide, or a fragment, portion, or variant thereof.

18. The pCAR as claimed in any of the preceding claims, wherein the CCR of (2) comprises two or more CCR polypeptides.

19. The pCAR as claimed in any of the preceding claims, wherein the CCR of (2) comprises three CCR polypeptides.

20. The pCAR of claim 18, wherein the CCR comprises two CCR peptides, the two CCR peptides dimerizing after the second binding element interacts with the second target antigen.

21. The pCAR of claim 19, wherein the three CCR peptides trimerize after the second binding element interacts with the second target antigen.

22. The pCAR as claimed in any of the preceding claims, wherein the second-generation CAR has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 59, and the CCR has at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 24, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, and SEQ ID NO:

83.

23. One or more polynucleotide constructs encoding a pCAR as described in any of the preceding claims.

24. One or more expression vectors comprising one or more polynucleotide constructs as described in claim 23.

25. An immune-response cell comprising pCAR as described in any one of claims 1-22, one or more polynucleotide constructs as described in claim 23, or one or more expression vectors as described in claim 24.

26. The immune response cell of claim 25, wherein the immune response cell is an αβ T cell, a γδ T cell, or a natural killer (NK) cell.

27. A pharmaceutical composition comprising one or more polynucleotide constructs as claimed in claim 23, one or more expression vectors as claimed in claim 24, or a population of immune-response cells as claimed in claim 25 or claim 26, and an excipient.

28. The pCAR of any one of claims 1-22, one or more polynucleotide constructs of claim 23, one or more expression vectors of claim 24, immune response cells of claim 25 or 26, or pharmaceutical compositions of claim 27 for therapeutic purposes.

29. The pCAR of any one of claims 1-22, one or more polynucleotide constructs of claim 23, one or more expression vectors of claim 24, immune response cells of claim 25 or 26, or pharmaceutical compositions of claim 27 for the treatment of cancer.

30. A fusion polypeptide comprising (i) a DNAX activator protein 10 (Dap10) polypeptide or a functional variant thereof, and (ii) a modified CD3ζ polypeptide comprising an unmodified immune receptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and / or ITAM3.

31. The fusion polypeptide of claim 30, wherein the Dap10 polypeptide has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

47.

32. The fusion polypeptide of claim 30 or claim 31, wherein the modified CD3ζ polypeptide contains mutations in both ITAM2 and ITAM3.

33. The fusion polypeptide according to any one of claims 30-32, wherein the ITAM2 and ITAM3 in the modified CD3ζ are nonfunctional.

34. The fusion polypeptide of any one of claims 30-33, wherein (i) each of the ITAM2 and the ITAM3 comprises at least one Tyr-to-Phe mutation or two Tyr-to-Phe mutations, or (ii) the modified CD3ζ polypeptide comprises a deletion of ITAM2 or a portion thereof and a deletion of ITAM3 or a portion thereof.

35. The fusion polypeptide of any one of claims 30-34, wherein the modified CD3ζ polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48.

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