Chimeric antigen receptors and methods of use thereof

Abstract

The present application discloses a dual chimeric antigen receptor (dual CAR) system targeting neuroendocrine lineage markers, the system comprising (1) a first CAR comprising a primary intracellular signaling domain of an immune cell, and (2) a second CAR not comprising a primary intracellular signaling domain derived from CD3 zeta or a primary intracellular signaling domain of an immune cell. The invention relates to a single CAR system targeting neuroendocrine lineage markers. Engineered immune cells comprising such a system and the use of these cells in the treatment of cancer are also described.

Description

[0001] Priority Statement

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 093263, filed on May 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application incorporates by reference a sequence list submitted with this application, which is an XML file titled "IEC240077PCT SEQUENCE LISTING.xml", created on May 7, 2024, and is 61,750 bytes in size. Technical Field

[0005] This disclosure relates to chimeric antigen receptors (CARs) targeting neuroendocrine lineage markers, dual chimeric antigen receptor (dual CAR) systems, engineered immune cells, and methods of using them. Background Technology

[0006] Neuroendocrine carcinoma is a spectrum-restricted tumor type in which neuroendocrine-related neuroendocrine and / or neuronal biomarkers are predominantly expressed. It mainly includes small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), and gastrointestinal neuroendocrine carcinoma.

[0007] Delta-like ligand 3 (DLL3) is a neuroendocrine lineage marker that is highly expressed in SCLC and other neuroendocrine tumors, but expressed at minimal levels in normal tissues. CD56 is another neuroendocrine lineage marker that has been widely used in the diagnosis of SCLC. In addition to DLL3 and CD56, other neuroendocrine lineage markers include the C-terminal subunit of mucin-1 (MUC1-C), cadherin-17 (CDH17), disialiacoganglioside GD2, Netrin-3, seizure protein 6 homolog (SEZ6), phosphatidylinositol proteoglycan 2 (GPC2), and B7 homolog 3 (B7-H3).

[0008] SCLC accounts for approximately 15% of all lung cancers. Although small cell lung cancer exhibits a high response rate to first-line chemotherapy and radiotherapy, patients with extensive-stage SCLC eventually relapse, and very few survive more than 5 years from the date of diagnosis. Treatment options for relapsed or refractory disease are limited, and existing treatments are accompanied by significant treatment-related toxicities. There is a need to develop therapies for SCLC and other neuroendocrine tumors. Summary of the Invention

[0009] This document provides compositions and methods for treating diseases associated with the expression of neuroendocrine lineage markers, such as DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, or B7-H3. In one aspect, this disclosure relates to engineered immune cells comprising a dual CAR system targeting neuroendocrine lineage markers, the dual CAR system comprising (1) a first CAR containing a primary intracellular signaling domain of an immune cell, and (2) a second CAR not containing a primary intracellular signaling domain derived from CD3ζ or a primary intracellular signaling domain of an immune cell. In some embodiments, the first CAR specifically recognizes a first antigen, and the second CAR specifically recognizes a second antigen. In some embodiments, the first and second antigens are selected from the group consisting of DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, and B7-H3. This disclosure also includes methods for administering engineered immune cells to a subject.

[0010] In one aspect, this disclosure relates to engineered immune cells comprising a dual chimeric antigen receptor (dual CAR) system, which includes:

[0011] (a) The first chimeric antigen receptor (CAR), which comprises:

[0012] i) The first antigen-binding domain that specifically recognizes the first antigen;

[0013] ii) The first transmembrane domain; and

[0014] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0015] (b) A second chimeric antigen receptor (CAR), which comprises:

[0016] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0017] ii) The second transmembrane domain; and

[0018] iii) A second intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the second intracellular signal transduction domain.

[0019] The first intracellular signal transduction domain further includes a first primary intracellular signal transduction domain, and the second intracellular signal transduction domain does not include a primary intracellular signal transduction domain derived from CD3ζ; and

[0020] The first and second antigens are selected from the following groups: DLL3, CD56, MUC1C, CDH17, GD2, neuro-guided factor 3, SEZ6, GPC2, and B7-H3.

[0021] In one aspect, this disclosure relates to engineered immune cells comprising a dual chimeric antigen receptor (dual CAR) system, which includes:

[0022] (a) The first chimeric antigen receptor (CAR), which comprises:

[0023] i) The first antigen-binding domain that specifically recognizes the first antigen;

[0024] ii) The first transmembrane domain; and

[0025] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0026] (b) A second chimeric antigen receptor (CAR), which comprises:

[0027] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0028] ii) The second transmembrane domain; and

[0029] iii) A second intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the second intracellular signal transduction domain.

[0030] The first intracellular signal transduction domain further includes a primary intracellular signal transduction domain of immune cells, and the second intracellular signal transduction domain does not include a primary intracellular signal transduction domain derived from CD3ζ; and

[0031] Each of the primary and secondary antigens is a marker of the neuroendocrine spectrum.

[0032] In some embodiments, the second intracellular signal transduction domain does not include the primary intracellular signal transduction domain of the immune cell.

[0033] In some embodiments, the first primary intracellular signal transduction domain is derived from CD3ζ.

[0034] In some embodiments, the co-stimulatory signal transduction domain of the first intracellular signal transduction domain and / or the co-stimulatory signal transduction domain of the second intracellular signal transduction domain are derived from molecules selected from the group consisting of: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands.

[0035] In some embodiments, the co-stimulatory signal transduction domain of the first intracellular signal transduction domain includes an intracellular signal transduction domain of 4-1BB, and the co-stimulatory signal transduction domain of the second intracellular signal transduction domain includes an intracellular signal transduction domain of CD28.

[0036] In some embodiments, the first antigen is the same as the second antigen, for example, where the first antigen is DLL3 and the second antigen is DLL3.

[0037] In some embodiments, the first antigen is different from the second antigen.

[0038] In some embodiments, the first antigen is selected from the group consisting of DLL3, GD2 and GPC2.

[0039] In some embodiments, the second antigen is selected from the group consisting of: CD56, MUC1C, CDH17, neuro-guided factor 3, SEZ6, and B7-H3.

[0040] In some embodiments, the first binding domain and / or the second binding domain includes a first antigen-binding portion and / or a second antigen-binding portion selected from the following: Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb), or VHH domain.

[0041] In some embodiments, the second antigen is CD56; and / or the second antigen-binding domain contains anti-CD56scFv.

[0042] In some embodiments, the second antigen is CD56, and the second antigen-binding domain comprises an anti-CD56 scFv containing: a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0043] In some embodiments, anti-CD56 scFv includes:

[0044] The VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these HCDRs are defined according to the Kabat numbering scheme; and

[0045] VL domains comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, wherein these LCDRs are defined according to the Kabat numbering scheme.

[0046] In some embodiments, anti-CD56 scFv includes:

[0047] The VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and HCDR3 containing the amino acid sequence of SEQ ID NO: 15, wherein these HCDRs are defined according to the AbM numbering scheme; and

[0048] VL domains comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 16, LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and LCDR3 containing the amino acid sequence of SEQ ID NO: 18, wherein these LCDRs are defined according to the AbM numbering scheme.

[0049] In some embodiments, the first antigen is DLL3; and / or the first antigen-binding domain contains one or more anti-DLL3 sdAbs.

[0050] In some embodiments, the first antigen-binding domain comprises two anti-DLL3 sdAbs, the two anti-DLL3 sdAbs comprising:

[0051] The first anti-DLL3 sdAb comprises CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 8, and CDR3 containing the amino acid sequence of SEQ ID NO: 9, wherein these CDRs are defined according to the Kabat numbering scheme; and

[0052] The second anti-DLL3 sdAb comprises CDR1 containing the amino acid sequence of SEQ ID NO: 10, CDR2 containing the amino acid sequence of SEQ ID NO: 11, and CDR3 containing the amino acid sequence of SEQ ID NO: 12, wherein these CDRs are defined according to the Kabat numbering scheme.

[0053] In some embodiments, the first antigen-binding domain comprises two anti-DLL3 sdAbs, the two anti-DLL3 sdAbs comprising:

[0054] The first anti-DLL3 sdAb comprises CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 20, and CDR3 containing the amino acid sequence of SEQ ID NO: 21, wherein these CDRs are defined according to the AbM numbering scheme; and

[0055] The second anti-DLL3 sdAb comprises CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 24, wherein these CDRs are defined according to the AbM numbering scheme.

[0056] In some embodiments, the first antigen-binding domain comprises a first anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO: 28 and a second anti-DLL3 sdAb containing the amino acid sequence of SEQ ID NO: 29.

[0057] In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises transmembrane domains derived from molecules selected from the group consisting of: CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1.

[0058] In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 34-36.

[0059] In some embodiments, the first CAR includes a hinge domain located between the C-terminus of the first antigen-binding domain and the N-terminus of the first transmembrane domain; and wherein the hinge domain of the first chimeric antigen receptor is derived from CD8α or CD28; and the second CAR includes a hinge domain located between the C-terminus of the second antigen-binding domain and the N-terminus of the second transmembrane domain; and wherein the hinge domain of the second chimeric antigen receptor is derived from CD8α or CD28.

[0060] In some embodiments, the hinge domain of the first CAR and / or the hinge domain of the second CAR contains an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 31-33.

[0061] In some embodiments, the hinge domain of the second CAR contains the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 32, and / or the second transmembrane domain contains the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 35.

[0062] In some embodiments, each first CAR contains a signal peptide located at the N-terminus of a first antigen-binding domain, optionally wherein the signal peptide is derived from CD8α; and / or the second CAR contains a signal peptide located at the N-terminus of a second antigen-binding domain, optionally wherein the signal peptide is derived from CD8α.

[0063] In some embodiments, the first CAR contains an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and / or the second CAR contains an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42.

[0064] In some embodiments, the engineered immune cells comprise a polypeptide comprising a first CAR and / or a second CAR having an amino acid sequence selected from the group consisting of SEQ ID NO:40-44.

[0065] In one respect, this disclosure relates to nucleic acids comprising one or more nucleic acid sequences encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR), wherein

[0066] (a) The first chimeric antigen receptor (CAR) includes:

[0067] i) An antigen-binding domain that specifically recognizes the first antigen;

[0068] ii) The first transmembrane domain; and

[0069] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0070] (b) The second chimeric antigen receptor (CAR) includes:

[0071] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0072] ii) The second transmembrane domain; and

[0073] iii) A second intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the second intracellular signal transduction domain.

[0074] The first intracellular signal transduction domain further includes a first primary intracellular signal transduction domain of the immune cell, and the second intracellular signal transduction domain does not include a primary intracellular signal transduction domain derived from CD3ζ; and

[0075] The first and second antigens are selected from the following groups: DLL3, CD56, MUC1C, CDH17, GD2, neuro-guided factor 3, SEZ6, GPC2, and B7-H3.

[0076] In some embodiments, the second intracellular signal transduction domain does not include the primary intracellular signal transduction domain of the immune cell.

[0077] In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the following:

[0078] a. A first CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and / or

[0079] b. A second CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acids in SEQ ID NO: 41 or SEQ ID NO: 42; or

[0080] c. A first CAR and a second CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acids in SEQ ID NO: 43 or SEQ ID NO: 44.

[0081] In some embodiments, the nucleic acid comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein

[0082] (i) The first and second nucleic acids are operatively linked to the same promoter;

[0083] (ii) The first nucleic acid is upstream of the second nucleic acid, or wherein the first nucleic acid is downstream of the second nucleic acid; and / or

[0084] (iii) The first and second nucleic acids are linked by a nucleic acid sequence encoding a linker sequence of P2A or T2A.

[0085] In one respect, this disclosure relates to a single chimeric antigen receptor (CAR), which includes:

[0086] (a) An antigen-binding domain containing an anti-CD56 binding moiety that specifically recognizes CD56;

[0087] (b) Transmembrane domains; and

[0088] (c) Intracellular signal transduction domains,

[0089] The anti-CD56 binding portion includes a VH domain containing the amino acid sequence of SEQ ID NO: 25 and a VL domain containing the amino acid sequence of SEQ ID NO: 26.

[0090] In some embodiments, the anti-CD56 binding portion includes:

[0091] The VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3, wherein these HCDRs are defined according to the Kabat numbering scheme; and

[0092] VL domains comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, wherein these LCDRs are defined according to the Kabat numbering scheme.

[0093] In some embodiments, the anti-CD56 binding portion includes:

[0094] The VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and HCDR3 containing the amino acid sequence of SEQ ID NO: 15, wherein these HCDRs are defined according to the AbM numbering scheme; and

[0095] VL domains comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 16, LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and LCDR3 containing the amino acid sequence of SEQ ID NO: 18, wherein these LCDRs are defined according to the AbM numbering scheme.

[0096] In some embodiments, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain and does not include the primary intracellular signal transduction domain of immune cells.

[0097] In some embodiments, the co-stimulatory signal transduction domain comprises an intracellular domain whose amino acid sequence is at least 95% identical to that of the intracellular domains of CD28 and / or 4-1BB.

[0098] In some embodiments, the transmembrane domain is derived from molecules selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0099] In some embodiments, a single CAR further includes a hinge domain derived from CD8α or CD28.

[0100] In some embodiments, a single CAR further includes a signal peptide domain derived from CD8α.

[0101] In some embodiments, the hinge domain is at least 95% identical to the amino acid sequence shown in any one of SEQ ID NO: 31-33, and / or the transmembrane domain is at least 95% identical to the amino acid sequence shown in any one of SEQ ID NO: 34-36, and / or the signal peptide domain is at least 95% identical to the amino acid sequence of SEQ ID NO: 30.

[0102] In some embodiments, a single CAR contains an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 41 or SEQ ID NO: 42.

[0103] In one respect, this disclosure relates to nucleic acids, which contain one or more nucleic acid sequences encoding the single CAR described herein.

[0104] In one respect, this disclosure relates to vectors containing the nucleic acids described herein.

[0105] In one respect, this disclosure relates to the use of the vectors described herein for the generation of engineered immune cells.

[0106] In one respect, this disclosure relates to engineered immune cells that comprise the single CAR described herein, or the nucleic acid described herein, or the vector described herein.

[0107] In some embodiments, the engineered immune cells are selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0108] In one respect, this disclosure relates to pharmaceutical compositions comprising engineered immune cells and pharmaceutically acceptable carriers as described herein.

[0109] In one respect, this disclosure relates to a method for generating the engineered immune cells described herein, the method comprising introducing the vector described herein into the cells.

[0110] In one aspect, this disclosure relates to a method of treating a subject who has cancer or is at risk of having cancer, the method comprising: administering the engineered immune cells described herein, the pharmaceutical composition described herein, or the engineered immune cells described herein to a subject who has cancer or is at risk of having cancer.

[0111] In some embodiments, the subject has a neuroendocrine tumor (NET).

[0112] In some embodiments, the cancer is small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), or gastrointestinal neuroendocrine carcinoma.

[0113] In one respect, this disclosure relates to a dual CAR system, which includes:

[0114] (a) The first CAR, which includes:

[0115] i) The first antigen-binding domain that specifically recognizes the first antigen;

[0116] ii) The first transmembrane domain; and

[0117] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0118] (b) The second CAR, which includes:

[0119] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0120] ii) The second transmembrane domain; and

[0121] iii) A second intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the second intracellular signal transduction domain.

[0122] The first intracellular signal transduction domain further includes a first primary intracellular signal transduction domain, and the second intracellular signal transduction domain does not include a primary intracellular signal transduction domain derived from CD3ζ; and

[0123] The first and second antigens are selected from the following groups: DLL3, CD56, MUC1C, CDH17, GD2, neuro-guided factor 3, SEZ6, GPC2, and B7-H3. Attached Figure Description

[0124] Figure 1A-1C A schematic diagram of the DLL3 bispecific chimeric antigen receptor (DLL3 CAR) and the DLL3 / CD56 bichimeric antigen receptor (DLL3 / CD56 dual CAR) is shown. Figure 1A An exemplary structure of a DLL3 CAR is shown. Figure 1B An exemplary structure of the DLL3 / CD56 dual CAR is shown. Figure 1C Another exemplary structure of the DLL3 / CD56 dual CAR is shown. DLL3 / CD56 dual CAR-1# ( Figure 1B ) and DLL3 / CD56 dual CAR-2# ( Figure 1C It is coupled with divergent downstream stimulus signals.

[0125] Figures 2A-2D It showed that human primary T cells responded to antibodies containing anti-DLL3 V. H H's CAR expression. Figure 2A The expression of CAR in T cells transfected with DLL3CAR is shown. Figure 2B The CAR expression in T cells transfected with DLL3 CAR / CD56 double CAR-1# is shown. Figure 2C The CAR expression in T cells transfected with DLL3 CAR / CD56 double CAR-2# is shown. Figure 2D This shows CAR expression in untransfected control T cells. The X-axis shows anti-V antibodies. H H signal (anti-V) H H), and the Y-axis shows the cell size signal (FSC-H).

[0126] Figures 3A-3C This study demonstrates the cytotoxicity of CAR-T cells against tumor cells at different E:T ratios. DLL3CAR or DLL3 / CD56 dual CAR was compared with wild-type SHP-77 (…). Figure 3A SHP-77-Luc-DLL3 KO ( Figure 3B ) or NK-92 cells ( Figure 3C CAR-T cells were co-cultured at E:T ratios of 0.5:1 and / or 2:1. CAR-T-mediated target cell lysis was analyzed by examining LDH release in the co-culture supernatant. SHP-77 cells were double-positive for DLL3 and CD56. SHP-77-Luc-DLL3 KO cells were DLL3-negative and CD56-positive. NK-92 cells were DLL3-positive and CD56-negative.

[0127] Figures 4A-4D The release of IFN-γ from CAR-T cells was observed. Analysis was performed separately with wild-type SHP-77 (…). Figure 4A SHP-77-Luc-DLL3 KO ( Figure 4B ) or NK-92 cells ( Figure 4C IFN-γ release from co-cultured DLL3 CAR-T cells and DLL3 / CD56 dual CAR-T cells. Baseline IFN-γ release from CAR-T and UnT cells was measured in single T cell cultures without any target cells. Figure 4D Δ represents below the lower limit of quantitation (< LLOQ).

[0128] Figures 5A-5C The persistence of CAR-T cells was demonstrated in DLL3 CAR and DLL3 / CD56 dual CAR-T cells. Figure 5A ) and CAR-T amplification ( Figures 5B-5C In repeated tumor challenge assays, CAR-T cells and UnT cells were cultured together with tumor cells at an E:T ratio of 1:5.

[0129] Figures 6A-6D The in vivo efficacy and pharmacokinetics of DLL3 CAR-T cells and DLL3 / CD56 dual CAR-T cells in a xenograft model based on the SCLC tumor cell line NCI-H82 were demonstrated. Figure 6A This study demonstrated that changes in tumor volume serve as an indicator of CAR-T cell efficacy. Figure 6B Body weight change was shown as an indicator of CAR-T cell safety. Expansion of total T cells and CAR-T cells in the peripheral blood of NCG mice was observed 14 days post-treatment, serving as an indicator of CAR-T cell pharmacokinetics. Figures 6C-6D ). Figure 6C Total T cell expansion was observed. Figure 6D CAR-T amplification was shown.

[0130] Figures 7A-7C The persistence of CAR-T cells was demonstrated in DLL3 biCAR and DLL3 / CD56 dual biCAR-T cells. Figure 7A ) and CAR-T amplification ( Figures 7B-7C In repeated tumor challenge assays, CAR-T cells and UnT cells were cultured together with tumor cells at an E:T ratio of 1:5.

[0131] Figure 8 The sequences listed in this application are shown. Detailed Implementation

[0132] This document provides compositions and methods for treating diseases associated with the expression of neuroendocrine lineage markers, such as DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, or B7-H3. In one aspect, this disclosure relates to a dual chimeric antigen receptor (dual CAR) system targeting neuroendocrine lineage markers, the dual chimeric antigen receptor system comprising (1) a first CAR containing a primary intracellular signaling domain of an immune cell, and (2) a second CAR not containing a primary intracellular signaling domain derived from CD3ζ or a primary intracellular signaling domain of an immune cell. The first CAR specifically recognizes a first antigen, and the second CAR specifically recognizes a second antigen. The first and second antigens may be selected from the group consisting of DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, and B7-H3. In another aspect, this disclosure relates to engineered immune cells comprising a dual CAR system.

[0133] In this disclosure, a dual CAR targeting strategy is employed to deliver multiple inputs to drive the expansion and cytotoxicity of immune cells, such as T cells. The dual CAR targeting strategy delivered by DLL3 and CD56 demonstrates superiority of dual CAR-T cells targeting two antigens (e.g., DLL3 and CD56) compared to CAR-T cells targeting a single antigen (e.g., DLL3). Furthermore, due to the dominant expression of CD56 in neuronal lineages, the classic approach of coupling CD56, which binds to co-stimulatory signaling domains, with the CD3ζ signaling domain can be optimized by removing the CD3ζ signaling domain.

[0134] As used herein, when referring to the domains described herein, the term "derived from" means a domain obtained from the relevant functional part of a protein (e.g., through recombinant expression or de novo synthesis). This term encompasses domains having naturally occurring sequences and domains with mutated sequences. Domains derived from a particular protein may have a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the relevant functional part of the particular protein. Domains derived from a particular protein may originate from natural or synthetic sources.

[0135] As used herein, a “vector” is any construct capable of delivering one or more nucleic acids of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more nucleic acids of interest as encoded polypeptides in a host cell that has already been introduced into the expression vector. Thus, in an expression vector, the nucleic acid of interest is operatively positioned for expression in the vector by means of regulatory elements such as promoters, enhancers, and / or poly-A tails, which are located within the vector or in the genome of the host cell at or near the integration site of the nucleic acid of interest, such that the nucleic acid of interest will be translated in the host cell in which the expression vector has been introduced.

[0136] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain extracellular ligand-binding domains or extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains that are specific to one or more ligands or antigens (such as tumor antigens). "CAR-T cells" refer to T cells that express CARs.

[0137] As used herein, the term "antibody" includes, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or single-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), single-chain antibodies and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies and / or affinity-matured antibodies, as well as antibodies derived from other species (e.g., mice, rabbits, llamas, etc.). Antibodies also include, but are not limited to, synthetic antibodies, recombinant antibodies, single-domain antibodies or humanized variants thereof, including those from camel species (e.g., llamas or alpacas), endoantibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), which are portions of the antibody heavy or light chain polypeptide that retain some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), VHH fragments, Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, biantibodies, triantibodies, tetraantibodies, and miniantibodies. In particular, the antibodies described herein comprise immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing antigen-binding sites (e.g., one or more CDRs of an antibody). Antibodies may contain the Fc region of human antibodies.

[0138] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment containing VH and VL domains linked to a single polypeptide chain. For example, an scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0139] As used herein, “sdAb” or “monolithic antibody” refers to a single monomeric variable antibody domain that is capable of binding an antigen (e.g., a single-domain antibody that binds DLL3 or CD56). Single-domain antibodies include the VHH domain as described herein. Examples of single-domain antibodies include, but are not limited to, antibodies naturally lacking the light chain, such as antibodies from camelid species (e.g., llamas), single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. For example, as described herein, single-domain antibodies can be derived from camelid species such as camels, llamas, dromedary camels, alpacas, and guanacos. Other species besides camelids can produce heavy-chain antibodies naturally lacking the light chain; VHHs derived from such other species are within the scope of this disclosure. The single-domain antibodies (e.g., VHHs or V) provided herein are... H H) has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., a pharmaceutical agent) as described herein. Single-domain antibodies may be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0140] As used herein, the terms “complementarity-determining region” and “CDR” are used interchangeably. “CDR” refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet frame of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet frame of an antibody. Thus, a CDR is a variable region sequence scattered within a framework sequence. CDR regions are well known to those skilled in the art and are defined by well-known numbering systems / schemes. For example, the Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (see, for example, Kabat et al. above). Chothia, instead, refers to the location of a structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When using the Kabat numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software of Oxford Molecular (see, for example, Antibody Engineering, Vol. 2 (edited by Kontermann and Dübel, 2nd ed., 2010)). The “Contact” hypervariable region is based on the analysis of available complex crystal structures. Another general-purpose numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System. ®(Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system specifically for the study of immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, references to CDRs refer to both their amino acid sequence and their position in the light or heavy chain. Since the “position” of CDRs within the structure of immunoglobulin variable domains is conserved across species and exists in structures called loops, CDRs and frame residues are easily identified by using a numbering system that aligns variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into the recipient frame, which is typically derived from human antibodies. Honegger and Plückthun, J. Mol. Biol. 309: 657-70 (2001) developed an additional numbering system (AHon). The correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat above; Chothia and Lesk above; Martin above; Lefranc et al. above). Residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0141] Table 1. Exemplary CDRs based on various numbering systems

[0142]

[0143] The boundaries of a given CDR can vary depending on the scheme used for identification. Therefore, unless otherwise stated, the terms “CDR” and “complementarity-determining region” for a given antibody or its regions (such as variable regions), and the individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or its regions, should be understood to encompass the complementarity-determining region as defined by any known scheme described above. In some cases, a scheme for identifying one or more specific CDRs is specified, such as CDRs defined by IMGT, Kabat, AbM, Chothia, or Contact methods. In some cases, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number. For exemplary numbering of the VHH domain according to Kabat, see, for example, Deschacht et al., 2010. J Immunol 184:5696-704. In other cases, a specific amino acid sequence of the CDR is given. It should be noted that CDR areas can also be defined by combinations of various numbering systems, such as combinations of the Kabat and Chothia numbering systems, combinations of the Kabat and AbM numbering systems, or combinations of the Kabat and IMGT numbering systems. Therefore, terms such as "CDR as described in a particular VH or VHH" include, but are not limited to, any CDR defined by the exemplary CDR numbering systems described above. Once a variable area (e.g., a VHH domain, a VH, or a VL domain) is given, those skilled in the art will understand that CDRs within that area can be defined by different numbering systems or combinations thereof.

[0144] As used herein, the terms "antigen-binding domain" or "extracellular antigen-binding domain" refer to a portion of a full-length antibody that is capable of specifically binding an antigen. An antigen-binding fragment may contain at least one variable domain (e.g., a variable domain of a heavy chain, a single-domain antibody, or a VHH). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0145] The "percentage (%) amino acid sequence identity" and "homology" of peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in various ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN™ (DNASTAR), Snapgene, or Clustal W software, or other similar software programs. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full-length sequences being compared.

[0146] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include those exhibiting an abnormal state or condition characterized by rapid proliferation of cell growth. The term implies the inclusion of cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and malignant transformations of cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as those of the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, endocrine, and neuroendocrine tumors (NETs); and adenocarcinomas, which include malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumors (PNETs), gastrointestinal neuroendocrine carcinomas, and small bowel cancers. “Naturally occurring” cancer includes any cancer not induced experimentally by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to carcinogens, cancer caused by insertion of transgenic oncogenes or knockout of tumor suppressor genes, and cancer caused by infection (e.g., viral infection). The term “cancer” is generally accepted to refer to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of both carcinomatous and sarcomatous tissue. “Adenocarcinoma” refers to cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures. The term “sarcoma” is generally accepted to refer to a malignant tumor derived from mesenchyme. The term “hematopoietic neoplastic disorder” includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can originate from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0147] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human, or non-human to whom treatment is administered according to the methods disclosed herein. This disclosure contemplates both veterinary and non-veterinary applications. Human subjects may be adults or adolescents (e.g., persons under the age of 18). In addition to humans, subjects include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include, for instance, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification, including the definitions, shall prevail.

[0149] 1. Neuroendocrine carcinomas and neuroendocrine spectrum markers

[0150] The neuroendocrine system contains various cells distributed within non-endocrine functional structures, capable of synthesizing amines and peptides that have both local (paracrine) and systemic (endocrine) effects. The presence of neuroendocrine lineage markers is often associated with the development of neuroendocrine carcinomas.

[0151] Delta-like ligand 3 (DLL3) is a neuroendocrine lineage marker. It is an inhibitory Notch ligand, highly expressed in neuroendocrine tumors and small cell lung cancer (SCLC), but expressed at minimal levels in normal tissues. DLL3 is an inhibitory Notch pathway ligand that is highly upregulated and aberrantly expressed on the cell surface of SCLC and other higher-order neuroendocrine tumors. Notch signaling is downregulated during neuroendocrine tumor growth and is inhibited by DLL3 expression. DLL3 expression is regulated by achaete-scute homolog 1 (ASCL1), a transcription factor required for normal development of lung neuroendocrine cells and an oncogenic driver in SCLC. In preclinical models, DLL3 expression promotes SCLC migration and invasion through mechanisms involving the control of the epithelial-mesenchymal transition protein Snail. DLL3 is specifically expressed on the surface of SCLC tumor cells. DLL3 is also expressed in other tumor types of neuroendocrine origin, including melanoma, glioblastoma multiforme, small cell bladder cancer, metastatic castration-resistant prostate cancer, and neuroendocrine lung tumors.

[0152] CD56, also known as the neural cell adhesion molecule (NCAM), is a member of the immunoglobulin superfamily involved in both homophilic and heterophilic interactions. It is a marker of neuroendocrine lineage. There are three main isoforms of CD56 (NCAM-120, NCAM-140, and NCAM-180), all generated through alternative splicing of a single gene, and they differ in the length of their intracellular domains. CD56 is generally considered a marker of neural lineage typing due to its discovery site. However, CD56 expression has also been found, particularly in the hematopoietic system. Furthermore, CD56 expression is most closely associated with natural killer (NK) cells, but is by no means limited to them. CD56 has been detected on other lymphoid cells, including γδ (gamma delta) T cells and activated CD8+ T cells, as well as dendritic cells (DCs). In addition, CD56 plays a crucial role in the bone marrow, the site of hematopoiesis. Mesenchymal stromal cells, in particular, maintain long-term hematopoiesis by providing a microenvironment for hematopoietic stem cells through the expression of adhesion molecules, including CD56.

[0153] Other neuroendocrine spectrum markers include the C-terminal subunit of mucin-1 (MUC1-C), cadherin-17 (CDH17), disialotetrahexosylganglioside GD2, neuroguide factor-3, seizure protein 6 homolog (SEZ6), phosphatidylinositol proteoglycan 2 (GPC2), and B7 homolog 3 (B7-H3).

[0154] More information on neuroendocrine carcinomas and neuroendocrine lineage markers can be found in, for example, Ferolla, P. et al., “The biological characterization of neuroendocrine tumors: the role of neuroendocrine markers.” Journal of endocrinological investigation 31 (2008):277-286; Rekhtman, Natasha. “Lung neuroendocrine neoplasms: recent progress and persistent challenges.” Modern Pathology 35. Supplement 1 (2022): 36-50; VanAcker, Heleen H. et al., “CD56 in the immune system: more than a marker for cytotoxicity?” Frontiers in immunology 8 (2017): 892; Owen, Dwight H. et al., “DLL3: an emerging target in small cell lung cancer.” Journal of hematology & oncology 12.1 (2019): 1-8; Yao, James et al., “DLL3 as an Emerging Target for the Treatment of Neuroendocrine Neoplasms. The Oncologist 27.11 (2022): 940-951; each is incorporated in its full text by reference.

[0155] 2. Engineered immune cells containing chimeric antigen receptors

[0156] One aspect of this application provides engineered immune cells comprising a chimeric antigen receptor (CAR) targeting a neuroendocrine lineage marker, wherein the chimeric antigen receptor may be a dual chimeric antigen receptor (dual CAR) comprising (1) a first CAR containing a primary intracellular signaling domain of an immune cell, and (2) a second CAR not containing a primary intracellular signaling domain derived from CD3ζ or a primary intracellular signaling domain of an immune cell. Another aspect of this application provides engineered immune cells comprising a single CAR targeting CD56.

[0157] 2.1 Chimeric antigen receptor

[0158] 2.1.1 Dual CAR

[0159] One aspect of this application provides a dual chimeric antigen receptor (dual CAR) system comprising: (a) a first chimeric antigen receptor (CAR) comprising:

[0160] i) The first antigen-binding domain that specifically recognizes the first antigen;

[0161] ii) The first transmembrane domain; and

[0162] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0163] (b) A second chimeric antigen receptor (CAR), which comprises:

[0164] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0165] ii) The second transmembrane domain; and

[0166] iii) A second intracellular signal transduction domain comprising a co-stimulatory signal transduction domain thereof; wherein the first intracellular signal transduction domain further comprises a primary intracellular signal transduction domain of the immune cell, and the second intracellular signal transduction domain does not comprise a primary intracellular signal transduction domain derived from CD3ζ. In some embodiments, each of the first antigen and the second antigen is a neuroendocrine lineage marker. In some embodiments, the first antigen and the second antigen are selected from the group consisting of: DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, and B7-H3.

[0167] In some embodiments, the first antigen and the second antigen are the same, for example, the first antigen and the second antigen are DLL3. In some embodiments, the first antigen and the second antigen are different. A dual-CAR system may include a first CAR that specifically binds to a neuroendocrine lineage marker selected from the group consisting of DLL3, GD2, and GPC2, such as DLL3. A dual-CAR system may include a second CAR that specifically binds to a neuroendocrine lineage marker selected from the group consisting of CD56, MUC1C, CDH17, neuroguide factor 3, SEZ6, and B7-H3, such as CD56. A dual-CAR system may include a first CAR that specifically binds to DLL3 and a second CAR that specifically binds to CD56.

[0168] Antigen-binding domain of dual CAR

[0169] In the dual CAR system described herein, the antigen-binding domain of each CAR may contain one or more antibodies or antibody fragments (such as any one of 1, 2, 3, 4, 5, 6 or more).

[0170] The first antigen-binding domain and / or the second antigen-binding domain may comprise a first antigen-binding moiety selected from the following: Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), mini antibody, biantibody, single-domain antibody (sdAb), or VHH domain. The first antigen-binding domain may comprise a first and a second antigen-binding moiety as an sdAb (e.g., a VHH domain). The second antigen-binding domain may comprise an antigen-binding moiety as a single-chain Fv (scFv, such as a VL-VH pair). In some embodiments, the first CAR comprises a first antigen-binding domain containing two anti-DLL3 sdAbs (e.g., VHH domains). In some embodiments, the second CAR comprises a second antigen-binding domain containing an anti-CD56 scFv (e.g., a VL-VH pair). The antigen-binding domains of the first and / or second CARs described herein may comprise one or more (such as any one of 1, 2, 3, 4, 5, 6, or more) sdAbs or scFvs. sdAb and / or scFv can be directly fused to each other via peptide bonds or via peptide linkers. An exemplary structure of a dual CAR is shown in... Figure 1A-1C middle.

[0171] In some embodiments, this disclosure provides a first CAR (also referred to herein as a “DLL3 CAR”) targeting DLL3 in a dual CAR system, the first CAR comprising: (a) a first antigen-binding domain including an anti-DLL3 binding moiety; (b) a first transmembrane domain; and (c) a first intracellular signaling domain, wherein the first intracellular signaling domain comprises a co-stimulatory signaling domain of an immune effector cell (such as a T cell) and a primary intracellular signaling domain. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The co-stimulatory signaling domain of the first intracellular signaling domain may be derived from a molecule selected from the group consisting of co-stimulatory molecules derived from ligands of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the DLL3 CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the DLL3 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the DLL3 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the antigen-binding domain. In some embodiments, the first CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a DLL3 antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signal transduction domain derived from 4-1BB, and a primary intracellular signal transduction domain derived from CD3ζ. The DLL3 CAR may be monospecific. The DLL3 CAR may be bispecific or bivalent. Multiple antigen-binding domain sequences can be used as antigen-binding domains for DLL3 CARs, such as AMG119 and AMG157, as well as those disclosed in various patents (e.g., WO 2019200007 A1, WO 2020180591 A1 and WO 2021008610 A1).

[0172] In some embodiments, this disclosure provides a first CAR (also referred to herein as a “DLL3 CAR”) targeting DLL3 in a dual CAR system, the first CAR comprising: (a) a first antigen-binding domain comprising an anti-DLL3 binding moiety containing two anti-DLL3 sdAbs (e.g., a VHH domain); (b) a first transmembrane domain; and (c) a first intracellular signaling domain, wherein the two anti-DLL3 sdAbs comprise any one of: (1) a first anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 7, a CDR2 containing the amino acid sequence of SEQ ID NO: 8, and a CDR3 containing the amino acid sequence of SEQ ID NO: 9; and a second anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 10, a CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a CDR3 containing the amino acid sequence of SEQ ID NO: 12, wherein these CDRs are defined according to the Kabat numbering scheme; or (2) a first ... containing the amino acid sequence of SEQ ID NO: 10, a CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a CDR3 The anti-DLL3 sdAb comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 19, a CDR2 containing the amino acid sequence of SEQ ID NO: 20, and a CDR3 containing the amino acid sequence of SEQ ID NO: 21; and a second anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 22, a CDR2 containing the amino acid sequence of SEQ ID NO: 23, and a CDR3 containing the amino acid sequence of SEQ ID NO: 24, wherein these CDRs are defined according to the AbM numbering scheme. The anti-DLL3 sdAb may be camel-derived, chimeric, human, or humanized. The VHH CDRs (CDR1-3) may be determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the anti-DLL3 sdAb comprises a VHH containing an amino acid sequence selected from the group consisting of SEQ ID NO: 28-29. HH domain. In some embodiments, the anti-DLL3 binding portion includes a first VHH domain comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 28; and a second VHH domain comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the first intracellular signaling domain comprises a co-stimulatory signaling domain of an immune effector cell (such as a T cell) and a primary intracellular signaling domain. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The co-stimulatory signal transduction domain of the first intracellular signal transduction domain can be derived from a group of co-stimulatory molecules derived from CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83 ligands. In some embodiments, the DLL3 CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the DLL3 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the DLL3 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the antigen-binding domain. In some embodiments, the first CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a DLL3 antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signal transduction domain derived from 4-1BB, and a primary intracellular signal transduction domain derived from CD3ζ.

[0173] In some embodiments, the first CAR (e.g., DLL3 CAR) comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the first CAR (e.g., DLL3 CAR) comprises the amino acid sequence of SEQ ID NO: 40. A polypeptide comprising the amino acid sequence of SEQ ID NO: 40 is also provided.

[0174] In some embodiments, this disclosure provides a second CAR (also referred to herein as a “CD56 CAR”) targeting CD56 in a dual CAR system, the second CAR comprising: (a) a second antigen-binding domain comprising an anti-CD56 binding portion; (b) a second transmembrane domain; and (c) a second intracellular signaling domain comprising a co-stimulatory signaling domain, wherein the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second intracellular signaling domain does not comprise a primary intracellular signaling domain of an immune effector cell, such as a T cell. The anti-CD56 binding portion may be derived from one or more variable regions of an anti-CD56 antibody, such as clone HCD56 (BioLegend), clone MEM-188 (BioLegend), the humanized anti-CD56 antibody lorvotuzumab (huN901), or those antibodies disclosed in patent applications (such as WO 2017023859 A1).

[0175] The co-stimulatory signal transduction domain of the second intracellular signal transduction domain can be derived from a group of co-stimulatory molecules derived from ligands of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the CD56 CAR further comprises a hinge domain (such as a CD28 hinge domain or a mutant CD28 hinge domain) located between the C-terminus of the CD56 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CD56 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the CD56 antigen-binding domain. In some embodiments, the second CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a CD56 antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a transmembrane domain derived from CD28, and a co-stimulatory signal transduction domain derived from CD28.

[0176] In some embodiments, this disclosure provides a second CAR (also referred to herein as a “CD56 CAR”) targeting CD56 in a dual CAR system, the second CAR comprising: (a) a second antigen-binding domain comprising an anti-CD56 binding moiety containing an anti-CD56 scFv (e.g., a VL-VH pair); (b) a second transmembrane domain; and (c) a second intracellular signaling domain comprising a co-stimulatory signaling domain, wherein the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3ζ, and wherein the anti-CD56 scFv comprises any of the following: (a) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and HCDR3 containing the amino acid sequence of SEQ ID NO: 4. The anti-CD56 scFv may be mouse, chimeric, human, or humanized. The VH CDRs (HCDR1-3) and VL CDRs (LCDR1-3) may be determined according to the Kabat numbering scheme, wherein these CDRs are defined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. The CDRs are defined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the anti-CD56 scFv comprises a VH domain containing the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 25; and a VL domain containing the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the second intracellular signaling domain does not contain the primary intracellular signaling domain of immune effector cells (such as T cells).The co-stimulatory signal transduction domain of the second intracellular signal transduction domain can be derived from a group of co-stimulatory molecules derived from ligands of CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the CD56 CAR further comprises a hinge domain (such as a CD28 hinge domain or a mutant CD28 hinge domain) located between the C-terminus of the CD56 antigen-binding domain and the N-terminus of the second transmembrane domain. In some embodiments, the CD56 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the CD56 antigen-binding domain. In some embodiments, the first CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a CD56 antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a transmembrane domain derived from CD28, and a co-stimulatory signal transduction domain derived from CD28.

[0177] In some embodiments, the second CAR in the dual CAR system (e.g., a CD56 CAR) comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 41-42. In some embodiments, the second CAR in the dual CAR system (e.g., a CD56 CAR) comprises an amino acid sequence shown in any one of SEQ ID NO: 41-42. Peptides comprising amino acid sequences selected from the group consisting of SEQ ID NO: 41-42 are also provided.

[0178] 2.1.2 Single CAR

[0179] In one aspect, this disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a transmembrane domain; and (c) an intracellular signal transduction domain, wherein the antigen is a neuroendocrine lineage marker. In some embodiments, the antigen is selected from DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, or B7-H3. In some embodiments, the antigen is CD56. In some embodiments, the antigen is DLL3.

[0180] single CAR antigen-binding domain

[0181] Antigen-binding domains can be derived from one or more variable regions of a linked monoclonal antibody, such as single-chain variable fragments (scFv) or VHH domains. scFv format antibodies consist of heavy chain (VH) and light chain (VL) variable regions linked together by flexible peptide linkers or disulfide bonds. An antigen-binding domain may contain one or more (e.g., 1, 2, 3, 4, 5, or 6) VH-VL pairs. An antigen-binding domain may contain one or more (e.g., 1, 2, 3, 4, 5, or 6) VL-VH pairs.

[0182] In some embodiments, this disclosure provides a single CAR (also referred to herein as a “CD56 CAR”) targeting CD56, the single CAR comprising: (a) an antigen-binding domain comprising an anti-CD56 binding moiety that specifically recognizes CD56; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD56 binding moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 26. Its CD56 antigen-binding domain may contain an anti-CD56 scFv sequence that is at least 80%, 85%, 90%, or 95% identical to the selected scFv sequence shown in SEQ ID NO: 27. Anti-CD56 scFv can be mouse, chimeric, human, or humanized.

[0183] CDRs of the anti-CD56 binding site or anti-CD56 scFv, such as VH CDRs (HCDR1-3) and VL CDRs (LCDR1-3), can be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or combinations thereof. The VH CDR of the anti-CD56 binding site or anti-CD56 scFv may contain the amino acid sequences of SEQ ID NO: 1-3, and the VL CDR of the anti-CD56 binding site or anti-CD56 scFv may contain the amino acid sequences of SEQ ID NO: 4-6 as defined by the Kabat numbering scheme. The VH CDR of the anti-CD56 binding site or anti-CD56 scFv may contain the amino acid sequences of SEQ ID NO: 13-15, and the VL CDR of the anti-CD56 binding site or anti-CD56 scFv may contain the amino acid sequences of SEQ ID NO: 16-18. The anti-CD56 binding portion or anti-CD56 scFv may comprise (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and (b) LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, wherein the CDRs are defined according to the Kabat numbering scheme. The anti-CD56 binding moiety or anti-CD56 scFv may comprise (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 15; and (b) LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, wherein these CDRs are defined according to the AbM numbering scheme. The anti-CD56 binding moiety described herein may contain a VH comprising HCDR1 having zero, one, or two amino acid insertions, deletions, and / or substitutions; HCDR2 having zero, one, or two amino acid insertions, deletions, and / or substitutions; and / or HCDR3 having zero, one, or two amino acid insertions, deletions, and / or substitutions.The anti-CD56 binding moiety described herein may contain a VL containing an LCDR1 having zero, one, or two amino acid insertions, deletions, and / or substitutions; an LCDR2 having zero, one, or two amino acid insertions, deletions, and / or substitutions; and / or an LCDR3 having zero, one, or two amino acid insertions, deletions, and / or substitutions.

[0184] In some embodiments, the CD56 CAR may comprise: (a) an antigen-binding domain comprising an anti-CD56 scFv (e.g., a VL-VH pair); (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD56 scFv comprises any one of the following: (a) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, wherein these CDRs are defined according to the Kabat numbering scheme; or (b) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 15, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16, wherein these CDRs are defined according to the Kabat numbering scheme; or (b) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 ... The HCDR3 comprises the amino acid sequence of SEQ ID NO: 15; and the VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, wherein these CDRs are defined according to the AbM numbering scheme. In some embodiments, the anti-CD56 scFv comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 26. The intracellular signal transduction domain may comprise a co-stimulatory signal transduction domain of immune effector cells (such as T cells) and a primary intracellular signal transduction domain. In some embodiments, the intracellular signaling domain may not include the primary intracellular signaling domain of immune effector cells (such as T cells). The co-stimulatory signaling domain of the intracellular signaling domain may be derived from ligands selected from the group consisting of: CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.In some embodiments, the CD56 CAR further comprises a hinge domain (such as a CD28 hinge domain or a mutant CD28 hinge domain) located between the C-terminus of the CD56 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CD56 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the CD56 antigen-binding domain. In some embodiments, the CD56 CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a CD56 antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a transmembrane domain derived from CD28, and a co-stimulatory signal transduction domain derived from CD28.

[0185] The CD56 CAR may contain at least 95%, 99%, or 100% of the same amino acid sequence as any one of the amino acid sequences shown in SEQ ID NO: 41-42. A polypeptide containing any one of the amino acid sequences shown in SEQ ID NO: 41-42 is also provided.

[0186] 2.2 Other CAR areas

[0187] The other components and regions of dual-CAR and single-CAR are described in more detail below.

[0188] Intracellular signal transduction domains

[0189] The dual CARs (including first and second CARs) and single CARs disclosed herein comprise at least one intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one normal effector function of CAR-expressing immune cells. As used herein, the terms “intracellular signaling domain” or “intracellular signaling region” are used interchangeably and refer to a portion of a protein that transduces effector function signals and directs the cell to perform a specific function. While the entire intracellular signaling domain can generally be used, in many cases it is not necessary to use the entire strand. With regard to the use of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the complete strand, provided it transduces effector function signals. Therefore, the term intracellular signaling domain means any truncated portion (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion) that is sufficient to transduce effector function signals. Intracellular signaling domains can be derived from wild-type membrane proteins (e.g., receptors) or their functional variants as intracellular or cytoplasmic signaling domains. Intracellular signaling domains can have one or more mutations, including, for example, insertions, deletions, and / or substitutions. Intracellular signaling domains can contain co-stimulatory signaling domains of immune effector cells and / or primary intracellular signaling domains. The term "effective function" refers to a cell's specialized function. Effector functions of T cells may include, for example, cytolytic activity or helper activities, including the secretion of cytokines.

[0190] In some embodiments, the intracellular signaling domain may comprise a primary intracellular signaling domain of an immune effector cell. The CAR may comprise an intracellular signaling domain that is substantially composed of primary intracellular signaling domains of immune effectors. A “primary intracellular signaling domain” refers to an intracellular signaling sequence that acts in a stimulatory manner to induce the function of an immune effector. The primary intracellular signaling domain may contain a signaling motif called an immune receptor tyrosine-based activation motif or ITAM. As used herein, “ITAM” is a conserved protein motif commonly found at the tail of signaling molecules expressed in many immune cells. The motif may comprise two repeats of the amino acid sequence YxxL / I, separated by 6–8 amino acids, where each x is independently any amino acid, resulting in the conserved motif YxxL / Ix(6–8)YxxL / I. The ITAM within the signaling molecule is important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. The ITAM may also serve as a docking site for other proteins involved in signal transduction pathways. Exemplary primary intracellular signaling sequences containing ITAM include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and / or CD66d.

[0191] The primary intracellular signal transduction domain of a single CAR and / or the primary intracellular signal transduction domain of the first CAR in a dual CAR system can be derived from CD3ζ and retain the relevant function of the primary intracellular signal transduction domain of CD3ζ. The primary intracellular signal transduction domain can have the same sequence as the primary intracellular signal transduction domain of CD3ζ, or a sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of the primary intracellular signal transduction domain of CD3ζ. The primary intracellular signal transduction domain can be composed of intracellular signal transduction domains of CD3ζ. The primary intracellular signal transduction domain can be a wild-type intracellular signal transduction domain of CD3ζ. The primary intracellular signal transduction domain can be a functional mutant of the intracellular signal transduction domain of CD3ζ containing one or more mutations (such as Q65K). The primary intracellular signal transduction domain of CD3ζ may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 54.

[0192] Co-stimulatory signal transduction domain

[0193] In addition to stimulating antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. A CAR may contain at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce immune responses, such as effector functions. The co-stimulatory signaling domain of a chimeric antigen receptor described herein may be an intracellular signaling domain derived from a co-stimulatory protein that transduces signals and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "co-stimulatory signaling domain" may be an intracellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a homologous binding partner on an immune cell, such as a T cell, that specifically binds to a co-stimulatory ligand, thereby mediating co-stimulatory responses of the immune cell, such as, but not limited to, proliferation and survival.

[0194] In some embodiments, the intracellular signaling domain comprises a single co-stimulatory signaling domain. The intracellular signaling domain may comprise two or more co-stimulatory signaling domains (such as any one of about 2, 3, 4, or more). The intracellular signaling domain may comprise two or more identical co-stimulatory signaling domains. The intracellular signaling domain may comprise two or more co-stimulatory signaling domains from different co-stimulatory proteins (such as any two or more co-stimulatory proteins described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as a primary intracellular signaling domain of CD3ζ) and one or more co-stimulatory signaling domains. In some embodiments, one or more co-stimulatory signaling domains and the primary intracellular signaling domain (such as a primary intracellular signaling domain of CD3ζ) are fused to each other via an optional peptide linker. The primary intracellular signaling domain and one or more co-stimulatory signaling domains may be arranged in any suitable order. In some embodiments, one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as a primary intracellular signaling domain of CD3ζ). Multiple co-stimulatory signal transduction domains can provide cumulative or synergistic stimulation.

[0195] Activation of costimulatory signaling domains in host cells (e.g., immune cells) can induce increased or decreased cellular production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be applicable to the CARs described herein. One or more types of costimulatory signaling domains are selected based on factors such as the type of immune effector cells in which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effector).Examples of co-stimulatory signaling domains for use in CARs can be intracellular signaling domains of co-stimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF). R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B; members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0196] In some embodiments, one or more co-stimulatory signal transduction domains are derived from one or more molecules selected from the group consisting of: CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0197] In some embodiments, the intracellular signal transduction domain of the CAR disclosed herein (e.g., a dual CAR or a single CAR) includes a co-stimulatory signal transduction domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signal transduction domain of the CAR (e.g., the first CAR in a dual CAR or a single CAR) includes a primary intracellular signal transduction domain of CD3ζ and a 4-1BB co-stimulatory signal transduction domain. In some embodiments, the intracellular signal transduction domain of the CAR (e.g., a dual CAR or a single CAR) includes a 4-1BB co-stimulatory signal transduction domain. The 4-1BB co-stimulatory signal transduction domain may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 37.

[0198] In some embodiments, the intracellular signal transduction domain of the CAR disclosed herein (e.g., a dual CAR or a single CAR) includes a co-stimulatory signal transduction domain derived from CD28. The intracellular signal transduction domain of the CAR (e.g., the first CAR in a dual CAR or a single CAR) may include a primary intracellular signal transduction domain of CD3ζ and a co-stimulatory signal transduction domain of CD28. In some embodiments, the intracellular signal transduction domain of the CAR (e.g., a dual CAR or a single CAR) includes a co-stimulatory signal transduction domain of CD28. The CD28 co-stimulatory signal transduction domain may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 38.

[0199] Any variants of the costimulatory signaling domain described herein are also within the scope of this disclosure, enabling the costimulatory signaling domain to modulate the immune response of immune cells. The costimulatory signaling domain may contain up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to the wild-type control. Such a costimulatory signaling domain containing one or more amino acid variations is referred to as a variant. Mutations of amino acid residues in the costimulatory signaling domain, relative to the non-mutated costimulatory signaling domain, can lead to increased signal transduction and enhanced stimulation of the immune response. Mutations of amino acid residues in the costimulatory signaling domain, relative to the non-mutated costimulatory signaling domain, can lead to decreased signal transduction and reduced stimulation of the immune response.

[0200] In a dual-CAR system, the first intracellular signaling domain of the first CAR can consist of 1, 2, 3, 4, 5, or more co-stimulatory signaling domains. The first intracellular signaling domain of the first CAR can also consist of a single co-stimulatory signaling domain. The first intracellular signaling domain of the first CAR can contain 1, 2, 3, 4, 5, or more primary intracellular signaling domains of immune cells (such as T cells). The first intracellular signaling domain of the first CAR can also consist of a primary intracellular signaling domain of an immune cell (such as a T cell) capable of inducing primary activation signals in the immune cell (such as a T cell). The primary intracellular signaling domain can be a T cell receptor (TCR) component. The primary intracellular signaling domain of the first intracellular signaling domain can contain an immune receptor tyrosine-based activation motif (ITAM). The primary intracellular signal transduction domain of the first intracellular signal transduction domain may comprise an amino acid sequence derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FceRI, CD66d, DAP10, DAP12, or combinations thereof. In some embodiments, the primary intracellular signal transduction domain of the first intracellular signal transduction domain is derived from CD3ζ. The primary intracellular signal transduction domain may consist of intracellular signal transduction domains of CD3ζ. The primary intracellular signal transduction domain may be an intracellular signal transduction domain of wild-type CD3ζ. The primary intracellular signal transduction domain may be a functional mutant of the intracellular signal transduction domain of CD3ζ containing one or more mutations (such as Q65K). The primary intracellular signal transduction domain of CD3ζ may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 39 or 54.

[0201] In a dual-CAR system, the second intracellular signal transduction domain of the second CAR can consist of 1, 2, 3, 4, 5, or more co-stimulatory signal transduction domains. Alternatively, the second intracellular signal transduction domain of the second CAR can consist of a single co-stimulatory signal transduction domain. Each of the second intracellular signal transduction domains of the second CAR can contain more than one co-stimulatory domain.

[0202] As described herein, each of the first and second CARs in the dual CAR system may contain at least one co-stimulatory signal transduction domain of a first intracellular signal transduction domain and / or one co-stimulatory signal transduction domain of a second intracellular signal transduction domain. This co-stimulatory signal transduction domain may contain a functional signal transduction domain from proteins selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a / CD18, 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, and LIGHT. , HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, IT GA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD 11b,ITGAX,CD 11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD16 0 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands. The co-stimulatory signal transduction domains of the first intracellular signal transduction domain and / or the co-stimulatory signal transduction domains of the second intracellular signal transduction domain may include functional signal transduction domains derived from OX40, CD28, 4-1BB, ICOS, or their signal transduction portions. The co-stimulatory signal transduction domains of the first intracellular signal transduction domain and / or the second intracellular signal transduction domain may be derived from 4-1BB and / or CD28.In some embodiments, the co-stimulatory signal transduction domain of the first intracellular signal transduction domain comprises a 4-1BB intracellular signal transduction domain, and the co-stimulatory signal transduction domain of the second intracellular signal transduction domain comprises a CD28 intracellular signal transduction domain. The 4-1BB co-stimulatory signal transduction domain may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 37. The CD28 co-stimulatory signal transduction domain may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 38.

[0203] Transmembrane domain

[0204] Each of the first and second CARs in the dual CAR system disclosed herein, as well as the single CAR, comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. As used herein, the terms “transmembrane domain” or “transmembrane region” are used interchangeably to refer to a portion of a membrane protein (e.g., a receptor) embedded in the cell membrane. A transmembrane region may be the entire portion of a protein embedded in the cell membrane or only a portion thereof (e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire portion). A transmembrane region may be derived from the transmembrane region of a wild-type receptor or a functional variant thereof. A transmembrane region may have one or more mutations, including, for example, insertions, deletions, and / or substitutions. A transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain suitable for the CARs described herein may be obtained from naturally occurring proteins. Alternatively, it may be a synthetic, non-naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment in the cell membrane.

[0205] Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of crossing the phospholipid bilayer of the cell. Alternatively, transmembrane domains can also be classified based on their topology, including the number of times the domain crosses the membrane and the protein's orientation. For example, a single-transmembrane protein crosses the cell membrane once, while a multi-transmembrane protein crosses the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III, depending on the topology of their terminals and one or more membrane-transfer segments relative to the cell's interior and exterior. Type I membrane proteins have a single transmembrane domain oriented such that the N-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell, while the C-terminus is located on the intracellular side. Type II membrane proteins also have a single transmembrane domain, but are oriented such that the C-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell, while the N-terminus is located on the intracellular side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided based on the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0206] Each transmembrane domain of the CAR described herein may be derived from a type I single-transmembrane protein. Transmembrane domains from multi-transmembrane proteins may also be applicable to the CARs described herein. Multi-transmembrane proteins may contain complex (at least 2, 3, 4, 5, 6, 7 or more) α-helical or β-sheet structures. The N-terminus and C-terminus of a multi-transmembrane protein may be located on opposite sides of the lipid bilayer; for example, the N-terminus of the protein may be located on the intracellular side of the lipid bilayer, while the C-terminus of the protein may be located on the extracellular side.

[0207] Each CAR transmembrane domain may contain transmembrane domains selected from the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD152, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, C D19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, PD1, and / or NKG2C. In some embodiments, the transmembrane domains are derived from molecules selected from the group consisting of: CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1.

[0208] In some embodiments, the transmembrane domain is derived from CD8α. The transmembrane domain may be a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO: 34 or a sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the transmembrane domain is derived from CD28. The transmembrane domain may be a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 35 or a sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 35. The transmembrane domain may be a mutant CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36 or a sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 36.

[0209] The transmembrane domain used in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring α-helix or β-sheet. The protein segment is at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0210] The transmembrane domains described herein may comprise a transmembrane region and an intracellular region located on the C-terminal side of the transmembrane domain. The intracellular region of the transmembrane domain may contain three or more amino acids and facilitates the orientation of the transmembrane domain within a lipid bilayer. One or more cysteine ​​residues may be present in the transmembrane region of the transmembrane domain. One or more cysteine ​​residues are present in the intracellular region of the transmembrane domain. The intracellular region of the transmembrane domain may contain positively charged amino acids. In some embodiments, the intracellular region of the transmembrane domain contains the amino acids arginine, serine, and lysine.

[0211] The transmembrane region of a transmembrane domain may contain hydrophobic amino acid residues. The transmembrane domain of each CAR presented herein may contain an artificially hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. The transmembrane region primarily contains hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. The transmembrane region may contain a polyleucine-alanine sequence. The hydrophilic or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed using any method known in the art, such as Kyte and Doolittle hydrophilicity analyses.

[0212] Hinge domain

[0213] Each of the first and second CARs in the dual-CAR system disclosed herein, as well as a single CAR, may include a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. As used herein, the terms "hinge domain" or "hinge region" are used interchangeably to refer to the portion of a membrane protein (e.g., a receptor) that connects the transmembrane domain and the extracellular domain. A hinge domain is a small domain located between the antigen-binding domain and the extracellular membrane. For example, a hinge domain may be located between the C-terminus of the antigen-binding domain and the N-terminus of the transmembrane domain. A hinge domain may be part of an extracellular domain. A hinge domain may be derived from a hinge domain of a wild-type receptor or a functional variant thereof. A hinge domain may have one or more mutations, including, for example, insertions, deletions, and / or substitutions.

[0214] Hinge domains can confer flexibility to proteins, as well as the ability for one or two domains to move relative to each other. Any amino acid sequence that provides this flexibility and movement of an extracellular antigen-binding domain relative to a transmembrane domain of an effector molecule can be used.

[0215] The hinge domain may contain approximately 10-100 amino acids, for example, approximately 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The length of the hinge domain may be at least approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0216] The hinge domain can be a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art that contains a hinge domain can be used for the chimeric receptor described herein. The hinge domain can be part of the hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor. The hinge domain can be derived from CD8α. The hinge domain can be part of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. The CD8α hinge domain can contain the amino acid sequence of SEQ ID NO: 31, or can contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. The hinge domain can be the juxtamembrane region of CD28. The hinge domain of CD28 can be a wild-type CD28 hinge domain or a mutant CD28 hinge domain. The CD28 hinge domain may contain the amino acid sequence of SEQ ID NO: 32, or may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. The mutant CD28 hinge domain may contain the amino acid sequence of SEQ ID NO: 33, or may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0217] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE, or IgD antibodies) are also applicable to the pH-dependent chimeric receptor systems described herein. A hinge domain can be a hinge domain connecting the constant domains CH1 and CH2 of an antibody. A hinge domain can be a hinge domain of an antibody and can include the hinge domain of the antibody and one or more constant regions of the antibody. A hinge domain can include the hinge domain of the antibody and the CH3 constant region of the antibody. A hinge domain can include the hinge domain of the antibody as well as the CH2 and CH3 constant regions of the antibody. The antibody can be an IgG, IgA, IgM, IgE, or IgD antibody. The antibody can be an IgG1, IgG2, IgG3, or IgG4 antibody. A hinge region can include the hinge region of an IgG1 antibody as well as the CH2 and CH3 constant regions. A hinge region can include the hinge region of an IgG1 antibody and the CH3 constant region.

[0218] Non-naturally occurring peptides can also be used as the hinge domain of the chimeric receptor described herein. The hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor is a peptide linker, such as a (GxS)n linker, where x and n can be independent integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or larger.

[0219] signal peptide

[0220] Each of the first and second CARs in the dual CAR system disclosed herein, as well as the single CAR, may include a signal peptide (also referred to as a signal sequence) at the N-terminus of the antigen-binding domain. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in the cell. In some embodiments, the signal peptide targets effector molecules to cellular secretory pathways and allows effector molecules to integrate and anchor into the lipid bilayer. Signal peptides suitable for use with the CARs described herein (including signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences) will be apparent to those skilled in the art. The signal peptide may be derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and the IgG1 heavy chain. The signal peptide may be a CD8α-derived signal peptide. The signal peptide may contain an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0221] peptide linkers

[0222] When multiple antibodies (e.g., multiple antibody fragments) are present in this CAR, the various antibodies or antibody fragments can fuse with each other through one or more peptide linkers (e.g., VHH-linker-VHH or VL-linker-VH). Antibodies or antibody fragments can also fuse directly with each other without any peptide linkers. The peptide linkers connecting different antibodies can be the same or different. Different domains of the CAR can also fuse with each other through peptide linkers.

[0223] Depending on the structural and / or functional characteristics of the antibody and / or various domains, each peptide linker in a CAR can have the same or different lengths and / or sequences. Each peptide linker can be selected and optimized independently. The length, flexibility, and / or other properties of one or more peptide linkers used in a CAR can have some influence on properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not spatially interfere with each other. Short peptide linkers can be positioned between the transmembrane domains and intracellular signaling domains of the CAR. Peptide linkers can contain flexible residues (such as glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine dinucleotide can be a suitable peptide linker.

[0224] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is no more than about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids.

[0225] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from the hinge region of heavy-chain-only antibodies can be used as linkers. See, for example, WO 1996 / 34103. Peptide linkers can be flexible linkers. Exemplary flexible linkers include, but are not limited to, glycine polymers (G). n Glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, threonine-serine polymers, and other flexible connectors known in the art. Other connectors known in the art (e.g., as described in WO 2016014789, WO 2015158671, WO 2016102965, US 20150299317, WO2018067992, US 7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988)) may also be included in the CARs provided herein, the disclosures of each of which are incorporated herein by reference.

[0226] Various adapter sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS adapters), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary adapter sequences may comprise amino acid sequences, including but not limited to GGGGSGGGGSGGGGS (SEQ ID NO: 47), GGSG (SEQ ID NO: 48), GGSGG (SEQ ID NO: 49), GGSSG (SEQ ID NO: 50), GGSGG (SEQ ID NO: 51), GGGSG (SEQ ID NO: 52), GSSSG (SEQ ID NO: 53), etc. Those skilled in the art will be able to select appropriate adapter sequences.

[0227] A dual-CAR system may include a first CAR and a second CAR linked by a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A linker" refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. The use of the term "self-cleaving" does not imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those skilled in the art, including but not limited to those found in members of the Picornaviridaevirus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), TaV, and porcine cerebrospinal viral-1 (PTV-1); and carioviruses such as Theilovirus and encephalocarditis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those skilled in the art will be able to select appropriate self-cleaving peptides. In some embodiments, the self-cleaving peptide is a P2A linker having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the self-cleaving peptide is a T2A linker having at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46.

[0228] A dual-CAR system may comprise a DLL3 CAR and a CD56 CAR linked by a self-cleaving peptide. The dual-CAR system may comprise a DLL3 CAR containing at least 95%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO: 40; and a CD56 CAR containing at least 95%, 99%, or 100% of the amino acid sequences identical to those of SEQ ID NO: 41-42. In some embodiments, the dual-CAR system may comprise a polypeptide encoding a DLL3 CAR and a CD56 CAR having a 2A linker, the polypeptide containing at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO: 43 or SEQ ID NO: 44.

[0229] The hinge domains, transmembrane domains, and / or intracellular signaling domains (e.g., co-stimulatory signaling domains and / or primary intracellular signaling domains) of CARs can be derived from first-, second-, third-, or fourth-generation CAR structures. Details of the structural features of CARs can be found, for example, in Jackson, Hollie J., et al., Nature Reviews ClinicalOncology 13.6 (2016): 370; and Subklewe, Marion, et al., Transfusion Medicine and Hemotherapy 46.1 (2019): 15–24; which are each incorporated herein by reference.

[0230] 2.3 Engineered Immune Cells

[0231] In one aspect, this disclosure provides engineered immune cells (e.g., T cells, NK cells, tumor-infiltrating lymphocytes) comprising the dual CARs described herein. The engineered immune cells may comprise a single CAR (e.g., CD56CAR) as described herein. In some embodiments, the engineered immune cells may be used to treat various conditions or diseases as described herein (e.g., neuroendocrine carcinoma).

[0232] The cells to be engineered can be obtained from, for example, humans or non-human animals. The cells to be engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. For example, the cells can be obtained from humans, rats, or mice. The cells can be mouse lymphocytes and engineered (e.g., transduced) to express the CAR described herein. The cells can be obtained from humans. The cells can be blood cells. The cells can be white blood cells (e.g., T cells), lymphocytes, or any other suitable blood cell type. The cells can be peripheral blood cells. The cells can be tumor-infiltrating lymphocytes (TILs). The cells can be T cells, B cells, or NK cells. The cells can be human peripheral blood mononuclear cells (PBMCs). Human PBMCs can be CD3+ cells. Human PBMCs can be CD8+ cells or CD4+ cells.

[0233] The cells can be T cells. T cells can express one or more CARs that recognize specific antigens on the surface of target cells. T cells can be obtained by various methods known in the art, such as in vitro culture of T cells isolated from a subject (e.g., tumor-infiltrating lymphocytes). Genetically engineered T cells can be obtained by transducing T cells (e.g., isolated from peripheral blood of a subject) with a vector (such as the vector provided herein). T cells can be CD4+ T cells, CD8+ T cells, or regulatory T cells. T cells can be T helper type 1 T cells and / or T helper type 2 T cells. T cells expressing a CAR can be αβ-T cells. T cells expressing the receptor can be γδ-T cells. T cells can be central memory T cells. T cells can be effector memory T cells. T cells can be naive T cells.

[0234] The preparation of engineered immune cells may include one or more culture and / or preparation steps. Cells to be engineered to express one or more CARs can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. The subject from whom the cells are isolated may have a disease or condition, or require or be subject to cell therapy. The subject may be someone who requires a specific therapeutic intervention, such as adoptive cell therapy of the cells being isolated, processed, and / or engineered.

[0235] The cells can be stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as cells isolated directly from the subject and / or cells isolated from the subject and frozen. Stem cells can be cultured with additional differentiation factors to obtain the desired cell type (e.g., T cells).

[0236] Different cell types can be obtained through appropriate isolation methods. Isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers, like surface proteins, intracellular markers, or nucleic acids. Any known isolation method based on such markers can be used. Isolation can be based on affinity or immunoaffinity. For example, in some aspects, isolation involves separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers), such as by incubating with antibodies or binding couplers that specifically bind to such markers, followed typically by a washing step and separating cells bound to the antibodies or binding couplers from those not bound to the antibodies or binding couplers.

[0237] Such separation steps can be based on positive selection (where cells bound to the reagent are retained for further use) and / or negative selection (where cells not bound to the antibody or bound to a partner are retained). Both portions can be retained for further use. Negative selection can be particularly useful when no antibody is available to specifically identify cell types within a heterogeneous population, allowing for optimal separation based on markers expressed by cells other than the desired cell population.

[0238] Methods, nucleic acids, compositions, and kits (reagents) for expressing one or more CARs and / or for generating genetically engineered immune cells expressing one or more CARs are also provided. Genetic engineering typically involves introducing nucleic acids encoding one or more CARs into cells, such as through retroviral transduction, transfection, or transformation. Gene transfer can be accomplished by first stimulating cells, such as by combining the cells with stimuli that induce responses (such as proliferation, survival, and / or activation) (e.g., as measured by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in culture to a quantity sufficient for clinical application.

[0239] Recombinant nucleic acids can be transferred into cells using recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). Recombinant nucleic acids can also be transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. Retroviral vectors have long terminal repeats (LTRs), such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. Retroviruses can include those derived from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they are capable of infecting host cells of several species, including humans. The vector can be a lentiviral vector. In some embodiments, the vector contains any of the nucleic acids encoding the CAR described herein. Nucleic acids can be cloned into vectors using any molecular cloning method known in the art, including, for example, the use of restriction endonuclease sites and one or more selectivity markers. Recombinant nucleic acids can be transferred to T cells via electroporation. Recombinant nucleic acids can also be transferred to T cells via transposition. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment; and strontium phosphate DNA coprecipitation. Many of these methods are described, for example, in WO 2019195486, which is incorporated herein by reference in its entirety. Prior to transduction, T cells can be pre-activated, for example, using anti-CD3 / CD28 particles for approximately 12 hours, 24 hours, 36 hours, 48 ​​hours, or 60 hours. Transduced T cells can be harvested on days 5, 6, 7, 8, 9, 10, 11, or 12 post-transduction.

[0240] One or more CARs disclosed herein can be expressed in at least 17%, for example 28%, of a cell population engineered to express one or more CARs, as measured by flow cytometry.

[0241] Transfection efficiency can be determined by flow cytometry (e.g., as described in Example 2) using anti-VHH antibody 4 days post-infection. The transfection efficiency of the vector can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

[0242] CAR expression can be determined by flow cytometry (e.g., as described in Example 2) using anti-VHH antibodies 4 days post-infection. The CAR positivity rate of engineered immune cells can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%.

[0243] Engineered immune cell populations, compositions containing such cells and / or enriched with such cells, such as those expressing CARs comprising at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the total cells in the composition or of a certain type of cells (such as T cells, CD8+ or CD4+ cells).

[0244] On days 0, 1, 2, 3, 4, or 5 post-transduction, the viability of the transduced T cells may be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. On days 0, 1, 2, 3, 4, or 5 post-transduction (e.g., day 5), the viability of the transduced T cells may be at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, or at least or about 120% compared to the viability of untransduced T cells.

[0245] On days 0, 1, 2, 3, 4, or 5 post-transduction, the T cell expansion fold can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times. On days 0, 1, 2, 3, 4, or 5 post-transduction (e.g., day 5), the T cell expansion fold of the transduced T cells compared to untransduced T cells can be at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90%.

[0246] The in vitro cytotoxicity of engineered immune cells can be determined using an LDH (lactate dehydrogenase)-based cytotoxicity assay (e.g., as described in Example 3). Engineered immune cells (e.g., CAR-T cells) can be co-cultured with target cells for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 16 hours, 18 hours, 1 day, 2 days, 3 days, or longer, allowing the engineered immune cells (e.g., CAR-T cells) to be activated. Engineered immune cells can be co-cultured with different tumor cell lines (e.g., DLL3-positive SHP-77 cells, SHP-77-Luc-DLL3 KO, or NK-92 cells) for 24 hours. The E:T ratio can be 0.5:1 or 2:1.

[0247] For example, using the method described in the examples, the in vitro cytotoxicity of engineered immune cells can be at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The in vitro cytotoxicity of engineered immune cells can be less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95%. The in vitro cytotoxicity of engineered immune cells can be about 40%-90%, about 40%-80%, or about 50%-80%. The cytotoxicity can be specific to target cells expressing a first or second antigen, which is, for example, a neuroendocrine lineage marker such as DLL3, CD56, MUC1C, CDH17, GD2, neuro-guided factor 3, SEZ6, GPC2, and / or B7-H3, such as DLL3 and CD56. Engineered immune cells can induce minimal or background cytotoxicity against target cells that do not express primary or secondary antigens, such as neuroendocrine lineage markers like DLL3, CD56, MUC1C, CDH17, GD2, neuroguide factor 3, SEZ6, GPC2, and / or B7-H3, such as DLL3 and CD56.

[0248] When engineered immune cells containing a dual CAR system are exposed to target cells containing the target antigen, these engineered immune cells can exhibit in vitro cytotoxicity (as measured by the method described in the examples) that is higher than that of engineered immune cells containing only one CAR containing a primary intracellular signaling domain, for example, by at least 30% or, for example, by at least 60%. Advantageously, higher cytotoxicity can lead to the killing of more target tumor cells.

[0249] Engineered immune cells comprising a dual CAR system, wherein the second CAR comprises a mutant hinge domain and a mutant transmembrane domain, such as a mutant CD28 hinge domain and a mutant CD28 transmembrane domain, for example, a hinge domain comprising the amino acid sequence of SEQ ID NO: 33 and a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, may exhibit lower cytotoxicity (as measured by methods according to examples), for example, at least 15%, compared to engineered immune cells comprising a dual CAR system containing wild-type hinge domains and transmembrane domains, when exposed to target cells containing the target antigen. Reduced cytotoxicity of engineered immune cells may be beneficial because it can reduce any risks associated with overactivation of the dual CAR system; for example, it can reduce the risk of cytokine storms by assisting in the control of immune cell activation.

[0250] Long-term cytotoxicity of engineered immune cells (e.g., CAR-T cells) can be determined, for example, by re-attacking the engineered immune cells. Exemplary re-attack procedures for CAR-T cells can be found, for example, Wang, Dongrui, et al., Journal of Visualized Experiments: JoVE 144 (2019); Wang D, et al., JCI Insight 2018, 3(10); Lange et al., Cancer Discov. 9 Feb 2021, candisc.0896.2020; each of which is incorporated herein by reference in its entirety.

[0251] Engineered immune cells can secrete cytokines (e.g., IFNγ) with or without exposure to antigens recognized by antigen-binding domains. Cytokine release can be measured in vitro in an in vitro cytokine release assay (e.g., as described in Example 4). The concentration of cytokines (e.g., IFN-γ) released by the engineered immune cells (e.g., CAR-T cells) described herein can be determined by homogeneous time-resolved fluorescence (HTRF) assay. Engineered immune cells can be co-cultured with tumor cells. The effector cell to target cell (E:T) ratio can be 0.5:1 or 2:1. Engineered immune cells can secrete amounts of IFNγ greater than 50 pg / ml, greater than 100 pg / ml, greater than 200 pg / ml, greater than 300 pg / ml, greater than 400 pg / ml, greater than 500 pg / ml, greater than 1000 pg / ml, greater than 1500 pg / ml, greater than 2000 pg / ml, greater than 2500 pg / ml, or greater than 3000 pg / ml. Engineered immune cells can secrete amounts of IFNγ less than 50 pg / ml, less than 100 pg / ml, less than 200 pg / ml, less than 300 pg / ml, less than 400 pg / ml, less than 500 pg / ml, less than 1000 pg / ml, less than 1500 pg / ml, less than 2000 pg / ml, less than 2500 pg / ml, or less than 3000 pg / ml. Engineered immune cells can secrete IFNγ in amounts of 500-5000 pg / ml, 1000-4000 pg / ml, 1000-3000 pg / ml, 1500-3000 pg / ml, 50-500 pg / ml, 50-400 pg / ml, 100-1000 pg / ml, 100-800 pg / ml, 100-600 pg / ml, 100-400 pg / ml, 200-400 pg / ml, 200-300 pg / ml, 200-1000 pg / ml, or 200-800 pg / ml. Compared to engineered immune cells containing a single CAR, engineered immune cells containing a dual CAR system secrete more IFNγ. Compared to untransfected control cells, engineered immune cells secrete more IFNγ. Compared to wild-type (i.e., non-engineered cells), engineered immune cells can have increased expression or secretion of cytokines (e.g., IFN-γ). Cytokine release can be increased by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000, or 10000 times.

[0252] When engineered immune cells containing a dual CAR system are exposed to target cells containing the target antigen, these engineered immune cells may exhibit in vitro IFNγ production (as measured by the method described in the examples) that is higher than that of engineered immune cells containing only one CAR containing a primary intracellular signaling domain.

[0253] According to the method described in the example, when exposed to target cells containing first and second target antigens at an E:T ratio of 0.5:1, engineered immune cells containing a dual CAR system can produce at least about 2000 pg / ml of IFNγ in vitro.

[0254] According to the example method, when exposed to target cells (containing one of the first antigen or the second antigen) at an E:T ratio of 2:1, engineered immune cells containing a dual CAR system can produce less than about 1000 pg / ml of IFNγ in vitro.

[0255] According to the method described in the example, when exposed to target cells (containing either the first antigen or the second antigen) at a ratio of 0.5:1, engineered immune cells containing a dual CAR system can produce less than about 500 pg / ml of IFNγ in vitro.

[0256] Engineered immune cells comprising a dual CAR system, wherein the second CAR comprises a mutant hinge domain and a mutant transmembrane domain, such as a mutant CD28 hinge domain and a mutant CD28 transmembrane domain, such as a hinge domain comprising the amino acid sequence of SEQ ID NO: 33 and a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 36, may exhibit lower in vitro IFNγ production (as measured according to the method of the example) when exposed to target cells expressing a first and / or second antigen, compared to dual CARs comprising wild-type hinge domains and transmembrane domains.

[0257] Engineered immune cells may be re-attacked at least 1, 2, 3, 4, 5, or 6 times. In some embodiments, the calculated cytotoxicity (cytotoxicity %) is determined after each re-attack. In some embodiments, after the first re-attack, the calculated cytotoxicity of the engineered immune cells described herein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. After the second re-attack, the calculated cytotoxicity of the engineered immune cells may be at least 80%, at least 90%, or at least 95%. After the third re-attack, the calculated cytotoxicity of the engineered immune cells may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. After the fourth re-attack, the calculated cytotoxicity of the engineered immune cells may be at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. After the fifth re-attack, the calculated cytotoxicity of the engineered immune cells may be at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. After the sixth re-attack, the calculated cytotoxicity of the engineered immune cells may be at least 0%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, the maximum number of re-attacks of the engineered immune cells described herein (i.e., the number of re-attacks before tumor cells grow) is at least 5, 6, 7, 8, 9, or 10.

[0258] The persistence and / or exhaustion of engineered immune cells in vitro can be assessed by repeatedly stimulating engineered immune cells with tumor cells (e.g., SHP-77 cells) several rounds in a re-challenge assay (e.g., as described in Example 5). Engineered immune cells can be first challenged (co-cultured) with tumor cells (e.g., SHP-77 cells) in the first round, and then re-challenged with fresh tumor cells (e.g., SHP-77 cells) in a subsequent round. The E:T ratio can be 1:5. In the re-challenge assay, after 1, 2, 3, or 4 rounds of stimulation, the percentage of CD3 positivity in total viable cells can be greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%. In the re-challenge assay, after 1, 2, 3, or 4 rounds of stimulation, total T cells could expand by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, or 250 times. In the re-challenge assay, after 1, 2, 3, or 4 rounds of stimulation, engineered immune cells can expand by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 500, 1000, 2000, 3000, or 4000 times. Each round of stimulation can last for 1, 2, 3, 4, or 5 days. In some embodiments, each round of stimulation lasts for 3 days. Engineered immune cells can exhibit better long-term durability than unengineered wild-type cells. Compared with control cells, the amount of engineered immune cells can be increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1,000%.Engineered immune cells containing a dual-CAR system can exhibit better long-term durability than engineered immune cells containing only a single CAR (e.g., the first CAR in a dual-CAR system). In some embodiments, the proliferation of dual-CAR engineered immune cells is increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1,000% compared to single-CAR engineered immune cells.

[0259] The inhibition of tumors by engineered immune cells in vivo can be assessed by in vivo tumor suppression assays (e.g., as described in Example 6). Tumor models can be established by subcutaneously injecting NCG mice with SCLC NCI-H82 cells. Engineered immune cells can be intravenously administered to tumor-transplanted mice 10 days after tumor inoculation. Tumor length (L) and width (W) can be measured every 3–4 days using calipers after CAR-T cell injection. Tumor volume can be estimated using the following formula: V = (W² × L) / 2. After 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, these engineered immune cells were able to inhibit tumor growth. More than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 1,000%. Throughout the experiment, the mice's body weight may not have changed significantly. After 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, the mice's body weight can change by less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 100%.After 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, engineered immune cells containing a dual CAR system can have higher efficacy than those containing only a single CAR (e.g., in a dual CAR system). Engineered immune cells from the first CAR showed more significant tumor suppression, exceeding 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000%.

[0260] After 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, engineered immune cells containing a dual-CAR system can increase the total number of immune cells (e.g., T cells) compared to engineered immune cells containing only a single CAR (e.g., the first CAR in a dual-CAR system). For example, they can increase the total number of immune cells (e.g., T cells) by more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, or more than 50 times. After 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, engineered immune cells containing a dual-CAR system can increase the number of immune cells (e.g., T cells) compared to engineered immune cells containing only a single CAR (e.g., the first CAR in a dual-CAR system). For example, they can increase the number of immune cells (e.g., T cells) by more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, or more than 50 times. Compared to untransduced immune cells (i.e., immune cells without CAR), engineered immune cells can exhibit better long-term persistence. In some embodiments, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, the proliferation rate of engineered immune cells can be more than 5% higher than that of untransduced immune cells (e.g., immune cells without CAR). 10% or higher, 15% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, 100% or higher, 150% or higher, 200% or higher, 250% or higher, 300% or higher, 400% or higher, 500% or higher, 600% or higher, 700% or higher, 800% or higher, 900% or higher, or 1,000% or higher. Engineered immune cells containing only a single CAR (e.g., the first CAR in a dual-CAR system) exhibit better long-term durability compared to engineered immune cells containing only a single CAR (e.g., the first CAR in a dual-CAR system).In some embodiments, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days, the proliferation rate of engineered immune cells can be 5% higher than that of engineered immune cells containing only a single CAR (e.g., the first CAR in a dual CAR system). % or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, 200% or more, 250% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, or 1,000% or more.

[0261] This disclosure provides methods or processes for preparing, manufacturing, and / or using engineered immune cells to treat pathological diseases or conditions.

[0262] Cells used for engineering can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. In some embodiments, the subject from whom cells are isolated is a subject suffering from a disease or condition or requiring or to whom cell therapy will be administered. The subject may be someone requiring a specific therapeutic intervention, such as adoptive cell therapy where cells are being isolated, processed, and / or engineered.

[0263] Cells can be primary cells, such as primary human cells. Samples can include tissues, fluids, and other samples taken directly from the subject, as well as samples generated by one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction using viral vectors), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, and tissue and organ samples, including processed samples derived therefrom.

[0264] 3. Nucleic acid

[0265] In one respect, this disclosure provides (i) nucleic acids encoding the dual CARs described herein, and / or (ii) nucleic acids encoding the single CARs described herein.

[0266] In one aspect, this disclosure provides nucleic acids comprising one or more nucleic acid sequences encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR), wherein

[0267] (a) The first chimeric antigen receptor (CAR) includes:

[0268] i) An antigen-binding domain that specifically recognizes the first antigen;

[0269] ii) The first transmembrane domain; and

[0270] iii) A first intracellular signal transduction domain, which includes a co-stimulatory signal transduction domain of the first intracellular signal transduction domain; and

[0271] (b) The second chimeric antigen receptor (CAR) includes:

[0272] i) A second antigen-binding domain that specifically recognizes a second antigen;

[0273] ii) The second transmembrane domain; and

[0274] iii) A second intracellular signal transduction domain comprising a co-stimulatory signal transduction domain thereof, wherein the first intracellular signal transduction domain further comprises a first primary intracellular signal transduction domain of the immune cell, and the second intracellular signal transduction domain does not comprise a primary intracellular signal transduction domain derived from CD3ζ; and wherein the first antigen and the second antigen are selected from the group consisting of: DLL3, CD56, MUC1C, CDH17, GD2, neurotrophic factor 3, SEZ6, GPC2, and B7-H3. In some embodiments, the second intracellular signal transduction domain does not comprise a primary intracellular signal transduction domain of the immune cell.

[0275] In some embodiments, the nucleic acid may comprise one or more nucleic acid sequences encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR). In some embodiments, the first chimeric antigen receptor (CAR) may be a DLL3 CAR comprising: (a) a first antigen-binding domain comprising an anti-DLL3 binding moiety containing two anti-DLL3 sdAbs (e.g., a VHH domain); (b) a first transmembrane domain; and (c) a first intracellular signal transduction domain, wherein the two anti-DLL3 sdAbs comprise any one of: (1) a first anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 7, a CDR2 containing the amino acid sequence of SEQ ID NO: 8, and a CDR3 containing the amino acid sequence of SEQ ID NO: 9; and a second anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 10, a CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a CDR3 containing the amino acid sequence of SEQ ID NO: 12, wherein these CDRs are defined according to the Kabat numbering scheme; or (2) a first ...2, and a CDR3 containing the amino acid sequence of SEQ ID NO: 12, wherein these CDRs are defined according to the Kabat numbering scheme; or (3) a first anti-DLL3 sdAb comprising a CDR1 containing the amino acid sequence of SEQ ID NO: 10, a CDR2 containing the amino acid sequence of SEQ ID NO: 12, and a CDR3 containing the amino acid sequence of SEQ ID NO: 1 The CDR1 containing the amino acid sequence of SEQ ID NO: 20, the CDR2 containing the amino acid sequence of SEQ ID NO: 21, and the CDR3 containing the amino acid sequence of SEQ ID NO: 21; and a second anti-DLL3 sdAb comprising CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 23, and CDR3 containing the amino acid sequence of SEQ ID NO: 24, wherein these CDRs are defined according to the AbM numbering scheme. The anti-DLL3 sdAb may be camel-derived, chimeric, human, or humanized. VHHCDRs (CDR1-3) may be determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof. In some embodiments, the anti-DLL3 sdAb comprises a VHHCDR containing an amino acid sequence selected from the group consisting of SEQ ID NO: 28-29. HH domain. In some embodiments, the anti-DLL3 binding portion includes a first VHH domain comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 28; and a second VHH domain comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the first intracellular signaling domain comprises a co-stimulatory signaling domain of an immune effector cell (such as a T cell) and a primary intracellular signaling domain. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The co-stimulatory signal transduction domain of the first intracellular signal transduction domain can be derived from molecules selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the DLL3 CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the DLL3 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the DLL3 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the DLL3 antigen-binding domain. In some embodiments, the first CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a DLL3 antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signal transduction domain derived from 4-1BB, and a primary intracellular signal transduction domain derived from CD3ζ.In some embodiments, the second CAR may be a CD56 CAR comprising: (a) a second antigen-binding domain comprising an anti-CD56 binding moiety containing an anti-CD56 scFv (e.g., VL-VH pair); (b) a second transmembrane domain; and (c) a second intracellular signaling domain comprising a co-stimulatory signaling domain, wherein the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3ζ, and wherein the anti-CD56 scFv comprises any of the following: (a) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and HCDR3 containing the amino acid sequence of SEQ ID NO: 4. The anti-CD56 scFv may be mouse, chimeric, human, or humanized. The CDRs of anti-CD56 scFv, such as VH CDRs (HCDR1-3) and VL CDRs (LCDR1-3), may be determined according to the Kabat numbering scheme; or (b) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 13, HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and HCDR3 containing the amino acid sequence of SEQ ID NO: 15; and a VL domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 16, LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and LCDR3 containing the amino acid sequence of SEQ ID NO: 18, wherein these CDRs are defined according to the AbM numbering scheme. The anti-CD56 scFv may be mouse, chimeric, human, or humanized. The CDRs of anti-CD56 scFv, such as VH CDRs (HCDR1-3) and VL CDRs (LCDR1-3), may be determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or combinations thereof. In some embodiments, the anti-CD56 scFv comprises a VH domain containing the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 25; and a VL domain containing the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the second intracellular signaling domain does not contain the primary intracellular signaling domain of immune effector cells (such as T cells).The co-stimulatory signal transduction domain of the second intracellular signal transduction domain can be derived from molecules selected from the group consisting of: CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In some embodiments, the CD56 CAR further comprises a hinge domain (such as a CD28 hinge domain or a mutant CD28 hinge domain) located between the C-terminus of the CD56 antigen-binding domain and the N-terminus of the second transmembrane domain. In some embodiments, the CD56 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the CD56 antigen-binding domain. In some embodiments, the first CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a CD56 antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a transmembrane domain derived from CD28, and a co-stimulatory signal transduction domain derived from CD28.

[0276] The nucleic acid disclosed herein may comprise a first nucleic acid sequence and a second nucleic acid sequence. The first nucleic acid may be upstream of or downstream of the second nucleic acid. The first and second nucleic acid sequences may be separated by an adapter. The adapter used in this disclosure allows multiple proteins encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), which are translated into polyproteins that dissociate into individual protein components. The nucleic acid may comprise a first nucleic acid sequence, an adapter, and a second nucleic acid sequence from its 5' to 3' end. Alternatively, the nucleic acid may comprise a second nucleic acid sequence, an adapter, and a first nucleic acid sequence from its 5' to 3' end. The first nucleic acid sequence may encode a first CAR as described herein, and the second nucleic acid sequence may encode a second CAR as described herein.

[0277] The adapter may contain a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a protein-coding region of a start codon (such as ATG), thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES obtained from viral or cellular mRNA sources such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtained from, for example, cardiogenic viruses, rhinoviruses, foot-and-mouth disease viruses, HCV, Friend mouse leukemia virus (FrMLV), and Moloney mouse leukemia virus (MoMLV). Those skilled in the art will be able to select appropriate IRES.

[0278] Linkers may contain nucleic acid sequences encoding self-cleaving peptides. As used herein, a “self-cleaving peptide” or “2A linker” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins that dissociate into component proteins upon translation. The use of the term “self-cleaving” does not imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), TaV, and porcine cephalomyelitis virus-1 (PTV-1); and BCG viruses, such as Theylvirus and encephalomyelitis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. The P2A connector may have the same sequence as SEQ ID NO: 45, at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The T2A connector may have the same sequence as SEQ ID NO: 46, at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0279] Various adapter sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS adapters), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary adapter sequences may comprise amino acid sequences, including but not limited to GGGGSGGGGSGGGGS (SEQ ID NO: 47), GGSG (SEQ ID NO: 48), GGSGG (SEQ ID NO: 49), GGSSG (SEQ ID NO: 50), GGSGG (SEQ ID NO: 51), GGGSG (SEQ ID NO: 52), GSSSG (SEQ ID NO: 53), etc. Those skilled in the art will be able to select appropriate adapter sequences.

[0280] In some embodiments, this disclosure provides a nucleic acid comprising a nucleic acid sequence encoding a single CAR (e.g., a CD56 CAR) comprising: (a) an antigen-binding domain comprising an anti-CD56 binding moiety containing an anti-CD56 scFv (e.g., a VL-VH pair); (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD56 scFv comprises any one of: (a) a VH domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and a VL domain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, wherein these CDRs are defined according to the Kabat numbering scheme; or (b) a VH domain comprising an anti-CD56 binding moiety containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 1, HCDR3 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, HCDR4 containing the amino acid sequence of SEQ ID NO: 4, HCDR56 containing the amino acid sequence of SEQ ID NO: 5, HCDR3 containing the amino acid sequence of SEQ ID NO: 6, HCDR4 containing the amino acid sequence of SEQ ID NO: 6, HCDR56 containing the amino acid sequence of SEQ ID NO: 6 ... The HCDR1 containing the amino acid sequence of SEQ ID NO: 13, the HCDR2 containing the amino acid sequence of SEQ ID NO: 14, and the HCDR3 containing the amino acid sequence of SEQ ID NO: 15; and the VL domain containing the LCDR1 containing the amino acid sequence of SEQ ID NO: 16, the LCDR2 containing the amino acid sequence of SEQ ID NO: 17, and the LCDR3 containing the amino acid sequence of SEQ ID NO: 18, wherein these CDRs are defined according to the AbM numbering scheme. In some embodiments, the anti-CD56 scFv comprises a VH domain containing the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 25; and a VL domain containing the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence shown in SEQ ID NO: 26. The intracellular signal transduction domain may include a co-stimulatory signal transduction domain of immune effector cells (such as T cells) and a primary intracellular signal transduction domain. In some embodiments, the intracellular signal transduction domain may not include a primary intracellular signal transduction domain of immune effector cells (such as T cells). The co-stimulatory signal transduction domain of the intracellular signal transduction domain may be derived from ligands selected from the group consisting of: CD27, CD28, CD137 (4-1BB), OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.In some embodiments, the CD56 CAR further comprises a hinge domain (such as a CD28 hinge domain or a mutant CD28 hinge domain) located between the C-terminus of the CD56 antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CD56 CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the CD56 antigen-binding domain. In some embodiments, the CD56 CAR comprises a polypeptide comprising, from the N-terminus to the C-terminus: a CD8α signal peptide, a CD56 antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a transmembrane domain derived from CD28, and a co-stimulatory signal transduction domain derived from CD28.

[0281] The nucleic acid disclosed herein may be a restriction endonuclease site sequence.

[0282] The nucleic acids disclosed herein can be operatively linked to transcriptional control elements, such as promoters and enhancers.

[0283] The promoter can be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, the CD4 gene promoter can be used; see, for example, Salmon et al., Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al., Blood (2011) 117:1565.

[0284] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any nucleic acid sequence operatively linked to it. Other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, the EF-1α promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, this disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch that can turn on the expression of a nucleic acid sequence operatively linked to it when expression is needed, or turn off expression when expression is not needed. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0285] The nucleic acids disclosed herein can be provided for generating CARs in dual CAR systems (e.g., in mammalian cells). The nucleic acids disclosed herein can be provided for nucleic acid amplification.

[0286] Vectors, such as expression vectors (e.g., lentiviral vectors), can be used to introduce nucleic acids encoding dual or single CARs into immune cells (e.g., T cells) or their precursors. Vectors disclosed herein (e.g., lentiviral vectors) may contain one or more nucleic acids encoding dual CARs disclosed herein. Vectors disclosed herein (e.g., lentiviral vectors) may contain one nucleic acid encoding a single CAR disclosed herein. Vectors (e.g., lentiviral vectors) may contain additional elements that contribute to the functional expression of the CAR encoded therein. Expression vectors containing nucleic acids encoding dual or single CARs may contain mammalian promoters. Vectors may contain an elongation factor-1-α promoter (EF-1α promoter). The use of an EF-1α promoter can improve the expression efficiency of downstream transgenes (e.g., nucleic acids encoding CARs). Physiological promoters (e.g., EF-1α promoters) are unlikely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and can be incorporated into vectors disclosed herein. Vectors (e.g., lentiviral vectors) may contain non-essential cis-acting sequences that can improve titers and gene expression.

[0287] The nucleic acid can encode a first CAR (in a dual CAR system) comprising, from the N-terminus to the C-terminus, a CD8α signal peptide, an antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3ζ primary intracellular signal transduction domain. The nucleic acid can also encode a DLL3 CAR. The nucleic acid can encode an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40.

[0288] The nucleic acid may encode a second CAR (in a dual CAR system) or a CD56 CAR, which comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an antigen-binding domain, a CD28 hinge domain or a mutant CD28 hinge domain, a CD28 transmembrane domain or a mutant CD28 transmembrane domain, and a CD28 co-stimulatory signal transduction domain. In some embodiments, the nucleic acid encodes a CD56 CAR. In some embodiments, the nucleic acid encodes an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41 or SEQ ID NO: 42.

[0289] In some embodiments, the nucleic acid encodes a dual CAR system comprising a DLL3 CAR and a CD56 CAR. In some embodiments, the nucleic acid comprises, from the N-terminus to the C-terminus, a first CD8α signal peptide, a first antigen-binding domain targeting DLL3, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, a CD3ζ primary intracellular signal transduction domain, a 2A linker, a second CD8α signal peptide, a second antigen-binding domain targeting CD56, a CD28 hinge domain (wild-type or mutant), a CD28 transmembrane domain (wild-type or mutant), and a CD28 co-stimulatory signal transduction domain. In some embodiments, the nucleic acid encodes an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43 or SEQ ID NO: 44.

[0290] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences may be ignored for comparison). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of a reference sequence, and in some embodiments at least 90%, 95%, or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of shared positions between the sequences, taking into account the number of vacancies and the length of each vacancy, requiring the introduction of vacancies to achieve optimal alignment between the two sequences. For the purposes of this disclosure, the comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix in which a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5 are used.

[0291] 4. Treatment methods

[0292] The CAR, dual CAR systems, nucleic acids, and engineered immune cells disclosed herein can be used for a variety of experimental, therapeutic, and commercial applications.

[0293] In one respect, this disclosure provides a method for modulating an immune response, which includes administering an effective amount of the engineered immune cells described herein to a subject in need.

[0294] As used in this article, the term "effective dose" means the dose and time period necessary to achieve the desired result.

[0295] On the other hand, this disclosure provides a method for treating cancer, which includes administering an effective amount of the engineered immune cells described herein to a subject in need.

[0296] On the one hand, the engineered immune cells described in this article can be used to treat subjects who have cancer or are at risk of developing cancer.

[0297] Subjects may have neuroendocrine lesions (NENs), such as neuroendocrine tumors (NETs) or neuroendocrine carcinomas (NECs).

[0298] Examples of treatable cancers include, but are not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), and gastrointestinal neuroendocrine carcinoma.

[0299] This disclosure further includes the use of the engineered immune cells described herein in the manufacture of medicaments or pharmaceutical compositions for modulating immune responses or treating cancers as described above.

[0300] Engineered immune cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0301] One or more of the engineered immune cells described herein can be administered to a subject in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). In some cases, the engineered immune cells can be expanded in the subject after administration. The engineered immune cells can be frozen to provide cells for multiple treatments with the same cell preparation. The engineered immune cells disclosed herein and pharmaceutical compositions comprising these engineered immune cells can be packaged as kits. Kits may include instructions for use of the engineered immune cells and compositions comprising these engineered immune cells (e.g., written instructions).

[0302] Methods for administering engineered immune cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, cell therapy (e.g., adoptive T-cell therapy) is performed via autologous transfer, wherein cells are isolated from and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some aspects, cells are derived from a subject requiring treatment (e.g., a patient), and these cells are administered to the same subject after isolation and processing.

[0303] Cell therapy (e.g., adoptive T-cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the subject to receive or ultimately receive the cell therapy (e.g., the first subject), and these cells are subsequently administered to a different subject of the same species (e.g., the second subject). The first and second subjects can be genetically identical. The first and second subjects can be genetically similar. The second subject can express the same HLA class or supertype as the first subject.

[0304] The engineered immune cells described herein can be administered to animals (e.g., mammals, and even humans) to treat cancer. Additionally, engineered immune cells can be used to treat any cancer-related condition, particularly cell-mediated immune responses against tumor cells, where treatment or mitigation of the disease is desired. Types of cancer for which engineered immune cells or pharmaceutical compositions may be used for treatment include, but are not limited to, neuroendocrine vesicles (NENs), such as neuroendocrine tumors (NETs), such as neuroendocrine carcinoma (NEC), small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), and gastrointestinal neuroendocrine carcinoma.

[0305] The engineered immune cells (e.g., T cells or NK cells) described herein may be included in a composition for immunotherapy. The composition may comprise a pharmaceutical composition and further comprise a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the engineered immune cells may be administered.

[0306] Engineered immune cells can be used immediately for the above-described treatments, experimental or commercial applications, or these cells can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0307] Any reference to treatment methods refers to the compounds, pharmaceutical compositions and / or drugs disclosed herein, used as medicines, used in methods of treating animals (e.g., humans), or used in the treatment of diseases, such as in the treatment of cancer.

[0308] Example

[0309] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0310] Example 1: Plasmid construction, virus preparation, and titer assessment

[0311] In short, the lentiviral vector PLVX-EF1A was created using pLVX-Puro (Clontech #632164) by replacing the original promoter with the human extension factor 1α promoter (hEF1α) and removing the puromycin resistance gene using EcoRI and BamHI via GenScript. PLVX-EF1A was further subjected to the lentiviral packaging procedure described below.

[0312] To generate viral particles containing nucleic acids encoding the CAR construct disclosed herein, a mixture of lentiviral packaging plasmids including pMDLg / pRRE (Addgene#11251), pRSV-Rev (Addgene#11253), and pMD2.G (Addgene#11259) was premixed with polyetherimide (PEI) at a pre-optimized ratio with the PLVX-EF1A vector (including the target system), thoroughly mixed, and incubated at room temperature for 5 min. The transfection mixture was added dropwise to 293-T cells and gently mixed. The transfected 293-T cells were incubated overnight at 37°C and 5% CO2. Twenty-four hours post-transfection, the supernatant was collected and centrifuged at 4°C, 500 g for 10 min to remove any cell debris. The centrifuged supernatant was filtered through a 0.45 μm PES filter to remove cell debris before ultracentrifugation. After ultracentrifugation, the supernatant was carefully discarded, and the viral pellet was washed with pre-cooled DPBS. Measure the viral concentration. Divide the virus into equal portions and store at -80°C. Determine the viral titer via functional transduction on T cell lines.

[0313] Example 2: Preparation of engineered immune cells expressing chimeric antigen receptors

[0314] The constructs of DLL3 bispecific CAR (DLL3 CAR, SEQ ID NO: 40) and DLL3 / CD56 bispecific CAR (DLL3 / CD56 dual CAR-1#, SEQ ID NO: 43, and DLL3 / CD56 dual CAR-2#, SEQ ID NO: 44) are as follows: Figure 1A-1C The design was carried out as shown. CAR backbone nucleotide sequences encoding DLL3 CAR and DLL3 / CD56 dual CAR peptides were chemically synthesized and cloned into a pre-modified lentiviral vector (PLVX-EF1A).

[0315] Specifically, the DLL3 CAR contains a polypeptide from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 30), and two anti-DLL3 V peptides. H The antigen-binding domain of the H domain (SEQ ID NO: 28-29), the CD8α hinge domain (SEQ ID NO: 31), the CD8α transmembrane domain (SEQ ID NO: 34), the 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 37), and the CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 39).

[0316] The DLL3 / CD56 dual CAR comprises nucleotides encoding a DLL3 CAR and a CD56 CAR. The nucleotides encoding the DLL3 CAR and the CD56 CAR are linked by nucleotides encoding a 2A linker. The 2A linker has the amino acid sequence shown in SEQ ID NO: 46. The CD56 CAR (in the DLL3 / CD56 dual CAR) may comprise a polypeptide from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 30), an antigen-binding domain having an anti-CD56 scFv domain (e.g., a VL-VH domain, SEQ ID NO: 27), a CD28 hinge domain (SEQ ID NO: 32) or a mutant CD28 hinge domain (SEQ ID NO: 33), a CD28 transmembrane domain (SEQ ID NO: 35) or a mutant CD28 transmembrane domain (SEQ ID NO: 36), and a CD28 co-stimulatory signal transduction domain (SEQ ID NO: 38). CD56 CAR-1# contains a polypeptide having the amino acid sequence of SEQ ID NO: 41, and CD56 CAR-2# contains a polypeptide having the amino acid sequence of SEQ ID NO: 42.

[0317] Table 2. Exemplary CDRs based on the Kabat and AbM numbering scheme

[0318]

[0319] Immune cells, in this case, T cells, were isolated from healthy donor PBMCs (HemaCare) using the pan T cell isolation kit (Miltenyi Biotec, 130096535). The isolated T cells were cultured in AIMV (Gibco, 31035025) medium containing 5% FBS (Gibco, 10099141) and further activated at 37°C and 5% CO2 at a 2:1 ratio with CD3 / CD28 activation beads (Miltenyi Biotec, 130091442). T cells were transduced with a lentivirus expressing DLL3-targeting CAR at an appropriate multiple of infection (MOI) in the presence of 8 µg / mL polyglobulin (SIGMA-ALDRICH, H9268-10G) at 24 h or 72 h after initial activation. Additional IL-2 was added to a final concentration of 300 IU / mL. The medium was replaced with fresh medium 24 h after lentiviral infection. Infected T cells were maintained in AIMV medium containing 5% FBS and 300 IU / mL IL-2 at a cell density of 5 × 10⁻⁶ cells / mL. 5 Up to 1 × 10 6 Between cells / mL.

[0320] Four days post-infection, rabbit anti-V antibodies were analyzed by flow cytometry (BD FACsCelesta). H CAR expression was determined using H antibody (Genscript). CAR positivity rate and geometric mean expression (mean fluorescence intensity, MFI) were further analyzed using Flowjo 7.6. Figures 2A-2D As shown, DLL3 CAR was successfully expressed in each CAR-T group, with CAR positivity rates of 55.9%, 17.7%, and 28%, respectively. Due to the lack of effective reagents to distinguish scFv from V... H H, no expression of the CD56-targeting moiety co-expressed with DLL3 CAR was detected in DLL3 / CD56 dual CAR-T cells.

[0321] Example 3: Cytotoxicity of engineered immune cells

[0322] To determine the cytotoxicity of engineered immune cells (CAR-T cells from Example 2 in this case) against tumor cells expressing tumor-associated antigens (TAAs), an LDH (lactate dehydrogenase)-based cytotoxicity assay was performed. Briefly, T cells were co-cultured with different tumor cell lines (DLL3-positive SHP-77 cells, SHP-77-Luc-DLL3 KO, or NK-92 cells) at E:T ratios of 0.5:1 and 2:1, respectively, for 24 hours.

[0323] SHP-77 cells are DLL3 and CD56 positive. SHP-77-Luc-DLL3 KO cells were generated by CRISPR-Cas9-mediated DLL3 knockout in parental SHP-77 cells via GenScript services. SHP-77-Luc-DLL3 KO cells are DLL3 negative and CD56 positive. NK-92 cells are DLL3 negative and CD56 positive.

[0324] According to the manufacturer's protocol (Roche, #11644793001), LDH release from dead and dying cells in the supernatant was detected using a PHERStar microplate reader. Baseline LDH release from target cells in the absence of effector cells and baseline LDH release from effector cells in the absence of target cells were subtracted from the total LDH amount. At the start of the cytotoxicity assay, maximum target release was obtained by adding a final concentration of 1% Triton-X100 to target cells in the absence of effector cells. Minimum target release was obtained from the supernatant from target cells in the absence of effector cells. Specific cytotoxicity was calculated using the following formula: Target cell lysis % = 100 × [(OD CAR-T cells + target cells) - (OD CAR-T cells) - (OD target cells) + (OD buffer background)] / (Maximum target release - Minimum target release).

[0325] like Figures 3A-3C As shown, UnT exhibited minimal cytotoxicity against tumor cells. Specific cytotoxicity against DLL3-positive SHP-77 cells was observed in the CAR-T cell group. Figure 3A DLL3 CAR cells exhibited approximately 50% cytotoxicity; DLL3 / CD56 dual CAR cells showed approximately 80% cytotoxicity against DLL3 / CD56 dual CAR-1# (60% higher than DLL3 single CAR cells); and approximately 65% ​​cytotoxicity against DLL3 / CD56 dual CAR-2# (30% higher than DLL3 single CAR cells). Notably, in response to DLL3-negative and CD56-positive SHP-77-DLL3 knockout cells (SHP-77-Luc-DLL3 KO)... Figure 3B ) or NK-92 cells ( Figure 3C In CAR-T cells, only minimal or background cell lysis was observed. These results indicate that functional DLL3-restricted cell lysis in the current dual-targeting strategy can significantly reduce the risk of introducing a second TAA.

[0326] Example 4: IFN-γ released by engineered immune cells

[0327] To further analyze the effects of engineered immune cells (in this case, engineered T cells from Example 2) on tumor cells, the release of interferon-γ (IFN-γ) from the culture supernatant was analyzed using the HTRF human IFNγ kit (Cisbio, #62HIFNGPEH).

[0328] like Figures 4A-4DAs shown, UnT cells exhibit very low IFN-γ secretion. DLL3 CAR and DLL3 / CD56 dual CAR T cells secreted varying amounts (2200-3300 pg / ml) of IFN-γ when co-cultured with DLL3-positive SHP-77 cells. Figure 4A Interestingly, consistent with the limited cytotoxicity against DLL3-negative and CD56-positive SHP-77-Luc-DLL3 KO cells, only baseline IFN-γ release was observed. Figure 4B This indicates limited CAR-T cell activation. Notably, significantly higher cell lysis and baseline cytokine expression were observed in DLL3 / CD56 dual CAR-1# compared to DLL3 / CD56 dual CAR-2#. Figures 4A-4D This suggests that wild-type CD28 may favor spontaneous activation of CAR-T cells. Further mutations in CD28 reduced cytokine release to levels similar to DLL3 CAR, indicating the contribution of the CD28 hinge domain and / or transmembrane domain to mediating baseline activation of CAR-T cells.

[0329] Example 5: Durable cytotoxicity as measured by repeated DLL3-mediated stimulation

[0330] Assess the persistence of CAR-T cells in repeated tumor attack assays.

[0331] In short, 1.2 × 10 5 CAR-T cells (DLL3 CAR, DLL3 / CD56 dual CAR-1#, or DLL3 / CD56 dual CAR-2#) were added to 6 × 10⁶ cells respectively. 5 SHP-77 cells were co-cultured in 24-well plates. After three days, cells were harvested to determine the relative ratio of live T cells to tumor cells, quantify CAR-T cells, and the plates were replated with fresh SHP-77 cells at an E:T ratio of 1:5 for the next round.

[0332] like Figures 5A-5C As shown, a significant advantage in durability was observed in DLL3 / CD56 dual CAR-T cells compared to DLL3 CAR-T cells, independent of transmembrane-mediated downstream signaling after binding of the wild-type or mutant CD28 hinge domain and CD56. By introducing another tumor-associated antigen, the CD56 binding moiety, CAR-T durability in response to repeated tumor-associated antigen attacks mediated by SHP-77 tumor cells was significantly enhanced (with at least one round of advantage). Figure 5A Therefore, for total T cells ( Figure 5B ) and CAR-positive T cells ( Figure 5CCompared with DLL3 CAR-T cells, the proliferation of DLL3 / CD56 dual CAR-T cells was significantly enhanced by approximately 8-fold and 4-fold, respectively.

[0333] Example 6: In vivo efficacy of DLL3 / CD56 dual CAR-T cells

[0334] To further demonstrate the antitumor activity of dual CAR-T cells in vivo, SCLC NCI-H82 cells were subcutaneously injected into NCG mice (NOD-PrkdcCd5I12rgCd / NjuCrl). Ten days after tumor inoculation, a single dose of untransduced T cells (8.8 × 10⁻⁶) was administered intravenously to the tumor-implanted mice. 6 ) or CAR-T cells (1.5 × 10 6 Tumor length (L) and width (W) were measured every 3–4 days after CAR-T cell injection. Tumor volume was estimated using the following formula: V = (W² × L) / 2. At 14 days post-treatment, tumor burden was reduced in all NCG mice in both the DLL3 CAR-T treatment group and the DLL3 / CD56 dual CAR-T treatment group compared to the UnT group. Figure 6A Compared to the DLL3 CAR-T therapy group, the DLL3 / CD56 dual CAR-T therapy group showed more significant tumor regression. All mice treated with DLL3 CAR-T cells (5 / 5) exhibited inhibited tumor growth, but xenograft tumors were not eliminated. Figure 6A Due to disease progression, mice treated with DLL3CAR-T were sacrificed starting on day 14 post-treatment. Figure 6A In contrast, 5 / 5 (100%) of mice treated with DLL3 / CD56 dual CAR-T cells exhibited sustained tumor remission. The data highlight the superiority of DLL3 / CD56 dual CAR-T cells in tumor regression and protection against disease progression in mice.

[0335] No significant weight loss was observed in NCG mice receiving DLL3 CAR-T and DLL3 / CD56 dual CAR-T cells, indicating that NCG mice tolerated both DLL3 CAR-T and DLL3 / CD56 dual CAR-T cells well. Figure 6B ).

[0336] Furthermore, the expansion and persistence of CAR-T cells in vivo are considered important predictors of sustained clinical tumor regression in cancer subjects. To understand the basic kinetics of the infused CAR-T cells, the percentage of CAR-T cells in the peripheral blood of NCG mice was assessed using flow cytometry. Figures 6C-6DAs shown, an increase in the percentage of total T cells and CAR-T cells in the peripheral blood of NCG mice was observed 14 days after treatment. On day 14, after treatment with 1.5 × 10⁻⁶ CAR-T cells... 6 4.67% ± 4.18% of CAR-positive T cells were found in the peripheral blood of NCG mice treated with DLL3 CAR-T cells. Figure 6D The percentage of CAR-positive T cells in the peripheral blood of mice treated with DLL3 / CD56 dual CAR-T cells (36.55% ± 13.48% on day 14) was at least 9 times higher than that of mice treated with DLL3 CAR-T cells. Figure 6D The data highlight the superiority of DLL3 / CD56 dual CAR-T cells in CAR-T persistence and proliferation.

[0337] Consistent with minimal weight loss, no TAA-independent CAR-T amplification was observed during the study, as reflected in the restricted proliferation on day 21.

[0338] Example 7: Generation and evaluation of DLL3 / CD56 dual CAR-T cells expressing another DLL3 bi-VHH CAR

[0339] Immune cells (such as T cells) expressing another DLL3 bi-VHH CAR (hereinafter referred to as DLL3 biCAR) were also generated, which includes two anti-DLL3 VHH domains in its antigen-binding domain. CAR backbone nucleotide sequences encoding the peptides DLL3 biCAR, DLL3 / CD56 bi-biCAR-1#, and DLL3 / CD56 bi-biCAR-2# were chemically synthesized and cloned into pre-modified lentiviral vectors (PLVX-EF1A). The anti-DLL3 VHH domains differ from those disclosed in the examples above. In summary, DLL3 biCAR comprises a polypeptide from the N-terminus to the C-terminus: a CD8α signal peptide (SEQ ID NO: 30), an antigen-binding domain having two additional anti-DLL3 VHH domains, a CD8α hinge domain (SEQ ID NO: 31), a CD8α transmembrane domain (SEQ ID NO: 34), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO: 37), and a CD3ζ primary intracellular signal transduction domain (SEQ ID NO: 54). DLL3 / CD56 biCAR-1# and DLL3 / CD56 biCAR-2# contain nucleotides encoding DLL3 biCAR and CD56 CAR. The nucleotides encoding DLL3 biCAR and CD56 CAR are linked by nucleotides encoding a 2A linker. The CD56 CAR in DLL3 / CD56 dual biCAR-1# comprises a polypeptide from the N-terminus to the C-terminus: a CD8α signal peptide, an antigen-binding domain with anti-CD56 scFv, a CD28 hinge domain, a CD28 transmembrane domain, and a CD28 co-stimulatory signal transduction domain, and contains the amino acid sequence of SEQ ID NO: 42. The CD56 CAR in DLL3 / CD56 dual biCAR-2# comprises a polypeptide from the N-terminus to the C-terminus: a CD8α signal peptide, an antigen-binding domain with anti-CD56 scFv, a mutant CD28 hinge domain, a mutant CD28 transmembrane domain, and a CD28 co-stimulatory signal transduction domain, and contains the amino acid sequence of SEQ ID NO: 42.

[0340] CAR positivity rates were analyzed using Flowjo v10.9.0. DLL3 biCAR, DLL3 / CD56 biCAR-1#, and DLL3 / CD56 biCAR-2# were successfully expressed in each CAR-T group, with CAR positivity rates of 23.2%, 52.2%, and 57.8%, respectively.

[0341] The persistence of CAR-T cells was assessed in repeated tumor attack assays. Briefly, 0.5 × 10⁻⁶ cells were used.5 CAR-T cells (DLL3 biCAR, DLL3 / CD56 biCAR-1#, and DLL3 / CD56 biCAR-2#) were added to 3 × 10⁻⁶ cells respectively. 5 SHP-77 cells were co-cultured in 24-well plates. After three days, cells were harvested to determine the relative ratio of live T cells to tumor cells, quantify CAR-T cells, and the cells were replated with fresh SHP-77 cells at an E:T ratio of 1:5 for the next round. Figures 7A-7C As shown, a significant advantage in durability was observed in DLL3 / CD56 dual biCAR-1# and DLL3 / CD56 dual biCAR-2# CAR-T cells compared to DLL3 biCAR. By introducing another tumor-associated antigen, the CD56 binding moiety, CAR-T durability in response to repeated tumor-associated antigen attacks mediated by SHP-77 tumor cells was significantly enhanced (with at least one round of advantage). Figure 7A Therefore, compared with DLL3 biCAR-T cells, the proliferation of DLL3 / CD56 biCAR-1# and DLL3 / CD56 biCAR-2# CAR-T cells was significantly enhanced, and the proliferation of total T cells was enhanced by approximately 1.5 to 3 times. Figure 7B ), and the proliferation of CAR-positive T cells increased by approximately 1.3 to 3 times ( Figure 7C ).

[0342] Other embodiments

[0343] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, and not limit, the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered immune cell comprising a dual chimeric antigen receptor (dual CAR) system, the dual chimeric antigen receptor system comprising: (a) a first chimeric antigen receptor (CAR) comprising: i) a first antigen binding domain that specifically recognizes a first antigen; ii) a first transmembrane domain; and iii) a first intracellular signaling domain comprising a costimulatory signaling domain of the first intracellular signaling domain; and (b) a second chimeric antigen receptor (CAR) comprising: i) a second antigen binding domain that specifically recognizes a second antigen; ii) a second transmembrane domain; and iii) a second intracellular signaling domain comprising a costimulatory signaling domain of the second intracellular signaling domain, wherein the first intracellular signaling domain further comprises a first primary intracellular signaling domain, and the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3 zeta; and wherein the first antigen and the second antigen are selected from the group consisting of DLL3, CD56, MUC1C, CDH17, GD2, Neuronal guidance factor 3, SEZ6, GPC2, and B7-H3.

2. An engineered immune cell comprising a dual chimeric antigen receptor (dual CAR) system, the dual chimeric antigen receptor system comprising: (a) a first chimeric antigen receptor (CAR) comprising: i) a first antigen binding domain that specifically recognizes a first antigen; ii) a first transmembrane domain; and iii) a first intracellular signaling domain comprising a costimulatory signaling domain of the first intracellular signaling domain; and (b) a second chimeric antigen receptor (CAR) comprising: i) a second antigen binding domain that specifically recognizes a second antigen; ii) a second transmembrane domain; and iii) a second intracellular signaling domain comprising a costimulatory signaling domain of the second intracellular signaling domain, wherein the first intracellular signaling domain further comprises a primary intracellular signaling domain of an immune cell, and the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3 zeta; and wherein each of the first antigen and the second antigen is a marker of a neuroendocrine lineage.

3. The engineered immune cell of claim 1 or claim 2, wherein the second intracellular signaling domain does not comprise a primary intracellular signaling domain of an immune cell.

4. The engineered immune cell of any one of claims 1-3, wherein the first primary intracellular signaling domain is derived from CD3 zeta.

5. The engineered immune cell of any one of claims 1-4, wherein the costimulatory signaling domain of the first intracellular signaling domain and / or the costimulatory signaling domain of the second intracellular signaling domain is derived from a molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand of CD83.

6. The engineered immune cell of any one of claims 1-5, wherein the costimulatory signaling domain of the first intracellular signaling domain comprises an intracellular signaling domain of 4-1BB, and the costimulatory signaling domain of the second intracellular signaling domain comprises an intracellular signaling domain of CD28.

7. The engineered immune cell of any one of claims 1-6, wherein the first antigen is the same as the second antigen, e.g., wherein the first antigen is DLL3 and the second antigen is DLL3.

8. The engineered immune cell of any one of claims 1-6, wherein the first antigen is different from the second antigen.

9. The engineered immune cell of claim 8, wherein the first antigen is selected from the group consisting of DLL3, GD2, and GPC2.

10. The engineered immune cell of any one of claims 8-9, wherein the second antigen is selected from the group consisting of CD56, MUC1C, CDH17, Neuronal guidance factor 3, SEZ6, and B7-H3.

11. The engineered immune cell of any one of claims 1-10, wherein the first binding domain and / or the second binding domain comprises a first antigen binding moiety and / or a second antigen binding moiety selected from a Fab, a Fab', a F(ab')2, a Fv, a single chain Fv (scFv), a minibody, a diabody, a single domain antibody (sdAb), or a VHH domain.

12. The engineered immune cell of any one of claims 1-11, wherein the second antigen is CD56; and / or wherein the second antigen binding domain comprises an anti-CD56 scFv.

13. The engineered immune cell of any one of claims 1-12, wherein the second antigen is CD56 and the second antigen binding domain comprises an anti-CD56 scFv comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

14. The engineered immune cell of any one of claims 12-13, wherein the anti-CD56 scFv comprises: (i) a VH domain comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the HCDRs are defined according to the Kabat numbering scheme; and (ii) a VL domain comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the LCDRs are defined according to the Kabat numbering scheme; or wherein the anti-CD56 scFv comprises: (iii) a VH domain comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, wherein the HCDRs are defined according to the AbM numbering scheme; and (iv) a VL domain comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, wherein the LCDRs are defined according to the AbM numbering scheme.

15. The engineered immune cell of any one of claims 1-14, wherein the first antigen is DLL3; and / or wherein the first antigen binding domain comprises one or more anti-DLL3 sdAbs.

16. The engineered immune cell of any one of claims 1-15, wherein the first antigen binding domain comprises two anti-DLL3 sdAbs comprising: (i) a first anti-DLL3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9, wherein the CDRs are defined according to the Kabat numbering scheme; and (ii) a second anti-DLL3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, wherein the CDRs are defined according to the Kabat numbering scheme; or wherein the two anti-DLL3 sdAbs comprise: (iii) a first anti-DLL3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 21, wherein the CDRs are defined according to the AbM numbering scheme; and (iv) a second anti-DLL3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24, wherein the CDRs are defined according to the AbM numbering scheme.

17. The engineered immune cell of any of claims 1-16, wherein the first antigen binding domain comprises a first anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO: 28, and a second anti-DLL3 sdAb comprising the amino acid sequence of SEQ ID NO:

29.

18. The engineered immune cell of any of claims 1-17, wherein the first transmembrane domain and / or the second transmembrane domain comprises a transmembrane domain derived from a molecule selected from the group consisting of CD8a, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1.

19. The engineered immune cell of any of claims 1-18, wherein the first transmembrane domain and / or the second transmembrane domain comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence set forth in any of SEQ ID NOS: 34-36.

20. The engineered immune cell of any of claims 1-19, wherein the first CAR comprises a hinge domain located between the C-terminus of the first antigen binding domain and the N-terminus of the first transmembrane domain; and wherein the hinge domain of the first chimeric antigen receptor is derived from CD8a or CD28; and the second CAR comprises a hinge domain located between the C-terminus of the second antigen binding domain and the N-terminus of the second transmembrane domain; and wherein the hinge domain of the second chimeric antigen receptor is derived from CD8a or CD28.

21. The engineered immune cell of claim 20, wherein the hinge domain of the first CAR and / or the hinge domain of the second CAR comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence set forth in any of SEQ ID NOS: 31-33.

22. The engineered immune cell of any one of claims 20-21, wherein the hinge domain of the second CAR comprises the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 32, and / or wherein the second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO:

35.

23. The engineered immune cell of any one of claims 1-22, wherein each of the first CARs comprises a signal peptide N-terminal to the first antigen binding domain, optionally wherein the signal peptide is derived from CD8a; and / or the second CAR comprises a signal peptide N-terminal to the second antigen binding domain, optionally wherein the signal peptide is derived from CD8a.

24. The engineered immune cell of any one of claims 1-23, wherein the first CAR comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40; and / or wherein the second CAR comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:

42.

25. The engineered immune cell of any one of claims 1-24, comprising a polypeptide comprising the first CAR and / or the second CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-44.

26. A nucleic acid comprising one or more nucleic acid sequences encoding a first chimeric antigen receptor (CAR) and a second chimeric antigen receptor (CAR), wherein (a) the first chimeric antigen receptor (CAR) comprises: i) an antigen binding domain that specifically recognizes a first antigen; ii) a first transmembrane domain; and iii) a first intracellular signaling domain comprising a costimulatory signaling domain of the first intracellular signaling domain; and (b) the second chimeric antigen receptor (CAR) comprises: i) a second antigen binding domain that specifically recognizes a second antigen; ii) a second transmembrane domain; and iii) a second intracellular signaling domain comprising a costimulatory signaling domain of the second intracellular signaling domain, wherein the first intracellular signaling domain further comprises a first primary intracellular signaling domain of an immune cell, and the second intracellular signaling domain does not comprise a primary intracellular signaling domain derived from CD3 zeta; and wherein the first antigen and the second antigen are selected from the group consisting of: DLL3, CD56, MUC1C, CDH17, GD2, Neuronal guidance factor 3, SEZ6, GPC2, and B7-H3.

27. The nucleic acid of claim 26, wherein the second intracellular signaling domain does not comprise a primary intracellular signaling domain of an immune cell.

28. The nucleic acid of claim 26 or claim 27, comprising one or more nucleic acid sequences encoding: a. the first CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid of SEQ ID NO: 40; and / or b. the second CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid of SEQ ID NO: 41 or SEQ ID NO: 42; or c. the first CAR and the second CAR having an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid of SEQ ID NO: 43 or SEQ ID NO:

44.

29. The nucleic acid of any one of claims 26-28, comprising a first nucleic acid sequence encoding the first CAR and a second nucleic acid sequence encoding the second CAR, wherein (i) the first nucleic acid and the second nucleic acid are operably linked to the same promoter; (ii) the first nucleic acid is upstream of the second nucleic acid, or wherein the first nucleic acid is downstream of the second nucleic acid; and / or (iii) the first nucleic acid and the second nucleic acid are linked by a nucleic acid sequence encoding a P2A or T2A linker sequence.

30. A single chimeric antigen receptor (CAR) comprising: (a) an antigen binding domain comprising an anti-CD56 binding moiety that specifically recognizes CD56; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-CD56 binding moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25; and a VL domain comprising the amino acid sequence of SEQ ID NO:

26.

31. The single CAR of claim 30, wherein the anti-CD56 binding moiety comprises: (i) a VH domain comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein the HCDRs are defined according to the Kabat numbering scheme; and (ii) a VL domain comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the LCDRs are defined according to the Kabat numbering scheme; or: (iii) a VH domain comprising a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 15, wherein the HCDRs are defined according to the AbM numbering scheme; and (iv) a VL domain comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, wherein the LCDRs are defined according to the AbM numbering scheme. (iv) a VL domain comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, wherein the LCDRs are defined according to the AbM numbering scheme.

32. The single CAR of any one of claims 30-31, wherein the intracellular signaling domain comprises a costimulatory signaling domain and does not comprise a primary intracellular signaling domain of an immune cell.

33. The single CAR of any one of claims 30-32, wherein the costimulatory signaling domain comprises an amino acid sequence of an intracellular domain that is at least 95% identical to an amino acid sequence of an intracellular domain of CD28 and / or 4-1BB.

34. The single CAR of any one of claims 30-33, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8a, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

35. The single CAR of any one of claims 30-34, wherein the single CAR further comprises a hinge domain derived from CD8a or CD28.

36. The single CAR of any one of claims 30-35, wherein the single CAR further comprises a signal peptide domain derived from CD8a.

37. The single CAR of any one of claims 30-36, wherein the hinge domain is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 31-33, and / or the transmembrane domain is at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 34-36, and / or the signal peptide domain is at least 95% identical to the amino acid sequence of SEQ ID NO:

30.

38. The single CAR of any one of claims 30-37, wherein the single CAR comprises an amino acid sequence that is at least 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO:

42.

39. A nucleic acid comprising one or more nucleic acid sequences encoding the single CAR of any one of claims 30-38.

40. A vector comprising the nucleic acid of claims 26-29 or the nucleic acid of claim 39.

41. Use of the vector of claim 40 for producing an engineered immune cell.

42. An engineered immune cell comprising the single CAR of any one of claims 30-38, or the nucleic acid of claims 26-29, or the nucleic acid of claim 39, or the vector of claim 40.

43. The engineered immune cell of any one of claims 1-25 and 42, wherein the engineered immune cell is selected from the group consisting of a T cell, an αβ T cell, a γδ T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, and a combination thereof.

44. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-25 and 42-43, and a pharmaceutically acceptable carrier.

45. A method for producing the engineered immune cell of any one of claims 1-25 and 42-43, the method comprising introducing the vector of claim 40 into a cell.

46. A method of treating a subject having or at risk of having cancer, the method comprising: administering the engineered immune cell of any one of claims 1-25, the pharmaceutical composition of claim 44, or the engineered immune cell of claims 42-43 to a subject having or at risk of having a cancer.

47. The method of claim 46, wherein the subject has a neuroendocrine tumor (NET).

48. The method of claim 46 or claim 47, wherein the cancer is small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), or a gastroenteric neuroendocrine carcinoma.

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