Development of CAR-linking molecular platforms that enhance function and / or persistence of CAR T cells

Abstract

Chimeric antigen receptor (CAR) linking molecules and protein entities and dimers comprising an extracellular domain of an antigen present on a cancer cell and a first immune cell effector domain are disclosed, as well as their use in co-treatment of cancer with CAR immune cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Serial No. 63 / 441,253, filed on January 26, 2023, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is incorporated by reference in its entirety. The XML copy, created on January 22, 2024, is named 52095_774001WO_ST.xml and is 109 KB in size.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] T cells expressing chimeric antigen receptors (CARs) have revolutionized the treatment of blood-borne malignancies and have shown promising results in the treatment of hematopoietic cancers. Six CAR T cell therapies targeting two antigens, CD19 and BCMA, have currently been approved by the FDA. CD19 is a B cell co-receptor expressed on B cells and a variety of blood-borne malignancies. CD19 CAR T cells were initially approved for the treatment of acute lymphoblastic leukemia (ALL) and subsequently approved for Burkitt lymphoma and mantle cell lymphoma. BCMA is a receptor expressed on the surface of B cell lineage cells and is a major marker for multiple myeloma (MM). MM is associated with the uncontrolled proliferation of plasma cells in the bone marrow, which can develop into extramedullary lesions that form elsewhere in the body. BCMA CAR T cell therapy has shown great promise for MM, with studies showing an overall response rate of 80% even in patients with extramedullary lesions (Gagelmann et al., Eur. J. Haematol. 104(4):318-327(2020)).

[0007] However, CAR T cell therapy still faces challenges. A recent meta-analysis of 22 CAR T cell clinical studies emphasized its ineffectiveness in solid tumors, with a poor average overall response rate of 9% (Hou et al., Dis. Markers 2019: 3425291 (2019)). Even in hematological cancers, the duration of response remains a challenge, and almost all MM patients treated with BCMACAR eventually relapse (Gagelmann et al., Eur. J. Haematol. 104 (4): 318-327 (2020); Roex et al., J. Hematol. Oncol. 13 (1): 164 (2020); Raje et al., N. Engl. J. Med. 380 (18): 1726-1737 (2019)). Treatment may produce serious side effects, including cytokine release syndrome (CRS) and neurotoxicity.

[0008] CAR T cells need to home to the tumor site, proliferate, and persist in the circulation, at least until they neutralize and kill the last remaining cancer cells. Therefore, methods to regulate the activity of CAR-T cells in a controllable manner are urgently needed.

[0009] Overview of the Disclosure

[0010] Chimeric antigen receptor (CAR)-adapter molecules disclosed herein and methods of use thereof are expected to address the above-mentioned needs. CAR adaptor molecules enhance the function and persistence of CAR immune cells in vivo. They can also reduce the cell dose required for CAR immune cell therapy, thereby reducing the adverse side effects (e.g., cytokine release syndrome) caused by the larger doses commonly used in the clinic, and therefore, CAR adaptor molecules are also referred to herein as CAR enhancers. In addition, since the binding of CAR adaptor molecules to CAR is reversible, CAR adaptor molecules do not induce the formation of CAR immune synapses on the surface of CAR immune cells. Therefore, CAR adaptor molecules do not block CAR-mediated cancer cell killing. During minimal residual disease (MRD), CAR immune cells generally do not persist in vivo, as is known in the art, which is related to limited cancer antigens. The disclosed CAR adaptor molecules can support the persistence, proliferation, and efficacy of CAR T cells in the MRD state.

[0011] A first aspect of the present disclosure relates to a chimeric antigen receptor (CAR) adapter molecule, which comprises an extracellular domain of an antigen present on a cancer cell that is connected to an immune cell effector domain. The connection between the portion comprising the extracellular domain and the immune effector domain can be a peptide connection or a non-peptide connection (covalent), so the CAR adapter molecule can be a continuous protein or polypeptide, or a portion comprising two protein entities connected by a covalent bond. The extracellular domain of the CAR adapter molecule is designed to bind the extracellular domain of CAR, which targets the extracellular domain of an antigen on a cancer cell. In some embodiments, the binding between CAR and a cancer antigen is direct. In some embodiments, the binding between CAR and a cancer antigen is indirect. In these embodiments, the CAR adapter molecule extracellular domain is a cancer-independent antigen present in a protein therapeutic. The cancer-independent antigen in the protein therapeutic and the CAR adapter molecule extracellular domain bind CAR. Protein therapeutics are used to redirect CAR immune cells from cancer-independent antigens to cancer antigens by a cancer antigen binding domain. This CAR immune cell is called a universal CAR or a binary activation CAR (BAT-CAR). A representative cancer-independent antigen is the small molecule fluorescein isothiocyanate (FITC) that targets universal anti-FITC CAR immune cells.

[0012] Another aspect of the present disclosure relates to heterodimeric CAR adapter molecules, which include a first entity containing an extracellular domain of an antigen present on a cancer cell connected to a first dimerization domain, and a second entity containing a first immune cell effector domain connected to a second dimerization domain, wherein the first dimerization domain and the second dimerization domain are combined to form a heterodimeric CAR adapter molecule. In embodiments where the connection is a peptide bond, the first entity and the second entity are referred to as first and second proteins or polypeptides.

[0013] Other aspects of the present disclosure relate to nucleic acids encoding a CAR adaptor molecule protein, nucleic acids encoding a first entity of a heterodimeric CAR adaptor molecule protein, and nucleic acids encoding a second entity of a heterodimeric CAR adaptor molecule protein.

[0014] Other aspects of the present disclosure relate to vectors containing nucleic acids encoding CAR adapter molecule proteins and vectors containing the first protein and / or the second protein of the heterodimeric CAR adapter molecule.

[0015] Another aspect of the disclosure relates to cells transformed with the vector.

[0016] Another aspect of the present disclosure relates to a pharmaceutical composition containing a CAR adapter molecule and a pharmaceutically acceptable carrier.

[0017] Another aspect of the present disclosure relates to a method for preparing a CAR adapter molecule protein. The method requires culturing cells transformed with a vector containing a nucleic acid encoding a CAR adapter molecule protein or its protein entity in a culture medium under conditions that allow the nucleic acid to be expressed, and isolating the CAR adapter molecule protein or its protein entity from the cells and / or culture medium. In embodiments where the nucleic acid encodes a protein entity, the protein entity is connected by click chemistry.

[0018] Another aspect of the present disclosure relates to a method for treating cancer. The method requires administering an effective amount of CAR adapter molecules to the subject. In some embodiments, the subject receives pre-administration of immune cells expressing CAR, wherein the CAR comprises an antigen (the cancer cell comprises the extracellular domain) present in combination with the extracellular domain of the extracellular domain of the CAR adapter molecule, a transmembrane domain, and an intracellular domain comprising a stimulating domain.

[0019] The working examples disclosed herein demonstrate that CAR adapter molecules enhance BCMA-targeting CAR immune cells, drive them to generate memory cells, and prevent the exhaustion of CAR immune cells.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematically illustrates the multiple domains of the CAR adapter molecule according to some embodiments, which includes the extracellular domain (Ag) of the antigen on the surface of the cancer cell, the CH3 dimerization domain and the immune cell effector domain (ICE). The CAR adapter molecule can be a monomer, a dimer or a multimer.

[0022] Figure 2A-Figure 2I is a set of schematic diagrams and line drawings showing three CAR adaptor molecules. Figure 2A Schematic illustration of a CAR adaptor molecule containing the BCMA extracellular domain and the Neo2 / 15 synthetic cytokine immune cell effector domain. Figure 2B Schematic illustration of a CAR adaptor molecule containing the BCMA extracellular domain and two synthetic cytokine immune cell effector domains of weak-affinity mutant IL-2 (mIL2). Figure 2C Schematic illustration of a CAR adaptor molecule comprising the BCMA extracellular domain and the 4-1BBL immune cell effector domain. Figure 2D are line graphs showing dose-dependent staining of CD19-binding CAR T cells or non-transduced T cells (NT T cells) with CAR adaptor molecules or control proteins. Figure 2E are line graphs showing dose-dependent staining of BCMA-binding CAR T cells or non-transduced T cells (NT T cells) with CAR adaptor molecules or control proteins. Figure 2F and Figure 2GFigures are line graphs and bar graphs, respectively, which together show the dose-dependent activation of CAR T cells after CAR adaptor molecule treatment. Figure 2H is a line graph showing that the BCMA-muIL2CAR adaptor molecule does not block the killing efficacy of CAR T cells. Figure 2I is a line graph showing phosphorylation of signal transducer and activator of transcription (STAT5) in BCMACAR T cells.

[0023] Figure 3A-3B is a set of schematic diagrams and line graphs showing the effects of CAR adaptor molecules on untransduced T cells. Figure 3A The experimental design is schematically illustrated. Figure 3B is a set of line graphs showing T cell counts and carboxyfluorescein succinimidyl ester (CFSE) staining of untransduced activated T cells treated with tesileukin, a CAR adapter molecule containing the BCMA ectodomain and two weak-affinity mutant IL-2 (mIL2), or a CAR adapter molecule containing the BCMA ectodomain and a Neoleukin domain.

[0024] Figures 4A-4C is a set of schematic diagrams and line graphs showing that CAR engager molecules specifically activate CAR T cells. Figure 4A The experimental design is schematically illustrated. Figure 4B is a bar graph showing the expression of CD69 after treatment with BCMA CAR adapter molecules, BCMA CAR adapter molecules without immune cell effector domain control, or non-antigen specific CAR adapter molecule control. + Percentage of activated T cells transduced with anti-BCMACAR. Figure 4C is a bar graph showing the percentage of CD69+ anti-CD19 CAR-transduced activated T cells after treatment with a CD19 CAR adapter molecule or a non-antigen-specific CAR adapter molecule control.

[0025] Figure 5 Figure 2 is a line graph showing that CAR adapter molecules do not inhibit BCMA CAR T cell killing and the percentage of OPM2 target cells that survive after incubation with CAR T cells and CAR adapter molecules (red) or non-transduced T cells (blue).

[0026] Figure 6A – Figure 6C is a set of schematics and photographs showing that CAR adapter molecules reduce tumor burden in vivo. Figure 6A The experimental design is schematically illustrated. Figure 6B-6C is a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR adaptor molecule treatment.

[0027] Figures 7A-7C Figure 2 is a set of flow cytometry images showing tumor burden in mice following CAR T cell infusion and CAR adaptor molecule treatment. Figure 7A is a set of flow cytometry images showing OPM2 tumor burden in blood, spleen, and lymph nodes. Figure 7B is a set of flow cytometry images showing OPM2 tumor burden in bone marrow and lung. Figure 7C is a set of flow cytometry graphs showing OPM2 tumor burden in liver, kidney, and eye tumor sites. eGFP (OPM2 cells) is shown on the y-axis, and PerCP signal control is shown on the x-axis.

[0028] Figures 8A-8C is a set of flow cytometry images showing human CD45 in mice after CAR T cell infusion and CAR adaptor molecule treatment. + CAR-T cells. Figure 8A is a set of flow cytometry images showing CAR T cells in blood, spleen, and lymph nodes. Figure 8B Figure 2 is a set of flow cytometry images showing CAR T cells in the bone marrow and lung. Figure 8C Figure 2 is a set of flow cytometry images showing CAR T cells in liver, kidney, and eye tumor sites. CD45 staining is shown on the y-axis, and CAR adapter molecules labeled with AF647 staining are shown on the x-axis.

[0029] Figures 9A-9E Figure 2 is a set of schematics, line graphs, and boxplots showing that CAR adaptor molecule therapy enhances the activity and persistence of CAR T cells in vivo. Figure 9A is a line graph showing the circulation half-life of the BCMACAR adaptor molecule. Figure 9B The experimental design is schematically illustrated. Figure 9C and Figure 9D is a set of flow cytometry graphs and boxplots showing the selective expansion and persistence of BCMACAR T cells. Figure 9E is a box plot showing the CD8 + Percentage of CAR T cells.

[0030] Figures 10A-10J A collection of schematics, survival plots, line graphs, bar graphs, t-distributed stochastic neighbor embedding (tSNE) plots, and photographs showing that CAR adaptor molecule therapy reduces the required CAR T cell dose. Figure 10A The experimental design is schematically illustrated. Figure 10B is a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR adaptor molecule treatment. Figure 10Cis a Kaplan-Meier plot showing the survival analysis. Figure 10D is a line graph showing flow cytometric analysis of CAR T cells in blood samples. Figure 10E is a set of bar graphs showing the generation of memory CAR T cells. Figure 10F and Figure 10G Figure 2 is a set of flow cytometry graphs and bar graphs showing a high number of CAR T cells 2 months after CAR T cell injection. Figure 10H is a line graph showing that the mice maintained consistent body weight throughout the experiment. Figure 10I Figure 2 is a set of flow cytometry images showing that CAR T cells from mice treated with CAR adapter molecules have a stem cell memory phenotype. Figure 10J is a set of tSNE plots showing FlowSOM-defined clusters in persisting BCMACAR T cells.

[0031] Figures 11A-11E is a collection of schematics, photographs, and line, bar, and tSNE plots showing that CAR adaptor molecule therapy leads to the persistence of CAR T cells in vivo. Figure 11A The experimental design is schematically illustrated. Figure 11B is a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR adaptor molecule treatment. Figure 11C Figure 2 is a set of flow cytometry images showing the persistence of CAR T cells. Figure 11D is a set of bar graphs showing in vitro killing assays of persistent T cells. Figure 11E is a set of t-SNE plots showing the + Immune cell marker for T cells.

[0032] Figures 12A-12B is a schematic diagram and a set of bar graphs showing that CAR-E treatment enables the expansion of CAR T cells in vivo in the absence of tumor cells. Figure 12A The experimental design is schematically illustrated. Figure 12B is a set of bar graphs showing CAR T cell counts 30 days after injection.

[0033] Figures 13A-13B Figure 2 is a set of flow cytometry images showing that neither BCMA-muIL2 nor VHH-muIL2 treatments showed binding to any specific population within human PBMCs. PBMCs were labeled with various markers to pre-gate for B cells (CD20), T cells (CD3), or myeloid cells (CD11b). Cells were stained with varying concentrations of treatments followed by secondary staining with anti-FLAG-Alexa647. Figure 13Ais a set of flow cytometry graphs showing that BCMA-muIL2 does not bind to human PBMCs. Figure 13A is a set of flow cytometry images showing that VHH-muIL2 does not bind to human PBMCs.

[0034] Figures 14A-14C is a set of micrographs and dot plots showing the specific binding and gradual internalization of BCMA-muIL2 in CAR T cells. Figure 14A Figure 2 is a panel of micrographs showing cells stained with CellTracker Blue CMAC, incubated with the indicated treatments, labeled with Alexa647 (BCMA-muIL2) or dsRed (VHH-muIL2) for 1 to 5 hours, and imaged. Micrographs are representative of images of >100 cells. Figure 14B are dot plots showing quantitative analysis of imaged cells. Figure 14C is a dot plot showing the correlation between the average intensity of Alexa647 (BCMA-muIL2) and the average intensity of dsRed (VHH-muIL2).

[0035] Figures 15A-15C is a set of flow cytometry graphs showing the Figure 9D Individual flow cytometry data corresponding to the merged data shown in . Figure 15A Figure 2 is a set of flow cytometry results from mice treated with CAR T cells alone. Figure 15B Figure 2 is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. Figure 15C Figure 2 is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[0036] Figures 16A-16C is a set of flow cytometry images showing Figures 10A-10J Individual flow cytometry data for the mice shown in . Figure 16A Figure 2 is a set of flow cytometry results from mice treated with CAR T cells alone. Figure 16B Figure 2 is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. Figure 16C Figure 2 is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[0037] Figures 17A-17C is a set of bar charts, line graphs, and tSNE plots showing human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low dose of CAR T cells. Figure 17AThe bar graph shows the levels of IFNγ, GM-CSF, and TNFα. Serum samples were diluted 1:40. Standard samples and serum samples were incubated on the same plate, and a standard curve for each cytokine was plotted. Figure 17B is a set of line graphs showing IFNγ levels between the BCMA-muIL2 group and the VHH-muIL2 group (error bars represent mean and standard deviation). Figure 17C Figure 2 is a set of Flt-SNE maps of CAR T cells from mice treated with PBS, BCMA-muIL2, and VHH-muIL2, showing the expression of ten immune cell markers.

[0038] Figure 18 A panel of CD4 T cells from five BCMA-muIL2 CAR-E treated mice + CAR + t-SNE plot of T cells showing the expression of nine immune cell markers.

[0039] Figures 19A-19G A set of flow cytometry plots, tSNE plots, bar charts, violin and pie charts, and heat maps showing single-cell RNA sequencing analysis illustrating the effects of BCMA-muIL2 on CAR T cells.

[0040] Figure 19A is a set of flow cytometry images showing CAR T cells analyzed 89 days after CAR-T administration. + cell. Figure 19B is a tSNE plot showing the data after the Harmony algorithm, which shows the proportion of CD4, CD8 and proliferating (CD4 and CD8) cells. Figure 19C is a tSNE plot showing the split between groups treated with BCMA-muIL2 or VHH-muIL2. Figure 19D After the indicated treatment, CD4 + CAR T cells and CD8 + A set of heat maps of significantly differentially expressed genes in CAR T cells. Figure 19E is a set of violin plots of the gene score between CD8 and CD4 cells, which was calculated using Figure 19D The normalized expression of different genes for each phenotype was constructed. Figure 19F is a set of pie charts showing the diversity of T cell receptor (TCR) clonotypes. Figure 19G is a bar graph showing clonotype diversity within the total cell count of the samples.

[0041] Figure 20A-Figure 20BFigure 2 is a set of schematics and flow cytometry images showing that CAR adaptor molecule therapy can enhance organ trafficking of CAR T cells in vivo. Figure 20A The experimental design is schematically illustrated. Figure 20B is a set of flow cytometry images showing the selective trafficking, expansion, and persistence of BCMACAR T cells.

[0042] Figures 21A-21C is a set of line and bar graphs showing the effects of CAR adaptor molecules on untransduced T cells and CAR T cells. Figure 21A and Figure 21B are line graphs and bar graphs, respectively, which together show the dose-dependent activation of CAR T cells after CAR adaptor molecule treatment ( Figure 21A ) and the CAR adapter molecule does not activate non-transduced T cells ( Figure 21B ). Figure 21C is a line graph showing that the CD19-muIL2 CAR adaptor molecule does not block the killing efficacy of CD19 CAR T cells or non-transduced T cells (NT T cells).

[0043] Detailed description of this disclosure

[0044] 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 subject matter belongs. Unless otherwise indicated, the following terms used in the specification and appended claims have the indicated meanings to facilitate understanding of this disclosure.

[0045] As used in this description and the appended claims, the singular forms "a," "an," and "the" mean "one or more" and thus include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an inhibitor" includes mixtures of two or more such inhibitors, and so forth.

[0046] Unless otherwise indicated, the term "about" is understood to mean within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0047] As used herein, the term "about" refers to a value that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) of the reference value, unless otherwise specified or obvious from the context (unless such a number would exceed 100% of the possible value).

[0048] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude other unrecited elements or method steps. In contrast, the transitional phrase "consisting of excludes any element or method step not specified in the claim (or the specified element or method step associated with the phrase "consisting of."). The transitional phrase "consisting essentially of" limits the scope of the claim to the specified elements and method steps and "unrecited elements and method steps that do not materially affect the basic and novel characteristics of the claimed disclosure."

[0049] CAR adaptor molecules

[0050] In one aspect, the present disclosure provides a CAR adapter molecule, also referred to herein as a CAR enhancing molecule, comprising a first protein entity and a second protein entity. The first protein entity comprises an extracellular domain (also referred to herein as a cancer antigen) of an antigen present on a cancer cell, and the second protein entity comprises an immune cell effector domain connected to the extracellular domain. The term "antigen" as used herein refers to a target protein expressed by a cancer (e.g., tumor) cell. The extracellular domain of an antigen is at least a portion of an antigen exposed to the surface of a cancer cell. The extracellular domain binds to the extracellular domain of the CAR presented on an immune cell, which is used together with the CAR adapter molecule used in the present method. The immune cell effector domain binds to a cognate receptor on the same immune cell.

[0051] In some embodiments, the CAR adapter molecule is a continuous protein, wherein the first protein entity and the second protein entity are connected by a peptide bond. In some embodiments, the first protein entity and the second protein entity are connected by click chemistry.

[0052] In some embodiments, the CAR adapter molecule is formulated and administered as a monomeric protein or protein entity. In other embodiments, the CAR adapter molecule is formulated and administered as a dimer or protein entity that is a homodimer or heterodimer protein.

[0053] Extracellular domain

[0054] The extracellular domain of the CAR adapter molecule binds to the extracellular domain of the CAR presented on the immune cell. As is known in the art, the extracellular domain of a cancer antigen is the part that the antigen binds to the T cell receptor or CAR on the immune cell on the surface of the cancer cell. The binding between CAR and the cancer antigen can be direct or indirect. In the embodiment of direct binding, the extracellular domain can be formed by continuous or discontinuous amino acid residues in the extracellular domain of the cancer antigen, or can be an antibody or antibody fragment (including nano antibodies and nano antibody fragments) of the CAR presented on the immune cell. In some embodiments, the CAR adapter molecule can include the entire extracellular domain of the cancer antigen. According to standard techniques, the extracellular domain can be derived from (for example, found in) cancer antigens. See, for example, Gershoni et al., Biodrugs 21 (3): 145-156 (2007) and Francino-Urdaniz and Whitehead, RSC Chem.Biol. 2 (6): 1580-1589 (2021). As used herein, the term "derived from" when referring to proteins and nucleic acids refers to sequences derived from and identified from the sequences of parent (e.g., wild-type or endogenous) proteins and nucleic acids, respectively. A sequence derived from a parent sequence can be identical to the parent sequence, can be a portion of the parent sequence, or can have at least one variant from the parent sequence. Variants can include substitutions, insertions, or deletions. Thus, for example, an amino acid sequence derived from a parent sequence may be identical to a particular amino acid range of the parent, but will not include amino acids outside of that particular region.

[0055] The amino acid sequences of representative cancer antigens from which the extracellular domains can be derived are provided in the NCBI accession numbers listed in Table 1 and are incorporated herein by reference.

[0056] Table 1: Gene names, symbols and NCBI accession numbers of representative cancer antigens

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] The extracellular domain is not limited to known cancer antigens. Unique cancer antigens (neoantigens) can be determined by known methods. For example, the cancer genome can be compared with the normal cell genome to identify new antigens. In some embodiments, the cancer transcriptome is compared with the normal cell transcriptome. Then, a computational method can be used to determine the appropriate binding site of CAR. In most cases, CAR binds to a part of the extracellular domain of the antigen. In some embodiments, CAR and the corresponding cancer antigen are known in the art.

[0066] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the entire extracellular domain of the cancer antigen. In some embodiments, the CAR adapter molecule comprises a portion of the extracellular domain of the cancer antigen targeted by the CAR.

[0067] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of BCMA. The amino acid sequence of a representative CAR adapter molecule containing the BCMA extracellular domain is MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

[0068] In some embodiments, the extracellular domain of the CAR adapter molecule comprises two repeats of the BCMA extracellular domain. The amino acid sequence of a representative CAR adapter molecule containing two repeats of the BCMA extracellular domain is shown below (SEQ ID NO: 2):

[0069]

[0070] In some embodiments, the extracellular domain of the CAR adapter molecule comprises a variant of the extracellular domain of CD 19. The amino acid sequence of a representative CAR adapter molecule containing a variant of the CD19 extracellular domain is shown below (SEQ ID NO: 3):

[0071]

[0072] In some embodiments, the extracellular domain has at least 85% sequence identity to SEQ ID NO: 3, at least 90% sequence identity to SEQ ID NO: 3, at least 95% sequence identity to SEQ ID NO: 3, at least 98% sequence identity to SEQ ID NO: 3, and at least 99% sequence identity to SEQ ID NO: 3.

[0073] The amino acid sequence of a representative CAR adapter molecule containing the second variant of the CD19 extracellular domain is shown below (SEQ ID NO: 4):

[0074]

[0075] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of CD 19. The amino acid sequence of a representative CAR adapter molecule containing the extracellular domain of CD 19 is shown below (SEQ ID NO: 5):

[0076]

[0077] In some embodiments, the extracellular domain of the CAR adapter molecule comprises a portion of the extracellular domain of CD 19. In some embodiments, the extracellular domain of the CAR adapter molecule is KDRPEIWEGEPP (SEQ ID NO: 103), which corresponds to positions 142-153 of SEQ ID NO: 5.

[0078] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of CD20. The amino acid sequence of a representative CAR adapter molecule containing the extracellular domain of CD20 is KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

[0079] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of CD22. The amino acid sequence of a representative CAR adapter molecule containing the extracellular domain of CD22 is shown below (SEQ ID NO: 7):

[0080]

[0081] In some embodiments, the extracellular domain of the CAR adapter molecule comprises a portion of the extracellular domain of CD22 (SEQ ID NO: 7). In some embodiments, the extracellular domain of the CAR adapter molecule comprises the Ig domains 2-3 of CD22. The amino acid sequence of a representative CAR adapter molecule containing the Ig domains 2-3 of CD22 is shown below (SEQ ID NO: 104):

[0082]

[0083] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the Ig domain 3 of CD22. The amino acid sequence of a representative CAR adapter molecule comprising the Ig domain 3 of CD22 is shown below (SEQ ID NO: 105):

[0084]

[0085] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR adapter molecule comprising the Ig domains 5-7 of CD22 is shown below (SEQ ID NO: 106):

[0086]

[0087] In some embodiments, the extracellular domain of the CAR adapter molecule comprises Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR adapter molecule comprising Ig domains 6-7 of CD22 is shown below (SEQ ID NO: 107):

[0088]

[0089] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of Claudin 18.2. The amino acid sequence of a representative CAR adapter molecule comprising the first extracellular domain of Claudin 18.2 is shown below (SEQ ID NO: 8):

[0090]

[0091] The amino acid sequence of a representative CAR adapter molecule comprising the second extracellular domain of Claudin 18.2 is shown below (SEQ ID NO: 9):

[0092]

[0093] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of SLAMF7. The amino acid sequence of a representative CAR adapter molecule comprising the extracellular domain of SLAMF7 is shown below (SEQ ID NO: 10):

[0094]

[0095] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of PD-1. The amino acid sequence of a representative CAR adapter molecule comprising the extracellular domain of PD-1 is shown below (SEQ ID NO: 11):

[0096]

[0097] In some embodiments, the extracellular domain of the CAR adapter molecule comprises a variant of the extracellular domain of PD-1. In some embodiments, the extracellular domain of the CAR adapter molecule comprises the N-loop of PD-1. The amino acid sequence of a representative CAR adapter molecule comprising the N-loop of the PD-1 extracellular domain is LDSPDRPWNP (SEQ ID NO: 108), which corresponds to positions 2 to 11 of SEQ ID NO: 11.

[0098] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the CD loop of PD-1. The amino acid sequence of a representative CAR adapter molecule comprising the CD loop of the PD-1 extracellular domain is NQTDKLAAFPEDRSQPGQDCRFRVTQ (SEQ ID NO: 109), which corresponds to positions 51 to 76 of SEQ ID NO: 11.

[0099] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of KIT. The amino acid sequence of a representative CAR adapter molecule comprising the extracellular domain of KIT is shown below (SEQ ID NO: 12):

[0100]

[0101] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of TROP2. The amino acid sequence of a representative CAR adapter molecule comprising the extracellular domain of TROP2 is shown below (SEQ ID NO: 13):

[0102]

[0103] In some embodiments, the extracellular domain of the CAR adapter molecule comprises the extracellular domain of CD38. The amino acid sequence of a representative CAR adapter molecule comprising the extracellular domain of CD38 is shown below (SEQ ID NO: 14):

[0104]

[0105] In some embodiments, the extracellular domain of the CAR adapter molecule is derived from mesothelin (MSLN). MSLN is a GPI-anchored protein, so the entire MSLN protein is extracellular. The amino acid sequence of a representative MSLN is shown below (SEQ ID NO: 15):

[0106]

[0107] In some embodiments, the extracellular domain of the CAR adapter molecule comprises a portion of the extracellular domain of a cancer antigen. In some embodiments, the extracellular domain of the CAR adapter molecule comprises a portion of an MSLN protein. In some embodiments, the extracellular domain of the CAR adapter molecule is IPNGYLVLDLSMQEALS (SEQ ID NO: 16). In some embodiments, the extracellular domain of the CAR adapter molecule is YNVNDLSMQEL (SEQ ID NO: 17), wherein N is any amino acid.

[0108] In some embodiments, the CAR adapter molecule extracellular domain is an antibody or antibody fragment that binds to the CAR present on the immune cell. In these embodiments, the CAR adapter molecule extracellular domain can be an antibody fragment, which includes a nano antibody fragment. In some embodiments, the CAR adapter molecule extracellular domain is an antibody or fragment thereof for the CAR extracellular domain. In some embodiments, the CAR adapter molecule extracellular domain is an anti-mouse antibody that is bound to an antibody fragment of mouse origin, which has been integrated into the CAR extracellular domain. See Kochenderfer et al., J. Immunother. 32 (7): 689-702 (2009) and Cheng et al., Cytometry A. 103 (1): 16-26 (2023). In some embodiments, the CAR adapter molecule extracellular domain is an anti-idiotypic antibody fragment that binds to the variable region of the CAR antibody fragment. In some embodiments, the CAR adapter molecule extracellular domain is an antibody or fragment thereof, which is described in U.S. Patent No. 9,701,758 and U.S. Patent Application Publication No. 2005 / 0287148, both of which are incorporated herein by reference in their entirety. In some embodiments, the CAR adapter molecule extracellular domain is Peptostreptococcus magnus protein L (NCBI Accession No. Q51918), which binds to the scFv VL region of an antibody and a kappa light chain.

[0109] Indirect combination implementation plan

[0110] The embodiment that relies on indirect binding between CAR and cancer antigen contains an extracellular domain comprising cancer-irrelevant antigen.Cancer-irrelevant antigen is not an antigen on the surface of cancer cells, but an exogenous antigen contained on a protein therapeutic. Protein therapeutics typically include at least cancer-irrelevant antigen and a cancer antigen binding domain (e.g., an antibody or antibody fragment), and are used to redirect CAR immune cells from cancer-irrelevant antigens to cancer antigens. CAR that binds to cancer-irrelevant antigens is called universal CAR or dual activation CAR (BAT-CAR). In some embodiments, the cancer-unrelated antigen is a fluorescent molecule (e.g., fluorescein, fluorescein isothiocyanate (FITC), anthracene, alexa fluor, rhodamine, rhodol, acridine, or xanthene), 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylic acid (AQ), anthraquinone-2-acid, cyclopentane tetraacetic acid (DOTA), amphetamine, benzodiazepine, benzoylecgonine, buprenorphine, an opioid, a cannabinoid, phencyclidine, a tricyclic antidepressant, dextromethorphan, fentanyl, meprobamate, methadone, methamphetamine, oxycodone, THC, tramadol, zolpidem, ketamine, LSD, MDMA, methaqualone, propoxyphene, norketimine, biotin, or a leucine zipper. Other cancer-unrelated antigens are described in, for example, U.S. Patent No. 11,225,520, U.S. Patent Application Publication Nos. 2020 / 0306376, 2021 / 0137987, and 2021 / 0228699, and International Patent Application Publication No. WO 2023 / 060126, all of which are incorporated herein by reference.

[0111] Immune cell effector domain

[0112] The immune cell effector domain of the CAR adapter molecule binds to the homologous receptor on the immune cell expressing the nucleic acid encoding CAR.This binding event regulates the activity of the CAR immune cell. As used herein, the terms "modulate (s)" and "modulation" include the activation and inhibition of CAR immune cells. Therefore, the immune cell effector domain can be a cytokine, an immune cell activation portion or an immune cell inhibition portion, and a variant and a fragment thereof that binds to a homologous target. As known in the art, the term "cytokine" includes low-molecular-weight extracellular polypeptides / glycoproteins that promote, regulate and control immune responses (i.e., increase or decrease activity, differentiation or proliferation). Representative examples of cytokines include chemokines, interferons (IFN), interleukins (IL), lymphokines, and tumor necrosis factors (TNF). The term "immune cell activation variant" of cytokines used herein refers to a non-natural variant of a cytokine that can bind to a cytokine receptor on an immune cell and initiate signal transduction through the receptor to achieve an effect substantially identical to that of a naturally occurring cytokine.

[0113] In some embodiments, the CAR adapter molecule comprises a plurality of (i.e., two or more) immune cell effector domains, any two or more of which may be identical or different from each other. In some embodiments, the CAR adapter molecule comprises two immune cell effector domains. In some embodiments, the CAR adapter molecule comprises three immune cell effector domains.

[0114] In some embodiments, the immune cell effector domain is an immune cell activating portion, for example, an immune cell activating cytokine and an immune cell activating variant and fragments thereof. The immune cell activating portion activates, promotes or maintains the activity of an immune cell.

[0115] In some embodiments, the immune cell effector domain is derived from CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-induced TNFR-related protein ligand (GITRL; TNFSF18), herpes virus entry mediator (HVEM; TNFSF14), semaphorin 3B (SEMAA; SEMA3B), signaling lymphocyte activation molecule family member 1 (SLAM; SLAMF1; CD150), T cell immunoglobulin and mucin domain-containing 4 (TIM4), TNF superfamily member 4 (TNFSF4; OX40L), TNF superfamily member 8 (TNFSF8; CD30L), interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, CCL21, 4-1BBL (also known as TNF superfamily member 9; TNFSF9), or an immune cell activating variant thereof.

[0116] The amino acid sequences of representative immune cell activating portions (e.g., cytokines) from which immune cell effector domains can be derived are provided by the NCBI accession numbers listed in Table 2 and are incorporated herein by reference.

[0117] Table 2: Gene names, symbols, and NCBI accession numbers of representative immune cell activation proteins

[0118]

[0119]

[0120]

[0121] In some embodiments, the immune cell effector domain is wild-type IL-2 having the amino acid sequence set forth below (SEQ ID NO: 102; NCBI Accession No. NP_000577):

[0122]

[0123] In some embodiments, the immune cell effector domain is a synthetic, ie, non-natural, IL-2 that is a variant of wild-type IL-2 (SEQ ID NO: 102) in that it has an amino acid substitution at position 16 and / or position 42.

[0124] In some embodiments, the immune cell effector domain has an H16A substitution relative to SEQ ID NO: 102 (i.e., alanine (A) is substituted for histidine (H) at position 16) and / or an F42A substitution relative to SEQ ID NO: 102 (i.e., alanine (A) is substituted for phenylalanine (F) at position 42), the two substituted alanine residues being shown as boxed amino acids in SEQ ID NO: 19. In some embodiments, the immune cell effector domain is a weak affinity variant of IL-2 (muIL2) having the amino acid sequence shown below (SEQ ID NO: 19), comprising the H16A and F42A substitutions, as follows:

[0125]

[0126] "Weak affinity" means that the natural (wild-type) sequence of human IL-2 (SEQ ID NO: 102) has a higher affinity for the IL-2 receptor (IL-2R) than SEQ ID NO: 19.

[0127] More specifically, the muIL2 (SEQ ID NO: 19) immune cell effector domain has a dissociation constant (K) of approximately 1200 nM for IL-2Rα (CD25). D ), indicating a 110-fold decrease compared to wild-type IL-2, and has a K of approximately 610 nM for IL-2Rβ. D , indicating a 3-fold decrease compared with wild-type IL-2.

[0128] In some embodiments, the CAR adapter molecule comprises more than one immune cell effector domain. The immune cell effector domains in the CAR adapter molecule can be the same or different.

[0129] In some embodiments, the CAR adapter molecule comprises two immune cell effector domains, e.g., a first immune cell effector domain and a second immune cell effector domain, which are weak affinity IL-2 variants, which together have the amino acid sequence shown below (SEQ ID NO: 20):

[0130]

[0131] In some embodiments, the immune cell effector domain can be derived from IL-7. The amino acid sequence of a representative IL-7 is shown below (SEQ ID NO: 21):

[0132]

[0133] In some embodiments, the immune cell effector domain can be derived from IL- 15. The amino acid sequence of a representative IL-15 is shown below (SEQ ID NO: 22):

[0134]

[0135] In some embodiments, the immune cell effector domain can be derived from IL- 18. The amino acid sequence of a representative IL-18 is shown below (SEQ ID NO: 23):

[0136]

[0137] In some embodiments, the immune cell effector domain can be derived from IL-21. The amino acid sequence of a representative IL-21 is shown below (SEQ ID NO: 24):

[0138]

[0139] In some embodiments, the immune cell effector domain can be derived from IL-27. The amino acid sequence of a representative IL-27 is shown below (SEQ ID NO: 25):

[0140]

[0141] In some embodiments, the immune cell effector domain is an immune cell activating variant of a cytokine. In some embodiments, the immune cell effector domain is a neo-interleukin-2 / 15 (Neo-2 / 15) that binds to IL-2R-β and has the amino acid sequence shown below (SEQ ID NO: 26).

[0142]

[0143] In some embodiments, the CAR adapter molecule comprises two immune cell effector domains of Neo-2 / 15, each having the amino acid sequence of SEQ ID NO:26.

[0144] In some embodiments, the immune cell effector domain can be derived from 4-1BBL. 4-1BBL is also known as TNF ligand superfamily member 9 (TNFSF9). The amino acid sequence of representative 4-1BBL is provided by NCBI accession number NP_003802, which is incorporated herein by reference. In some embodiments, the immune cell effector domain can be derived from the extracellular domain of 4-1BBL. In some embodiments, the immune cell effector domain comprises a portion of the extracellular domain of 4-1BBL, having the amino acid sequence listed below (SEQ ID NO: 27):

[0145]

[0146] In some embodiments, the CAR adapter molecule comprises three immune cell effector domains, for example, wherein the first immune cell effector domain, the second immune cell effector domain, and the third immune cell effector domain are all extracellular domains of 4-1BBL, each having the amino acid sequence of SEQ ID NO: 27.

[0147] In some embodiments, the immune cell effector domain can be a fragment that combines and activates CAR immune cells, such as a single-chain variable antibody fragment (scFv). In some embodiments, the immune cell effector domain is scFv, which is combined with 4-1BB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM or TIM1.

[0148] In some embodiments, the immune cell effector domain is a scFv that binds to CTLA4. In some embodiments, the immune cell effector domain is derived from a commercially available anti-CTLA4 antibody, antibody fragment, or derivative thereof, e.g., bavunalimab (formerly pavunalimab / XmAb 22841), botensilimab, cadonilimab, ipilimumab, or quavonlimab, tremelimumab Volrustomib, vudalimab, or zalifrelimab. The amino acid sequences of representative heavy and light chain antibodies that bind to CTLA4 are shown in Table 3.

[0149] Table 3: Amino acid sequences of heavy and light chains of representative anti-CTLA antibodies

[0150]

[0151]

[0152] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 36):

[0153]

[0154] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 37):

[0155]

[0156] In some embodiments, the immune cell effector domain binds to OX40. In some embodiments, the immune cell effector domain is derived from a commercially available anti-OX40 antibody, an antibody fragment (e.g., scFv), or derivative thereof, such as tavolimab or vonlerolizumab (Pogalizumab; MOXR 0916). Representative heavy and light chain amino acid sequences are shown in Table 4.

[0157] Table 4: Amino acid sequences of heavy and light chains of representative anti-OX40 antibodies

[0158]

[0159]

[0160] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 42):

[0161]

[0162] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 43):

[0163]

[0164] In some embodiments, the immune cell effector domain binds to PD-1. In some embodiments, the immune cell effector domain is derived from a commercially available anti-PD-1 antibody, antibody fragment (e.g., scFv), or derivative thereof, such as atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, durvalumab, or

[0165] Inbakicept, lodapolimab, pimivalimab, or socazolimab. The amino acid sequences of their representative heavy and light chains are shown in Table 5.

[0166] Table 5: Amino acid sequences of heavy and light chains of representative anti-PD-1 antibodies

[0167]

[0168]

[0169] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 52):

[0170]

[0171] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 53):

[0172]

[0173] In some embodiments, the immune cell effector domain is an immune cell inhibitory portion, and its representative types include immune cell inhibitory cytokines and immune cell inhibitory variants and fragments thereof. The immune cell inhibitory portion inhibits or blocks immune cell activity and function. In some embodiments, the immune cell inhibitory portion can be derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galectin 3, HLA-E, HVEM, PD-L1, PD-L2, VISTA or VTCN1 (B7-H4). The amino acid sequence of the representative immune cell inhibitory protein from which the immune cell effector domain can be derived is provided by the NCBI accession number shown in Table 6, and is incorporated herein by reference.

[0174] Table 6: Gene names, symbols and NCBI accession numbers of immune cell inhibitory proteins

[0175]

[0176]

[0177] In some embodiments, the immune cell effector domain is the extracellular domain of CD80. The amino acid sequence of a representative CD80 extracellular domain is shown below (SEQ ID NO: 54):

[0178]

[0179] In some embodiments, the immune cell effector domain is the extracellular domain of CD86. The amino acid sequence of a representative CD86 extracellular domain is shown below (SEQ ID NO: 55):

[0180]

[0181] In some embodiments, the immune cell effector domain is the extracellular domain of CD155 (fibronectin-5; PVR). The amino acid sequence of a representative CD155 extracellular domain is shown below (SEQ ID NO: 56):

[0182]

[0183] In some embodiments, the immune cell effector domain is the extracellular domain of CD276 (B7-H3). The amino acid sequence of a representative CD276 extracellular domain is shown below (SEQ ID NO: 57):

[0184]

[0185] In some embodiments, the immune cell effector domain is the extracellular domain of Ceacam-1. The amino acid sequence of a representative extracellular domain of Ceacam-1 is shown below (SEQ ID NO: 58):

[0186]

[0187] In some embodiments, the immune cell effector domain is the extracellular domain of FGL1. The amino acid sequence of a representative FGL1 extracellular domain is shown below (SEQ ID NO: 59):

[0188]

[0189] In some embodiments, the immune cell effector domain is the extracellular domain of Galectin-3. The amino acid sequence of a representative extracellular domain of Galectin-3 is shown below (SEQ ID NO: 60):

[0190]

[0191]

[0192] In some embodiments, the immune cell effector domain is the extracellular domain of HLA-E. The amino acid sequence of a representative HLA-E extracellular domain is shown below (SEQ ID NO: 61):

[0193]

[0194] In some embodiments, the immune cell effector domain is the extracellular domain of HVEM (CD270). The amino acid sequence of a representative HVEM extracellular domain is shown below (SEQ ID NO: 62):

[0195] In some embodiments, the immune cell effector domain is the extracellular domain of laminin-2 (CD112, HVEB). The amino acid sequence of a representative laminin-2 extracellular domain is shown below (SEQ ID NO: 63):

[0196]

[0197] In some embodiments, the immune cell effector domain is the extracellular domain of PD-L1. The amino acid sequence of a representative PD-L1 extracellular domain is shown below (SEQ ID NO: 64):

[0198]

[0199] In some embodiments, the immune cell effector domain is the extracellular domain of PD-L2. The amino acid sequence of a representative PD-L2 extracellular domain is shown below (SEQ ID NO: 65):

[0200]

[0201] In some embodiments, the immune cell effector domain is the extracellular domain of VTCN1 (B7-H4). The amino acid sequence of a representative VTCN1 is shown below (SEQ ID NO: 66):

[0202]

[0203] Dimerization domain

[0204] In some embodiments, the CAR adapter molecule further comprises a dimerization domain. In these cases, the CAR adapter molecule is formed and administered in the form of a homodimer or a homomultimer. Thus, the homodimer comprises two CAR adapter molecule entities. The order of the extracellular domain, the immune effector domain, and the dimerization domain is not important. In some embodiments, the dimerization domain is located between the extracellular domain and the immune cell effector domain.

[0205] In some embodiments, the CAR adapter molecule is in the form of a heterodimer containing a first entity comprising an extracellular domain of an antigen present on a cancer cell connected to a first dimerization domain and a second entity comprising an immune cell effector domain connected to a second dimerization domain. In these embodiments, the first dimerization domain and the second dimerization domain dimerize the first entity and the second entity to form a heterodimer.

[0206] In some embodiments, the first dimerization domain and the second dimerization domain comprise a knob-in-hole configuration. One of the dimerization domains comprises a protuberance (knob) and the other dimerization domain comprises a cavity (hole) that sterically compensates for the protuberance, wherein the tertiary structure of the protuberance can be positioned within the tertiary structure of the cavity. The dimerization domain with a knob-in-hole configuration may have targeted amino acid mutations, wherein the protuberance is an amino acid with a larger side chain volume than an amino acid on a dimerization domain from a natural source (such as IgA, IgD, IgG, IgM, or IgE), and the cavity is an amino acid with a smaller side chain volume than the side chain volume of an amino acid present on a dimerization domain from a natural source.

[0207] In some embodiments, the protuberance is an amino acid change from threonine (T) to lysine (K), and the corresponding cavity is an amino acid change from leucine (L) to aspartic acid (D) or lysine (K). In some embodiments, the first dimerization domain comprises two amino acid substitutions, e.g., threonine (T) to lysine (K) and leucine (L) to lysine (K), and the second dimerization domain comprises leucine (L) to aspartic acid (D) or glutamic acid (E) and tyrosine (Y) to glutamic acid (E) or aspartic acid (D).

[0208] In some embodiments, the knob-to-hole dimerization domains are based on opposite charges. In some embodiments, a first dimerization domain comprises positively charged amino acids and a second dimerization domain comprises negatively charged amino acids that are spatially opposed to the positively charged amino acids on the first dimerization domain.

[0209] Other protuberance and cavity arrangements are known in the art. See, for example, U.S. Patents 5,821,333, 7,183,076, 8,642,745, 9,248,182, 9,309,311, 9,527,927, 9,562,109, 9,890,204, 10,138,303, and 11,168,344 and U.S. Patent Application Publications 2005 / 0079170, 2006 / 0025576, 2013 / 0089554, and 2014 / 0024111.

[0210] In some embodiments, the dimerization domain can be derived from IgA, IgD, IgG, IgM or IgE. The first dimerization domain and the second dimerization domain can comprise the same or different amino acid sequences, provided that they bind to each other. In some embodiments, the first dimerization domain and the second dimerization domain are IgG1 heavy chain constant region (CH) 3 domains. The amino acid sequence of a representative IgG1 CH3 domain is shown below (SEQ ID NO: 67):

[0211]

[0212] In some embodiments, the first dimerization domain and the second dimerization domain are IgG1 heavy chain constant region CH2 domains. The amino acid sequence of a representative IgG1 CH2 domain is shown below (SEQ ID NO: 68):

[0213]

[0214] In some embodiments, the first dimerization domain and the second dimerization domain are the CH2 and CH3 domains of IgG1. The CH2 and CH3 domains can be connected to each other via a linker.

[0215] connector

[0216] In some embodiments, the CAR adapter molecule includes one or more connectors. In terms of allowing the extracellular domain and immune cell effector domain to bind to their respective cognate receptors on the immune cells expressing CAR or the spatial interval (i.e., spacer) between the extracellular domain and immune cell effector domain, connectors can provide flexibility.

[0217] A linker can be positioned between any two CAR adapter molecule components (also referred to herein as domains, moieties, or portions) (e.g., an extracellular domain and an immune cell effector domain).

[0218] The connector can be located between the dimerization domain and the adjacent domain. In some embodiments, the connector can be located between the dimerization domain and the immune cell effector domain. In some embodiments, the CAR adapter molecule comprises two connectors, wherein the first connector is located between the extracellular domain and the dimerization domain, and the second connector is located between the dimerization domain and the immune cell effector domain.

[0219] In some embodiments, the linker comprises amino acids having the sequence GGGX, GGGGX (SEQ ID NO: 69), or GSSGSX (SEQ ID NO: 70), where X is any nucleotide, typically cysteine ​​(C) or serine (S), or repeats thereof. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO:71), GSPRG (SEQ ID NO:72), GGGGSGGGGS (SEQ ID NO:73), GGGGSGGGGSGGGGS (SEQ ID NO:74), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:75), GSPRGGGGSGGGGSGGGGS (SEQ ID NO:76), GSTSGSGKPGSGEGSTKG (SEQ ID NO:77), KESGSVSSEQLAQFRSLD (SEQ ID NO:78), EGKSSGSGSESKST (SEQ ID NO:79), or GSAGSAAGSGEF (SEQ ID NO:80).

[0220] In some embodiments, the linker can be derived from IgA, IgD, IgE, IgG or IgM. In some embodiments, the linker can be derived from the hinge region of CD3ζ, CD4, CD8α, CD28, IgG1, IgG2 or IgG4. The amino acid sequences of representative linkers are listed in Table 7.

[0221] Table 7: Amino acid sequences of representative linkers

[0222]

[0223] In some embodiments, CAR adapter molecules are in the form of fusion proteins, wherein components are connected by peptide bonds. In other embodiments, CAR adapter molecules include protein entities connected to each other by click chemistry (chemical connection formed by a method for controlled protein connection). Connections can be azide-alkyne connections, oxime or hydrazine connections, tetrazine-trans cyclooctene connections, azide-nitrone connections, thiol-olefin connections, olefin-tetrazole connections, olefin-tetrazine connections, olefin-azide connections, conjugated diene-olefin connections or isonitrile-tetrazine connections.

[0224] Other controlled protein ligation chemistries, systems, and methods are known in the art. See, for example, U.S. Patents 7,375,234, 7,763,736, 8,101,238, 8,372,986, 8,394,914, 8,877,170, 8,927,682, 8,927,736, 9,302,997, 9,896,547, 11,028,185, 11,091,588, and 11,352,460 and U.S. Patent Application Publication 2009 / 0069561.

[0225] Nucleic Acids

[0226] On the other hand, the present disclosure provides nucleic acids encoding CAR adapter molecule proteins. The term "nucleic acid" as used herein refers to a polymer of nucleotides, each of which is an organic molecule consisting of nucleosides (nucleobases and pentose) and phosphates. The term nucleotide, unless otherwise specified or apparent from the context, includes nucleosides with ribose (i.e., ribonucleotides forming ribonucleic acid (RNA)) or 2'-deoxyribose (i.e., deoxyribonucleotides forming deoxyribonucleic acid (DNA)). Nucleotides are used as monomeric units of nucleic acid polymers or polynucleotides. The four nucleobases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). Nucleic acids are nucleotides (e.g., at least 3 nucleotides) linear chains chemically bound by a series of ester bonds between the phosphate group of a nucleotide and the hydroxyl group of the sugar in the adjacent nucleotide (i.e., ribose or 2'-deoxyribose).

[0227] In some embodiments, the CAR adapter molecule is encoded by two nucleic acids, e.g., the extracellular domain is encoded by a first nucleic acid and the immune cell effector domain is encoded by a second nucleic acid.

[0228] In some embodiments, the nucleic acid encoding the CAR adapter molecule includes a nucleic acid encoding a signal peptide, which is located at the 5' end of the nucleic acid encoding the extracellular domain. The term "signal peptide" as used herein refers to a short segment (e.g., 5-30 or 10-100 amino acids long) of amino acids that guides the transport of proteins during translation. The CAR adapter molecule comprising a signal peptide will be secreted from the cell. Typically, the signal peptide is cleaved from the CAR adapter molecule before secretion. The signal peptide can be connected to a nucleic acid encoding an extracellular domain or a nucleic acid encoding an immune cell effector domain.

[0229] In some embodiments, the signal peptide can be derived from Ig-γ-3 heavy chain (IGHG3), albumin, CD8α, CD33, erythropoietin (EPO), IL-2, human or mouse Ig-κ chain V-III (IgKVIII), tissue plasminogen activator (tPA), or secretory alkaline phosphatase (SEAP). The signal peptide can also be synthetic (i.e., non-natural). The amino acid sequences of representative signal peptides are listed in Table 8.

[0230] Table 8: Amino acid sequences of representative signal peptides

[0231] signal peptide sequence IGHG3 (SEQ ID NO: 88) MKHLWFFLLLVAAPRWVLS Albumin (SEQ ID NO: 89) MKWVTFISLLFLFSSAYS Synthetic, modified albumin (SEQ ID NO: 90) MKWVTFISLLFLFSSSSRA CD8α (SEQ ID NO:91) MALPVTALLLPLALLLHAARP CD33 (SEQ ID NO:92) MPLLLLLPLLWAGALA EPO (SEQ ID NO: 93) MGVHECPAWLWLLLSLLSLPLGLPVLG IL-2 (SEQ ID NO:94) MYRMQLLSCIALSLALVTNS Mouse IgK VIII (SEQ ID NO: 95) METDTLLLWVLLLWVPGSTG Human IgK VII (SEQ ID NO: 96) MEAPAQLLFLLLLWLPDTTG Synthetic, modified human IgK VIII (SEQ ID NO: 97) MEAPAQLLFLLLLWLPSSRA tPA (SEQ ID NO: 98) MDAMKRGLCCVLLLCGAVFVSPS SEAP (SEQ ID NO:99) MLLLLLLLGLRLQLSLG Synthetic consensus sequence (SEQ ID NO: 100) MLLLLLLLLLLALALA Synthetic secreton (SEQ ID NO: 101) MWWRLWWLLLLLLLLWPMVWA

[0232] carrier

[0233] The nucleic acid encoding CAR adapter molecules can be imported into cells by suitable carriers.In embodiments, wherein the extracellular domain and immune effector domain are connected chemically (for example, by click chemistry), the nucleic acid encoding CAR can be imported into one or more cells by different carriers.Carrier is configured to include and realizes transport to immune cells and realizes the element required for expression after nucleic acid conversion.These elements include replication origin, poly A tail sequence, selective marker and one or more suitable sites for inserting nucleic acid sequence, such as multiple cloning site (MCS), one or more suitable promoters, each promoter is operably connected to the insertion site and selective marker of nucleic acid sequence, and other optional regulatory elements.

[0234] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcription of a corresponding nucleic acid coding sequence to which it is operably connected, and in the context of the present disclosure, it is operably linked to a CAR adapter molecule protein. A promoter can play a role in regulating transcription alone, or it can act synergistically with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in a nucleic acid sequence or a vector). The promoter is located near the transcription start site of a gene, on the same chain and upstream of DNA (towards the 5' region of the sense strand). The length of a promoter is generally in the range of about 100 to 1000 base pairs.

[0235] As used herein, the term "operably linked" is understood to mean that a nucleic acid sequence is located or positioned in a vector spatially relative to another nucleic acid sequence, for example, a promoter is operably linked to drive expression of a nucleic acid coding sequence (e.g., a nucleic acid sequence encoding a CAR adapter molecule).

[0236] In some embodiments, a single vector comprises a single promoter operably connected to the nucleic acid encoding the CAR adapter molecule. In some embodiments, a single vector comprises a single promoter operably connected to the nucleic acid encoding the extracellular domain and the nucleic acid encoding the immune cell effector domain. In some of these embodiments, these nucleic acids are separated by nucleic acids encoded from cleavage peptides or internal ribosome entry sites (IRES). In some embodiments, a single vector comprises a first promoter operably connected to the nucleic acid encoding the extracellular domain and a second promoter operably connected to the nucleic acid encoding the immune cell effector domain.

[0237] In some embodiments, two vectors are provided. In some embodiments, the first vector comprises a promoter operably linked to a nucleic acid encoding an extracellular domain, and the second vector comprises a promoter operably linked to a nucleic acid encoding an immune cell effector domain.

[0238] In some embodiments, the vector comprises a strong mammalian promoter, such as the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, a synthetic promoter (e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, splice acceptor of chicken β-actin and rabbit β-globin)), or a promoter derived from the β-actin, phosphoglycerate kinase (PGK), or factor EF1α genes. In some embodiments, the promoter may comprise a core region located adjacent to the nucleic acid coding sequence. In some embodiments, the promoter is engineered to remove methylation-sensitive motifs (e.g., a cytosine nucleotide followed by a guanine nucleotide or "CpG"), or by adding regulatory sequences that bind transcription factors that inhibit DNA methylation. In some embodiments, the vector includes an A / T-rich matrix interaction sequence, termed a scaffold matrix attachment region (S / MAR), which increases transformation efficiency and improves the stability of transgene expression.

[0239] In some embodiments, the vector is a viral vector, e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, a herpes virus vector, an adenovirus, or an adeno-associated virus (AAV) vector. For example, the construction of lentiviral vectors has been described in, e.g., U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530.

[0240] In other embodiments, the vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single-stranded (ss) DNA or linear double-stranded (ds) DNA, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac (PB)-based vectors. In yet other embodiments, the vector may include both viral and non-viral elements.

[0241] In some embodiments, the vector is a plasmid. In addition to the promoter operably connected to the nucleic acid, the plasmid may also include other elements, such as elements that promote the transport and expression of nucleic acids in immune cells. The plasmid can be linearized with restriction endonucleases, transcribed in vitro to produce mRNA, and then modified with 5' caps and 3' poly A tails. In some embodiments, the vector has multiple plasmids, a first plasmid encoding a first protein entity (e.g., an extracellular domain of a CAR adapter molecule) and a second plasmid encoding a second protein entity (e.g., an immune effector domain of a CAR adapter molecule).

[0242] cell

[0243] One aspect of the present disclosure is a genetically modified (or transformed) cell comprising a vector comprising a nucleic acid encoding a CAR adapter molecule or a CAR adapter molecule component for producing and purifying the CAR adapter molecule protein.

[0244] Cells that can be used for cloning and other manipulations of these vectors are conventional. Cells from different strains of E. coli can be used for replication of the vectors and other steps in the construction of the CAR adapter molecules of the present disclosure.

[0245] Suitable host cells or cell lines for expressing nucleic acids encoding CAR adapter molecules include eukaryotic cells. In some embodiments, the cell is a mammalian cell line. In some embodiments, the cell is a mammalian cell, such as CHO (e.g., DG44, CHO-S), fibroblasts (e.g., 3T3, COS), embryonic cells (e.g., PER.C6, HEK (e.g., HEK.293)), somatic cell hybrids (e.g., Sp2 / 0) and cancer cells, such as myeloma cells (e.g., NS0 (NS zero)). In some embodiments, the nucleic acid encoding CAR adapter molecules is expressed in CHO or myeloma cells. Human cells can be used so that the expressed CAR adapter molecules can be modified with human glycosylation patterns. Selection of suitable mammalian cells and methods for conversion, culture, amplification, screening and product production and purification are known in the art. See, for example, Green et al., eds., Molecular Cloning: A Laboratory Manual, 5 thed., Cold Spring Harbor Laboratory Press, New York, 2012.

[0246] In some embodiments, the cell is prokaryotic. Prokaryotic (i.e., bacterial) cells can prove to be useful host cells, suitable for expressing nucleic acids encoding CAR adapter molecules (see, e.g., Pluckthun, Immunol. Rev. 130: 151-188 (1992)). However, since proteins expressed in bacterial cells tend to exist in unfolded or incorrectly folded forms or non-glycosylated forms, any CAR adapter molecules produced in bacterial cells will be screened to retain function (e.g., CAR binding ability). If the CAR adapter molecules expressed by bacterial cells are produced in correctly folded form, the bacterial cells will be ideal hosts, or in optional embodiments, CAR adapter molecules can be expressed in bacterial hosts and then refolded. For example, various Escherichia coli strains used for expression are well known in the field of biotechnology as host cells. Various strains of Bacillus subtilis (B. Subtilis), Streptomyces (Streptomyces), other Bacillus, etc. can also be used.

[0247] After expression in the cell, the CAR adapter molecule is isolated from the culture medium of the cell (e.g., cell lysate) or cultured cells. Protein separation techniques are known in the art. Representative separation techniques include chromatography, affinity chromatography, nickel-nitrotriacetic acid (Ni-NTA) affinity chromatography, high performance liquid chromatography (HPLC), hydroxyapatite chromatography, protein A-agarose gel, gel electrophoresis and dialysis. In some embodiments, the affinity chromatography resin is protein A affinity chromatography resin or protein G affinity chromatography resin. Other protein separation systems and methods are known in the art. See, e.g., U.S. Patents 516,9936, 6,267,958, 8,357,778, 9,630,165, 9,708,399, 10,023,608, 10,207,229, 11,369,703, and 11,390,668, U.S. Patent Application Publications 2008 / 0090995, 2012 / 0244075, 2017 / 0158760, 2019 / 0276492, and 2021 / 0206815, and Traunecker et al., Embo J. 10(12):3655-9 (1991).

[0248] In some embodiments, the CAR adapter molecule is encoded by two or more nucleic acids, for example, the portion containing the extracellular domain is encoded by one nucleic acid, and the immune cell effector domain is encoded by a second nucleic acid. In these embodiments, after expression and purification in suitable cells, the purified extracellular domain and the purified immune cell effector can be connected by the above-mentioned suitable chemical connection reaction.

[0249] Pharmaceutical composition

[0250] The pharmaceutical composition of the present disclosure includes an effective amount of CAR adapter molecules and a pharmaceutically acceptable carrier. The term "effective amount" as used herein refers to the amount of CAR adapter molecules sufficient to provide the desired effect, for example, the amount of CAR adapter molecules bound to immune cells expressing CAR. Depending on the location, type and severity of the cancer, the age, weight and physical condition of the patient, the amount of CAR adapter molecules administered to the subject will vary over a wide range. The doctor will ultimately determine the appropriate dose to be used. As described herein, the CAR adapter molecules in the pharmaceutical composition can be in the form of monomers (in the absence of a dimerization domain), homodimers or heterodimers.

[0251] The amount of CAR adapter molecule administered to a subject can vary widely depending on many factors, including the location, type, and severity of the cancer, as well as the age, weight, and physical condition of the subject being treated. The physician will ultimately determine the appropriate amount of CAR adapter molecule and the dosage to be used. Typically, the CAR adapter molecule will be administered in a range of doses. In some embodiments, the effective amount of the CAR adapter molecule is from about 50 mg to about 180 mg per dose per subject. In some embodiments, the effective amount of the CAR adapter molecule is between about 1 mg and about 18 mg per kg of subject body weight.

[0252] The composition can be provided as a sterile solid or liquid preparation. The solid preparation can be redissolved and diluted into a liquid preparation before use, for example, using a carrier containing an isotonic aqueous solution, suspension, emulsion, dispersion or viscous solution, which can be buffered to a selected pH. Liquid carriers include aqueous carriers or non-aqueous carriers, etc. Representative examples of liquid carriers include sterile water for injection, saline, lactated Ringer's injection, phosphate buffer, soluble proteins, soluble sugars (e.g., glucose), dimethyl sulfoxide (DMSO), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), ethanol and suitable mixtures thereof. In some embodiments, the liquid carrier includes proteins dissolved or dispersed therein, and representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin and casein. The compositions are generally isotonic, that is, they have the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyols and isotonic electrolyte solutions (e.g., Plasma- ) can be used to achieve the desired isotonicity. Depending on the carrier, other excipients may be added, for example, wetting agents, dispersants or emulsifiers, gelling agents and viscosity increasing agents, preservatives, etc. as known in the art. In some embodiments, the composition includes citric acid, ethylenediaminetetraacetic acid (EDTA) and polysorbate 20, and the pH range is about 6.8 to about 7.2.

[0253] cancer

[0254] In some aspects, the present disclosure relates to treating cancer in a subject. The method requires administering a pharmaceutical composition containing a CAR adapter molecule described herein to a subject in need thereof. The term "cancer" as used herein refers to a disease or condition characterized by excessive proliferation or decreased apoptosis in a subject. Cancers that can be treated with the CAR adapter molecules disclosed herein include hematopoietic cancers and cancers characterized by the presence of solid tumors.

[0255] The term "subject" (or "patient") as used herein includes all members of the animal kingdom that are susceptible to or have the cancer. In some embodiments, the subject is a human. Therefore, a subject "suffering from cancer" or "in need of" treatment according to the present disclosure broadly includes subjects who have been positively diagnosed, including subjects with active disease who may have previously received one or more rounds of treatment, and subjects who are not currently receiving treatment (e.g., in remission) but are still at risk of recurrence, as well as subjects who have not been positively diagnosed but are susceptible to cancer (e.g., due to past medical history and / or family history, or otherwise the presence of one or more risk factors, such that a medical professional may reasonably suspect that the subject is susceptible to cancer).

[0256] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention, procedure, or administration of an active agent to a subject in need thereof, with the therapeutic purpose ("therapeutic effect") to reverse, alleviate, ameliorate, inhibit, reduce, slow, halt, stabilize, or prevent the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical marker associated with cancer.

[0257] In some embodiments, cancer is a hematopoietic cancer. Representative hematologic cancers include plasma cell neoplasms (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of undetermined significance (MUGS), asymptomatic smoldering multiple myeloma or solitary plasmacytoma), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, plasmablastic lymphoma, plasmacytoid lymphoma or diffuse large B-cell lymphoma), leukemia (e.g., relapsed or refractory acute B-cell leukemia, or relapsed or refractory acute lymphoblastic leukemia) and carcinoma (e.g., Waldenstrom's macroglobulinemia or glioblastoma (astrocytoma)). In these embodiments, the therapeutic effect may include one or more art-recognized therapeutic efficacy indicators, representative examples of which include prevention or prolongation of metastasis, improvement in survival time, full / complete or partial remission of cancer, such as no detectable cancer cells and fewer tumor cells or smaller tumors or a reduction in the number of tumor cells, respectively. In some embodiments, the hematopoietic cancer is multiple myeloma, lymphoma, or leukemia.

[0258] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is bladder cancer (e.g., transitional cell carcinoma, also known as urothelial carcinoma), kidney cancer (e.g., renal cell carcinoma (RCC), clear cell renal carcinoma (KIRC), transitional cell carcinoma, or Wilms' tumor), skin cancer (e.g., melanoma, cutaneous melanoma (SKCM), basal cell carcinoma, and cutaneous squamous cell carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN), also known as head and neck squamous cell carcinoma), or Cancers of the lining of the lungs, ovaries, and pancreas include ovarian cancer (e.g., cystic ovarian cancer, embryonal ovarian cancer, epithelial ovarian cancer, fallopian tube cancer, and primary peritoneal cancer), endometrial cancer, cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)), prostate cancer (e.g., prostate adenocarcinoma (PRAD)), and stomach cancer (e.g., stomach adenocarcinoma (STAD)).

[0259] In some embodiments, the cancer is characterized as being in a minimal residual disease (MRD) state. MRD is a state in which a small number of cancer cells remain in a cancer patient's body after treatment. The number of remaining cells may be so small that they do not cause any signs or symptoms of cancer and may generally not be detected by traditional methods (such as observing cells under a microscope and / or by tracking abnormal serum proteins in the blood).

[0260] The amount of cancer antigens present in subjects in an MRD state is limited. And this limited presence of cancer antigens may not be sufficient to support the proliferation and efficacy of CAR immune cells. The additional presence of CAR adapter molecules not only presents additional cancer antigens to CAR immune cells, but also provides supportive immune cell effector domains that can modulate the activity of CAR immune cells to promote proliferation, efficacy and / or persistence.

[0261] In some embodiments, the subject to whom the CAR adapter molecule is administered is in an MRD state. In some embodiments, the method for treating cancer includes treating the minimal residual disease (MRD) state of the subject. In some embodiments, the method for treating cancer includes eliminating MRD in the subject.

[0262] To detect MRD, a sample from a blood draw or bone marrow aspiration can be used. The most widely used tests for measuring MRD are flow cytometry, polymerase chain reaction (PCR), and next-generation sequencing. For example, U.S. Patents 8,124,353, 9,528,160, 10,280,462, 11,618,787, and 11,633,426 and U.S. Patent Application Publications 2011 / 0294148 and 2022 / 0380852 describe methods that may be suitable for measuring MRD.

[0263] Application

[0264] In some embodiments, the methods of the present disclosure require administering an effective amount of a CAR adapter molecule to a cancer patient who has previously received administration of an immune cell comprising a CAR comprising an extracellular domain that binds to the extracellular domain of the CAR adapter molecule, a transmembrane domain, and an intracellular domain comprising a stimulatory domain. In some embodiments, after determining that the CAR immune cell has lost viability or persistence in the subject, the CAR adapter molecule is administered to the subject. This determination can be performed according to known techniques. In some embodiments, for example, a sample is obtained from the subject after administering the immune cell. The concentration of the immune cells present in the sample can be used to calculate the difference between the concentration of the immune cells administered to the subject and the concentration of the immune cells measured in the sample. Once the measured immune cell concentration is less than the administered immune cell concentration, the CAR adapter molecule can be administered. In some embodiments, once the measured immune cell concentration is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 25%, less than 10% or less than 5% of the administered immune cell concentration, the CAR adapter molecule is administered.

[0265] In some embodiments, the CAR adapter molecule is administered at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, or at least about 1 year after administration of the CAR-immune cell.

[0266] In some embodiments, the CAR adapter molecule is administered as an infusion once every 3 weeks (21-day cycle) for about 30 minutes to about 90 minutes. In some embodiments, the CAR adapter molecule is administered for 5 consecutive days every 21 days, and repeated for 8 cycles.

[0267] In other embodiments, the method also requires co-administration of an effective number of CAR-expressing immune cells (also referred to as CAR immune cells) in the same course of treatment in which the CAR adapter molecule is administered. The term "effective number of CAR immune cells" as used herein (indirectly including a corresponding number of CARs) refers to a number of CAR immune cells sufficient to provide the desired effect.

[0268] More generally, the order in which the CAR adapter molecule and the CAR immune cell are administered during the same treatment process may not be important, as long as they are able to interact in vivo and produce the desired effect. In some embodiments, the CAR adapter molecule and the CAR immune cell are co-administered to the subject at substantially the same time. In some embodiments, the CAR adapter molecule is contacted with the CAR immune cell in vitro prior to co-administration to the subject. In some embodiments, the CAR adapter molecule is administered to the subject after the CAR immune cell is administered. In some embodiments, the CAR adapter molecule is administered to the subject before the CAR immune cell is administered.

[0269] The extracellular domain of a CAR that binds to the extracellular domain of a cancer antigen may comprise an antibody fragment. In some embodiments, the CAR binds to BCMA. Extracellular domains of CARs that bind to BCMA are known in the art. See, for example, the FDA-approved CAR-expressing immune cell, Cidaciolinsel. and Ekiviron U.S. Patents 10,072,088, 10,683,369, 11,084,880, and 10,174,095, and U.S. Patent Application Publications 2016 / 0131655, 2017 / 0226216, 2018 / 0133296, 2019 / 0151365, 2019 / 0359727, 2019 / 0381171, 2020 / 0339699, 2020 / 0360431, 2020 / 0055948, and 2022 / 0064316. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-BCMA antibody, BCMA-binding fragment, or derivative thereof, e.g., mabetuzumab. Linvoseltamab (REGN5458), pacanatuzumab (AMG 420), parvulumab (AMG 701), and terituzumab In some embodiments, the extracellular domain of the CAR will bind to the BCMA extracellular domain of a CAR adapter molecule having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0270] In some embodiments, the CAR binds to CD19. Extracellular domains of CARs that bind to CD19 are known in the art. See, for example, FDA-approved immune cells expressing CARs, lisocabtagene maraleucel. tisagenlecleucel Brexucabtagene autoleucel and axicabtagene ciloleucel U.S. Patents 9,629,877, 10,273,300, and 10,533,055, and U.S. Patent Application Publication Nos. 2020 / 0392248 and 2021 / 0238253. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD19 antibody, anti-CD19 binding fragment, or derivative thereof, such as loncastuximab. tafasitamab Denintuzumab (SGN-CD19A) and inebilizumab In some embodiments, the extracellular domain of the CAR binds to the CD19 extracellular domain of a CAR adapter molecule having the amino acid sequence of any one of SEQ ID NOs: 3-5 or 103.

[0271] In some embodiments, the CAR binds to CD20. CAR extracellular domains that bind to CD20 are known in the art. See, for example, U.S. Patents 10,189,903, 10,442,867, 10,934,363, 11,066,457, 11,160,833, and 11,439,665, and U.S. Patent Application Publication 2018 / 0187149. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD20 antibody, anti-CD20 binding fragment, or derivative thereof, such as ofatumumab. Velutuzumab (IMMU-106), tositumomab and rituximab In some embodiments, the extracellular domain of the CAR binds to the CD20 extracellular domain of a CAR adapter molecule having the amino acid sequence of SEQ ID NO:6.

[0272] In some embodiments, CAR binds to CD22. In some embodiments, the extracellular domain of CAR binds to the CD22 extracellular domain of the CAR adapter molecule. CAR extracellular domains that bind to CD22 are known in the art. See, for example, U.S. Patents 9,139,649, 9,181,343, and 10,494,435, U.S. Patent Application Publications 2015 / 0175711, 2018 / 0086843, 2021 / 0047402, 2021 / 0095022, 2022 / 0220198, and 2022 / 0273710, and Fry et al., Nat. Med. 24 (1): 20-28 (2018). In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD22 antibody, an anti-CD22 binding fragment, or a derivative thereof, such as bectumomab, epratuzumab, inotuzumab, moxetumomab, and epratuzumab. In some embodiments, the CAR adapter molecule comprises a CD22 extracellular domain having an amino acid sequence of SEQ ID NO: 7. In some embodiments, the CAR adapter molecule comprises a CD22 extracellular domain having an amino acid sequence of SEQ ID No: 104-107.

[0273] In some embodiments, the CAR binds to SLAMF7. The extracellular domain of CAR that binds to SLAMF7 is known in the art. See, for example, U.S. Patent No. 10,799,536 and U.S. Patent Application Publication Nos. 2020 / 0024342, 2020 / 0283534, 2021 / 0230548, and 2021 / 0253729. In some embodiments, the extracellular domain of CAR is derived from a commercially available anti-SLAMF7 antibody, an anti-SLAMF7 binding fragment, or a derivative thereof, such as elotuzumab. In some embodiments, the extracellular domain of the CAR binds to the SLAMF7 extracellular domain of a CAR adapter molecule having the amino acid sequence of SEQ ID NO: 10.

[0274] In some embodiments, the CAR binds to PD-1. The CAR extracellular domain that binds to PD-1 is known in the art. See, for example, U.S. Patents 10,124,023 and 11,136,392 and U.S. Patent Application Publications 2021 / 0061877, 2020 / 0281974, and 2022 / 0064595. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-PD-1 antibody, an anti-PD-1 binding fragment, or a derivative thereof, for example, batilimab, bruglimab, cadonilimab, cemiplimab, or the like. cetrelimab, dostalimab

[0275] Izuralimab, nivolumab Pacmizumab, pembrolizumab Penpulimab, peresolimab, pidilizumab, retifanlimab, rosnilimab, sintilimab, spartalizumab, tislelizumab, toripalimab, volrustomig, vodalizumab, zeluvalimab and zimberelimab. Therefore, in some embodiments, the extracellular domain of CAR binds to the PD-1 extracellular domain of the CAR adapter molecule having the amino acid sequence SEQ ID NO: 11.

[0276] In some embodiments, CAR binds to the receptor tyrosine kinase KIT proto-oncogene (KIT). The CAR extracellular domain binding to KIT is known in the art. See, for example, U.S. Patent Application Publications 2017 / 0335281, 2020 / 0048359, 2020 / 0071397, and 2021 / 0299177. In some embodiments, the CAR extracellular domain is derived from commercially available anti-KIT antibodies, anti-KIT binding fragments, or derivatives thereof, such as barzolvolimab. In some embodiments, the extracellular domain of CAR binds to the KIT extracellular domain of a CAR adapter molecule having an amino acid sequence of SEQ ID NO: 12.

[0277] In some embodiments, the CAR binds to CD38. CAR extracellular domains that bind to CD38 are known in the art. See, for example, U.S. Patents 10,709,775, 10,799,536, 10,836,998, and 11,365,394 and U.S. Patent Application Publications 2017 / 0296623, 2019 / 0135894, 2019 / 0135937, 2020 / 0308541, 2021 / 0046118, and 2022 / 0202859. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD38 antibody, an anti-CD38 binding fragment, or a derivative thereof, such as daratumumab. Isatuximab In some embodiments, the extracellular domain of the CAR binds to the CD38 extracellular domain of the CAR adapter molecule having the amino acid sequence of SEQ ID NO: 14.

[0278] The intracellular domain of CAR includes a signal transduction domain that can realize intracellular signal transduction and immune cell function.The signal transduction domain may include a primary signal transduction domain and / or a costimulatory signal transduction domain.In some embodiments, the intracellular domain can transmit a signal close to the natural connection of a molecule or receptor complex (such as a TCR receptor complex) containing ITAM.

[0279] In some embodiments, the signaling domain includes multiple, for example, two or three, costimulatory signaling domains, selected from, for example, 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the signaling domain may include a CD3ζ domain as the primary signaling domain and any of the following pairs of costimulatory signaling domains in the extracellular to intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; 4-1BB-CD3ζ; CD3ζ-4-1BB; CD28-CD3ζ; CD3ζ-CD28; CD28-4-1BB and 4-1BB-CD28. In some embodiments, the primary signaling domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some embodiments, the costimulatory signaling domain is derived from one or more of CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD68, CD72, CD80, CD86, CD137 (4-1BB; TNFRSF9), CD154, CLEC-1, 4-1BB, DAP10 (hematopoietic cell signal transducer (HCST)), DAP12 (TYROBP), Dectin-1, FcαRI, FcγRI, FcγRII, FcγRIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70.

[0280] A representative CAR with a CD3ζ stimulatory signaling domain is the FDA-approved CAR-expressing immune cell tisagencleucel. A representative CAR with CD3ζ and 4-1BB costimulatory signaling domains is the FDA-approved CAR-expressing immune cell elkivironide. Leakey Myron and Cedar Oaks A representative CAR with CD28 and CD3ζ costimulatory signaling domains is the FDA-approved CAR-expressing immune cell BRIC. and Akilum

[0281] In some embodiments, CAR immune cells are T cells. In some embodiments, CAR immune cells are NK cells. Other CAR immune cells are known in the art, for example, U.S. Patents 5,906,936, 7,446,190, 7,741,465, 8,389,282, 8,399,645, 9,422,351, 9,790,267, 9,885,298, 10,124,023, 10,815,301 and 11,433,100 and U.S. Patent Application Publications 2019 / 0375815, 2020 / 0281973, 2021 / 0300986, 2022 / 0056101 and 2022 / 0193138.

[0282] Without being bound by theory, the interaction between the CAR adapter molecule and the CAR does not result in aggregation and synapse formation. In contrast, cancer antigens cause aggregation and synapse formation between the CAR and the cancer antigens on the surface of the CAR immune cell. Therefore, the binding of the CAR adapter molecule to the CAR immune cell is short-term and reversible, which ensures that the CAR immune cell can bind to the cancer cell through the CAR in the presence of the CAR adapter molecule.

[0283] The number of CAR immune cells administered to a subject will vary widely depending on the location, type, and severity of the cancer, the age, weight, and physical condition of the patient, and other factors. The physician will ultimately determine the appropriate number and dose of cells to be used. Typically, CAR immune cells will be administered in a single dose.

[0284] In some embodiments, the effective number of CAR immune cells is about 1×10 5 to about 1×10 10 In some embodiments, the effective number of CAR immune cells is about 1×10 per kg of subject body weight. 5 about 6×10 8 Between cells.

[0285] Because the CAR adapter molecule enhances the function and in vivo persistence of CAR immune cells, it can reduce the cell dose required for CAR immune cell therapy, which in turn can reduce adverse side effects (e.g., cytokine release syndrome) caused by the larger doses commonly used in the clinic. Therefore, in some embodiments, the effective number of CAR immune cells is about 1×10 per subject. 4 to about 1×10 7 In some embodiments, the effective number of CAR immune cells is about 1×10 per kg of subject body weight. 4 about 6×10 5 between cells.

[0286] The composition comprising an effective amount of CAR adapter molecules and an effective number of CAR immune cells can be administered to a subject by any medically acceptable route to treat cancer. CAR adapter molecules and CAR immune cells are typically delivered intravenously, but they may also be introduced into other convenient sites (e.g., to an affected organ or tissue) or modes, as determined by the attending physician.

[0287] Expanders and differentiators can be provided before, during, or after cell administration to increase the differentiation, expansion, and / or persistence of CAR immune cells (e.g., T cells and NK cells).

[0288] Administration of CAR immune cells can be autologous or allogeneic. For example, immune cells or their progenitor cells can be isolated from a subject's bodily fluid tissue prior to administration to the same subject (autologous) or to a different compatible subject (allogeneic).

[0289] In some embodiments, the CAR adapter molecule is administered periodically, for example, once a week, once every two weeks, or once every three weeks. The cycle is repeated, for example, 2 cycles, 3 cycles, 5 cycles, or 8 cycles. In some embodiments, the CAR adapter molecule is administered for several consecutive days prior to the cyclic administration, for example, once a day for five consecutive days, and once every three weeks thereafter. In some embodiments, the CAR adapter molecule is administered as an intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some embodiments, the infusion time is between 30 minutes and 60 minutes. In some embodiments, the first administration is injected into the patient over 90 minutes, and the other administrations are injected into the patient over 30 minutes.

[0290] Combination therapy

[0291] In some embodiments, the present method may include administering another anticancer agent in conjunction with a CAR adapter molecule or a CAR adapter molecule and a CAR immune cell. The term "combined administration" includes administration by the same or different dosage forms substantially simultaneously, or sequentially, for example, as part of the same treatment regimen or by a sequential treatment regimen. Therefore, if the sequence is administered, at the start of administration of the second therapy, in some cases, the first of the two therapies is still detectable at an effective concentration at the treatment site. The order and time interval can be determined so that they can act together (e.g., synergistically to provide a greater benefit than otherwise administered). For example, therapeutic agents can be administered sequentially in any order, simultaneously or at different time points; however, if not administered simultaneously, they can be administered within a sufficiently close time to provide the desired therapeutic effect, which can be a collaborative approach. Therefore, these terms are not limited to administering active agents at exactly the same time.

[0292] The anticancer agents that can be used in combination with the cells of the present invention are known in the art.See, for example, U.S. Patent number 9,101,622 (its Section 5.2). For example, by killing cancer cells, inducing cancer cell apoptosis, reducing cancer cell growth rate, reducing metastasis incidence or quantity, reducing tumor volume, inhibiting tumor growth, reducing the blood supply to tumor or cancer cells, promoting the immune response to cancer cells or tumors, preventing or inhibiting cancer progression or increasing the life span of cancer subjects, "anticancer" agents can have a negative impact on the cancer of the subject. More generally, these other compositions will be provided with a combined amount that effectively kills or inhibits cancer cell proliferation. The process can be related to contacting cancer cells with receptor cells and one or more agents or multiple factors simultaneously. This can be by contacting cancer cells with a single composition or pharmacological preparation comprising two agents, or by contacting cancer cells with two different compositions or preparations simultaneously to achieve, wherein a composition comprises receptor cells, and another comprises a second agent.

[0293] In some embodiments, the CAR adapter molecules and CAR immune cells of the present disclosure are used in combination with or following prior therapy such as chemotherapy, radiotherapy, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.

[0294] In some embodiments, before administering genetically modified immune cells, the CAR adapter molecules of the present disclosure and CAR immune cells are used in combination with high-dose chemotherapeutic agents. In some embodiments, bone marrow cells or peripheral blood stem cells are administered after high-dose chemotherapeutic agents.

[0295] In some embodiments, the CAR adapter molecules and CAR immune cells of the present disclosure are used in combination with an effective amount of thalidomide, lenalidomide, bortezomib, or a combination thereof.

[0296] Other enhancement treatments that can be used in conjunction with the genetically modified immune cells of the present disclosure include melphalan. ) is an alkylating antineoplastic agent used for high-dose conditioning prior to hematopoietic stem cell transplantation in patients with multiple myeloma, as well as for the palliative treatment of multiple myeloma and palliative treatment of unresectable epithelial ovarian cancer. Melphalan is also used to treat AL amyloidosis, neuroblastoma, rhabdomyosarcoma, breast cancer, retinoblastoma of the eye, some conditioning regimens prior to bone marrow transplantation, and in some cases, malignant melanoma. Melphalan can be administered in the form of an oral pill. Typically, a 2 mg dose is taken on an empty stomach. In some cases, melphalan can be administered by injection or intravenous infusion. The dosage depends on weight, height, disease and condition state, and the general health of the subject.

[0297] Immunotherapy

[0298] Immunotherapy, including immune checkpoint inhibitors, can be used to treat established cancers. For example, immune checkpoint molecules include PD-1, PDL1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Examples of clinically available immune checkpoint inhibitors include durvalumab Atezolizumab and avelumab Clinically available examples of PD-1 inhibitors include nivolumab Pembrolizumab and cemipril imab Other inhibitors that may be useful in the practice of the present disclosure are known in the art. See, for example, U.S. Patent Application Publications 2012 / 0321637, 2014 / 0194442, and 2020 / 0155520.

[0299] chemotherapy

[0300] Anticancer therapy also includes combination therapy with various chemotherapeutic agents and radiation therapy. Combination chemotherapy includes, for example, Hexamethylmelamine, docetaxel, methotrexate, Cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, actinomycin D, daunorubicin, doxorubicin, bleomycin, mithramycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, Gemcitabine, Farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant thereof and combinations thereof.

[0301] Radiation therapy

[0302] Anticancer therapy also includes radiation-based DNA-damaging therapies. Combined radiation therapy, typically involving gamma radiation, X-rays, and / or the targeted delivery of radioisotopes to tumor cells, causes extensive damage to DNA, its replication and repair, and chromosome assembly and maintenance. The dosage range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumor cells. It will be determined by the attending physician.

[0303] Radiation therapy can include external radiation therapy or internal radiation therapy. External radiation therapy involves a radioactive source outside the body and delivers radiation to the cancerous area inside the body. Internal radiation therapy uses radioactive material sealed in needles, seeds, wires, or catheters that are placed directly in or near the cancer.

[0304] These and other aspects of the present application will be further understood upon consideration of the following examples, which are intended to illustrate certain embodiments of the application but are not intended to limit the scope as defined by the claims. Example

[0305] Example 1: Materials and Methods

[0306] Protein cloning and expression were performed according to standard methods. As briefly described below, other procedures, including flow cytometry analysis, BLI imaging, CAR T cell generation, cell culture, and animal treatment, were performed according to standard protocols.

[0307] Generation of CAR adapter molecules. All genes are codon-optimized, synthesized, and inserted into a vector expression system with a signal sequence for protein secretion into the supernatant for mammalian expression in HEK293 cells. In order to promote the production of products, a stable HEK293 cell line was generated. Therefore, HEK293 cells were transfected with pPAX2, pVSVG (packaging vector), and a lentiviral plasmid containing the sequence of interest. The lentivirus was harvested 48 hours, 72 hours, and 96 hours after transfection, precipitated at 20,000xg for 2h, and resuspended in optiMEM culture medium. Three rounds of viral transduction were then performed on a new batch of HEK293 cells. Cells were allowed to recover in DMEM complete medium and puromycin was selected to retain only cells that had integrated the lentiviral plasmid. Cells were then amplified in four 15cm culture dishes until they reached confluence, carefully washed with PBS, and incubated in serum-free DMEM for 24h to 48h. Supernatant was harvested, and protein expression was confirmed by SDS-PAGE and immunoblotting. The protein was purified by adsorption onto a nickel-nitrilotriacetic acid (Ni-NTA) metal affinity column. Nonspecifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM to allow recovery of the target protein. The protein was further purified by size exclusion chromatography and stored in 50 mM HEPES buffer, pH 7.5, at -80°C until use.

[0308] Typically, the adapter molecule is passed through an affinity chromatography resin in the presence of a neutral phosphate buffer to separate a portion of the CAR adapter molecule. The affinity chromatography resin is then placed in an acidic buffer having a pH of about 3 to about 4 to wash the CAR adapter molecule from the affinity chromatography resin. The acidic buffer can be neutralized with an alkaline buffer, and the neutralized buffer can then be subjected to tangential flow filtration using a formulation buffer to isolate a concentrated and purified solution containing the CAR adapter molecule.

[0309] Generation of CAR T cells. The CAR construct binding to human CD19 includes scFv derived from the anti-human CD19 antibody clone FMC69, followed by human CD28 and CD3ζ intracellular signaling domains. The CAR construct binding to human BCMA includes scFv derived from the anti-human BCMA antibody clone MSK54, followed by human 41BB and CD3ζ intracellular signaling domains. The human signaling CAR construct is transduced into HeLa cells, which stably produce pseudotyped γ-retrovirus with a feline endogenous virus (RD114) envelope, which has been shown to efficiently transduce human hematopoietic cells (HSC) (Ward et al., Mol. Ther. 8 (5): 804-12 (2003)). High virus titer clones are isolated by limiting dilution. High expression clones are inoculated and grown in DMEM complete medium containing 10% FBS until 80% confluence. The culture medium is replaced with RPMI complete medium containing 10% FBS. After 24 hours, the virus-containing medium was harvested, sterile-filtered using a 0.45 μm PES filter, and used to generate CAR T cells.

[0310] The generation of CAR T cells was adapted from previous studies. See, for example, Li et al., Methods Mol. Biol. 1514: 111-118 (2017). Briefly, whole blood was obtained from apheresis rings of healthy platelet donors because of the presence of a large number of viable leukocytes. Whole blood was centrifuged through a Ficoll gradient to isolate PBMCs. Intact PBMCs were used without selecting for CD8 + T cells. PBMCs were resuspended in 3 mL of culture medium per well of a 6-well plate. The culture medium was RPMI medium containing 10% fetal bovine serum (FBS), 200 IU / mL IL-2, 60 ng / mL IL-7, 10 ng / mL IL-15, 2 μg / mL anti-human CD3 (OKT3 clone), and 0.5 μg / mL anti-human CD28 (CD28.1 clone). The cell concentration was 4 × 10 6 cells / mL. After 24 hours, the cells were harvested, spun down, and resuspended in the same volume of fresh medium containing FBS, IL-2, IL-15, and IL-7, which also contained medium harvested from cells producing anti-human BCMACAR γ-retrovirus, resulting in γ-retrovirus-inoculated PBMCs. The PBMCs were then plated at 4 × 10 6Cells were plated at a density of 3 mL / mL in 6-well plates coated with 20 μg of recombinant human fibronectin (retronectin) (coated with 1 mL of PBS containing 20 μg / mL of recombinant human fibronectin at 4°C for 24 hours). PBMCs were centrifuged at 2000 × g for 1 hour at 30°C and cultured at 37°C. The transduction step was repeated with fresh medium containing γ-retrovirus, cytokines, and spin-inoculation. Flow cytometry analysis was used to assess the transduction efficiency of the CAR transgene using a dsRed reporter gene and recombinant BCMA labeled with AlexaFlour-647.

[0311] In vivo experiments. In all experiments, NOD / SCID / Gamma (NSG; NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) mice because of their immunocompromised state and ability to efficiently engraft human cancer cell lines. A multiple myeloma mouse model was established in NSG mice using cells from the human multiple myeloma OPM2 cell line. 1 × 10 6 In vivo experiments were initiated with OPM2 cells expressing GFP and firefly luciferase, followed by biweekly bioluminescence imaging (BLI). Three weeks after effective engraftment, mice were intravenously injected with CAR T cells via the tail vein. Tumor burden was assessed by BLI every two weeks thereafter. Quantification was performed using Aura software using photons / sec.

[0312] Organ analysis. At the end of the experiment, surviving mice were sacrificed and spleen, bone marrow, blood, liver, kidney and lung were harvested and weighed. The liver, kidney and lung were minced, digested with collagenase (final concentration of 1 μg / mL collagenase), and incubated at 37°C for 1 hour. The spleen was crushed and the bone marrow was aspirated using a 30-gauge insulin needle. All treated cells were pushed through a 70 μm filter to produce a single-cell suspension of cells. The cells were resuspended in 1 mL of ammonium-chloride-potassium (ACK) lysis buffer to consume 2 mL of red blood cells in the sample at room temperature. The resulting single-cell suspension was washed with fluorescence-activated single-cell sorting (FACS) buffer containing PBS and 0.5% BSA, stained, and analyzed using a flow cytometer.

[0313] Cell lines and culture. The OPM2 cell line, which endogenously expresses BCMA, was engineered to express green fluorescent protein (GFP) and firefly luciferase. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy platelet donors by apheresis using Ficoll gradients with leukocyte-depleted rings. HEK293T cells were cultured in complete DMEM (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), and 1% penicillin and streptomycin (Cytiva), with 10% fetal bovine serum (FBS). OPM2 and PBMCs were cultured in complete RPMI-1640 (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), and 1% penicillin and streptomycin (Cytiva), with 10% fetal bovine serum (FBS). All cells were grown in a 37°C, 5% CO2, 95% air humidified incubator.

[0314] Mouse studies. All experiments adhered to relevant ethical and safety protocols. These studies were conducted under the supervision of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (Protocol No. 20-006). The xenograft model used in this article is described in Smith et al., Mol. Ther. 26(6): 1447-1456 (2018). Briefly, 8- to 12-week-old NOD-scid IL2Rγ mice were purchased from Jackson Laboratory or cultured in-house. null (NSG) and NOD-scid H2-K1 null H2-Ab1 null H2-D1 null IL2Rg null (NSG-MHC I / II double knockout (DKO)). All mice were grouped by sex and age. 1×10 6 Xenograft models were established by inoculating OPM2 or Nalm6 cells expressing GFP and luciferase. Mice received the indicated treatments (in 300 mL PBS) via intraperitoneal injection. D-luciferin (150 mg / kg, from a 15 mg / ml solution) was injected intraperitoneally at the indicated times and then Lumina Series III (PerkinElmer) assesses tumor burden. A maximum of 5 mice were grouped, and each mouse was imaged in the supine position at the same time point (5 min). BLI intensity was analyzed by Aura imaging analysis software (Spectral Instruments Imaging). Mouse peripheral blood was obtained by submandibular bleeding into EDTA-coated tubes, and CAR T cell detection and amplification were analyzed. In short, the blood volume was determined to calculate the absolute value. The sample was then centrifuged and serum was collected. The cell pellet was resuspended in 500 mL-1000 mL ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 1 minute. The cells were then washed twice in FACS buffer, PBS + 1% bovine serum albumin (BSA). The samples were then stained with anti-CD45-Pacific Blue (1:50, Biolegend), anti-CD4-PE / Dazzle594 (1:50, Biolegend), anti-CD8-FITC (1:50, Biolegend), anti-CCR7-AlexaFluor700 (1:50, Biolegend), anti-CD62L-PE (1:50, Biolegend), anti-CD45RO-PerCP / Cy5.5 (1:50, Biolegend), anti-CD45RA-APC / Fire750 (1:50, Biolegend), anti-PD1-BV605 (1:50, Biolegend), anti-HLA-DR-PE / Cy7 (1:50, Biolegend), anti-CD69-BV421 (1:50, Biolegend) and recombinant BCMA-AlexaFlour647 (homemade). Samples were processed on a Sony SP6800 spectrometer. Flow rate and acquisition time were recorded to calculate absolute values. All experiments were performed in a blinded and randomized manner. Animals were euthanized at the end of the experiment or when the predetermined endpoint was reached according to the IACUC protocol. At the endpoint, major immune organs (spleen and bone marrow) and vital organs (liver, lungs, and kidneys) that may form metastatic lesions were collected, weighed, and analyzed. In brief, the spleen was crushed using a plunger and passed through a 40 μm filter to obtain a single cell suspension. Bone marrow was aspirated using a 30-gauge insulin needle. The liver, lungs, and kidneys were cut open with surgical scissors in 3 mL of digestion buffer (1 mL RPMI + 2 mL PBS). Type I collagenase (Worthington) was added at a final concentration of 100 mg / mL and incubated at 37°C for 1 hour. The resulting sample was passed through a 40 μm filter to obtain a single cell suspension. The sample was then stained with the same antibody used to stain the blood sample and analyzed using a Sony SP6800 spectrometer.The flow rate and acquisition time were recorded to calculate the absolute value.

[0315] Microscopy. Cells were first stained with CellTracker Blue CMAC and seeded on poly-D-lysine coated coverslips. Subsequently, samples were incubated with the indicated therapeutics labeled with Alexa647 for the indicated times and temperatures. After fixation with BD Cytofix buffer, cells were imaged using a Leica THUNDERImager. The intensity threshold of the AlexaFluor647 channel was set to 2000 for all images except those shown on the right, with the exception of the VHH-muIL2 sample (condition 4) on the right, where the image sensitivity was enhanced 25-fold (intensity threshold 80) to visualize the Alexa647 signal. Quantitative analysis of fluorescence intensity was analyzed by aligning the base 2 logarithm of the ratio of membrane to cytoplasm integrated intensity (see Figure 14B For BCMA-muIL2 and BCMA-CH3 (conditions 1 and 2), only cells with a mean intensity to background ratio higher than 4 based on the dsRed channel were analyzed, as they were identified as CAR + For VHH-muIL2, cells with a mean intensity to background ratio greater than 2 in the Alexa 647 channel were selected to eliminate background artifacts. Image quantification was performed using ImageJ software.

[0316] ELISA. Figure 17A ELISA analysis was performed to measure the levels of human T cell-derived cytokines in the serum of mice that received low-dose CAR T cells after receiving OPM2 cancer cells as shown. ELISAMAX was used according to the protein provided by the manufacturer. TM The cytokines analyzed using the Standard Set (Biolegend) include IFN-γ, GM-CSF, and TNF-α; however, only IFN-γ was detectable in the collected samples. Serum samples were diluted 1:40. Standard samples and serum samples were incubated on the same plate, and a standard curve for each cytokine was generated.

[0317] Example 2: In vitro characterization of BCMA-containing CAR adapter molecules.

[0318] A fusion protein consisting of the extracellular domain of human BCMA was fused to two low-affinity mutant human IL-2 (muIL2) domains, and in order to improve pharmacokinetics and enhance stability, the CH3 domain of human IgG1 (approximately 14 kDa) (Feige et al., Trends Biochem. Sci. 35(4):189-198 (2010)) was incorporated between the antigen and muIL2 ( Figure 2B)(Quayle et al., Clin. Cancer Res. 26(8):1953-1964(2020)). Therefore, the BCMA-muIL2 CAR adapter molecule preferentially delivers low-affinity IL-2 to the surface of CAR T cells through antigen-CAR specific binding, thereby minimizing the impact on normal T cells, Tregs or systemic toxicity.

[0319] Recently, it was demonstrated that IL-2 induces an alternative differentiation pathway for T cells, leading to the generation of a unique “better effector” CD8 + T cells (Hashimoto et al., Nature 610(7930):173-181(2022)). This process may be at least partially dependent on the binding of IL-2 to IL-2Rα. In addition, IL-2Rβγ-biased agonists can drive T cells toward a terminally differentiated state (Codarri et al., Nature 610(7930):161-172(2022)). CAR adapter molecules may be able to anchor low-affinity IL-2 on the surface of CAR T cells through antigen-CAR binding to overcome the need for IL-2Rα in another differentiation pathway, thereby promoting the generation of memory CAR T cells. There may also be potential synergistic effects between CAR signaling and IL-2 signaling.

[0320] To evaluate the binding affinity of BCMA-containing CAR adapter molecules, flow cytometric analysis was performed by staining BCMA CART T cells with different concentrations of BCMA CART adapter molecules. 50 is approximately 0.21 nM, which is comparable to that of dimer BCMA lacking muIL2(BCMA-CH3) ( Figure 2E ), indicating that the binding is mainly due to the BCMA ectodomain rather than muIL2. Figure 2EDose-dependent staining of BCMA CAR T cells using CAR adapter molecules using flow cytometry is shown (n=3 for each point), untransduced T cells were used as controls, and Alexa647-labeled anti-FLAG secondary antibodies were used for staining. Error bars represent mean values ​​and 95% confidence intervals. Minimal binding of the BCMACAR adapter molecule to untransduced T cells and minimal binding to VHH-muIL2 were observed. VHH-muIL2 is a control construct that replaces the BCMA extracellular domain in the CAR adapter molecule CH3-muIL2 construct with an unrelated nanobody (VHH). This observation further indicates that the binding of the CAR adapter molecule to CAR T cells is mainly driven by the BCMA antigen, and muIL2 exhibits weak binding to CAR T and untransduced T cells. The BCMA-muIL2 CAR adapter molecule did not exhibit binding to any immune cell population in human peripheral blood mononuclear cells (PBMCs) ( Figure 13A – Figure 13B ).

[0321] Next, the functional effects of BCMACAR adapter molecules on BCMACAR T cells were evaluated. After a 24-hour resting period without cytokines, BCMACAR T cells were incubated with different concentrations of BCMA-muIL2 CAR adapter molecules for 24 hours, and then the expression of CD69 activation markers was evaluated using flow cytometric analysis (Cibrián and Sánchez-Madrid, Eur. J. Immunol. 47(6):946-953(2017)). The results showed that CD69 expression on CAR T cells increased in a dose-dependent and selective manner ( Figure 2F ), whereas no effect was observed on non-transduced T cells ( Figure 2G ). In addition, BCMACAR adapter molecule treatment resulted in a significant increase in CD69 expression compared to VHH-muIL2, BCMA-CH3, or their combination, indicating that the observed effect was only significant when low-affinity IL-2 was fused to the antigen. Unpaired t-tests showed that at concentrations of 0.1 nM or higher, the increased activation in the BCMA-muIL2-treated group compared to the VHH-muIL2, BCMA-CH3, or their combination control group was statistically significant (P ≤ 0.0001) (error bars represent mean values ​​and 95% confidence intervals) ( Figure 2F ). Figure 2G Zero treatment and CD3 / CD28 activation in were used as negative and positive controls, respectively (error bars represent mean values ​​and 95% confidence intervals).

[0322] BCMACAR-E did not inhibit the killing efficacy of BCMACAR T cells.Since both CAR adapter molecules and cancer antigens bind to CAR, the potential inhibitory effect of BCMACAR adapter molecules on the killing activity of BCMACAR T cells was investigated. To conduct this study, BCMACAR T cells and patient-derived BCMA were used in the presence of different concentrations of BCMACAR adapter molecules. + OPM2 cancer cell killing assay was performed. Notably, the results showed that even at the highest tested concentration (100 nM of CAR adapter molecule) Figure 2H ), nor did it inhibit killing. OPM2 cells were co-incubated with BCMA CAR T cells (shown in red) or untransduced T cells (shown in gray) in the presence of different concentrations of BCMA-muIL2 CAR-E therapeutic (E:T ratio 1:1; 30,000 cells each). After 48 hours, viable (PI - ) OPM2 cells were counted, and N for each experiment was 3. Figure 2H The error bars in represent mean and standard deviation. Without being bound by theory, this result may be attributed to the reversibility of the binding of CAR adapter molecules to CAR, and the killing process involving the aggregation effect and synapse formation between CAR and cancer antigens is an irreversible event. In addition, the binding affinity of CAR to membrane-bound BCMA may exceed the binding affinity of CAR to soluble antigens, thereby contributing to this result. It is worth noting that due to the shedding caused by gamma-secretase, multiple myeloma patients show high levels of soluble BCMA (Laurent et al., Nat. Commun. 6:7333 1-12 (2015)) in their circulation. Nevertheless, BCMACAR T cells produce a striking initial response in patients, which shows that soluble BCMA antigens do not suppress the activity of CAR T cells. The experimental results disclosed herein are consistent with these early results.

[0323] BCMACAR adaptor molecules selectively enhance the efficacy of IL-2 by delivering low-affinity IL-2 in cis to the same targeted CAR T cells. induced STAT5 activity in CAR T cells. It is known that IL-2 exhibits strong activity on T cells, and STAT5 phosphorylation is both a reliable indicator of IL-2 / IL-2R binding and is associated with downstream effects such as phenotypic marker expression and cell proliferation (Jones et al., J. Immunol. 205(7):1721-1730(2020)). To evaluate the effect of BCMACAR adapter molecules on STAT5 activity, BCMACAR T cells were exposed to different concentrations of BCMACAR adapter molecules. After incubation at 37°C for 5 minutes, the cells were fixed and stained for pY694 STAT5. The results showed that BCMA-muIL2 CAR adapter molecules induced STAT5 phosphorylation in CAR T cells, EC 50 About 0.014nM ( Figure 2I In contrast, the VHH-muIL2 control required a higher concentration (EC 50 =3.9nM) to induce STAT5 phosphorylation, indicating that BCMA-mediated low-affinity IL-2 delivery to the surface of CAR T cells significantly increased the sensitivity of muIL-2 by more than 200 times. Wild-type IL-2 showed a lower EC 50 (approximately 0.001 nM), suggesting differences in signaling kinetics. Without being bound by theory, the two-step process involved in STAT5 activity mediated by the BCMA-muIL2 CAR adaptor molecule includes: (i) binding of the antigen on the T cell surface to the CAR, and (ii) the subsequent interaction of low-affinity IL-2 with the adjacent IL-2R, which may account for the measured differences. In contrast, wild-type IL-2 only requires binding to the IL-2R, enabling it to induce STAT5 activity more rapidly. The fact that VHH-muIL2 can activate STAT5 solely through low-affinity IL-2 may explain why it requires higher concentrations to induce STAT5 activity in T cells.

[0324] BCMA CART cells pre-blocked with BCMA-CH3 showed a significant reduction in pSTAT5 levels to the same extent as the control VHH-muIL2, confirming that the efficacy of the CAR adapter molecule is mediated by antigen binding to the CAR ( Figure 2I ). To determine whether the binding of CAR adapter molecules to target cells will lead to STAT5 signaling (transactivation) in adjacent cells, unblocked and pre-blocked BCMA C AR T cells were co-cultured in the presence of different concentrations of CAR adapter molecules. Compared with co-cultured unblocked CAR T cells, the pSTAT5 level of pre-blocked CAR T cells was lower, indicating that CAR adapter molecules affect targeted CAR T cells (cis activation) but do not affect adjacent cells. BCMA C AR T cells were treated with the indicated therapeutic agents for 5 min at 37 ° C, and STAT5 phosphorylation was assessed. For the pre-blocking experiment, BCMA CAR T cells were treated with BCMA-CH3 (100 nM) at 4 ° C for 20 min and then exposed to CAR adapter molecules for 5 min at 37 ° C (n = 3 for each condition). Figure 2I The error bars represent the mean and standard deviation. In summary, the analysis of STAT5 activity supports the view that the BCMA CAR adaptor molecule exerts its effect on targeted CAR T cells through cis delivery of low-affinity IL-2, an effect mediated by antigen binding to the CAR.

[0325] Example 3: The immune cell effector domain of the CAR adapter molecule stimulates T cells independently of the CAR.

[0326] To demonstrate that the CAR adaptor molecule immune cell effector domain stimulates immune cells, the following experiment was performed. Figure 3A As shown, peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3, anti-CD28, IL-2, IL-7 and IL-15 to generate activated T cells that were not transduced with any exogenous transgenes. Activated T cells were treated with the following reagents for 4 days: interleukin (recombinant human IL-2 without a glycosyl unit), a weak affinity variant CAR adapter molecule comprising an N-terminal extracellular domain, a CH3 domain (about 14 kDa) and two repeated IL-2 immune cell effector domains that bind BCMA (about 7 kDa), having the overall structure of BCMA-CH3-muIL2-muIL2, and referred to herein as BCMA-muIL2, or a CAR adapter molecule comprising an extracellular domain, a CH3 domain and a Neo-2 / 15 immune cell effector domain that binds BCMA, whose overall structure is BCMA-CH3-Neo-2 / 15, referred to herein as BCMA-Neo-2 / 15. After treatment, T cells were counted and stained with carboxyfluorescein succinimidyl ester (CFSE), and the mean fluorescence intensity (MFI) of CFSE was analyzed to determine T cell division.

[0327] T cell counts and divisions (CFSE staining) achieved by T cells treated with the CAR adapter molecules BCMA-muIL2 or BCMA-Neo-2 / 15 were similar to those achieved with tesileukin, which is known to activate T cells; however, CAR adapter molecule treatment was less sensitive than tesileukin treatment. Systemic administration of IL-2 is associated with serious side effects (Rosenberg, J. Immunol. 192(12):5451-5458(2014); Dutcher et al., J. Immunother. Cancer. 2(1):26 1-23(2014); Pachella et al., J. Adv. Pract. Oncol. 6(3):212-221(2015)), including vascular leak syndrome and CD4 + CD25 + The preferential expansion of regulatory T (Treg) cells is known to cause immunosuppression. The results published so far show that when used at low concentrations, the CAR adapter molecule does not activate normal T cells, and when attached to the extracellular domain of the CAR adapter molecule, the stimulatory immune cell effector domain retains its normal function of activating T cells.

[0328] Example 4: CAR adapter molecules specifically activate CAR T cells through the extracellular domain

[0329] To demonstrate that the immune cell effector domain of the CAR adaptor molecule stimulates immune cells, the following experiments were performed. Figure 4A As shown, PBMCs are stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to produce activated T cells, which are then transduced with a vector containing CAR. Activated CAR-expressing T cells (CAR T cells) are left to rest for 24 hours and then treated with a CAR adapter molecule containing an immune cell effector domain or a CAR adapter molecule lacking an immune cell effector domain as an extracellular domain control.

[0330] T cell activation tests were performed on CAR adapter molecules containing a BCMA extracellular domain, a CH3 domain, and an immune cell effector domain comprising 4-1BBL (BCMA-41BBL), weak affinity IL-2 (BCMA-muIL2), or Neo-2 / 15 (BCMA-Neo-2 / 15). In order to test the extracellular domain specificity of the CAR adapter molecule for CAR T cells (as a control), T cell activation of unrelated nanobodies of the intracellular protein UBC6E (VHH6E) fused to the CH3 domain and 4-1BBL (VHH6E-41BBL) or weak affinity IL-2 (VHH6E-muIL2) was tested. Another extracellular domain specific control contained a nanobody (clone NJB2, abbreviated as NJB2-VHH) that bound FN1 and was fused to the CH3 domain and the Neo-2 / 15 stimulator (NJB2-VHH-Neo-2 / 15) to perform a T cell activation test. The extracellular domain-specific control has a similar overall structure to the CAR adapter molecule used in this experiment (protein domain-CH3-muIL2-muIL2 or protein domain-CH3-Neo-2 / 15). The extracellular domain-specific control and CAR adapter molecule were incubated with CART cells for 10 hours, and the cells were stained for CD69 (as an activation marker) and measured by flow cytometry.

[0331] All BCMA extracellular domain CAR adapter molecules induced CD69 expression in CAR T cells ( Figure 4B ). BCMA-CH3-Neo-2 / 15 has the lowest threshold for inducing CD69 expression in CAR T cells (0.01nM CAR adapter molecule). BCMA-CH3 protein (lacking the immune cell effector domain) still has little effect on the expression of CD69 on CAR T cells at the highest tested concentration (10nM BCMA-CH3 protein). None of the extracellular domain-specific control proteins induced CAR T cell expression of CD69. These results indicate that CAR adapter molecules containing cancer antigen extracellular domains specifically activate CAR T cells that express CARs that recognize the cancer antigen extracellular domain of the CAR adapter molecule.

[0332] Example 5: CAR adapter molecules stimulate CAR T cells to kill target cells

[0333] To demonstrate that CAR adapter molecules do not inhibit CAR T1 cell killing, the following experiments were performed. CAR T cells were generated as described above and co-expressed with CAR adapter molecules and BCMA. + Multiple myeloma cancer cells were co-cultured with CAR T cells and OPM2BCMA + The cells were incubated at an E:T ratio of 1:1 for 1 day, and the target cell survival rate was analyzed compared with that of target cells incubated without T cells ( Figure 5 A).

[0334] BCMA extracellular domain CAR adapter molecules with muIL2 (BCMA-CH3-muIL2) or 4-1BBL (BCMA-CH3-41BBL) immune cell effector domains did not inhibit the killing of OPM2 cells ( Figure 5 B) These results indicate that CAR adapter molecules comprising a cancer antigen extracellular domain and a stimulatory immune cell effector domain do not inhibit CAR T cell killing of target cells that also express the same cancer antigen as the CAR adapter molecule.

[0335] Example 6: CAR adapter molecules reduce tumor burden, prolong survival, and extend the persistence of CART cells in vivo sex

[0336] To demonstrate that CAR adaptor molecules reduce tumor burden, prolong CAR T cell persistence in vivo, and extend survival, the following experiments were performed. 5 10 days before the onset of anti-BCMACAR T cells, 1×10 6 OPM2BCMA + Multiple myeloma cancer cells were injected intravenously (iv) into NOD-scid IL2Rγ null (NSG) mice. After CAR T cell infusion, mice were treated twice a week for two weeks, followed by weekly intraperitoneal (ip) injections of 200 μg / mouse of a CAR adapter molecule containing the BCMA extracellular domain, the CH3 domain, and two weak-affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) ( Figure 6A ).

[0337] exist Figure 6B- On the days shown in Figure 6D, mice were imaged for luciferase bioluminescence (BLI) (indicating tumor burden of luciferase+OPM2 cells). During the experiment, the tumor burden of the control group of mice that received OPM2 cells without CAR T cell infusion gradually increased and reached humane endpoints on days 39 and 46. Tumor growth in mice that received OPM2 cells and suboptimal doses of CAR T cells was controlled during the experiment until day 32, when they also showed a gradual increase in tumor burden and reached humane endpoints on day 46. Mice treated with OPM2 cells, CAR T cells, and CAR adapter molecules had a reduced tumor burden ( Figure 6B (Figure 6D). In the CAR adapter molecule treatment group, all mice completely cleared OPM2 tumor cells from the bone marrow, as no signal was detected by imaging. One mouse in this group had significant OPM2 cell growth due to solid tumor formation near the eye and reached the humane endpoint on day 42. The remaining two mice completely cleared OPM2 tumor cells, as no signal was detected by imaging, and survived the experiment.

[0338] Next, the in vivo persistence of OPM2 and CAR T cells in these mice was analyzed by flow cytometry. One control mouse (OPM2 cells, no CAR T cell infusion) was sacrificed on day 46, two CAR-only treated mice were sacrificed on day 46, and one CAR T cell and CAR adapter molecule treated mouse was sacrificed on day 42. The blood, spleen, lymph nodes, bone marrow, and lungs of the sacrificed mice were analyzed for GFP+OPM2 cells ( Figures 7A-7C ) and CD45 + CAR + T cells ( Figures 8A-8C ). We also analyzed GFP+OPM2 cells and CD45+CAR in the ocular tumor sites of mice treated with CART cells and CAR adapter molecules. + T cells.

[0339] 7A to 7C Flow cytometry is shown with GFP on the y-axis. In the bone marrow, lung ( Figure 7B ) and liver( Figure 7C ) were detected at similar levels of GFP + OPM2 cells. A CAR-only mouse had extremely high levels of OPM2 cells in the blood and spleen ( Figure 7A ).

[0340] One mouse treated with CAR T cells and a CAR adaptor molecule developed eye tumors and no tumors in the blood or spleen ( Figure 7A ), bone marrow or lung ( Figure 7B ) and liver( Figure 7C) had almost no OPM2 cells. This mouse had more OPM2 cells in the kidney (3.18% GFP + cells), and the majority of cells in the ocular tumor site were OPM2 cells (96.5% GFP + cell).

[0341] Figures 8A-8C Flow cytometry is shown with anti-CD45 on the y-axis and Alexa Fluor TM 647 (AF647)-labeled BCMA + -CH3 is on the x-axis. CD45 + CAR + T cells were present only in mice treated with both CAR T cells and the CAR adaptor molecule. Mice treated with CAR alone had almost no CD45 in all organs tested. + cell( Figures 8A-8C However, mice treated with CAR T cells + CAR adaptor molecules had CD45 + cells, which also stained positive for the AF647-labeled BCMA cancer antigen (also a CAR binding target), as shown on the x-axis. Figure 8A ), bone marrow and lungs ( Figure 8B ) and liver and kidneys ( Figure 8C ) + AF647 + Double-positive CAR T cells. CD45 was detected abundantly only in the liver and kidney. + Single positive cells ( Figure 8C CD45 was barely detected in ocular tumors. + AF647 + Double-positive CAR T cells ( Figure 8C These results suggest that the CAR adaptor molecule reduces tumor burden, prolongs the persistence of CAR T cells in vivo, and extends survival.

[0342] Example 7: CAR Adapter Molecule Outcomes

[0343] The CAR adapter molecule binds to CAR T cells on the cell surface at 4°C and is slowly internalized at 37°C.The internalization of the CAR adapter molecule was assessed using a fluorescently labeled BCMA-muIL2 CAR adapter molecule. BCMACAR T cells were exposed to BCMA-muIL2, BCMA-CH3, or VHH-muIL2 labeled with AlexaFluor647 at a concentration of 2 nM. The cells were incubated at 4°C or 37°C for different time intervals, then fixed and subsequently imaged under a microscope. It was observed that the control VHH-muIL2 was rapidly internalized within 30 minutes at 37°C, while the internalization of the BCMA-muIL2 CAR adapter molecule was significantly slower ( Figures 14A-14C ). The internalization rate of BCMA-CH3 is also slow, even slower than that of the BCMA-muIL2 CAR adapter molecule. Figure 2C In the CAR and dsRed transcripts are encoded within the transgene, and therefore the dsRed signal reflects the expression level of CAR. The average intensity above background is reported for all imaged cells. For the dsRed channel, the cytoplasmic average intensity is reported because dsRed is expressed intracellularly, while for the AlexaFluor 647 channel, the average intensity of the entire cell is measured.

[0344] The CAR adaptor molecule is rapidly cleared from the circulation. Stimulating CAR T cells with a CAR adapter molecule therapeutic (wherein the stimulation includes a stimulation period and a subsequent resting period) is superior to prolonged exposure to the CAR adapter molecule, as prolonged exposure can lead to exhaustion or the production of terminally differentiated CAR T cells. CAR adapter molecules with a short circulating half-life can more effectively expand CAR T cells, drive the generation of memory CAR T cells, reduce potential competition for tumor antigen binding to CAR, and improve patient safety. Therefore, the CH3 domain of IgG1 is used in the CAR adapter molecule platform. Pharmacokinetic studies have shown that the circulating half-life of CAR adapter molecules is short (1 hour-1.5 hours) ( Figure 9A). NSG mice were administered 8 mg / kg of BCMA-muil 2CAR adapter molecules (intraperitoneal delivery, N=3 mice). Blood samples were collected by tail vein puncture at five different time points after administration (30 min, 2 h, 8 h, 24 h, 48 h). Serum was then obtained by centrifugation and used for subsequent analysis. ELISA was performed to determine the concentration of the therapeutic agent in the serum. The ELISA plate was coated overnight with 5 μg / ml anti-His6 antibody and then incubated with serum at room temperature for 2 h. Next, anti-FLAG HRP antibody was used for detection; the C-terminus of the CAR adapter molecule was engineered to have a FLAG tag and a His6 tag. Based on the five time points collected, the initial concentration of the treatment in the serum was estimated to be 20% higher than the time point collected at the first (30 min). BCMACAR-E was cleared from the circulation by >90% and >99% at 8 h and 24 h, respectively. The circulation half-life of the BCMACAR adapter molecule is estimated to be approximately 1.5 hours. The error bars represent the mean and standard deviation.

[0345] BCMACAR adaptor molecules enhance the activity and persistence of CAR T cells in multiple myeloma (MM) models. Transplantation of OPM2 cells into immunocompromised NOD-SCID IL-2Rγ null (NSG) MM xenograft mouse model in mice. Accordingly, NSG mice were intravenously injected with OPM2 cells (human MM, 1 million cells) via the tail vein. After two weeks of injection of OPM2 cells, freshly prepared BCMACAR T cells (500,000 CARs containing 41BB-CD3 CAR constructs) were intravenously administered. + cells). One group of mice received the BCMA-muIL2 CAR adaptor molecule ( Figure 9B ). NSG mice (n=5) were injected with OPM2 (human MM) cells and then BCMA CAR (human) T cells were administered according to the schedule. BCMA-muIL2 CAR-E therapeutic agent (200 μg) was administered twice a week for two weeks and then once a week until the end point. After one month or more, the mice were euthanized and the harvested organs were analyzed by flow cytometry. These results revealed a great expansion of CAR T cells in the spleen and bone marrow of the BCMA-muIL2 CAR adapter molecule treatment group, indicating that CAR T cells were activated in the spleen ( Figure 9C , left) and bone marrow ( Figure 9C , right panel) compared to untreated animals that received only CAR T cells by more than 100-fold. These experiments were repeated multiple times with similar results ( Figure 9D and Figures 15A-15C CAR T cells only n=12, CAR T cells plus CAR adaptor molecule therapeutic n=22).

[0346] BCMA CART cells were detected by co-staining with anti-human CD45 antibody and Alexa 647-labeled BCMA antigen. Similar results were obtained in repeated experiments. Other control groups received VHH-muIL2 therapeutic agents at the same dose and schedule as BCMA-muIL2. Figure 9D Pooled data from these experiments are shown. Data were analyzed by group mean comparison using one-way ANOVA followed by Tukey post hoc analysis. The individual flow diagrams for the pooled data are shown in Figures 15A to 15C Error bars represent mean and standard deviation.

[0347] Compared with the control group that received only CAR T cells without therapeutic agents, the control group (n=7) that received CAR T cells + VHH-muIL2 therapeutic agents did not show significant expansion or persistence of CAR T cells. These results indicate that the BCMA-muIL2 CAR adapter molecule can expand CAR T cells in vivo. Further analysis revealed that the BCMA-muIL2 therapeutic agent had a more significant specific effect on CD8+ CAR T cells, resulting in their + and CD8 + The proportion of CAR T cells in the population unexpectedly increased significantly from an initial approximately 30% to approximately 70% ( Figure 9E Groups receiving only CAR T cells or CART cells with VHH-muIL2 control treatment did not generate sufficient numbers of persistent CAR cells for similar analysis. Data were analyzed by group mean comparison using one-way ANOVA followed by Tukey post hoc analysis. Error bars represent mean and standard deviation.

[0348] BCMACAR adaptor molecules enhance CAR T cell trafficking in MM models. The MM xenograft mouse model was developed by transplanting OPM2 cells into immunocompromised NSG mice. Accordingly, OPM2 cells (human MM, 1 million cells) were injected intravenously into NSG mice via the tail vein. Ten days after the injection of OPM2 cells, freshly prepared BCMACAR T cells (500,000 CARs containing the 41BB-CD3 CAR construct) were administered intravenously. + cells). One group of mice received BCMA-muIL2 CAR-adapter molecule therapy ( Figure 20A ). NSG mice (n=5) were injected with OPM2 (human MM) cells and then BCMA CAR (human) T cells were administered according to the schedule. BCMA-muIL2 CAR-E therapeutic agent (200 μg) was administered twice a week for two weeks and then once a week until the end point. After one month or more, the mice were euthanized and the harvested organs were analyzed by flow cytometry. These results ( Figure 20A) revealed that many CAR T cells trafficked to the spleen, bone marrow, liver, kidney, and lungs of the BCMA-muIL2 CAR adapter molecule-treated group, demonstrating the presence and persistence of CAR T cells in all major organs tested compared to untreated animals that received CAR T cells alone ( Figure 20B ).

[0349] Example 8: BCMACAR adapter molecule therapy enables low-dose CAR T cell CAR T therapy

[0350] To further demonstrate the efficacy of CAR adaptor therapy and the clearance of tumor cells by CAR T cells, a similar protocol as described above was performed. However, in this study, a lower dose of only 100,000 CAR T cells ( Figure 10A All mice treated with the BCMA-muIL2 CAR adaptor achieved complete tumor clearance (5 / 5), whereas none of the control mice that received CAR T cells alone (n=4) or CAR T cells combined with VHH-muIL2 (n=4) were able to eliminate their tumors ( Figure 10B-10C ).

[0351] Analysis of blood samples collected at different time points showed that circulating CAR T cells expanded significantly after CAR adaptor molecule treatment, with peak expansion observed at week 4 ( Figure 10D ). Flow cytometric analysis of blood samples showed that CAR T cells in the treatment group expanded significantly compared with the PBS or VHH-muIL2 groups. In contrast, although VHH-muIL2 treatment slightly enhanced the initial response, it did not induce a significant expansion of CAR T cells. The data on days 7, 14, and 21 were analyzed using a two-way ANOVA. After all mice in the PBS group were euthanized, BCMA-muIL2 and VHH-muIL2 were compared on days 28 and 35 using a multiplex Mann-Whitney test. The error bars represent the mean and standard error. This expansion was correlated with the level of IFN-γ detected in the circulation ( Figures 17A-17C Furthermore, the treatment promoted the generation of memory CAR T cells, demonstrating long-term effects ( Figure 10E and Figures 17A-17C ). * P<0.05, ** P < 0.01. Error bars represent the mean and standard error.

[0352] Mice treated with the CAR adaptor molecule showed no signs of toxicity based on clinical observations and body weight measurements ( Figure 10H Subsequent analysis two months after CAR T cell injection showed that a large number of CAR T cells, including memory CAR T cells, were present in the CAR adapter molecule-treated mice. Figure 10F-10J 、 Figures 16A-16C and Figures 17A-17C In the CAR+PBS group, CAR T cells were detected in the spleen; however, these mice succumbed to tumor growth approximately 20 days after CAR T cell injection. BCMA-muIL2 treatment also increased the presence of CAR T cells in the bone marrow compared with the PBS or VHH-muIL2 groups, but the difference was less significant than in the spleen. Data were analyzed by two-way ANOVA with Tukey's multiple comparison test. * P<0.05, *** P<0.001, **** P < 0.0001. Figures 16A-16C Error bars represent mean and standard error. Figure 10I Data from the study showed that CAR T cells persisting in the spleens of BCMA-muIL2-treated mice expressed CCR7 + CD45RA + CD62L + stem cell memory phenotype, which was absent in the CAR+VHH-muIL2 or CAR+PBS groups.

[0353] tSNE analysis revealed the presence of distinct memory T cell populations based on surface marker expression of CD8a, CD4, CD45, CD45RA, CD45RO, CD62L, CD69, PD-1, HLA-DR, CCR7, and BCMA-CAR. CAR T cells were detected in the bone marrow but not in the spleen of the VHH-muIL2-treated group. In the BCMA-muIL2 group, the persisting CAR T cells were primarily CD8 T cells, whereas in the VHH-muIL2 group, the majority of bone marrow CAR T cells were CD4 T cells. Further analyses, such as Figures 17A-17C shown. Figure 17C The Flt-SNE map shown in the figure is derived from Figure 10A CAR T cells of mice treated with PBS, BCMA-muIL2 and VHH-muIL2 as shown in . The expression of ten immune cell markers (CD45-Pacific Blue, CD8-FITC, CD4-PE Dazzle594, BCMA-CAR (antigen)-AlexaFluor647, CD69-BV421, PD-1-BV605, CD45RA-APC-Cy7, CD45RO-PerCP-Cy5.5, CD62L-PE, CCR7-AlexaFluor700) on splenocytes and bone marrow from 3 PBS mice, 3 BCMA-muIL2 mice and 4 VHH-muIL2 mice was analyzed by flow cytometry.+ , α-BCMA-CAR + The total number of immune cell connections was approximately 9800 (PBS spleen), approximately 8100 (BCMA-muIL2 spleen), approximately 7600 (PBS bone marrow), approximately 14200 (BCMA-muIL2 bone marrow), and approximately 1420 (VHH-muIL2 bone marrow). The entire high-dimensional dataset was merged to create a single Flt-SNE map for each case, where the signal intensity of various phenotypic markers defined the specific immune phenotype expressed with a blue-green-yellow-red continuous color scale. FltSNE was performed using the following parameters: maximum number of iterations: 1000, theta: 0.5, learning rate: 200, perplexity: 20. The number of CAR T cells in the spleen of the VHH-muIL2 group was insufficient to perform Flt-SNE. To improve visibility, the points representing the VHH-muIL2 bone marrow samples were enlarged because fewer cells were detected in these mice. In the BCMA-muIL2-treated mice, most CAR + The cells are CD8 + cells, whereas in the VHH-muIL2 samples, CD4 + Cells account for CAR + It is worth noting that CAR in the CAR+PBS group + Cells showed low or no expression of CD45RA, CD45RO, or CD62L, whereas BCMA-muIL2-treated mice showed CARs with increased expression of these memory markers. + group.

[0354] Thus, this treatment not only promoted robust proliferation and tumor cell eradication using low-dose CAR T cells but also promoted the development of persistent memory cells, demonstrating the efficacy of BCMA-muIL2 CAR adaptor molecule therapy in enhancing CAR T cell clearance of tumor cells and generating persistent memory cells.

[0355] Example 9: Persistent CAR T cells treated with CAR adapter molecule therapeutics remain functional three months after infusion able

[0356] Mice received 1 million OPM2 cells followed by 500,000 BCMACAR T cells ( Figure 11AOne group of mice received the CAR adapter molecule twice a week for two weeks, followed by weekly administration for another two weeks (6 doses, 200 μg each on days 4, 10, 14, 17, 21, and 28; n=5). A control group received VHH-muIL2 at the same dose and schedule (n=5), while another control group received tumor cells alone (n=3). All mice that received CAR T cells showed an initial response compared to control mice without CAR T cells ( Figure 11B All mice treated with the CAR adaptor molecule (5 out of 5) and 3 out of 5 in the VHH-muIL2 group survived for more than three months, which included the duration of the experiment. One VHH-muIL2 mouse died within about a month, and a second mouse died from recurrence of cancer cells with liver metastasis ( Figure 11B , day 77). Surviving mice were euthanized three months after CAR T cell injection, and spleen and bone marrow cells were analyzed to assess the presence of CAR T cells. Notably, compared with mice receiving CAR T cells treated with VHH-muIL2, mice treated with the CAR adaptor molecule showed a large number of CAR T cells homing to the bone marrow and spleen and persisting ( Figure 11C Given that the mice had not received treatment for two months before being sacrificed, these results further suggest that treatment promoted the generation of memory cells among the CAR T cells.

[0357] To demonstrate the functionality of the CAR T cell adaptor molecules that persist in mice treated with CAR adaptor molecules, an in vitro killing assay was performed using BCMACAR T cells collected from the bone marrow and spleen. Bone marrow and spleen cells were analyzed by flow cytometry to detect and quantify CAR-expressing T cells, and bone marrow cells or spleen cells were co-incubated with OPM2 target cells at different E:T ratios (1:1 and 2:1) based on the cells expressing CAR. Survival was determined using flow cytometric analysis after 24 hours, 48 ​​hours, and 72 hours. Three-month-old CAR T cells showed effective killing of tumor cells and long-term function ( Figure 11D Only one VHH-muIL2-treated mouse exhibited sufficient CAR T cells to perform similar killing assays, and although it exhibited tumor cell killing, the efficiency was lower than that observed with CAR T lymphocytes treated with the CAR adapter molecule ( Figure 11D ) (Error bars represent mean and standard deviation.) Thus, CAR adaptor molecule therapy robustly expanded and drove the persistence of CAR T cells while maintaining their killing potential.

[0358] To further characterize the phenotype of these persistent CAR T cells, flow cytometric analysis was performed to evaluate the expression of a series of T cell markers (CD45, BCMACAR, CD4, CD8, CD62L, CD45RO, CD45RA, CD69, and PD-1). For ease of interpretation, t-SNE maps of splenocytes and bone marrow cells were generated ( Figure 11E Splenocytes and bone marrow from five BCMA-muIL2 treated mice were analyzed by flow cytometry for anti-CD45-Pacific Blue, anti-CD8-FITC, anti-CD4-PE-Dazzle594, BCMA (antigen)-AlexaFluor647, anti-CD69-BV421, anti-PD-1-BV605, anti-CD45RA-APC-Cy7, anti-CD45AR-PerCP-Cy5.5, anti-CD62L-PE, and CCR7-AlexaFluor700. + 、CD8 + , α-BCMA-CAR + The cells were concatenated to form a total of approximately 17,600 (spleen) and approximately 10,800 (bone marrow) cells. The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to create a single tSNE map with the signal intensities of six phenotypic markers that define specific immune phenotypes with a blue-green-yellow-red continuous color scale. tSNE analysis was performed on the spleen and bone marrow using 1,000 iterations, a perplexity of 30, and learning rates of 1,237 and 756, respectively. The population labeled 1 appears to exhibit a memory-like phenotype, expressing higher levels of CD45RO, CD62L, and CD45RA. The population labeled 2 appears to exhibit an effector cell-like phenotype, expressing low levels of CD45RO, CD62L, and CD45RA.

[0359] Flow cytometric analysis showed that the persistent CAR T cells were composed of CD4 + and CD8 + Group composition. CD8 + CAR cells appear to exhibit two distinct populations: effector cells and CD45RA + CD62L + Memory cells. The memory population showed higher expression levels of BCMACAR and CD45 ( Figure 11E ). Similarly, CD4 + CAR T cells demonstrate two populations of effector and memory cells ( Figure 18). Sufficient numbers of CAR T cells could not be detected from VHH-muIL2-treated mice to perform similar flow cytometric analysis. Therefore, CAR adaptor molecule treatment resulted in the generation of long-lasting memory CAR T cells.

[0360] Figure 18 Shows the source Figures 11A-11E CD4 T cells of the five BCMA-muIL2 CAR-E treated mice shown + CAR + t-SNE map of T cells. The expression of nine immune cell markers (aCD45-Pacific Blue, aCD8-FITC, aCD4-PE Dazzle594, BCMA (antigen)-AlexaFluor647, aCD69-BV421, aPD-1-BV605, aCD45RA-APC-Cy7, aCD45RO-PerCP-Cy5.5, aCD62L-PE) on splenocytes and bone marrow from five BCMA-muIL2-treated mice was analyzed by flow cytometry. + 、CD4 + , α-BCMA-CAR + Immune cells were connected to form a total of approximately 9,000 (spleen) and approximately 6,600 (bone marrow) cells. The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to create a single t-SNE map, in which the signal intensity of the six phenotypic markers defined a specific immune phenotype expressed with a blue-green-yellow-red continuous color scale. tSNE analysis was performed on the spleen and bone marrow using 1,000 iterations, a perplexity of 30, and learning rates of 630 and 466, respectively. The population labeled "1" appears to exhibit a memory-like phenotype, expressing higher levels of CD45RO, CD62L, and CD45RA. The population labeled "2" appears to exhibit an effector cell-like phenotype, expressing low levels of CD45RO, CD62L, and CD45RA.

[0361] Single-cell RNA sequencing (scRNAseq) analysis was performed on CAR+ T cells isolated from mice treated with BCMA-muIL2 or VHH-muIL2 control. Although the presence of CAR T cells was limited in VHH-muIL2-treated mice, sufficient numbers of cells were obtained from one VHH-treated mouse for the experiment ( Figure 19A CAR T cells were stained with BCMA-AlexaFluor647 and TotalSeq-C hashing antibodies from BCMA-muIL2 or VHH-muIL2 treated mice. + Cells were sorted as Figure 19AAs shown in the red and green boxes in the figure, 5000 CARs from the bone marrow and spleen of BCMA-muIL2 mice were injected into the + cells and 2500 CARs from the bone marrow and spleen of VHH-muIL2 mice + Cells were loaded onto the 10X channel. scRNAseq analysis showed that in BCMA-muIL2-treated mice, the major population of persistent CAR T cells consisted of CD8+ T cells ( Figure 19B-Figure 19C ), which showed enrichment of genes associated with activated T cell status ( Figure 19D-19E ). Figure 19D The heatmap in Figure 3 shows significantly differentially expressed genes between CAR adapter-treated and VHH-conditioned CD8 and CD4 CAR T cells, separated across the different relevant conditions. Genes marked with an * are significantly differentially expressed between BCMA-muIL2- and VHH-muIL2-treated mice in the subset of interest. This was demonstrated by increased expression of granzyme family genes, other cytotoxicity-related genes, and MHC class II genes. No significant differences in activation markers were observed between CAR T cells obtained from BCMA-muIL2- or VHH-muIL2-treated mice, as these mice had been tumor-free 60 days prior. BCMA-muIL2 treatment did not induce upregulation of exhaustion markers, indicating that this treatment did not induce sustained CAR T cell depletion.

[0362] Next, we evaluated the diversity of T cell receptor (TCR) clonotypes in BCMA-muIL2- and VHH-muIL2-treated mice ( Figure 19F-19G Both groups showed similar diversity in the presence of clonotypes, suggesting that BCMA-muIL2 CAR adapter therapy can effectively promote the generation of a diverse TCR repertoire in persistent CAR T cells, rather than promoting the dominance of a limited set of TCR clones. Figure 19F The pie chart in Figure 2 shows the diversity of TCR clonotypes. Each piece of the pie chart represents the proportion of different TCR clonotypes present; colors are randomly assigned to different clone types. The clonotype diversity within the total cell count of each sample is shown as a stacked bar chart ( Figure 19G ), where similar clonotype combinations with counts below 50 were considered. To assess diversity within each sample, the Simpson index was calculated, with higher values ​​indicating greater diversity. Overall, the results suggest that BCMACAR adapter molecules not only help CAR T cells completely eliminate tumor cells but also potently induce the generation of persistent and functional memory CAR T cells.

[0363] Example 10: CAR adapter molecules expand CAR T cells in the absence of tumor antigens

[0364] CAR T cell expansion typically occurs after patient infusion, with peak expansion observed approximately 10-14 days after infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002-1014(2023)).

[0365] Eradication of minimal residual disease (MRD) contributes to long-term and complete remission. However, the limited presence of corresponding antigens associated with MRD may not be sufficient to support the proliferation and efficacy of conventional CAR T cells. To demonstrate efficacy in the absence of tumor antigens, NSG mice were injected with only 250,000 BCMA CAR T cells in the absence of tumor cells. These mice received two 25 μg doses of BCMA-muIL2 on days 1 and 8 after injection of CAR T cells. The control group received VHH-muIL2 treatment (n=4 per group). On day 30, the mice were euthanized and evaluated for the presence of CART cells in their spleen and bone marrow. BCMA-muIL2-treated mice showed higher numbers of CARs in the spleen (approximately 6.8 times higher) and bone marrow (approximately 5.5 times higher) + T cells, indicating that BCMACAR adaptor molecules can expand CAR T cells in vivo even in the absence of tumor cells ( Figures 12A-12B ); error bars represent mean and standard deviation. Overall, these findings demonstrate that CAR adapter molecules are capable of expanding CAR T cells even in the absence of tumor antigens. Furthermore, therapeutic efficacy was evident even at lower doses and frequencies.

[0366] Example 11: CD19 CAR-E does not inhibit the killing efficacy of CD19 CAR T cells

[0367] It was observed that BCMA-binding CAR adaptor molecules bound to BCMA CART cells but did not inhibit the killing efficacy of BCMA CART cells ( Figure 2D-2H To investigate the effects of CAR-E containing other cancer antigens on antigen-specific CAR T cells, CD19 CAR T cells and patient-derived CD19 + Leukemia cell killing assays were performed. Notably, the results showed that even at the highest tested concentration (1000 nM of CAR adapter molecule) Figure 21C ), nor did it inhibit killing. Nalm6 cells were co-incubated with CD19 CAR T cells (filled) or non-transduced T cells (open) (E:T ratio 1:1; 30,000 cells each) in the presence of different concentrations of CD19-muIL2 CAR-E treatment. After 48 hours, the viable (PI -) Nalm6 cells, N for each experiment was 3. The experimental results disclosed herein for CD19 are consistent with the results of the above-mentioned BCMA cancer model and BCMACAR-E.

[0368] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All of these publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0369] Although the present disclosure has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It should be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A chimeric antigen receptor (CAR) adaptor protein comprising: The first part comprises the extracellular domain of an antigen present on cancer cells, The second part comprises a first immune cell effector domain, wherein the first part is connected to the second part.

2. The CAR adapter molecule of claim 1 , wherein the extracellular domain is derived from AFP, AXL, B4GALNT1, B cell maturation antigen (BCMA), CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, cancer / testis antigen 1B (CTAG1B), carcinoembryonic antigen (CEA), CLEC12A, claudin 18.2 (CLDN 18.2), CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, HPV E7, IL1RAP, IL3RA, IL13Rα2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PROM1, PSCA, ROR1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNF receptor superfamily member (TNFRSF) 8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, or ULBP2.

3. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from BCMA.

4. The CAR adapter molecule of claim 3, wherein the extracellular domain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

5. The CAR adapter molecule of claim 4, wherein the extracellular domain comprises the amino acid sequence 6. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from CD19.

7. The CAR adapter molecule of claim 6, wherein the extracellular domain comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDMTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGNLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 3).

8. The CAR adapter molecule of claim 7, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO:

3.

9. The CAR adapter molecule of claim 8, wherein the extracellular domain comprises the amino acid sequence PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISQVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWQVSDLGGLGCGLKQRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLQQSLSRDMTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGQLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 4) or PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSP KLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK(SEQ ID NO: 5).

10. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from CD20.

11. The CAR adapter molecule of claim 10, wherein the extracellular domain comprises the amino acid sequence KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

12. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from SLAMF7.

13. The CAR adapter molecule of claim 12, wherein the extracellular domain comprises the amino acid sequence 14. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from PD-1.

15. The CAR adapter molecule of claim 14, wherein the extracellular domain comprises the amino acid sequence 16. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from KIT.

17. The CAR adapter molecule of claim 16, wherein the extracellular domain comprises the amino acid sequence 18. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from CD38.

19. The CAR adapter molecule of claim 18, wherein the extracellular domain comprises the amino acid sequence 20. The CAR adapter molecule of claim 2, wherein the extracellular domain is derived from CD22.

21. The CAR adapter molecule of claim 20, wherein the extracellular domain comprises the amino acid sequence 22. The CAR adapter molecule of claim 20, wherein the extracellular domain comprises the amino acid sequence 23. The CAR adapter molecule of any one of claims 1-22, further comprising a first linker connecting the first portion and the second portion.

24. The CAR adapter molecule of claim 23, further comprising a dimerization domain positioned between the first linker and the second portion comprising the first immune cell effector domain.

25. The CAR adapter molecule of claim 24, further comprising a second linker connecting the dimerization domain and the second portion, wherein the first linker and the second linker may be the same or different.

26. The CAR adapter molecule of claim 25, wherein the first linker and the second linker are flexible.

27. The CAR adapter molecule of claim 26, wherein the first linker and / or the second linker are derived from the hinge region of CD3ζ, CD4, CD8α, CD28, IgG1, IgG2 or IgG4.

28. The CAR adapter molecule of claim 26, wherein the first linker and / or the second linker comprises the amino acid sequence GGGX, GGGGX (SEQ ID NO: 69), GSSGSX (SEQ ID NO: 70), GGGGS (SEQ ID NO: 71) or GSPRG (SEQ ID NO: 72), wherein X is C or S.

29. The CAR adapter molecule of claim 26, wherein the first linker has the amino acid sequence GGGGS (SEQ ID NO: 71) or GSPRG (SEQ ID NO: 72), and the second linker has the amino acid sequence GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 76).

30. The CAR adapter molecule of any one of claims 24-29, wherein the dimerization domain is derived from IgA, IgD, IgG, IgM, or IgE.

31. The CAR adapter molecule of claim 30, wherein the dimerization domain comprises an IgG1 heavy chain constant region (CH)3 domain.

32. The CAR adapter molecule of claim 30, wherein the dimerization domain further comprises an IgG CH2 domain and an IgG CH3 domain.

33. The CAR adapter molecule of any one of claims 1-32, wherein the first immune cell effector domain comprises a cytokine or immune cell activation portion.

34. The CAR adapter molecule of claim 33, wherein the first immune cell effector domain is derived from CD30L, CD40, CD48, CD58, CD70, CD80, CD86, CD112, GITRL, HVEM, OX40L, SEMAA, SLAM, TIM4, interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-15, IL-18, IL-21, IL-27, 4-1BBL, or an immune cell activating variant thereof.

35. The CAR adapter molecule of any one of claims 1-33, wherein the second portion further comprises a plurality of immune cell effector domains.

36. The CAR adapter molecule of claim 35, wherein the second portion further comprises a second immune cell effector domain, wherein the first immune cell effector domain and the second immune cell effector domain may be the same or different.

37. The CAR adapter molecule of claim 36, wherein the second immune cell effector domain comprises a weak affinity variant of IL-2 having the amino acid sequence 38. The CAR adapter molecule of claim 37, wherein the first immune cell effector domain and the second immune cell effector domain each have an amino acid sequence 39. The CAR adapter molecule of claim 36, wherein the first immune cell effector domain and the second immune cell effector domain each have an amino acid sequence of SEQ ID NO: 102 carrying an H16A substitution.

40. The CAR adapter molecule of claim 36, wherein the first immune cell effector domain and the second immune cell effector domain each have an amino acid sequence of SEQ ID NO: 102 carrying an F42A substitution.

41. The CAR adapter molecule of claim 36, wherein the first immune cell effector domain or the second immune cell effector domain comprises the amino acid sequence 42. The CAR adapter molecule of claim 41, wherein the first immune cell effector domain and the second immune cell effector domain each comprise the amino acid sequence of SEQ ID NO:

26.

43. The CAR adapter molecule of claim 35, wherein the first immune cell effector domain comprises 4-1BBL or an immune cell activating variant thereof.

44. The CAR adapter molecule of claim 43, wherein the first immune cell effector domain comprises the amino acid sequence 45. The CAR adapter molecule of claim 35, wherein the second portion further comprises a third immune cell effector domain, wherein any two or more of the first immune cell effector domain, the second immune cell effector domain, and the third immune cell effector domain may be the same or different.

46. ​​The CAR adapter molecule of claim 44, wherein the first immune cell effector domain, the second immune cell effector domain, and the third immune cell effector domain each comprise the amino acid sequence of SEQ ID NO:

27.

47. The CAR adapter molecule of claim 33, wherein the first cellular effector domain is a single-chain variable antibody fragment (scFv) that binds to and activates immune cells.

48. The CAR adapter molecule of claim 47, wherein the first cellular effector domain binds 4-1BB, CD2, CD27, CD28, CD30, CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1.

49. The CAR adapter molecule of claim 48, wherein the first cellular effector domain comprises a scFv that binds CTLA-4.

50. The CAR adapter molecule of claim 49, wherein the scFv comprises a VL domain having the amino acid sequence EIVLTQSPGTLSLSPGERATLSCRAQSVSRYLGWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 36) and a VH domain having the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLFGPFDIWGQGTLVTVSS (SEQ ID NO: 37).

51. The CAR adapter molecule of claim 48, wherein the first cellular effector domain binds OX40.

52. The CAR adapter molecule of claim 51 , wherein the scFv comprises a VL domain having the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIK (SEQ ID NO: 42) and a VH domain having the amino acid sequence EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS (SEQ ID NO: 43).

53. The CAR adapter molecule of claim 48, wherein the first cellular effector domain binds PD-1.

54. The CAR adapter molecule of claim 53, wherein the scFv comprises a VL domain having the amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK (SEQ ID NO: 52) and a VH domain having the amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVVRQAPGKGLEWVAVIWYDGSNKYYADSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQGTLVTVSS (SEQ ID NO: 53).

55. The CAR adapter molecule of any one of claims 1-54, wherein the first immune cell effector domain comprises an immune cell inhibitory portion.

56. The CAR adapter molecule of claim 55, wherein the first immune cell effector domain is derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, Galectin-3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4).

57. The CAR adapter molecule of claim 56, wherein the first immune cell effector domain is derived from PD-L1.

58. The CAR adapter molecule of claim 57, wherein the first immune cell effector domain comprises the amino acid sequence 59. The CAR adapter molecule of claim 56, wherein the first immune cell effector domain is derived from CD80.

60. The CAR adapter molecule of claim 59, wherein the first immune cell effector domain comprises the amino acid sequence 61. The CAR adapter molecule of claim 56, wherein the first immune cell effector domain is derived from CD276 (B7-H3).

62. The CAR adapter molecule of claim 61, wherein the first immune cell effector domain comprises the amino acid sequence 63. The CAR adapter molecule of claim 56, wherein the first immune cell effector domain is derived from VTCN1 (B7-H4).

64. The CAR adapter molecule of claim 63, wherein the first immune cell effector domain comprises the amino acid sequence 65. The CAR adapter molecule of any one of claims 1-64, wherein the CAR adapter molecule is in the form of a fusion protein and the first portion and the second portion are linked by a peptide bond.

66. The CAR adapter molecule of any one of claims 1-64, wherein the first portion comprising the extracellular domain is linked to the second portion comprising the first immune cell effector domain or the first linker via an azide-alkyne linkage, an oxime or hydrazine linkage, a tetrazine-trans-cyclooctene linkage, an azide-nitrone linkage, a thiol-olefin linkage, an olefin-tetrazole linkage, an olefin-tetrazine linkage, an olefin-azide linkage, a conjugated diene-olefin linkage, or an isonitrile-tetrazine linkage.

67. The CAR adapter molecule of any one of claims 24-66, which is in the form of a homodimer comprising two of said CAR adapter molecules.

68. A heterodimeric CAR adapter molecule comprising: a first portion comprising an extracellular domain of an antigen present on a cancer cell, said extracellular domain being linked to a first dimerization domain; and a second portion comprising a first immune cell effector domain connected to a second dimerization domain; The first dimerization domain and the second dimerization domain dimerize to form a heterodimer.

69. The CAR adapter molecule of claim 68, wherein the first dimerization domain comprises a protrusion, the second dimerization domain comprises a cavity that is spatially complementary to the protrusion, and wherein the protrusion is capable of being positioned within the cavity.

70. A nucleic acid encoding the CAR adapter molecule of claim 65.

71. A nucleic acid encoding the extracellular domain of an antigen present on a cancer cell fused to a first dimerization domain.

72. A nucleic acid encoding an immune cell effector domain fused to a second dimerization domain.

73. The nucleic acid of any one of claims 70-72, further comprising a sequence encoding a signal peptide.

74. A vector comprising the nucleic acid of any one of claims 70-73.

75. A cell comprising the vector of claim 74.

76. The cell of claim 75, which is a mammalian cell.

77. The cell of claim 76, which is a bacterial cell.

78. A pharmaceutical composition comprising the CAR adapter molecule of any one of claims 1-69 and a pharmaceutically acceptable carrier.

79. A pharmaceutical composition according to claim 78, comprising an effective number of immune cells comprising a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain comprising a stimulatory domain, and the extracellular domain is bound to the extracellular domain of a CAR adapter molecule.

80. A method of making a CAR adapter molecule, comprising: Culturing the cell of any one of claims 75-77 in culture medium under conditions such that a nucleic acid encoding the CAR adapter molecule is expressed; and The CAR adapter molecule is separated from the cells and / or culture medium.

81. A method for treating cancer, comprising: administering to a subject an effective amount of the CAR adapter molecule of any one of claims 1-69, wherein an effective number of immune cells comprising a chimeric antigen receptor (CAR) (CAR immune cells) are administered to the subject before, substantially simultaneously with, or after administration of the CAR adapter molecule, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain comprising a stimulatory domain, the extracellular domain binding to the extracellular domain of the CAR adapter molecule.

82. The method of claim 81, wherein the first immune cell effector domain of the CAR adapter molecule comprises a cytokine or immune cell activation portion.

83. The method of claim 81, wherein the first immune cell effector domain of the CAR adapter molecule comprises an immune cell inhibitory portion.

84. The method of any one of claims 81-83, wherein the CAR immune cell is administered to the subject prior to administration of the CAR adapter molecule.

85. The method of claim 84, wherein the CAR immune cell is administered at least about 6 months, at least about 9 months, or at least about one year prior to administration of the CAR adapter molecule.

86. The method of any one of claims 81-83, wherein the CAR adapter molecule and the CAR immune cell are administered in combination.

87. The method of claim 86, wherein the combined administration comprises administering the CAR immune cell and the CAR adapter molecule substantially simultaneously.

88. The method of claim 86, further comprising contacting the CAR adapter molecule with the CAR immune cell in vitro prior to administering the CAR adapter molecule and the immune cell.

89. The method of claim 86, further comprising the steps of: measuring the concentration of immune cells present in a sample obtained from the subject after administering the immune cells; and The difference between the concentration of immune cells administered to the subject and the measured immune cell concentration is calculated.

90. The method of claim 89, wherein the combined administration comprises administering the CAR adapter molecule when the measured CAR immune cell concentration is lower than the administered immune cell concentration.

91. The method of any one of claims 81-90, wherein the CAR immune cell is a T cell or a NK cell.

92. The method of claim 91, wherein the T cells are CD8 + T cells.

93. The method of any one of claims 81-92, wherein the cancer is a hematopoietic cancer.

94. The method of claim 93, wherein the cancer is leukemia, multiple myeloma, or lymphoma.

95. The method of claim 94, wherein the cancer is multiple myeloma.

96. The method of any one of claims 81-92, wherein the cancer is characterized as a solid tumor.

97. The method of claim 96, wherein the cancer is renal cell carcinoma, breast cancer, ovarian cancer, neuroblastoma, glioblastoma, gliosarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, pancreatic cancer, lung cancer, fallopian tube cancer, prostate cancer, kidney cancer, bladder cancer, gastrointestinal cancer, melanoma, colorectal cancer, or esophageal cancer.

98. The method of any one of claims 81-97, further comprising administering a high dose of a chemotherapeutic agent to the subject prior to administering the CAR immune cells.

99. The method of claim 98, further comprising administering bone marrow cells or peripheral blood stem cells to the subject.

100. The method of any one of claims 81-99, further comprising administering to the subject an additional active agent comprising one or more of thalidomide, lenalidomide, and bortezomib.

101. The method of any one of claims 81-100, wherein the effective number of CAR immune cells is about 1×10 4 about 6×10 5 cells.

102. The method of any one of claims 81-101, wherein the subject is in a state of minimal residual disease.

103. A chimeric antigen receptor (CAR) adapter molecule comprising: A first portion comprises the extracellular domain of the antigen linked to a second portion comprising a first immune cell effector domain.

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