Car-adapters containing il-2 variants to enhance function of car t cells

Abstract

Disclosed are novel weak affinity IL-2 variants and uses thereof, including adoptive cell therapies by enhancing the efficacy and duration of CAR-immune cells in the treatment of cancer.

Description

CAR adaptor containing IL-2 variants to enhance CAR T cell function

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 664,020, filed June 25, 2024, and U.S. Provisional Application No. 63 / 537,660, filed September 11, 2023, pursuant to 35 USC § 119(e), the entire contents of each of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML copy created on August 7, 2024, is named 046094_788001WO_ST.xml and is 133 KB in size. Background Technology

[0005] The homologous IL-2 receptor (IL-2R) consists of three subunits: IL-2 subunit α (IL-2Rα; CD25), IL-2 subunit β (IL-2Rβ; CD122), and IL-2 subunit γ (IL-2Rγ; CD132).

[0006] IL-2 can also induce alternative differentiation pathways of T cells, leading to the generation of different "better" effector CD8 cells. + T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may depend at least in part on the binding of IL-2 to IL-2Rα. In addition, agonists that favor IL-2Rβγ can drive T cells toward a terminal differentiation state (Codarri et al., Nature 610(7930):161-172 (2022)).

[0007] Known IL-2 variants may have different affinities for different IL-2R subunits and can offer therapy-related advantages. For example, weak-affinity IL-2 containing amino acid substitutions of H16A and F42A (muIL2) exhibits increased T-cell selectivity and less toxicity compared to wild-type IL-2 by reducing global IL-2 binding and activation of all IL-2R-expressing cells. Compared to wild-type IL-2, muIL2 has reduced binding affinity for human IL2Rα and IL2Rβ to 1 / 110 and 1 / 3, respectively (Quayle et al., Clin. Cancer Res. 26(8):1953-1964 (2020)). IL-2-based therapies remain challenging in clinical practice, with many clinical trials failing to meet their primary endpoints (Raeber et al., Ebiomedicine 90:104539, pp. 1-25 (2023)). For example, the PIVOT IO-001 trial (NCT03635983) tested NKTR-214 (a pegylated prodrug of recombinant IL2) in metastatic melanoma in contrast to NKTR-214 in combination with pembrolizumab, but the trial failed to meet its primary endpoints of ORR, PFS and OS (Diab et al., J. Clin. Oncol.41(30):4756-4767 (2023)).

[0008] T cells expressing chimeric antigen receptors (CARs) have revolutionized the treatment of hematopoietic malignancies and have shown promising results in the treatment of cancers of the hematopoietic system. Six CAR T-cell therapies targeting two antigens, CD19 and BCMA, are currently FDA-approved. CD19 is a B-cell co-receptor expressed on B cells and in a variety of hematopoietic malignancies. CD19 CAR T cells were initially approved for the treatment of acute lymphoblastic leukemia (ALL) and subsequently 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 of multiple myeloma (MM). MM is associated with 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)).

[0009] However, CAR T-cell therapy remains challenging. A recent meta-analysis of 22 CAR T-cell clinical studies highlighted its ineffectiveness in solid tumors, with a poor average overall response rate of 9% (Hou et al., Dis. Markers 2019:3425291, pp. 1-11 (2019)). Even in hematologic malignancies, the duration of response remains a challenge, with almost all MM patients treated with BCMA CAR eventually relapsing (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)). In addition, treatment can have serious side effects, including cytokine release syndrome (CRS) and neurotoxicity.

[0010] CAR T cells need to home to the tumor site, proliferate, and persist in circulation, at least until they neutralize and kill the last remaining cancer cells. Therefore, there is an urgent need for methods to enhance and prolong CAR T cell activity in a controlled manner. Summary of the Invention

[0011] The disclosed IL-2 variant, chimeric antigen receptor (CAR) adaptor containing the IL-2 variant, and method for enhancing the activity of chimeric CAR immune cells are expected to address the aforementioned needs. The IL-2 variant enhances anticancer therapy either alone or in connection with another active motif, such as an antibody or its binding fragment. The CAR adaptor containing the IL-2 variant enhances CAR immune cells, thereby driving them toward the generation of memory cells and preventing depletion. Furthermore, since the binding of the CAR adaptor to the CAR is reversible, the CAR adaptor does not induce immune synapse formation on the surface of CAR-NK cells. Therefore, the CAR adaptor does not block CAR-mediated target cell killing.

[0012] A first aspect of this disclosure relates to an IL-2 variant that differs from wild-type IL-2 (SEQ ID NO: 102) in respect of three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E62, L72, and V91 of SEQ ID NO: 1. In some embodiments, the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E62N, L72G, and V91H.

[0013] Related aspects include nucleic acids encoding IL-2 variants, vectors containing such nucleic acids, cells transformed with such vectors, methods for preparing IL-2 variants, pharmaceutical compositions containing IL-2 variants, and their use in treating cancer.

[0014] One such use relates to enhancing adoptive cell therapies, such as CAR T therapy. Therefore, another aspect of this disclosure relates to a chimeric antigen receptor (CAR) adaptor designed for use with CAR-immunotherapy (such as CAR-T therapy), wherein the CAR adaptor contains a first portion comprising an epitope on an extracellular domain (ED) that binds to the CAR, the first portion being linked to a second portion comprising an immune effector domain containing an IL-2 variant. The ED comprises one or more extracellular binding domains (EBDs) of the CAR and any other extracellular portion of the CAR, such as a linker connecting an antibody fragment or an EBD. The link between the first and second portions can be a peptide link or a non-peptide (covalent) link, such that the CAR adaptor can be a continuous protein or polypeptide, or a portion containing two proteinaceous entities linked by covalent bonds.

[0015] Another aspect of this disclosure relates to a heterodimeric CAR-adaptor comprising: a first portion that binds an epitope on the EB of a CAR linked to a first dimerizing domain; and a second portion that comprises an immune effector domain comprising an IL-2 variant linked to a second dimerizing domain, wherein the first dimerizing domain and the second dimerizing domain bind to form the heterodimeric CAR-adaptor.

[0016] Related aspects include nucleic acids encoding CAR-adaptors (in embodiments where the CAR-adaptor is a continuous protein), nucleic acids encoding a first portion of a heterodimeric CAR-adaptor protein, nucleic acids encoding a second portion of a heterodimeric CAR-adaptor protein, vectors containing nucleic acids encoding CAR-adaptor proteins, vectors containing first and / or second proteins of heterodimeric CAR-adaptors, cells transformed with the vectors, pharmaceutical compositions containing CAR-adaptors and pharmaceutically acceptable carriers, and methods for preparing CAR-adaptor proteins.

[0017] Another aspect of this disclosure relates to a method for treating cancer. This method requires administering a first course of an effective dose of CAR-adaptor therapy to a subject. In some embodiments, the subject has previously received an administration of immune cells expressing a CAR containing an extracellular domain (which contains an extracellular domain and an extracellular domain of the CAR-adaptor) that binds to an antigen present on cancer cells, a transmembrane domain, and an intracellular domain containing a stimulating domain.

[0018] The working examples disclosed herein (e.g., Example 11) demonstrate that CAR-adaptors with different IL-2 variants enhance CAR immune cells targeting BCMA and CD19, driving them toward the generation of memory cells and preventing the depletion of CAR immune cells. The working examples also demonstrate that the timing and dosage of CAR-adaptor therapy can optimize its effect on previous CAR immune cell therapies by enhancing them in terms of driving the generation of CAR immune cells targeting cancer antigens toward memory immune cells and preventing the depletion-preventive proliferation of CAR immune cells. Therefore, the working examples propose a hypothesis of a fundamental mechanism of action between CAR-adaptors and CAR immune cells. More specifically, the working examples demonstrate that two binding events—the binding between the immune effector domain of the CAR-adaptor and its homologous receptor on the immune cell, and the binding between the CAR-adaptor and the EB portion of the CAR—create synergistic molecular “crosstalk” between the intracellular domain (intramolecular domain) of the homologous receptor and the intracellular domain-stimulated region of the CAR, respectively, leading to the production of IFN-γ and TNF-α, and ultimately resulting in the generation of memory immune cells and the depletion-preventive proliferation of CAR immune cells. Attached Figure Description

[0019] Figure 1 schematically illustrates the structural domains of a CAR-adaptor according to some embodiments, the CAR-adaptor comprising: a first portion containing an antigen serving as a target for CAR (such as the extracellular domain of CD19 or BCMA on the surface of cancer cells) and a dimerizing domain (such as CH3); and a second portion containing an immune cell effector domain (ICE). The CAR-adaptor can be a monomer, dimer, or multimer.

[0020] Figures 2A to 2I are a set of illustrations and line graphs showing three CAR-protocols. Figure 2A schematically shows a CAR-protocol containing the BCMA extracellular domain and the Neo2 / 15 synthetic cytokine immune cell effector domain. Figure 2B schematically shows a CAR-protocol containing the BCMA extracellular domain and two weakly affinity mutated IL-2 (mIL2) synthetic cytokine immune cell effector domains. Figure 2C schematically shows a CAR-protocol containing the BCMA extracellular domain and the 4-1BBL immune cell effector domain. Figure 2D is a line graph showing dose-dependent staining of CD19-binding CAR T cells or untransduced T cells (NT T cells) by the CAR-protocol or control protein. Figure 2E is a line graph showing dose-dependent staining of BCMA-binding CAR T cells or untransduced T cells (NT T cells) by the CAR-protocol or control protein. Figures 2F and 2G are line graphs and bar graphs, respectively, which together show the dose-dependent activation of CAR T cells after CAR-protocol treatment. Figure 2H is a line graph showing that the BCMA-muIL2 CAR-adaptor does not block the killing efficacy of CAR T cells. Figure 2I is a line graph showing the phosphorylation of signal transducers and transcription activator (STAT5) in BCMA CAR T cells.

[0021] Figures 3A and 3B are a set of illustrations and line graphs showing the effect of CAR-adaptors on untransduced T cells. Figure 3A schematically illustrates the experimental design. Figure 3B is a set of line graphs showing T cell counts and carboxyfluorescein succinimide (CFSE) staining of untransduced activated T cells treated with CAR-adaptors containing tecoleukin, the BCMA extracellular domain and two mutant weak-affinity IL-2 (muIL2), or the BCMA extracellular domain and Neoleukin domain.

[0022] Figures 4A to 4C are a set of illustrations and line graphs showing the specific activation of CAR T cells by the CAR-adaptor. Figure 4A schematically illustrates the experimental design. Figure 4B is a bar graph showing CD69 cells treated with the BCMA CAR-adaptor, a BCMA CAR-adaptor control without an immune cell effector domain, or a non-antigen-specific CAR-adaptor control. + The percentage of activated T cells transduced with anti-BCMACAR. Figure 4C is a bar graph showing the percentage of CD69 cells after treatment with either the CD19 CAR-adaptor or a non-antigen-specific CAR-adaptor control. + Percentage of activated T cells transduced with anti-CD19 CAR.

[0023] Figure 5 is a line graph showing that the CAR-adaptor does not inhibit BCMA CAR T cell killing and shows the percentage of OPM2 target cells that survive after incubation with CAR T cells and CAR-adaptor (red) or untransduced T cells (blue).

[0024] Figures 6A through 6C are a set of illustrations and line graphs showing the reduction of tumor burden in vivo by the CAR-adaptor. Figure 6A schematically illustrates the experimental design. Figures 6B through 6C are a set of photographs showing the tumor burden in mice before and after CAR T cell infusion and CAR-adaptor treatment.

[0025] Figures 7A through 7C are a series of flow cytometry plots showing tumor burden in mice following CAR T cell infusion and CAR-adaptor treatment. Figure 7A is a series of flow cytometry plots showing OPM2 tumor burden in blood, spleen, and lymph nodes. Figure 7B is a series of flow cytometry plots showing OPM2 tumor burden in bone marrow and lung. Figure 7C is a series of flow cytometry plots showing OPM2 tumor burden in liver, kidney, and eye tumor sites. eGFP (OPM2 cells) are shown on the y-axis, and PerCP signal controls are shown on the x-axis.

[0026] Figures 8A to 8C are a set of flow cytometry images showing human CD45 in mice after CAR T cell infusion and CAR-adaptor 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 is a set of flow cytometry images showing CAR T cells in bone marrow and lung. Figure 8C 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-adaptor labeled with AF647 is shown on the x-axis.

[0027] Figures 9A through 9E are a set of schematic diagrams, line graphs, and box plots showing that CAR-adaptor treatment leads to enhanced in vivo activity and persistence of CAR T cells. Figure 9A is a line graph showing the circulating half-life of the BCMA CAR-adaptor. Figure 9B schematically illustrates the experimental design. Figures 9C and 9D are a set of flow cytometry plots and box plots showing the selective expansion and persistence of BCMA CAR T cells. Figure 9E shows the CD8+ after CAR-adaptor treatment. + Box plot of the percentage of CAR T cells.

[0028] Figures 10A through 10J are a set of schematic diagrams, survival plots, line graphs, bar graphs, and t-distributed random nearest neighbor embeddings (tSNE) plots and photographs showing that CAR-adaptor treatment reduced the required dose of CAR T cells. Figure 10A schematically illustrates the experimental design. Figure 10B is a set of photographs showing the tumor burden in mice before and after CAR T cell infusion and CAR-adaptor treatment. Figure 10C is a Kaplan-Meier plot showing the survival analysis. Figure 10D is a line graph showing the flow cytometry analysis of CAR T cells in blood samples. Figure 10E is a set of bar graphs showing the generation of memory CAR T cells. Figures 10F and 10G are a set of flow cytometry plots and bar graphs showing a large number of CAR T cells remaining two months after CAR T cell injection. Figure 10H is a line graph showing that mice maintained a consistent body weight throughout the experiment. Figure 10I is a set of flow cytometry plots showing that CAR T cells from mice treated with the CAR-adaptor have a stem cell memory phenotype. Figure 10J is a set of tSNE plots that show the clusters defined by FlowSOM in persistent BCMA CAR T cells.

[0029] Figures 11A through 11E are a set of schematic diagrams, photographs, line graphs, bar graphs, and tSNE plots showing the in vivo persistence of CAR T cells resulting from CAR-adaptor treatment. Figure 11A schematically illustrates the experimental design. Figure 11B is a set of photographs showing the tumor burden in mice before and after CAR T cell infusion and CAR-adaptor treatment. Figure 11C is a set of flow cytometry plots showing the persistence of CAR T cells. Figure 11D is a set of bar graphs showing the in vitro killing assay of persistent T cells. Figure 11E is a set of t-SNE plots showing the tumor burden from CD8+. + T cell immune cell markers.

[0030] Figures 12A and 12B are schematic diagrams and a set of bar graphs illustrating the in vivo expansion of CAR T cells by CAR-E treatment in the absence of tumor cells. Figure 12A schematically illustrates the experimental design. Figure 12B is a set of bar graphs showing the CAR T cell counts 30 days after injection.

[0031] Figures 13A and 13B are a series 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-gated B cells (CD20), T cells (CD3), or myeloid cells (CD11b). Cells were stained with different concentrations of treatment followed by secondary staining with anti-FLAG-Alexa647. Figure 13A shows a series of flow cytometry images demonstrating that BCMA-muIL2 does not bind to human PBMCs. Figure 13B shows a series of flow cytometry images demonstrating that VHH-muIL2 does not bind to human PBMCs.

[0032] Figures 14A through 14C are a set of photomicrographs and dot plots illustrating the specific binding and gradual internalization of BCMA-muIL2 in CAR T cells. Figure 14A is a set of photomicrographs showing cells stained with CellTracker Blue CMAC, incubated with specified treatments, and labeled with either Alexa647 (BCMA-muIL2) or dsRed (VHH-muIL2) for 1 to 5 hours before imaging. The photomicrographs represent images of >100 cells. Figure 14B is a dot plot illustrating quantitative analysis of the imaged cells. Figure 14C is a dot plot showing the correlation between the mean intensity of Alexa647 (BCMA-muIL2) and the mean intensity of dsRed (VHH-muIL2).

[0033] Figures 15A through 15C are a set of flow cytometry plots showing individual flow cytometry data corresponding to the pooled data presented in Figure 9D. Figure 15A is a set of flow cytometry results from mice treated with CAR T cells alone. Figure 15B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. Figure 15C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[0034] Figures 16A to 16C are a set of flow cytometry plots showing individual flow cytometry data from the mice shown in Figures 10A to 10J. Figure 16A is a set of flow cytometry results from mice treated with CAR T cells alone. Figure 16B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. Figure 16C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[0035] Figures 17A through 17C are 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 and subsequently low-dose CAR T cells. Figure 17A is a bar chart showing the levels of IFNγ, GM-CSF, and TNFα. Serum samples were diluted at a ratio of 1:40. Both standard and serum samples were incubated using the same plates, and standard curves were plotted for each cytokine. Figure 17B is a set of line graphs showing the IFNγ levels between the BCMA-muIL2 and VHH-muIL2 groups (error bars represent mean ± standard deviation). Figure 17C is a set of Flt-SNE maps derived from CAR T cells from mice treated with PBS, BCMA-muIL2, and VHH-muIL2, showing the expression of ten immune cell markers.

[0036] Figure 18 shows CD4 derived from five mice treated with BCMA-muIL2 CAR-E. + CAR + A set of Flt-SNE mappings of T cells shows the expression of nine immune cell markers.

[0037] Figures 19A to 19G are a set of flow cytometry plots, tSNE plots, bar graphs, violin plots, pie charts, and heatmaps showing that single-cell RNA sequencing analysis elucidates the effect of BCMA-muIL2 on CAR T cells. Figure 19A is a set of flow cytometry plots showing CAR T cells analyzed 89 days after CAR-T administration. + Cells. Figure 19B is a tSNE plot showing the data after the Harmony algorithm, illustrating the proportions of CD4, CD8, and proliferating (CD4 and CD8) cells. Figure 19C is a tSNE plot showing the division between groups treated with BCMA-muIL2 or VHH-muIL2. Figure 19D shows the CD4 cells after the specified treatment. + CAR T cells and CD8 + A set of heatmaps showing significantly differentially expressed genes in CAT T cells. Figure 19E is a set of violin plots showing gene scores between CD8 and CD4 cells, constructed using normalized expression of different genes for each phenotype in Figure 19D. Figure 19F is a set of pie charts showing the diversity of T cell receptor (TCR) clonal types. Figure 19G is a bar chart showing clonal diversity within the total cell count of the sample.

[0038] Figures 20A and 20B are a set of schematic diagrams and flow cytometry plots showing that CAR-adaptor treatment leads to enhanced in vivo organ transport of CAR T cells. Figure 20A schematically illustrates the experimental design. Figure 20B is a set of flow cytometry plots showing the selective transport, expansion, and persistence of BCMA CAR T cells.

[0039] Figures 21A to 21C are a set of line and bar graphs illustrating the effects of the CAR-adaptor on untransduced T cells and CAR-T cells. Figures 21A and 21B are line and bar graphs, respectively, showing together the dose-dependent activation of CAR T cells after CAR-adaptor treatment (Figure 21A) and demonstrating that the CAR-adaptor does not activate untransduced T cells (Figure 21B). Figure 21C is a line graph showing that the CD19-muIL2 CAR-adaptor does not block the killing efficacy of CD19 CAR T cells or untransduced T cells (NT T cells).

[0040] Figures 22A and 22B are a set of line graphs illustrating the activation of CAR T cells by CAR-adaptor variants. Figure 22A is a line graph showing STAT5 phosphorylation resulting from CAR-adaptor variant treatment. Figure 22B is a line graph showing CD69 expression on CAR T cells resulting from CAR-adaptor variant treatment.

[0041] Figures 23A and 23B are a set of line graphs illustrating the activation of resting CAR T cells by CAR-adaptor variants. Figure 23A is a line graph showing STAT5 phosphorylation in resting CAR T cells caused by CAR-adaptor variant treatment. Figure 23B is a line graph showing CD69 expression on resting CAR T cells caused by CAR-adaptor variant treatment.

[0042] Figures 24A and 24B are a set of line graphs showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T cells induced by the BCMA CAR-adaptor and containing FDA-approved CAR constructs. Figure 24A is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel (Ide-cel) construct. Figure 24B is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved ciltacabtagene autoleucel (Cilta-cel) construct. BCMA CAR T cells were subjected to specified treatments at 37°C at different doses for 30 minutes, followed by assessment of pSTAT5 by cell counting (n=3 for each condition).

[0043] Figures 25A through 25F are a set of line graphs showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T cells containing the FDA-approved CAR construct induced by the BCMA-muIL2 CAR-adaptor. Figures 25A through 25C are a set of line graphs showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ide-cel construct. Figures 25D through 25F are a set of line graphs showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved Cilta-cel construct. After 24 hours of incubation, the CAR T cells were washed at 2 hours to remove any unbound CAR-adaptor.

[0044] Figures 26A and 26B are a set of line graphs illustrating dose-dependent activation of the BCMACAR-adaptor in resting BCMA CAR T cells via CD69 expression. Figure 26A is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved Ide-cel construct. Figure 26B is a line graph showing pSTAT5-positive BCMACAR T cells containing the FDA-approved cilta-cel construct. CD69 acts as a biomarker for human T cell activation.

[0045] Figures 27A and 27B are a set of line graphs illustrating dose-dependent activation of the BCMACAR-adaptor in resting BCMA CAR T cells expressed via CD69. Figure 27A is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved Cital construct. Figure 27B is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved Cilta-cel construct.

[0046] Figures 28A and 28B are a set of line graphs illustrating dose-dependent activation of the BCMACAR-adaptor in resting BCMA CAR T cells expressed via CD69. Figure 28A is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved acevironmental construct. Figure 28B is a line graph showing pSTAT5-positive BCMA CAR T cells containing the FDA-approved acevironmental construct.

[0047] Figures 29A to 29C are a set of line graphs illustrating the in vitro induction of CAR T cell proliferation by CAR-adaptor treatment, showing a reduced effect on untransduced T cells compared to wild-type IL-2. Figures 29A to 29B are a set of line graphs illustrating the incubation of activated BCMA CAR T cells with a Cidario lorense CAR construct, and treatment with different concentrations of BCMA CAR-adaptor, followed by flow cytometry assessment of CAR T cell proliferation after 3 days. Dashed lines represent the number of untreated CAR T cells. Figure 29C is a line graph illustrating the effect of the CAR-adaptor on the expansion of normal, untransduced T cells. The CAR-adaptor proliferated significantly fewer untransduced T cells than those treated with wild-type IL-2. The CAR-adaptor containing triple mutant IL-2 (V7, V9, Y2, and Y9) induced significantly less proliferation in normal T cells compared to the CAR-adaptor containing double mutant IL-2 (U4).

[0048] Figures 30A to 30C are a set of line graphs showing that biomembrane layer interferometry (BLI) association and dissociation between CAR-adaptors containing the triple mutant IL-2 V7 or the double mutant IL-2 U4 exhibit lower affinity for IL-2Ra compared to wild-type IL-2. Figure 30A is a line graph showing the BLI between wild-type IL-2 (tesi-interleukin) and IL-2Ra. Figure 30B is a line graph showing the BLI between the V7 CAR-adaptor and IL-2Ra. Figure 30C is a line graph showing the BLI between the CAR-adaptor containing the U4 CAR-adaptor and IL-2Ra.

[0049] Figures 31A and 31B are a set of schematic and line graphs showing the in vivo expansion of CAR-T cells by a CAR-adaptor containing an IL-2 variant, and the correlation between the expansion level and in vitro pSTAT5 signaling. Figure 31A schematically illustrates the experimental design. Mice received OPM2 cancer cells, followed by 0.5 × 10⁻⁶ cells one week later. 6 CAR-T cells (Cidexo-Lens). Figure 31B is a set of line graphs showing CD4 and CD8 CAR T cells circulating in the blood. Mice were bled at days 11, 18, 28, 35, and 42 post-injection for 5 weeks, and CD4 and CD8 CAR T cells were counted. Compared to the control PBS-treated group, all three tested CAR-adaptors expanded CAR T cells in vivo. Compared to the X12 CAR-adaptor, the V7 and Y2 CAR-adaptors expanded more CAR T cells.

[0050] Figures 32A through 32C are a set of schematic diagrams, photographs, and line graphs showing that the BCMA-V7 CAR-adaptor enhances CAR-T cell activity when administered on day 3 or even day 14 post-CAR T-cell injection. Figure 32A schematically illustrates the experimental design. One cohort of mice received CAR T-cells only (n=4). Another cohort of mice received six doses of the V7 CAR-adaptor starting on day 3 post-CAR-T cell injection, administered on days 3, 6, 10, 14, 21, and 28. A third cohort of mice received six doses starting on day 14 (which is the post-CRS window in patients), and on days 14, 18, 21, 28, 35, and 42. Figure 32B is a set of photographs showing BLI imaging at specified days to assess tumor growth in different cohorts. Figure 32C is a line graph showing CAR T cells circulating in the blood. Starting on day 14, mice were bled weekly for 6 weeks, and CAR T cells were counted. Compared to the PBS-treated group, V7 CAR-adaptor treatment expanded CAR T cells in both treated cohorts.

[0051] Figures 33A through 33M are a set of schematic diagrams, line graphs, bar graphs, and dot plots illustrating substantial activation and transcriptomic changes in CAR T cells after CAR-E treatment. Figure 33A is a line graph showing the induction of pSTAT5 activity in CAR T cells with a complete CAR construct or a CAR-ICD-Δ construct by CAR-E. Figures 33B through 33D are a set of line graphs showing CAR-E CD69 (Figure 33B), IFN-γ (Figure 33C), and TNF-α (Figure 33D) staining in BCMA CAR T cells and BCMA CAR-ICD-Δ T cells. Figures 33E through 33G are a set of line graphs showing CD69 (Figure 33E), IFN-γ (Figure 33F), and TNF-α (Figure 33G) staining in CAR T cells after CAR-E treatment with dasatinib or ruxolitinib. Figure 33H schematically illustrates the experimental design used to evaluate the efficacy of CAR-E on BCMA CAR T cells and BCMA CAR-ICD-Δ T cells in vivo. Figure 33I is a set of bar graphs showing the expansion and persistence of BCMA CAR T cells and BCMA CAR-ICD-Δ T cells in mouse organs one month after CAR T cell injection; P < 0.0001. Figure 33J is a volcano plot, showing the CD8+ levels 4 hours after CAR-E treatment. + The gene most highly upregulated in CAR T cells. Figure 33K is a volcano plot showing CD8 upregulation 4 hours after CAR-E treatment. +The gene most highly upregulated in CAR-ICD-Δ T cells. Figure 33L shows CD8 after 4 hours of CAR-E treatment. + and CD4 + A heatmap showing changes in gene expression in T cells. Figure 33M shows CD8 expression changes after 2 hours and 24 hours of CAR-E treatment. + and CD4 + Heatmap of gene expression changes in T cells.

[0052] Figure 34 is a set of bar charts showing the transcriptomic changes in CAR T cells after CAR-E treatment.

[0053] Figures 35A through 35G are a set of schematic diagrams, photographs, line graphs, and pie charts showing that lower doses of the CAR-adaptor enhance CAR T cell activity and promote functional memory. Figure 35A schematically illustrates the experimental design. Figure 35B is a set of photographs showing bioluminescence imaging (BLI) of the monitored tumor burden. Figure 35C is a survival analysis showing that all mice treated with the CAR-adaptor survived the duration of the experiment. Figure 35D is a line graph showing the quantification of the BLI analysis from Figure 35B. Figure 35E is a line graph showing a flow cytometry analysis of the presence of CAR T cells in the blood. Figure 35F is a line graph showing IFN-γ levels. Figure 35G is a set of pie charts showing CAR T cells in the bone marrow and spleen of mice treated with the CAR-adaptor.

[0054] Figures 36A through 36G are a set of schematic, bar, and line graphs showing the dose-dependent expansion of CAR T cells in vivo in the absence of tumor cells after treatment with the CAR-adaptor. Figure 36A schematically illustrates the experimental design. Figures 36B through 36C are a set of bar and line graphs showing flow cytometry analysis of spleen and bone marrow tissues harvested 30 days after CAR T cell injection. Figures 36D through 36E are a set of bar graphs showing the analysis of persistent CAR T cells and different subsets of memory CAR T cells in the spleen and bone marrow of CAR-E treated mice. Figure 36F is a bar graph showing that both the antigen and the low-affinity IL-2 component of the CAR-adaptor are essential for its effect.

[0055] Figure 37 is a set of flow cytometry images showing anti-human CD45 and BCMA-CAR staining in blood samples from mice that received human CAR T cells (with and without CAR-E treatment).

[0056] Figures 38A and 38B are a series of flow cytometry images showing anti-human CD45 and BCMA-CAR staining in individual mice that received human CAR T cells (with and without CAR-E treatment). Figure 38A is a series of flow cytometry images of mouse organs collected at different time points after CAR T cell administration. Figure 38B is a series of flow cytometry images of mouse organs collected at different time points after CAR T cell and CAR-E administration.

[0057] Figures 39A to 39D are a set of heatmaps and tSNE plots showing the phenotypic diversity of CAR-E-promoted bone marrow and spleen-derived CAR T cells. Figure 39A is a heatmap showing eight FLOWSOM-derived metaclusters in a bone marrow sample. Figure 39B is a heatmap showing eight FLOWSOM-derived metaclusters in a spleen sample. Figure 39C is a tSNE plot showing the CAR T population in a bone marrow sample. Figure 39D is a tSNE plot showing the CAR T population in a spleen sample.

[0058] Figures 40A to 40D are a series of flow cytometry images showing anti-human CD45 and BCMA-CAR staining in individual mice treated with human CAR T cells and CAR-E. Figure 40A is a series of flow cytometry images of mice receiving CAR T cells and PBS controls. Figure 40B is a series of flow cytometry images of mice receiving CAR T cells and 2 mg / kg BCMA-muIL2. Figure 40C is a series of flow cytometry images of mice receiving CAR T cells and 4 mg / kg BCMA-muIL2. Figure 40D is a series of flow cytometry images of mice receiving CAR T cells and 8 mg / kg BCMA-muIL2.

[0059] Figures 41A through 41E are a series of flow cytometry images showing anti-human CD45 and BCMA-CAR staining in individual mice treated with human CAR T cells and CAR-E. Figure 41A is a series of flow cytometry images of mice receiving CAR T cells and BCMA-CH3 controls. Figure 41B is a series of flow cytometry images of mice receiving CAR T cells and low-dose IL-2. Figure 41C is a series of flow cytometry images of mice receiving CAR T cells and VHH-muIL2. Figure 41D is a series of flow cytometry images of mice receiving CAR T cells and BCMA-muIL2. Figure 41E is a series of flow cytometry images of mice receiving only CAR T cells.

[0060] Figure 42 is a set of line graphs showing CAR-adaptors, each containing an immune cell effector domain containing a variant of U4, V6, V7, or Y2 IL-2, and their selective and efficient proliferation induction in CAR T cells (rather than untransduced T cells).

[0061] Figure 43 is a set of line graphs showing STAT5 phosphorylation in CAR T cells and untransduced T cells after CAR-E treatment.

[0062] Figure 44 is a set of line graphs showing CD69 expression on CAR T cells and untransduced T cells after CAR-E treatment.

[0063] Figure 45 is a set of line graphs showing the secretion of TNF-α and IFN-γ from CAR T cells and untransduced T cells after CAR-E treatment.

[0064] Figures 46A and 46B are a set of line graphs illustrating the binding of CAR-E with IL-2Rα and IL-2Rβγ via biomembrane interferometry. Detailed Implementation

[0065] 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 the subject matter pertains. As used in the specification and appended claims, unless otherwise indicated, the following terms have the meanings indicated to facilitate understanding of this disclosure.

[0066] As used in the specification and appended claims, the singular forms “a” and “the” mean “a or more” and therefore include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to “composition” includes a mixture of two or more such compositions, reference to “inhibitor” includes a mixture of two or more such inhibitors, and so on.

[0067] Unless otherwise stated, the term "about" is understood to mean within the normal tolerance range in this field, for example, within 2 standard deviations of the mean. "About" can be understood as 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 indicates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0068] As used herein, the term “approximately” means a range of values ​​falling within or less than 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction of the stated reference value, unless otherwise stated or otherwise obvious from the context (except where such a number would exceed 100% of the possible value).

[0069] The transitional term "comprising," synonymous with "comprising," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unlisted elements or method steps. Conversely, the transitional phrase "consisting of..." excludes any elements or method steps not specified in the claim (or specific elements or method steps associated with the phrase "consisting of..."). The transitional phrase "substantially constitutes..." limits the scope of the claim to the specified elements and methods or steps, as well as "unlisted elements and method steps that do not substantially affect the basis and novelty of the claimed disclosure."

[0070] IL-2 variants

[0071] In one aspect, this disclosure provides an IL-2 variant that differs from wild-type IL-2 (SEQ ID NO: 102) in that three to eight amino acid substitutions of amino acid residues H16, D20, R38, F42, Y45, E61, E62, L72, and V91 selected from SEQ ID NO: 102. In some embodiments, the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E61A, E62N, L72G, and V91H. As used herein, the term "IL-2 variant" refers to a non-naturally occurring variant of IL-2 that is capable of binding to a homologous IL-2 receptor on immune cells and initiating signal transduction through that receptor to achieve substantially the same effects as naturally occurring cytokines.

[0072] The amino acid sequence of wild-type IL-2 is shown below (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt All descriptions of the IL-2 variants disclosed herein are based on SEQ ID NO: 102. In some embodiments, the IL-2 variants comprise a first amino acid substitution (three to eight amino acid substitutions) selected from H16A, H16R, H16S, D20A, or D20Q from the N-terminus to the C-terminus, a second amino acid substitution selected from R38D, F42A, and V91H, and a third amino acid substitution selected from Y45A and E62N.

[0073] In some embodiments, the IL-2 variant contains a first amino acid substitution of H16A, H16R, or H16S. In other embodiments, the IL-2 variant contains a first amino acid substitution of D20A or D20Q.

[0074] In some embodiments, the IL-2 variant contains a second amino acid substitution of F42A. In some embodiments, the IL-2 variant contains a first amino acid substitution of H16A, H16R, or H16S and a second amino acid substitution of F42A. In some embodiments, the IL-2 variant contains a first amino acid substitution of D20A or D20Q and a second amino acid substitution of F42A.

[0075] In some embodiments, the IL-2 variant contains a third amino acid substitution of Y45A. In other embodiments, the third amino acid substitution is E62N.

[0076] In some embodiments, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. In some embodiments, the IL-2 variant comprises a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A.

[0077] In some embodiments, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. In some embodiments, the IL-2 variant comprises a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A.

[0078] In some implementations, the IL-2 variant contains a fourth amino acid substitution selected from R38D, E61A, L72G, and V91H.

[0079] In some embodiments, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, and a fourth amino acid substitution selected from D20A, D20Q, R38D, Y45A, E61A, L72G, and V91H.

[0080] In some embodiments, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, L72G, and V91H, and a fifth amino acid substitution of R38D.

[0081] In some implementations, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, and a sixth amino acid substitution of L72G.

[0082] In some embodiments, the IL-2 variant comprises a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A, a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, a sixth amino acid substitution of L72G, and a seventh amino acid substitution of E62N.

[0083] In some embodiments, the IL-2 variant has amino acid substitutions D20Q, F42A, and Y45A, and has the amino acid sequence shown below (SEQ ID NO: 112). The amino acid substitutions of SEQ ID NO: 112-123 relative to wild-type IL-2 are boxed in the following sequences.

[0084] 1 aptssstkkt qlqlehlllq lqmilnginn yknpkltrml tafampkka telkhlqcle

[0085] 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr

[0086] 121 witfcqsiis tlt

[0087] In some embodiments, the IL-2 variant has amino acid substitutions for H16A, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 113): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt In some embodiments, the IL-2 variant has amino acid substitutions for H16A, F42A, and Y45A, and has the following amino acid sequence (SEQ ID NO: 114): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfampkka telkhlqcle61 eelkpleevl nlaqsknfhl rprdlisnin In some embodiments, the IL-2 variant has amino acid substitutions for H16R, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 115): 1 aptssstkkt qlqlerllld lqmilnginn yknpkltrml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt In some embodiments, the IL-2 variant has amino acid substitutions for H16S, F42A, and Y45A, and has the following amino acid sequence (SEQ ID NO: 116): 1 aptssstkkt qlqlesllld lqmilnginn yknpkltrml In some embodiments, the IL-2 variant has amino acid substitutions D20Q, V91H, F42A, and E62N.And has the following amino acid sequence (SEQ ID NO: 1171 aptssstkkt qlqlehlllq lqmilnginn yknpkltrml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin hivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt). In some embodiments, the IL-2 variant has amino acid substitutions for H16A, F42A, Y45A, and L72G, and has the following amino acid sequence (SEQ ID NO: 118): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfampkka telkhlqcle61 eelkpleevl ngaqsknfhl rprdlisnin vivlelkgse ttfmceyade In some embodiments, the IL-2 variant has amino acid substitutions at H16R, D20Q, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 119): 1 aptssstkkt qlqlerlllq lqmilnginn yknpkltrml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt In some embodiments, the IL-2 variant has amino acid substitutions at H16S, D20Q, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 120): 1 aptssstkkt qlqleslllq lqmilnginn yknpkltrml In some embodiments, the IL-2 variant has amino acid substitutions at H16S, F42A, Y45A, and L72G.And has the following amino acid sequence (SEQ ID NO: 121): 1 aptssstkkt qlqlesllld lqmilnginn yknpkltrml takfampkka telkhlqcle61 eelkpleevl ngaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt In some embodiments, the IL-2 variant has amino acid substitutions at H16A, D20A, R38D, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 122): 1 aptssstkkt qlqleallla lqmilnginn yknpkltdml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade In some embodiments, the IL-2 variant has amino acid substitutions at H16R, D20Q, R38D, F42A, and E62N, and has the following amino acid sequence (SEQ ID NO: 123): 1 aptssstkkt qlqlerlllq lqmilnginn yknpkltdml takfympkka telkhlqcle61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt,

[0088] CAR-E (CAR-Connector)

[0089] The IL-2 variants disclosed herein can be used in adoptive cell therapy, particularly CAR T therapy, as an immune effector domain of a therapeutic component known as a CAR-encoder platform (also called a CAR enhancer), as disclosed in the applicant's PCT application PCT / US2024 / 013091, entitled "Developing a CAR-Encoder Platform to Enhance the Functionality and / or Persistence of CAR T Cells". As disclosed therein, CAR-encoders can enhance the in vivo functionality and persistence of CAR immune cells (e.g., CAR T cells). They can also reduce the cell dose required for CAR immune cell therapy, which can lead to a reduction in adverse side effects (e.g., cytokine release syndrome) caused by the larger doses commonly used in clinical practice, and therefore, CAR-encoders are also referred to herein as CAR-encoders or CAR-E. Furthermore, since the binding of the CAR-adaptor to CAR is reversible, the CAR-adaptor does not induce the formation of immune synapses on the surface of CAR immune cells. Therefore, the CAR-adaptor does not block CAR-mediated killing of cancer cells. CAR immune cells typically do not persist in vivo during minimal residual disease (MRD), which, as is known in the art, is associated with limited cancer antigens. The disclosed CAR-adaptor supports the persistence, proliferation, and efficacy of CAR T cells during MRD.

[0090] Therefore, in another aspect, this disclosure provides a CAR-E comprising a first proteinaceous portion and a second proteinaceous portion. The first proteinaceous portion binds to an epitope on the extracellular binding domain (ED) of the CAR. In some embodiments, the first proteinaceous portion binds to an epitope present on the extracellular binding domain (EBD) of the CAR, which binds to cancer antigens on the surface of cancer cells. The second proteinaceous portion, as an immune effector domain, comprises an IL-2 variant of this disclosure.

[0091] In some embodiments, the CAR-adaptor is a continuous protein, wherein the first protein entity and the second protein entity are linked by peptide bonds. In some embodiments, the first protein entity and the second protein entity are linked by click chemistry.

[0092] In some embodiments, the CAR-adaptor is formulated and administered as a monomeric protein or a proteinic entity. In other embodiments, the CAR-adaptor is formulated and administered as a homodimeric or heterodimeric protein or a proteinic entity, in dimer form.

[0093] CAR-E – First Protein Part

[0094] The first proteinaceous portion of the CAR-adaptor is designed to bind an epitope present on the extracellular binding domain of the CAR that targets an antigen on the surface of a cancer cell. In some embodiments, the first portion of the CAR-adaptor is the extracellular domain of the EBD that binds to the CAR presented on an immune cell. As is known in the art, the extracellular domain of a cancer antigen is a portion of an antigen on the surface of a cancer cell that binds to a T-cell receptor or CAR on an immune cell. In some embodiments, the CAR-adaptor may comprise the entire extracellular domain of the cancer antigen. According to standard techniques, the extracellular domain may be derived from the cancer antigen (e.g., identified in the cancer antigen). See, for example, Gershoni et al., Biodrugs21(3):145-156 (2007) and Francino-Urdaniz and Whitehead, RSC Chem. Biol.2(6):1580-1589 (2021). As used herein, when referring to proteins and nucleic acids, the term “derived from” means a sequence that is derived from and identified as a parental (e.g., wild-type or endogenous) protein and nucleic acid sequence, respectively. A sequence derived from a parental sequence can be a portion (fragment) of the parental sequence and / or may differ from the parental amino acid or nucleotide sequence by at least one position. Protein variants may contain amino acid substitutions, insertions, and / or deletions. For example, an amino acid sequence derived from a parental sequence may constitute a fragment of the parental sequence and be identical for a specific range of amino acids in the parent, but excluding amino acids outside that specific region. Nucleic acid variants may contain substitutions or in-frame insertions or deletions (i.e., insertions or deletions that do not result in a downstream frameshift of a nucleic acid codon).

[0095] The amino acid sequences of representative cancer antigens that can be targeted by CAR immune cells and from which extracellular domains can be derived are provided by the NCBI accession numbers shown in Table 1 and are incorporated herein by reference.

[0096] Table 1: Gene names, symbols, and NCBI accession numbers of representative cancer antigens

[0097] The extracellular domain is not limited to known cancer antigens. Unique cancer antigens (neoantigens) can be identified using known methods. For example, a cancer genome can be compared with a normal cell genome to identify a neoantigen. In some embodiments, the cancer transcriptome is compared with a normal cell transcriptome. Computational methods can then be used to identify suitable binding sites for the CAR. Most commonly, the CAR binds to a portion of the extracellular domain of the antigen. In some embodiments, the CAR and the corresponding cancer antigen are known in the art.

[0098] In some implementations, the extracellular domain of the CAR-adaptor contains the entire extracellular domain of the cancer antigen. In some implementations, the CAR-adaptor contains a portion of the extracellular domain of the cancer antigen targeted by the CAR.

[0099] In some implementations, the CAR targets BCMA, and the extracellular domain of the CAR-adaptor contains the extracellular domain of BCMA. A representative amino acid sequence of a CAR-adaptor containing the extracellular domain of BCMA is MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

[0100] In some embodiments, the extracellular domain of the CAR-adaptor contains two repeats of the extracellular domain of BCMA. A representative amino acid sequence of a CAR-adaptor containing two repeats of the extracellular domain of BCMA is shown below (SEQ ID NO: 2): 1 mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk gtnagggsgg61 gsprgsgggs mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk121 gtn In some embodiments where the CAR targets CD19, the extracellular domain of the CAR-adaptor contains a variant of the extracellular domain of CD19. The amino acid sequence of a representative CAR-adaptor for a variant containing the CD19 extracellular domain is shown below (SEQ ID NO: 3): 1 peeplvvkve egdeawlpcl kgtsdgptqq ltwsresplk pflkvsfgvp glgvhvrpna61 vslvisnvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwnvsdl gglgcglknr121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lnqslsrdmt vapgstlwls181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvtgtrlflp rataqdagky241 ychrgnltms fhlevkarpv sahtklrtgg In some embodiments, the extracellular domain of wk has at least 85% sequence identity with SEQ ID NO: 3, at least 90% sequence identity with SEQ ID NO: 3, at least 95% sequence identity with SEQ ID NO: 3, at least 98% sequence identity with SEQ ID NO: 3, and at least 99% sequence identity with SEQ ID NO: 3.

[0101] The amino acid sequence of the representative CAR-adaptor of the second variant containing the CD19 extracellular domain is shown below (SEQ ID NO: 4): 1 peeplvvkve egdeawlpcl kgtsdgptqq ltwsresplk pflkvsfgvp glgvhvrpna61 vslvisqvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwqvsdl gglgcglkqr121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lqqslsrdmt vapgstlwls181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvtgtrlflp rataqdagky241 ychrgqltms fhlevkarpv sahtklrtgg In some implementations, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of CD19. The amino acid sequence of a representative CAR-adaptor containing the CD19 extracellular domain is shown below (SEQ ID NO: 5): 1 peeplvvkve egdnavlqcl kgtsdgptqq ltwsresplk pflklslglp glgihmrpla61 iwlfifnvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwnvsdl gglgcglknr121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lnqslsqdlt mapgstlwls181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvmetglllp rataqdagky241 ychrgnltms fhleitarpv lwhwllrtgg In some embodiments, the extracellular domain of the CAR-adaptor contains a portion of the extracellular domain of CD19. In some embodiments, the extracellular domain of the CAR-adaptor is KDRPEIWEGEPP (SEQ ID NO: 106), which corresponds to amino acid residues 142-153 of SEQ ID NO: 5.

[0102] In some implementations, the CAR targets CD20, and the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of CD20. A representative amino acid sequence of a CAR-adaptor containing the CD20 extracellular domain is KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

[0103] In some implementations, the CAR targets CD22, and the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of CD22. The amino acid sequence of a representative CAR-adaptor containing the CD22 extracellular domain is shown below (SEQ ID NO: 7): 1 dsskwvfehp etlyawegac vwipctyral dgdlesfilf hnpeynknts kfdgtrlyes61 tkdgkvpseq krvqflgdkn knctlsihpv hlndsgqlgl rmesktekwm erihlnvser121 pfpphiqlpp eiqesqevtl tcllnfscyg ypiqlqwlle gvpmrqaavt stsltiksvf181 trselkfspq wshhgkivtc qlqdadgkfl sndtvqlnvk htpkleikvt psdaivregd241 svtmtcevss snpeyttvsw lkdgtslkkq ntftlnlrev tkdqsgkycc qvsndvgpgr301 seevflqvqy apepstvqil hspavegsqv eflcmslanp lptnytwyhn gkemqgrtee361 kvhipkilpw hagtyscvae nilgtgqrgp gaeldvqypp kkvttviqnp mpiregdtvt421 lscnynssnp svtryewkph gaweepslgv lkiqnvgwdn ttiacaacns wcswaspval481 nvqyaprdvr vrkikplsei hsgnsvslqc dfssshpkev qffwekngrl lgkesqlnfd541 sispedagsy scwvnnsigq taskawtlev lyaprrlrvs In some embodiments, the extracellular domain of the CAR-adaptor contains a portion of the extracellular domain of CD22 (SEQ ID NO: 7). In some embodiments, the extracellular domain of the CAR-adaptor contains Ig domains 2 and 3 (2-3) of CD22.The amino acid sequences of a representative CAR-adaptor containing CD22 Ig domains 2-3 are shown below (SEQ ID NO: 103): 1 phiqlppeiq esqevtltcl lnfscygypi qlqwllegvp mrqaavtsts ltiksvftrs61 elkfspqwsh hgkivtcqlq dadgkflsnd tvqpkleikv tpsdaivreg dsvtmtcevs121 ssnpeyttvs wlkdgtslkk qntftlnlre vtkdqsgkyc cqvsndvgpg rseevflq In some embodiments, the extracellular domain of the CAR-adaptor contains CD22 Ig domain 3. The amino acid sequence of a representative CAR-adaptor containing the Ig domain 3 of CD22 is shown below (SEQ ID NO: 104): 1 pkleikvtps daivregdsv tmtcevsssn peyttvswlk dgtslkkqnt ftlnlrevtk61 dqsgkyccqv sndvgpgrse evflq In some embodiments, the extracellular domain of the CAR-adaptor contains the Ig domains 5 to 7 (5-7) of CD22. The amino acid sequences of the representative CAR-adaptor containing the Ig domains 5-7 of CD22 are shown below (SEQ ID NO: 105): 1 pkkvttviqn pmpiregdtv tlscnynssn psvtryewkp hgaweepslg vlkiqnvgwd61 nttiacaacn swcswaspva lnprdvrvrk ikplseihsg nsvslqcdfs sshpkevqff121 wekngrllgk esqlnfdsis pedagsyscw vnnsigqtas prrlrvsmsp gdqvmegksa181 tltcesdanp pvshytwfdw nnqslpyhsq klrlepvkvq hsgaywcqgt nsvgkgrspl241 In some embodiments of the CAR-targeted sealing protein 18.2, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of the sealing protein 18.2.The amino acid sequence of a representative CAR-adaptor containing the first extracellular domain of sealing protein 18.2 is shown below (SEQ ID NO: 8): 1 dqwstqdlyn npvtavfnyq glwrscvres sgftecrgyf tllglpamlq avr The amino acid sequence of a representative CAR-adaptor containing the second extracellular domain of sealing protein 18.2 is shown below (SEQ ID NO: 9): 1 vtnfwmstan mytgmggmvq tvqtrytfga a In some embodiments where the CAR targets SLAMF7, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of SLAMF7. The amino acid sequence of a representative CAR-adaptor containing the SLAMF7 extracellular domain is shown below (SEQ ID NO: 10): 1 sgpvkelvgs vggavtfplk skvkqvdsiv wtfnttplvt iqpeggtiiv tqnrnrervd61 fpdggyslkl sklkkndsgi yyvgiysssl qqpstqeyvl hvyehlskpk vtmglqsnkn121 gtcvtnltcc mehgeedviy twkalgqaan eshngsilpi swrwgesdmt ficvarnpvs181 rnfsspilar klcegaaddp dssm In some embodiments of CAR targeting PD-1, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of PD-1. The amino acid sequence of a representative CAR-adaptor containing the PD-1 extracellular domain is shown below (SEQ ID NO: 11): 1 fldspdrpwn pptfspallv vtegdnatft csfsntsesf vlnwyrmsps nqtdklaafp61 edrsqpgqdc rfrvtqlpng rdfhmsvvra rrndsgtylc gaislapkaq ikeslraelr121 vterraevpt ahpspsprpa gqfqtlv In some embodiments, the extracellular domain of the CAR-adaptor contains a variant of the PD-1 extracellular domain. In some embodiments, the extracellular domain of the CAR-adaptor contains the N-loop of PD-1. The amino acid sequence of a representative CAR-adaptor containing the N-loop of the PD-1 extracellular domain is LDSPDRPWNP (SEQ ID NO: 107), which corresponds to amino acid residues 2-11 of SEQ ID NO: 11.

[0104] In some implementations, the extracellular domain of the CAR-adaptor contains a CD loop of PD-1. A representative amino acid sequence of a CAR-adaptor containing a CD loop of the PD-1 extracellular domain is NQTDKLAAFPEDRSQPGQDCRFRVTQ (SEQ ID NO:108), which corresponds to amino acid residues 51-76 of SEQ ID NO: 11.

[0105] In some implementations of CAR-targeted KIT, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of KIT. The amino acid sequence of a representative CAR-adaptor containing the KIT extracellular domain is shown below (SEQ ID NO: 12): 1 qpsvspgeps ppsihpgksd livrvgdeir llctdpgfvk wtfeildetn enkqnewite61 kaeatntgky tctnkhglsn siyvfvrdpa klflvdrsly gkedndtlvr cpltdpevtn121 yslkgcqgkp lpkdlrfipd pkagimiksv krayhrlclh csvdqegksv lsekfilkvr181 pafkavpvvs vskasyllre geeftvtcti kdvsssvyst wkrensqtkl qekynswhhg241 dfnyerqatl tissarvnds gvfmcyannt fgsanvtttl evvdkgfini In some embodiments of CAR-targeting TROP2, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of TROP2.The amino acid sequence of a representative CAR-adaptor containing the TROP2 extracellular domain is shown below (SEQ ID NO: 13): 1 htaaqdnctc ptnkmtvcsp dgpggrcqcr algsgmavdc stltskclll karmsapkna61 rtlvrpseha lvdndglydp dcdpegrfka rqcnqtsvcw cvnsvgvrrt dkgdlslrcd121 elvrthhili dlrhrptaga fnhsdldael rrlfreryrl hpkfvaavhy eqptiqielr181 qntsqkaagd vdigdaayyf erdikgeslf qgrggldlrv rgeplqvert liyyldeipp241 In some embodiments of CAR-targeting CD38, the extracellular domain of the CAR-adaptor contains at least a portion of the extracellular domain of CD38. The amino acid sequence of a representative CAR-adaptor containing the CD38 extracellular domain is shown below (SEQ ID NO: 14): 1 vprwrqqwsg pgttkrfpet vlarcvkyte ihpemrhvdc qsvwdafkga fiskhpcnit61 eedyqplmkl gtqtvpcnki llwsrikdla hqftqvqrdm ftledtllgy laddltwcge121 fntskinyqs cpdwrkdcsn npvsvfwktv srrfaeaacd vvhvmlngsr skifdknstf181 gsvevhnlqp ekvqtleawv ihggredsrd lcqdptikel esiiskrniq fsckniyrpd241 kflqcvknpe In some embodiments of dssctsei's CAR-targeted mesothelin (MSLN), the extracellular domain of the CAR-adaptor contains at least a portion of the MSLN, which is a GPI-anchored protein, and thus the entire MSLN protein is extracellular.The amino acid sequence of a representative CAR-adaptor containing MSLN is shown below (SEQ ID NO: 15): 1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss61 lsprqllgfp caevsglste rvrelavala qknvklsteq lrclahrlse ppedldalpl121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw241 svstmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq aprrplpqva421 tlidrfvkgr gqldkdtldt ltafypgylc slspeelssv ppssiwavrp qdldtcdprq481 ldvlypkarl afqnmngsey fvkiqsflgg aptedlkals qqnvsmdlat fmklrtdavl541 pltvaevqkl lgphveglka eerhrpvrdw ilrqrqddld tlglglqggi pngylvldls601 mqealsgtpc llgpgpvltv In some embodiments, the extracellular domain of the CAR-adaptor contains a portion of the extracellular domain of a cancer antigen. In some embodiments, the extracellular domain of the CAR-adaptor contains a portion of an MSLN protein. In some embodiments, the extracellular domain of the CAR-adaptor is IPXGYLVLDLSMQEALS (SEQ ID NO: 17), where X is any amino acid. In some embodiments, the extracellular domain of the CAR-adaptor is YXVXDLSMQEL (SEQ ID NO: 18), where X is any amino acid.

[0106] CAR-E Part 1: CAR-binding antibodies and their derivatives

[0107] In some implementations, the first part of the CAR-adaptor may be an antibody that binds to an epitope on the ED of the CAR, or an ED-binding derivative thereof. Antibody derivatives include antibody fragments (e.g., scFv and nanobody fragments).

[0108] In some embodiments of CAR targeting CD19, the first part of the CAR-adaptor is an anti-CD19 antibody binding portion that binds to an epitope on the CAR whose EBD binds to CD19. A representative anti-CD19 binding moiety is shown below (SEQ ID NO: 124): 1 qvqlqqpgae lvrpgasvkl scktsgysft rywmnwvkqr pgqglewigm ihpsdsetrl61 nqkfkdkatl tvdnssstay mqlssptsed savyycasiy yeeawgqgtl vtvsaggggs121 ggggsggggs diqmtqspas lsasvgetvt itcrasgnih nylawyqqkq gkspqllvyn181 aktladsvps rfsgsgsgtq yslkinslqp edfgsyycqh fwstpytfgg In some embodiments, the first portion of the CAR-adaptor binds to an epitope on a portion of the ED that does not directly bind to a cancer antigen, such as a linker (e.g., a linker between the VH and VL regions of the ED). In some embodiments, for example, where the CAR's EBD contains a linker containing a G4S motif, the first portion of the CAR-adaptor may be an anti-(G4S) binding portion that binds to an epitope on the CAR, the CAR's linker having at least two repeats of GGGGS (SEQ ID NO: 71). Representative heavy chain variable regions (VH) of the anti-G4S binding site are shown below (SEQ ID NO: 125): 1 qsvkesggrl vtpgtpltlt ctvsgfslss naidwvrqap gkglewigil grsgstyyas61 wakgrftisr tssttvdlki tspttedtat yfcarhfylw gpgtlvtvss Representative light chain variable regions (VL) of the anti-G4S binding site are shown below (SEQ ID NO: 126): 1 aqvltqtasp vsaavggtvt incqasqsvy snylswyqqk pgqppkllma ttstlepgvp61 srfkgsgsgt qftltisdle cddaatyyca ggysvdiwvf In some embodiments, the first part of the CAR-adaptor is an anti-κ light chain antibody-binding moiety that binds to an epitope on the CAR whose EBD is the κ light chain. A representative anti-κ light chain binding moiety is shown below (SEQ ID NO: 110).

[0109] 1 mkinkkllma alagaivvgg ganayaaeed ntdnnlsmde isdayfdyhg dvsdsvdpve

[0110] 61 views hidslnhlse takklakndi dsattinain divaradvme

[0111] 121 rktaekeeae claaketak khidelkhla dktkelakrd idsattinai ndivaradvm

[0112] 181 erktaekeea eklaaaketa kkhidelkhl adktkelakr didsattida indivaradv

[0113] 241 merklseket pepeeevtik anlifadgst qnaefkgtfa kavsdayya dalkkdngey

[0114] 301 tvdvadkglt lnikfagkke kpeepkeevt ikvnlifadg ktqtaefkgt feeatakaya

[0115] 361 yadllaken eytadledgg ntinifagk etpetpeepk eevtikvnli fadgkiqtae

[0116] 421 fkgtfeeata kayayanlla kengeytadl edggntinik fagketpetp eepkeevtik

[0117] 481 vnlifadgkt qtaefkgtfe eataeayrya dllakvngey tadledggyt inikfagkeq

[0118] 541 pgenpgitid ewllknakee aikelkeagi tsdlyfslin kaktvegvea lkneilkaha

[0119] 601 geetpelkdg yatyeeaeaa akealknddv nnayeivqga dgryyyvlki evadeeepge

[0120] 661 dtpevqegya tyeeaeaaak ealkedkvnn ayevvqgadg ryyyvlkied kedeqpgeep

[0121] 721 genpgitide wllknakeda ikelkeagis sdiyfdaink aktvegveal kneilkahae

[0122] 781 kpgenpgiti dewllknake aaikelkeag itaeylfnli nkaktvegve slkneilkah

[0123] 841 aekpgenpgi tidewllkna kedaikelke agitsdiyfd ainkaktieg vealkneilk

[0124] 901 ahkkdeepgk kpgedkkped kkpgedkkpe dkkpgedkkp edkkpgktdk dspnkkkkak

[0125] 961 lpkagseaei ltlaaaalst aagayvslkk rk

[0126] In some implementations, the first part of the CAR-adaptor is an anti-mouse antibody-binding moiety that binds to an epitope on the CAR whose EBD is derived from a mouse-derived antibody. Representative anti-mouse binding moieties are known in the art; see Kochenderfer et al., J. Immunother.32(7):689-702 (2009) and Cheng et al., Cytometry A.103(1):16-26 (2023).

[0127] Other applicable antibodies and their derivatives that can be combined with CAR ED are known in the art, see, for example, U.S. Patent 9,701,758 and U.S. Patent Application Publication 2005 / 0287148, both of which are incorporated herein by reference in their entirety.

[0128] CAR-E – Part Two: Immune Cell Effector Domains

[0129] The second part comprises the immune cell effector domain containing the IL-2 variant as described herein. The IL-2 variant binds to one or more of the homologous receptor subunits of IL-2Rα, IL-2Rβ, and IL-2Rγ on immune cells (containing CARs). This binding event modulates the activity of CAR-immune cells. As used herein, the terms “modulate” and “modulation” encompass both the activation and inhibition of CAR-immune cells.

[0130] In some embodiments, the CAR-adaptor contains multiple (i.e., two, three, or more) immune cell effector domains, wherein at least one of these immune cell effector domains is an IL-2 variant as described herein, and wherein any two or more of these immune cell effector domains may be identical or different from each other. In some embodiments, the CAR-adaptor contains two immune cell effector domains containing two IL-2 variants as disclosed herein. In some embodiments, the CAR-adaptor contains three immune cell effector domains containing three IL-2 variants as disclosed herein.

[0131] In some embodiments, the second immune cell effector domain is a cytokine, an immune cell activating portion, or an immune cell suppressor portion, as well as variants and fragments thereof, that binds to its homologous target on an immune cell. As is known in the art, the term "cytokine" includes low-molecular-weight extracellular polypeptides / glycoproteins that promote, regulate, and modulate immune responses (i.e., increase or decrease activity, differentiation, or proliferation). Representative examples of cytokines include chemokines, interferons (IFNs), interleukins (ILs), lymphokines, and tumor necrosis factor (TNF). As used herein, the term "immune cell activating variant" of a cytokine refers to a non-naturally occurring variant of a cytokine that is capable of binding to a cytokine receptor on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effects as naturally occurring cytokines.

[0132] In some implementations, the second immune cell effector domain is an immune cell activation portion, such as immune cell activating cytokines and their immune cell activating variants and fragments. The immune cell activation portion activates, promotes, or maintains the activity of immune cells.

[0133] In some implementations, the second immune cell effector domain is derived from CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-induced TNFR-associated protein ligand (GITRL; TNFSF18), herpesvirus entry mediator (HVEM; TNFSF14), semaphorin 3B (SEMAA; SEMA3B), member 1 of the signal transduction lymphocyte activation molecule family (SLAM; SLAMF1; CD150), member 4 containing T cell immunoglobulin and mucin domain (TIM4), member 4 of the TNF superfamily (TNFSF4; OX40L), member 8 of the TNF superfamily (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 member 9 of the TNF superfamily; TNFSF9), or their immune cell activation variants.

[0134] The amino acid sequences of representative immune cell activation portions (e.g., cytokines) from which the second immune cell effector domain can be derived are provided with the NCBI accession numbers shown in Table 2 and are incorporated herein by reference.

[0135] Table 2: Gene names, symbols, and NCBI accession numbers of representative immune cell activation proteins

[0136] In some implementations, the second immune cell effector domain may be derived from wild-type IL-2, but differs from the IL-2 variants disclosed herein. The amino acid sequences of representative IL-2 are provided under NCBI accession number NP_000577 and are incorporated herein by reference. In some embodiments, the immune cell effector domain contains a weak affinity variant of IL-2 (muIL2) having the following amino acid sequence (SEQ ID NO: 19): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tlt In some embodiments, the second immune cell effector domain is an IL-2 variant having an H16A substitution (i.e., alanine (A) at amino acid residue 16 is replaced with histidine (H)) and / or an F42A substitution (i.e., alanine (A) at amino acid residue 42 is replaced with phenylalanine (F)), both substituted alanine residues being shown as the framed amino acids in SEQ ID NO: 19.

[0137] In some embodiments, the second immune cell effector domain contains at least a portion of a weak-affinity IL-2 variant, which together have the following amino acid sequence (SEQ ID NO: 20): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tltggggsgg ggsggggsgg ggsaptssst kktqlqleal lldlqmilng181 innyknpklt rmltakfymp kkatelkhlq cleeelkple evlnlaqskn fhlrprdlis241 ninvivlelk gsettfmcey adetativef lnrwitfcqs iistlt with SEQ ID Compared to NO: 19, the natural (wild-type) sequence of human IL-2 (NCBI accession number NP_000577) has a higher affinity for the IL-2 receptor (IL-2R). In some embodiments, the second immune cell effector domain contains at least a portion of wild-type IL-2, having the following amino acid sequence (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr121 witfcqsiis tltmuIL2 (SEQ ID NO: 19) The second immune cell effector domain has a dissociation constant (K) of approximately 1200 nM for IL-2Rα (CD25). D (Compared to wild-type IL-2, it showed a reduction to 1 / 110), and had a K+ of approximately 610 nM against IL-2Rβ. D (Compared to wild-type IL-2, it showed a reduction of up to 1 / 3).

[0138] In some embodiments, the second immune cell effector contains at least a portion of IL-7. A representative amino acid sequence of IL-7 is shown below (SEQ ID NO: 21): 1 mfhvsfryif glpplilvll pvassdcdie gkdgkqyesv lmvsidqlld smkeigsncl61 nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgq121 vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh In some embodiments, the second immune cell effector domain contains at least a portion of IL-15. The amino acid sequence of a representative IL-15 is shown below (SEQ ID NO: 22): 1 mriskphlrs isiqcylcll lnshflteag ihvfilgcfs aglpkteanw vnvisdlkki61 edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann121 slssngnvte sgckeceele eknikeflqs fvhivqmfin ts In some embodiments, the second immune cell effector domain contains at least a portion of IL-18. The amino acid sequence of a representative IL-18 is shown below (SEQ ID NO: 23): 1 maaepvednc infvamkfid ntlyfiaedd enlesdyfgk lesklsvirn lndqvlfidq61 gnrplfedmt dsdcrdnapr tifiismykd sqprgmavti svkcekistl scenkiisfk121 emnppdnikd tksdiiffqr svpghdnkmq fesssyegyf lacekerdlf klilkkedel181 gdrsimftvq ned In some embodiments, the second immune cell effector domain contains at least a portion of IL-21.The amino acid sequence of a representative IL-21 is shown below (SEQ ID NO: 24): 1 mrsspgnmer iviclmvifl gtlvhksssq gqdrhmirmr qlidivdqlk nyvndlvpef61 lpapedvetn cewsafscfq kaqlksantg nneriinvsi kklkrkppst nagrrqkhrl121 tcpscdsyek kppkeflerf ksllqkmihq hlssrthgse ds In some embodiments, the second immune cell effector domain contains at least a portion of IL-27. The amino acid sequence of a representative IL-27 is shown below (SEQ ID NO: 25): 1 mgqtagdlgw rlsllllpll lvqagvwgfp rppgrpqlsl qelrreftvs lhlarkllse61 vrgqahrfae shlpgvnlyl lplgeqlpdv sltfqawrrl sdperlcfis ttlqpfhall121 gglgtqgrwt nmermqlwam rldlrdlqrh lrfqvlaagf nlpeeeeeee eeeeeerkgl181 lpgalgsalq gpaqvswpql lstyrllhsl elvlsravre llllskaghs vwplgfptls241 pqp In some embodiments, the second immune cell effector domain contains an amino acid sequence that binds to IL-2R-β, having the following amino acid sequence (SEQ ID NO: 25). At least a portion of neoleukin-2 / 15 (Neo-2 / 15) of 26).

[0139] 1 gshmpkkkiq lhaehalyda lmilnivktn sppaeekled yafnfelile eiarlfesgd

[0140] 61 qkdeaekakr mkewmkrikt tasedeqeem anaiitilqs wifs

[0141] In some embodiments, the second immune cell effector domain contains two repeats of Neo-2 / 15 having the amino acid sequence of SEQ ID NO: 26.

[0142] In some embodiments, the second immune cell effector domain contains at least a portion of 4-1BBL. 4-1BBL is also known as TNF ligand superfamily member 9 (TNFSF9). The amino acid sequence of a representative 4-1BBL is provided with NCBI accession number NP_003802 and is incorporated herein by reference. In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of 4-1BBL. In some embodiments, the second immune cell effector domain contains a portion of the extracellular domain of 4-1BBL, having the following amino acid sequence (SEQ ID NO: 27): 1 dpaglldlrq gmfaqlvaqn vllidgplsw ysdpglagvs ltgglsyked tkelvvakag61 vyyvffqlel rrvvagegsg svslalhlqp lrsaagaaal altvdlppas searnsafgf121 qgrllhlsag qrlgvhlhte ararhawqlt qgatvlglfr vtpeipa In some embodiments, the CAR-adaptor contains three immune cell effector domains, for example, wherein the first immune cell effector domain is an IL-2 variant, and the second and third immune cell effector domains are the extracellular domains of 4-1BBL, each having the amino acid sequence of SEQ ID NO: 27.

[0143] In some embodiments, the second immune cell effector domain may be a fragment that binds to and activates CAR immune cells, such as a single-chain variable antibody fragment (scFv). In some embodiments, the second immune cell effector domain is an scFv that binds to epitopes on 4-1BB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1.

[0144] In some implementations, the second immune cell effector domain is derived from commercially available anti-CTLA4 antibodies, antibody fragments, or derivatives thereof, such as bavunalimab (formerly known as pavunalimab / XmAb 22841), botensilimab, cadonilimab, and yervoy. ® ), quavonlimab, imjudo ®), volrustomig, vudalimab, or zalifrelimab. In some embodiments, the second immune cell effector domain is a scFv that binds to CTLA4. The representative heavy and light chain amino acid sequences of antibodies that bind to CTLA4 are shown in Table 3.

[0145] Table 3: Amino acid sequences of representative anti-CTLA antibody heavy and light chains

[0146] In some embodiments, the second immune cell effector domain contains VL, which has the following amino acid sequence (SEQ ID NO 36): 1 eivltqspgt lslspgerat lscraqsvsr ylgwyqqkpg qaprlliyga stratgipdr61 fsgsgsgtdf tltitrlepe dfavyycqqy gsspwtfgqg tkveik In some embodiments, the second immune cell effector domain contains VH, which has the following amino acid sequence (SEQ ID NO 37): 1 evqlvesggg lvkpggslrl scaasgftfs sysmnwvrqa pgkglewvss isssssyiyy61 aesvkgrfti srdnaknsly lqmnslraed tavyycarvg lfgpfdiwgq In some embodiments, the second immune cell effector domain binds to OX40. In some embodiments, the second immune cell effector domain is derived from a commercially available anti-OX40 antibody, an antibody fragment (e.g., scFv), or a derivative thereof, such as tavolimab or vonlerolizumab (Pogalizumab; MOXR 0916). The amino acid sequences of its representative heavy and light chains are shown in Table 4.

[0147] Table 4: Amino acid sequences of representative anti-OX40 antibody heavy and light chains

[0148] In some embodiments, the immune cell effector domain contains VL, which has the following amino acid sequence (SEQ ID NO 42): 1 diqmtqspss lsasvgdrvt itcrasqdis nylnwyqqkp gkapklliyy tsrlrsgvps61 rfsgsgsgtd ftltisslqp edfatyycqq ghtlpptfgq gtkveik In some embodiments, the immune cell effector domain contains VH, which has the following amino acid sequence (SEQ ID NO 43): 1 evqlvqsgae vkkpgasvkv sckasgytft dsymswvrqa pgqglewigd mypdngdssy61 nqkfrervti trdtststay lelsslrsed tavyycvlap rwyfsvwgqg In some embodiments, the second immune cell effector domain of tlvtvss binds to PD-1. In some embodiments, the second immune cell effector domain is derived from a commercially available anti-PD-1 antibody, antibody fragment (e.g., scFv), or a derivative thereof, such as atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, and imfinzi. ® (i) inbakicept, lodapolimab, pimivalimab, or socazolimab. The representative heavy and light chain amino acid sequences are shown in Table 5.

[0149] Table 5: Amino acid sequences of representative anti-PD-1 antibody heavy and light chains

[0150] In some embodiments, the second immune cell effector domain contains VL, which has the following amino acid sequence (SEQ ID NO 52): 1 eivmtqspat lsvspgerat lscrasqsvs snlawyqqkp gqaprlliyg astratgipa61 rfsgsgsgte ftltisslqs edfavyycqq ynnwprtfgq ​​gtkveik In some embodiments, the second immune cell effector domain contains VH, which has the following amino acid sequence (SEQ ID NO 53): 1 qvqlvesggg vvqpgrslrl scaasgftfs sygmhwvrqa pgkglewvav iwydgsnkyy61 adsvmgrfti srdnskntly lqmnslraed tavyycasng dhwgqgtlvt In some embodiments, the second immune cell effector domain is an immunosuppressive portion, representative types of which include immunosuppressive cytokines and their immunosuppressive variants and fragments. The immunosuppressive portion inhibits or blocks immune cell activity and function. In some embodiments, the immunosuppressive portion may be derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galactolectin-3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4). The amino acid sequences of representative immunosuppressive proteins from which the second immune cell effector domain can be derived are provided with the NCBI accession numbers shown in Table 6 and are incorporated herein by reference.

[0151] Table 6: Gene names, symbols, and NCBI accession numbers of immune cell inhibitory proteins

[0152] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD80. A representative amino acid sequence of the CD80 extracellular domain is shown below (SEQ ID NO: 54): 1 vihvtkevke vatlscghnv sveelaqtri ywqkekkmvl tmmsgdmniw peyknrtifd61 itnnlsivil alrpsdegty ecvvlkyekd afkrehlaev tlsvkadfpt psisdfeipt121 snirriicst sggfpephls wlengeelna inttvsqdpe telyavsskl dfnmttnhsf181 mclikyghlr vnqtfnwntt kqehfpdn In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD86. The amino acid sequence of a representative CD86 extracellular domain is shown below (SEQ ID NO: 55): 1 aplkiqayfn etadlpcqfa nsqnqslsel vvfwqdqenl vlnevylgke kfdsvhskym61 grtsfdsdsw tlrlhnlqik dkglyqciih hkkptgmiri hqmnselsvl anfsqpeivp121 isnitenvyi nltcssihgy pepkkmsvll rtknstieyd gvmqksqdnv telydvsisl181 svsfpdvtsn mtifciletd ktrllsspfs ieledpqppp dhip In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD155 (nectin-5; PVR).The amino acid sequence of the representative CD155 extracellular domain is shown below (SEQ ID NO:56): 1 wpppgtgdvv vqaptqvpgf lgdsvtlpcy lqvpnmevth vsqltwarhg esgsmavfhq61 tqgpsysesk rlefvaarlg aelrnaslrm fglrvedegn ytclfvtfpq gsrsvdiwlr121 vlakpqntae vqkvqltgep vpmarcvstg grppaqitwh sdlggmpnts qvpgflsgtv181 tvtslwilvp ssqvdgknvt ckvehesfek pqlltvnltv yyppevsisg ydnnwylgqn241 eatltcdars npeptgynws ttmgplppfa In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD276 (B7-H3).The amino acid sequence of the representative CD276 extracellular domain is shown below (SEQ ID NO: 57): 1 levqvpedpv valvgtdatl ccsfspepgf slaqlnliwq ltdtkqlvhs faegqdqgsa61 yanrtalfpd llaqgnaslr lqrvrvadeg sftcfvsird fgsaavslqv aapyskpsmt121 lepnkdlrpg dtvtitcssy qgypeaevfw qdgqgvpltg nvttsqmane qglfdvhsil181 rvvlgangty sclvrnpvlq qdahssvtit pqrsptgave vqvpedpvva lvgtdatlrc241 sfspepgfsl aqlnliwqlt In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of Ceacam-1. The amino acid sequence of a representative Ceacma-1 extracellular domain is shown below (SEQ ID NO: 58): 1 kltiesmpls vaegkevlll vhnlpqhlfg yswykgervd gnslivgyvi gtqqatpgaa61 ysgretiytn aslliqnvtq ndigfytlqv iksdlvneea tgqfhvyqen apglpvgava121 g In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of FGL1.The amino acid sequence of the representative FGL1 extracellular domain is shown below (SEQ ID NO: 59): 1 makvfsfilv ttaltmgrei saledcaqeq mrlraqvrll etrvkqqqvk ikqllqenev61 qfldkgdent vidlgskrqy adcseifndg yklsgfykik plqspaefsv ycdmsdgggw121 tviqrrsdgs enfnrgwkdy engfgnfvqk hgeywlgnkn lhflttqedy tlkidladfe181 knsryaqykn fkvgdeknfy elnigeysgt agdslagnfh pevqwwashq rmkfstwdrd241 hdnyegncae edqsgwwfnr chsanlngvy ysgpytaktd In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of galactoglobulin-3. The amino acid sequence of the representative extracellular domain of galactolectin-3 is shown below (SEQ ID NO: 60): 1 madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp61 gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl121 plpggvvprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnn181 wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi241 dltsasytmi In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of HLA-E.The amino acid sequence of the representative HLA-E extracellular domain is shown below (SEQ ID NO: 61): 1 gshslkyfht svsrpgrgep rfisvgyvdd tqfvrfdnda asprmvprap wmeqegseyw61 dretrsardt aqifrvnlrt lrgyynqsea gshtlqwmhg celgpdgrfl rgyeqfaydg121 kdyltlnedl rswtavdtaa qiseqksnda seaehqrayl edtcvewlhk ylekgketll181 hleppkthvt hhpisdheat lrcwalgfyp aeitltwqqd geghtqdtel vetrpagdgt241 fqkwaavvvp sgeeqrytch vqheglpepv tlrwkpasqp In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of HVEM (CD270). A representative amino acid sequence of the HVEM extracellular domain is shown below (SEQ ID NO: 62): 1 lpsckedeyp vgseccpkcs pgyrvkeacg eltgtvcepc ppgtyiahln glskclqcqm61 cdpamglras rncsrtenav cgcspghfci vqdgdhcaac rayatsspgq rvqkggtesq121 dtlcqncppg tfspngtlee cqhqtkcswl vtkagagtss shwv In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of nectin-2 (CD112, HVEB).The amino acid sequence of the representative extracellular domain of nectin-2 is shown below (SEQ ID NO: 63): 1 qdvrvqvlpe vrgqlggtve lpchllppvp glyislvtwq rpdapanhqn vaafhpkmgp61 sfpspkpgse rlsfvsakqs tgqdteaelq datlalhglt vedegnytce fatfpkgsvr121 gmtwlrviak pknqaeaqkv tfsqdpttva lciskegrpp ariswlssld weaketqvsg181 tlagtvtvts rftlvpsgra dgvtvtckve hesfeepali pvtlsvrypp evsisgyddn241 wylgrtdatl scdvrsnpep tgydwsttsg tfptsavaqg In some implementations, the second immune cell effector domain contains at least a portion of the extracellular domain of PD-L1. The amino acid sequence of a representative PD-L1 extracellular domain is shown below (SEQ ID NO: 64): 1 ftvtvpkdly vveygsnmti eckfpvekql dlaalivywe medkniiqfv hgeedlkvqh61 ssyrqrarll kdqlslgnaa lqitdvklqd agvyrcmisy ggadykritv kvnapynkin121 qrilvvdpvt seheltcqae gypkaeviwt ssdhqvlsgk ttttnskree klfnvtstlr181 intttneify ctfrrldpee nhtaelvipe lplahppner In some embodiments, the second immune cell effector domain contains at least a portion of the PD-L2 extracellular domain.The amino acid sequence of a representative PD-L2 extracellular domain is shown below (SEQ ID NO: 65): 1 lftvtvpkel yiiehgsnvt lecnfdtgsh vnlgaitasl qkvendtsph reratlleeq61 lplgkasfhi pqvqvrdegq yqciiiygva wdykyltlkv kasyrkinth ilkvpetdev121 eltcqatgyp laevswpnvs vpantshsrt peglyqvtsv lrlkpppgrn fscvfwnthv181 reltlasidl qsqmeprthp t In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of VTCN1 (B7-H4). The amino acid sequence of a representative VTCN1 is shown below (SEQ ID NO: 66): 1 liigfgisgr hsitvttvas agnigedgil sctfepdikl sdiviqwlke gvlglvhefk61 egkdelseqd emfrgrtavf adqvivgnas lrlknvqltd agtykcyiit skgkgnanle121 yktgafsmpe vnvdynasse tlrceaprwf pqptvvwasq vdqganfsev sntsfelnse181 nvtmkvvsvl ynvtinntys cmiendiaka tgdikvtese ikrrshlqll nskas.

[0153] dimerizing domain

[0154] In some embodiments, the CAR-integrator also includes a dimerizing domain. In these cases, the CAR-integrator is formed and applied in the form of a homodimer or a homopolymer. Thus, the homodimer contains two CAR-integrator entities. The order of the first part, the second part, and the dimerizing domain is not critical. In some embodiments, the dimerizing domain is positioned between the first part and the second part.

[0155] In some embodiments, the CAR-adaptor is in the form of a heterodimer, comprising a first portion connected to a first dimerizing domain and a second portion connected to a second dimerizing domain. In these embodiments, the first and second dimerizing domains dimerize the first and second portions to form a heterodimer.

[0156] In some embodiments, the first and second dimerizing domains contain a knock-in-hole configuration. One of these dimerizing domains contains a protrusion (knob), and the other dimerizing domain contains a cavity (cavity) that spatially compensates for the protrusion, wherein the tertiary structure of the protrusion can be positioned within the tertiary structure of the cavity. The dimerizing domain with the knock-in-hole configuration may have directional amino acid mutations, wherein the protrusion is an amino acid with a side chain volume larger than that present on dimerizing domains derived from natural sources (e.g., IgA, IgD, IgG, IgM, or IgE), and the cavity is an amino acid with a side chain volume smaller than that present on dimerizing domains derived from natural sources.

[0157] In some embodiments, the protrusion 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 dimerizing domain contains two amino acid substitutions, such as threonine (T) to lysine (K) and leucine (L) to lysine (K), while the second dimerizing domain contains leucine (L) to aspartic acid (D) or glutamic acid (E) and tyrosine (Y) to glutamic acid (E) or aspartic acid (D).

[0158] In some embodiments, the dimerizing domains are based on opposite charges. In some embodiments, the first dimerizing domain contains a positively charged amino acid, and the second dimerizing domain contains a negatively charged amino acid spatially opposite the positively charged amino acid in the first dimerizing domain.

[0159] Other arrangements of protrusions and cavities 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.

[0160] In some embodiments, the dimerizing domain may be derived from IgA, IgD, IgG, IgM, or IgE. The first and second dimerizing domains may contain the same or different amino acid sequences, provided they bind to each other. In some embodiments, the first and second dimerizing domains are IgG1 constant weight (CH)3 domains. The amino acid sequence of a representative IgG1 CH3 domain is shown below (SEQ ID NO: 67): 1 epkspksadk thtapqprep qvytlppsrd eltknqvslt clvkgfypsd iavewesngq61 pennykttpp vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg121 k In some embodiments, the first and second dimerizing domains are IgG1 constant weight CH2 domains. The amino acid sequence of a representative IgG1 CH2 domain is shown below (SEQ ID NO: 68): 1 pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna61 ktkpreeqyn styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kak In some embodiments, the first dimerizing domain and the second dimerizing domain are IgG1 CH2 and CH3 domains. The CH2 and CH3 domains can be interconnected via linkers. In some embodiments, the first dimerizing domain, the second dimerizing domain, or both the first dimerizing domain and the second dimerizing domain contain a crystallizable fragment region (Fc). In some embodiments, the Fc contains L234A and L235A substitutions relative to the wild-type Fc, which eliminates the binding of the Fc to (1) the Fc-γ receptor and (2) the complement component 1q (C1q), and is referred to herein as the “silent Fc”. The silent Fc maintains binding to the neonatal Fc receptor (FcRn) (and thus extends the circulating half-life of the CAR-E containing the silent Fc to several days). The silent Fc also provides stabilizing effects for the CAR-E (comparable to the stabilizing effects of the wild-type Fc). The two L234A and L235A substitutions are also commonly referred to as “LALA”. In some embodiments, the silent Fc also has a P329G substitution; these three L234A, L235A, and P329G substitutions are also commonly referred to as “PG-LALA”.

[0161] connector

[0162] In some implementations, the CAR-adaptor contains one or more adapters. The adapter is “flexible” because the peptide bonds allow amino acid residues within the adapter to rotate and allow the first and second portions to move and bind to their respective homologous receptors on the CAR-expressing immune cells or to the spatial space between the first and second portions (i.e., spacers).

[0163] The connector can be placed between any two CAR-connector components (also referred to in this document as domain, solid, part, or subunit).

[0164] The connector can be disposed between the dimerizing domain and the adjacent domain. In some embodiments, the connector can be disposed between the dimerizing domain and the second part. In some embodiments, the CAR-connector contains two connectors, wherein a first connector is disposed between the first part and the dimerizing domain, and a second connector is disposed between the dimerizing domain and the second part.

[0165] In some embodiments, the adapter comprises an amino acid 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 a repeating sequence 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), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 75), GSPRGGGGGSGGGGSGGGGS (SEQ ID NO: 76), GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 77), KESGSVSSEQLAQFRSLD (SEQ ID NO: 78), EGKSSGSGSESKST (SEQ ID NO: 79), or GSAGSAAGSGEF (SEQ ID NO: 80).

[0166] In some embodiments, the linker may be derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the linker may 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.

[0167] Table 7: Amino acid sequences of representative linkers

[0168] In some embodiments, the CAR-adaptor is in the form of a fusion protein, wherein the components are linked by peptide bonds. In other embodiments, the CAR-adaptor contains proteinaceous entities that can be covalently linked by click chemistry, a type of chemical link formed by a controlled chemical linking method. The links can be azide-alkyne links, oxime or hydrazine links, tetrazine-trans-cyclooctene links, azide-nitroketone links, thiols-olefin links, olefin-tetrazole links, olefin-tetrazine links, olefin-azide links, conjugated diene-olefin links, or isonitrile-tetrazine links.

[0169] Other controlled protein-linking chemistry, 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.

[0170] Methods for generating CAR-E

[0171] In some implementations, CAR-E or IL-2 variants may be encoded in a nucleic acid that is expressed in cells to produce CAR-E. As used herein, the term "nucleic acid" refers to a polymer of nucleotides, each of which is an organic molecule composed of a nucleoside (a nucleotide and a pentose sugar) and a phosphate ester. Unless specifically stated or obvious from the context, the term nucleotide includes nucleosides having either a ribose (i.e., a ribonucleotide that forms RNA) or a 2'-deoxyribose (i.e., a deoxyribonucleotide that forms DNA). Nucleotides act as monomeric units of nucleic acid polymers or polynucleotides. The four nucleotides in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleotides in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). Nucleic acids are linear chains of nucleotides (e.g., at least 3 nucleotides) that are chemically bonded by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2'-deoxyribose) in the adjacent nucleotide.

[0172] Given the number of nucleic acid sequences encoding wild-type IL-2 due to codon redundancy and codon preference, those skilled in the art will be able to prepare nucleic acid sequences encoding the IL-2 variants disclosed herein, taking into account specific amino acid substitutions relative to SEQ ID NO: 102, according to standard techniques. The nucleic acid sequence of a representative wild-type IL-2 is shown below (NCBI accession number NM_000586.4; SEQ ID NO: 111): 1 ctatcaccta agtgtgggct aatgtaacaa agagggattt cacctacatc cattcagtca61 gtctttgggg gtttaaagaa attccaaaga gtcatcagaa gaggaaaaat gaaggtaatg121 ttttttcaga caggtaaagt ctttgaaaat atgtgtaata tgtaaaacat tttgacaccc181 ccataatatt tttccagaat taacagtata aattgcatct cttgttcaag agttccctat241 cactctcttt aatcactact cacagtaacc tcaactcctg ccacaatgta caggatgcaa301 ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagtgcacc tacttcaagt361 tctacaaaga aaacacagct acaactggag catttactgc tggatttaca gatgattttg421 aatggaatta ataattacaa gaatcccaaa ctcaccagga tgctcacatt taagttttac481 atgcccaaga aggccacaga actgaaacat cttcagtgtc tagaagaaga actcaaacct541 ctggaggaag tgctaaattt agctcaaagc aaaaactttc acttaagacc cagggactta601 atcagcaata tcaacgtaat agttctggaa ctaaagggat ctgaaacaac attcatgtgt661 gaatatgctg atgagacagc aaccattgta gaatttctga acagatggat taccttttgt721 caaagcatcaIn some implementations, the CAR-adaptor is encoded by two nucleic acids, for example, the first part is encoded by a first nucleic acid and the second part is encoded by a second nucleic acid.

[0173] In some implementations, the nucleic acid encoding the CAR-adaptor includes a nucleic acid encoding a signal peptide located at the 5' of the nucleic acid encoding the first part. As used herein, the term "signal peptide" refers to a short (e.g., 5 to 30 or 10 to 100 amino acid-long) segment of amino acids that guides protein transport during translation. The CAR-adaptor containing the signal peptide will be secreted from the cell. Typically, the signal peptide is cleaved from the CAR-adaptor prior to secretion. The signal peptide may be linked to either the nucleic acid encoding the first part or the nucleic acid encoding the second part.

[0174] In some implementations, the signal peptide may be derived from the Ig-γ-3 heavy chain (IGHG3), albumin, CD8α, CD33, erythropoietin (EPO), IL-2, human or mouse Ig-κ chain V-III (IgK VIII), tissue plasminogen activator (tPA), or secretory alkaline phosphatase (SEAP). The signal peptide may also be synthetic (i.e., not naturally occurring). The amino acid sequences of representative signal peptides are listed in Table 8.

[0175] Table 8: Amino acid sequences of representative signal peptides

[0176] carrier

[0177] Nucleic acids encoding IL-2 variants and CAR-adaptor nucleic acids can be introduced into cells via suitable vectors. In embodiments where the first and second parts are chemically linked (e.g., via click chemistry), the CAR-encoding nucleic acid can be introduced into one or more cells via separate vectors. The vector is configured to contain elements necessary to achieve transport into immune cells and to achieve nucleic acid expression after transformation. Such elements include an origin of replication, a poly-A tail sequence, optional markers and one or more suitable sites for inserting the nucleic acid sequence (such as multiple cloning sites (MCS)), one or more suitable promoters (each promoter operatively linked to the insertion site of the nucleic acid sequence and the optional markers), and additional optional regulatory elements.

[0178] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates transcription of a corresponding nucleic acid coding sequence to which it is operatively linked, in the context of this disclosure being an IL-2 variant, and otherwise a CAR-adaptor protein containing that variant. A promoter can function alone to regulate transcription, or it can work 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 vector. Promoters are located near the transcription start site of a gene, on the same strand, and upstream of the DNA (towards the 5' region of the sense strand). Promoter length typically ranges from about 100 to 1000 base pairs.

[0179] As used herein, the term “operably linked” should be understood as a nucleic acid sequence being spatially located or situated in a vector relative to another nucleic acid sequence, for example, a promoter being operably linked to drive the expression of a nucleic acid coding sequence (e.g., a nucleic acid sequence encoding a CAR-adaptor).

[0180] In some embodiments, a single vector contains a single promoter operatively linked to a nucleic acid encoding a CAR-adaptor of an enhancer. In some embodiments, a single vector contains a single promoter operatively linked to both a nucleic acid encoding a first portion and a nucleic acid encoding a second portion. In some of these embodiments, the nucleic acids are separated by nucleic acids encoding a self-cleaving peptide or an internal ribosome entry site (IRES). In some embodiments, a single vector contains a first promoter operatively linked to a nucleic acid encoding the first portion and a second promoter operatively linked to a nucleic acid encoding the first portion.

[0181] In some implementations, two vectors are constructed. In some implementations, the first vector contains a promoter operatively linked to a nucleic acid encoding a first portion, and the second vector contains a promoter operatively linked to a nucleic acid encoding a second portion.

[0182] In some embodiments, the vector contains a strong mammalian promoter, such as a cytomegalovirus (CMV) promoter, an early simian virus 40 (SV40) promoter, a synthetic promoter (e.g., RPBSA (synthetic, derived from Sleeping Beauty) or CAG (synthetic, a CMV early enhancer element, a splice acceptor of chicken β-actin and rabbit β-globin)), or a promoter derived from β-actin, phosphoglycerate kinase (PGK), or factor EF1α genes. In some embodiments, the promoter may contain a core region located near the nucleic acid coding sequence. In some embodiments, the promoter is modified to remove a methylation-sensitive motif (e.g., a cytosine nucleotide followed by a guanine nucleotide, or "CpG"), or by adding a regulatory sequence that binds to transcription factors that inhibit DNA methylation. In some embodiments, the vector contains an A / T-rich nuclear matrix interaction sequence, called a scaffold matrix attachment region (S / MAR), which enhances transformation efficiency and improves the stability of transgene expression.

[0183] In some embodiments, the vector is a viral vector, such as a retroviral vector, lentiviral vector, adenoviral vector, herpesvirus vector, adenovirus, or adeno-associated virus (AAV) vector. The construction of lentiviral vectors has been described, for example, in U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530.

[0184] 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, mini-circles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and PiggyBac (PB)-based vectors. In yet another embodiment, the vector may include viral and non-viral elements.

[0185] In some embodiments, the vector is a plasmid. In addition to a promoter operatively linked to a nucleic acid, the plasmid may also contain other elements, such as those promoting the transport and expression of the nucleic acid in immune cells. The plasmid can be linearized with a restriction enzyme, transcribed in vitro to produce mRNA, and then modified with a 5' cap and a 3' poly-A tail. In some embodiments, the vector comprises multiple plasmids, with a first plasmid encoding a first protein entity (e.g., the extracellular domain of a CAR-adaptor) and a second plasmid encoding a second protein entity (e.g., the immune effector domain of a CAR-adaptor).

[0186] cell

[0187] IL-2 variants and CAR-E can be expressed in genetically modified (or transformed) cells containing a vector that encodes a nucleic acid that encodes an IL-2 variant, CAR-E, or a component of CAR-E for the purpose of protein preparation and purification.

[0188] Cells used for cloning and other manipulations of these vectors are routine. Cells from various strains of Escherichia coli (E. coli) can be used for vector replication and other steps in the construction of the CAR-adaptor disclosed herein.

[0189] Suitable host cells or cell lines for expressing nucleic acids encoding IL-2 variants and nucleic acids encoding CAR-adaptors include eukaryotic cells. In some embodiments, the cells are mammalian cell lines. In some embodiments, the cells are mammalian cells 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 (NS0)). In some embodiments, the nucleic acid encoding the CAR-adaptor is expressed in CHO or myeloma cells. Human cells can be used, thereby enabling the expressed CAR-adaptor to be modified with a human glycosylation pattern. The selection of suitable mammalian cells and methods for transformation, culture, amplification, screening, and product generation and purification is known in the art. See, for example, Green et al. (eds.), Molecular Cloning: A Laboratory Manual, 5th edition, ColdSpring Harbor Laboratory Press, New York, 2012.

[0190] In some embodiments, the cells are prokaryotic cells. Prokaryotic (i.e., bacterial) cells have proven suitable as host cells for expressing nucleic acids encoding CAR-adaptors (see, for example, Pluckthun, Immunol. Rev. 130:151-188 (1992)). However, since proteins expressed in bacterial cells tend to be in unfolded or misfolded forms or in non-glycosylated forms, the functional preservation (e.g., CAR-binding capacity of the expressed CAR-E) of any CAR-adaptor produced in bacterial cells will be screened. If a CAR-adaptor expressed by a bacterial cell is produced in a correctly folded form, that bacterial cell will be the desired host, or in alternative embodiments, the CAR-adaptor may be expressed in a bacterial host and then subsequently refolded. For example, various bacteria, such as *Escherichia coli*, are well-known host cells in the field of biotechnology. Various strains of *Bacillus subtilis*, *Streptomyces*, other bacilli, etc., may also be used.

[0191] After expression in cells, the IL-2 variant or CAR-adaptor protein is isolated from the cells (e.g., cell lysates) or from the culture medium containing the 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 electrophoresis, gel electrophoresis, and dialysis. In some embodiments, the affinity chromatography resin is a protein A affinity chromatography resin or a protein G affinity chromatography resin. Other protein separation systems and methods are known in the art. See, for example, 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).

[0192] Pharmaceutical compositions containing IL-2 variants or CAR-E

[0193] To implement the disclosed methods, IL-2 variants and / or CAR-adaptors can be formulated in pharmaceutically acceptable carriers. As used herein, the term "effective amount" refers to an amount of IL-2 variant or CAR-adaptor sufficient to provide the desired effect, which may include, for example, any one or more of the following: killing at least cancer cells; reversing, alleviating, improving, inhibiting, reducing, slowing, blocking, stabilizing, achieving relief or prevention of the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or cancer-related biochemical markers. The amount of IL-2 variant and CAR-adaptor administered to a subject will vary over a wide range, depending on the location, type, and severity of the cancer, the age, weight, and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dose to be used. The IL-2 variant and / or CAR-adaptor in the pharmaceutical composition may be in the form of monomers (in embodiments lacking dimerizing domains), homodimers, or heterodimers as described herein.

[0194] The IL-2 variants disclosed herein can be used alone or in combination with another active agent to treat cancer. See, for example, Ren et al., J. Clin. Invest. 132(3):e153604 pp. 1–13 (2022). High-dose IL-2 therapy can overcome regulatory T cell (Treg)-associated IL-2 capture and allow additional IL-2 activation of tumor-infiltrating lymphocytes (TILs) for the treatment of cancers such as metastatic renal cell carcinoma and melanoma. However, patients who respond to high-dose IL-2 therapy often suffer from intolerable toxicity (see, for example, Li et al., Nat. Commun. 8(1):1762 (2017)), which limits its clinical application. Therefore, the IL-2 variants of this disclosure could constitute more attractive alternatives in terms of binding to IL-2Rα on Tregs (see, for example, Mott et al., J. Mol. Biol. 247(5):979-94 (1995)) or increasing binding to IL-2Rβ on effector cells (see, for example, Levin et al., Nature 484(7395):529-33 (2012) and Sun et al., Nat. Commun. 10(1):3874 pp. 1-12 (2019)). The IL-2 variants of this disclosure can be conjugated to other active moieties, such as antibodies. See, Ren et al., ibid. Because tumor-infiltrating lymphocytes express more PD-1 than other cells, the IL-2 variants of this disclosure can be fused to anti-PD-1 antibodies. These fusion proteins can exhibit better intratumoral T cell binding, more potent antitumor activity, and can also overcome PD-L1 therapy resistance. The IL-2 variants can also be conjugated to antitumor antibodies. See, for example, Sun et al., Nat. Commun. 10(1):3874, pp. 1-12 (2019).

[0195] The composition can be provided as a sterile solid or liquid formulation. Solid formulations can be reconstituted and diluted into a liquid formulation prior to use, for example using a carrier comprising isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which can be buffered to a selected pH. Liquid carriers include aqueous and non-aqueous carriers, etc. Representative examples of liquid carriers include sterile water for injection, saline, lactated Ringer's solution, phosphate-buffered saline, soluble sugars (e.g., dextrose), dimethyl sulfoxide (DMSO), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier comprises a protein dissolved or dispersed therein; representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. In some embodiments, the liquid carrier comprises a water-miscible polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.). The compositions are typically isotonic, i.e., they have the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyols, and isotonic electrolyte solutions (e.g., Plasma-Lyte) can be used. ® To achieve the desired isotonicity, other excipients may be added, depending on the carrier, such as wetting agents, dispersants or emulsifiers, gelling agents and viscosity enhancers, preservatives, etc., known in the art. In some embodiments, the composition comprises citric acid, ethylenediaminetetraacetic acid (EDTA), and polysorbate 20, with a pH range of about 6.8 to about 7.2.

[0196] Cancer subjects

[0197] In some aspects, this disclosure relates to methods of treating a subject with cancer. In some embodiments, these methods may require administering an IL-2 variant itself, or in a form fused with another active portion, such as an antibody or a binding fragment thereof that binds to a receptor on immune cells (e.g., TILs) or an antibody that binds to an epitope on an antigen present on tumor cells. In some embodiments, the administration of the IL-2 variant is for the treatment of renal cancer (e.g., metastatic renal cell carcinoma), melanoma, colon cancer, lung cancer, or ovarian cancer.

[0198] In other respects, this method requires administering a pharmaceutical composition containing the CAR-adaptor described herein to a subject in need. The CAR-adaptor can be administered before, after, or substantially simultaneously with the administration of CAR immune cells. Cancers that can be treated according to the disclosed method broadly include hematopoietic system cancers and cancers characterized by the presence of solid tumors.

[0199] As used herein, the term “subject” (or “patient”) includes all members of the animal kingdom who are susceptible to (or prone to) or have a specified cancer. In some embodiments, the subject is a human being. Thus, a subject who is “suffering from cancer” or “requires” treatment according to this disclosure broadly encompasses subjects who have been diagnosed as positive, including subjects with active disease who may have previously been treated with one or more rounds of therapy, subjects who are currently not receiving treatment (e.g., in remission) but may still be at risk of relapse, and subjects who have not yet been diagnosed as positive but are susceptible to cancer (e.g., due to prior medical history and / or family history, or otherwise presenting one or more risk factors that a medical professional may reasonably suspect are susceptible to cancer).

[0200] As used herein, the term "treatment" refers to a recognized indicator of therapeutic efficacy, intervention, procedure performed on a subject in need, or administration of an active agent to a subject in need, wherein the therapeutic goal ("therapeutic effect") is to reverse, alleviate, improve, inhibit, reduce, slow, stop, stabilize, or prevent the onset, progression, development, metastasis, severity, or recurrence of symptoms, improve survival time, achieve complete or partial remission, reduce complications or symptoms, or improve biochemical indicators related to cancer. Remission may include undetectable cancer cells, a reduction in tumor cells, tumor shrinkage, or a decrease in the number of tumor cells.

[0201] In some implementations, the cancer is a hematopoietic system cancer. Representative hematologic cancers include plasmacytomas (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasmacytic myeloma, extramedullary multiple myeloma, monoclonal immunoglobulinemia of undetermined significance (MUGS), asymptomatic stagnant multiple myeloma, or solitary plasmacytoma), lymphomas (e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma, Burkitt lymphoma, Waldenstrom macroglobulinemia). Macroglobulinemia, plasmablastic lymphoma, plasmacytoid lymphoma, B-cell lymphoma, high-grade B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBL), follicular lymphoma (FL), and mantle cell lymphoma (MCL)), and leukemias (e.g., plasma cell leukemia, relapsed or refractory acute B-lymphoblastic leukemia (ALL), relapsed or refractory acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or chronic lymphocytic leukemia (CLL)).

[0202] In some implementations, the cancer is characterized by the presence of a solid tumor. In some implementations, 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). Cancers include tumors, skin cancers (e.g., melanoma, cutaneous melanoma (SKCM), basal cell carcinoma, and cutaneous squamous cell carcinoma), lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)), head and neck cancers (e.g., head and neck squamous cell carcinoma (SCCHN), also known as head and neck squamous cell carcinoma (HNSC), laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, oral and oropharyngeal cancer, and salivary gland cancer), colon or rectal cancers (e.g., colorectal cancer (CRC), colonic adenocarcinoma (COAD), rectal adenocarcinoma (READ)), ovarian cancers (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, ovarian epithelial carcinoma, fallopian tube cancer, and primary peritoneal cancer), breast cancers (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, liver cancer, brain cancers (e.g., astrocytoma and glioma, such as glioblastoma), stomach cancer, and biliary tract cancers. Cancer, serous carcinoma of the uterus, cholangiocarcinoma, neuroblastoma, sarcoma, endometrial cancer, cervical cancer (e.g., cervical squamous cell carcinoma and cervical adenocarcinoma (CESC)), prostate cancer (e.g., prostate cancer (PRAD)) and gastric cancer (e.g., gastric adenocarcinoma (STAD)).

[0203] In some implementations, the disclosed methods treat malignant mesothelioma, ovarian cancer, breast cancer (e.g., TNBC), pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary tract cancer, serous uterine carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, or melanoma.

[0204] In some implementations, the disclosed methods also include anti-CD19 immunotherapy, CD19-binding CAR-E, and are used to treat HL, non-Hodgkin lymphoma, ALL, CLL, chronic lymphocytic leukemia, Burkitt lymphoma, DLBCL, PMBL, high-grade B-cell lymphoma, FL, MCL, or multiple myeloma (MM).

[0205] In some implementations, the disclosed methods also include anti-BCMA immunotherapy and BCMA-bound CAR-E, and are used to treat MM, HL, non-Hodgkin lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), plasma cell leukemia, SLE, acute AMR, chronic AMR, or AL-amyloidosis.

[0206] In some implementations, cancer is characterized by 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 physical signs or symptoms of cancer and are often undetectable by conventional methods such as examining cells under a microscope and / or tracking abnormal serum proteins in the blood.

[0207] The amount of cancer antigens present in subjects in MRD status is limited. Furthermore, this limited presence of cancer antigens may be insufficient to support the proliferation and efficacy of CAR immune cells. The additional presence of the CAR-adaptor not only presents additional cancer antigens to CAR immune cells but also presents a supporting IL-2 variant, and in some embodiments, presents a second immune cell effector domain that can modulate the activity of CAR immune cells to promote proliferation, efficacy, and / or persistence.

[0208] In some embodiments, the subject receiving CAR-adaptor administration is in a state of minimal residual disease (MRD). In some embodiments, the method of treating cancer involves treating the subject's minimal residual disease (MRD) state. In some embodiments, the method of treating cancer involves eliminating MRD in the subject.

[0209] To test for MRD, samples from blood draws or bone marrow aspirations can be used. The most widely used assays for measuring MRD are flow cytometry, polymerase chain reaction (PCR), and next-generation sequencing. Methods applicable to measuring MRD are described, for example, in 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.

[0210] CAR Immunotherapy

[0211] In some embodiments, the methods of treating cancer disclosed herein involve administering an IL-2 variant and / or CAR-E to a subject who has received CAR immunotherapy. As is known in the art, CAR immunotherapy cells contain synthetic CAR molecules that bind to cancer antigens. Typically, CARs contain an extracellular domain that binds to CAR-E, a transmembrane domain, and an intracellular domain that includes a stimulating domain.

[0212] The extracellular domain of a CAR that binds to the extracellular domain of a cancer antigen may contain an antibody fragment. In some embodiments, the CAR binds to BCMA. CAR extracellular domains that bind to BCMA are known in the art. See, for example, FDA-approved CAR-expressing immune cell line Carvykti.® (also referred to in this article as "Cilta-cel") and Abecma ® The CAR (also referred to herein as "Ide-cel") is associated with 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 extracellular domain of the CAR is derived from a commercially available anti-BCMA antibody, a BCMA-binding fragment, or a derivative thereof, such as belantamab (Blenrep). ® ), linvoseltamab (REGN5458), pacanalotamab (AMG 420), pavurutamab (AMG 701), and teritolamab (Tecvayli) ® In some implementations, the extracellular domain of the CAR binds to the BCMA extracellular domain of the CAR-adaptor having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0213] In some implementations, the CAR extracellular domain contains a single variable heavy chain (VHH) or a variant thereof. In some embodiments, VHH has the following amino acid sequence (SEQ ID NO: 127): 1 qvkleesggg lvqagrslrl scaasehtfs shvmgwfrqa pgkeresvav igwrdistsy61 adsvkgrfti srdnakktly lqmnslkped tavyycaarr idaadfdswg qgtqvtvss In some embodiments, VHH has the following amino acid sequence (SEQ ID NO: 128): 1 evqlvesggg lvqaggslrl scaasgrtft mgwfrqapgk erefvaaisl sptlayyaes61 vkgrftisrd nakntvvlqm nslkpedtal yycaadrksv msirpdywgq gtqvtvss In some embodiments, the CAR extracellular domain contains VH, which has the following amino acid sequence (SEQ ID NO: 128): 1 evqlvesggg lvqaggslrl scaasgrtft mgwfrqapgk erefvaaisl sptlayyaes61 vkgrftisrd nakntvvlqm nslkpedtal yycaadrksv msirpdywgq gtqvtvss In some embodiments, the CAR extracellular domain contains VH, which has the following amino acid sequence (SEQ ID NO: 128): 1 qvkleesggg lvqagrslrl scaasehtfs shvmgwfrqa pgkeresvav igwrdistsy61 adsvkgrfti srdnakktly lqmnslkpedtal yycaadrksv msirpdywgq gtqvtvss 129): 1 divltqspps lamslgkrat iscrasesvt ilgshlihwy qqkpgqpptl liqlasnvqt61 gvparfsgsg srtdftltid pveeddvavy yclqsrtipr tfgggtklei k In some embodiments, the CAR extracellular domain contains a VL having the following amino acid sequence (SEQ ID NO: 130): 1 qiqlvqsgpe lkkpgetvki sckasgytft dysinwvkra pgkglkwmgw intetrepay61 aydfrgrfaf sletsastay lqinnlkyed tatyfcaldy syamdywgqg tsvtvss In some embodiments, the CAR binds to CD19. CAR extracellular domains that bind to CD19 are known in the art. See, for example, the FDA-approved CAR-expressing immune cell niche myroncis (lisocabtagene maraleucel, Breyanzi). ® ), Tisagenlecleucel, Kymriah ® ), Breki Orensey (brexucabtageneautoleucel, Tecartus) ®) and axicabtagene ciloleucel, Yescarta ® U.S. Patents 9,629,877, 10,273,300, and 10,533,055, and U.S. Patent Application Publications 2020 / 0392248 and 2021 / 0238253. In some embodiments, the CAR extracellular domain is derived from commercially available anti-CD19 antibodies, anti-CD19 binding fragments, or derivatives thereof, such as loncastuximab (Zynlonta). ® ), tancituzumab (Tafasitamab, Monjuvi) ® ), denintuzumab (SGN-CD19A) and inebilizumab (Uplizna) ® In some embodiments, the extracellular domain of the CAR binds to the CD19 extracellular domain of the CAR-adaptor having an amino acid sequence of any one of SEQ ID NO:3-5 or 103.

[0214] In some embodiments, the CAR extracellular domain contains VH, which has the following amino acid sequence (SEQ ID NO: 131): 1 diqmtqttss lsaslgdrvt iscrasqdis kylnwyqqkp dgtvklliyh tsrlhsgvps61 rfsgsgsgtd ysltisnleq ediatyfcqq gntlpytfgg gtkleit In some embodiments, the CAR extracellular domain contains VL, which has the following amino acid sequence (SEQ ID NO: 132): 1 evklqesgpg lvapsqslsv tctvsgvslp dygvswirqp prkglewlgv iwgsettyyn61 salksrltii kdnsksqvfl kmnslqtddt aiyycakhyy yggsyamdyw In some embodiments, the CAR binds to CD20. The extracellular domain of the CAR that binds to CD20 is 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 extracellular domain of the CAR is derived from commercially available anti-CD20 antibodies, anti-CD20 binding fragments, or derivatives thereof, such as arzerra (or arzerra).® Kesimpta ® ), Vetuzumab (IMMU-106), Tosimomab (Bexxar) ® ) and rituximab (Rituxan ® ,Riabni ® Truximab ® In some implementations, the extracellular domain of the CAR binds to the CD20 extracellular domain of the CAR-adaptor having the amino acid sequence SEQ ID NO: 6.

[0215] In some embodiments, the CAR binds to CD22. The extracellular domain of the CD22-binding CAR is 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 extracellular domain of the CAR is derived from commercially available anti-CD22 antibodies, anti-CD22 binding fragments, or derivatives thereof, such as betuzumab, epazolizumab, inotuzumab, mosetumumab, and epazolizumab. In some embodiments, the extracellular domain of the CAR binds to the CD22 extracellular domain of the CAR-adaptor. In some embodiments, the CAR-adaptor contains the CD22 extracellular domain having the amino acid sequence SEQ ID NO: 7. In some embodiments, the CAR-adaptor contains the CD22 extracellular domain having the amino acid sequences SEQ ID NO: 104-107.

[0216] In some embodiments, the CAR binds to sealing protein 18.2. The extracellular domain of the CAR that binds to sealing protein 18.2 is known in the art. See, for example, U.S. Patents 10,421,817, 11,098,118, 11,485,782, 11,541,127, and 11,713,346, and U.S. Patent Application Publications 2022 / 0073643, 2023 / 0192840, and 2023 / 0242877. In some embodiments, the extracellular domain of the CAR is derived from commercially available anti-sealing protein 18.2 antibodies, anti-sealing protein 18.2 binding fragments, or derivatives thereof, such as oxetinemab and zotuximab (Vyloy). ®In some embodiments, the extracellular domain of the CAR binds to the sealing protein 18.2 extracellular domain of the CAR-adaptor. In some embodiments, the CAR-adaptor contains the sealing protein 18.2 extracellular domain having any of the amino acid sequences SEQ ID NO: 8-9.

[0217] In some embodiments, the CAR binds to SLAMF7. The extracellular domain of the CAR that binds to SLAMF7 is known in the art. See, for example, U.S. Patent 10,799,536 and U.S. Patent Application Publications 2020 / 0024342, 2020 / 0283534, 2021 / 0230548, and 2021 / 0253729. In some embodiments, the extracellular domain of the CAR is derived from a commercially available anti-SLAMF7 antibody, an anti-SLAMF7 binding fragment, or a derivative thereof, such as erlotinib (Empliciti). ® In some embodiments, the extracellular domain of the CAR binds to the SLAMF7 extracellular domain of the CAR-adaptor. In some embodiments, the extracellular domain of the CAR binds to the SLAMF7 extracellular domain of the CAR-adaptor having the amino acid sequence SEQ ID NO: 10.

[0218] In some embodiments, the CAR binds to PD-1. The extracellular domain of the CAR 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 extracellular domain of the CAR is derived from commercially available anti-PD-1 antibodies, anti-PD-1 binding fragments, or derivatives thereof, such as batitinimab, budesonide, cantulimab, cimetidine, and libtayopicana. ® ), cialis, dotalimab (Jemperli ® ), ijulizumab, nivolumab (Opdivo) ® ), paparimizumab, pembrolizumab (Keytruda) ®The CARs include pembrolizumab, perisolimab, pidilizumab, riverilimab, rosnilimab, sintilimab, spartazumab, tislelizumab, toripalimab, vorsumiumab, vordalilimab, zeluvalimab, and cepalimumab. In some embodiments, the extracellular domain of the CAR binds to the PD-1 extracellular domain of the CAR-adaptor. In some embodiments, the extracellular domain of the CAR binds to the PD-1 extracellular domain of the CAR-adaptor having any of the amino acid sequences SEQ ID NO: 11 and 107-108.

[0219] In some embodiments, the CAR binds to mast cell / stem cell growth factor receptor Kit (KIT; also known as receptor tyrosine kinase KIT proto-oncogene). The extracellular domain of the CAR that binds 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 extracellular domain of the CAR is derived from a commercially available anti-KIT antibody, an anti-KIT binding fragment, or a derivative thereof, such as bazolimumab. In some embodiments, the extracellular domain of the CAR binds to the KIT extracellular domain of a CAR-adaptor. In some embodiments, the extracellular domain of the CAR binds to the KIT extracellular domain of a CAR-adaptor having the amino acid sequence SEQ ID NO: 12.

[0220] In some embodiments, the CAR binds to TROP2. The extracellular domain of the CAR that binds to TROP2 is known in the art. See, for example, U.S. Patents 11,602,525 and 11,768,203, and U.S. Patent Application Publications 2018 / 0296689, 2021 / 0169852, and 2022 / 0204582. In some embodiments, the extracellular domain of the CAR is derived from commercially available anti-TROP2 antibodies, anti-TROP2 binding fragments, or derivatives thereof, such as datopotamab and sacituzumab. In some embodiments, the extracellular domain of the CAR binds to the TROP2 extracellular domain of a CAR-adaptor. In some embodiments, the CAR-adaptor contains the TROP2 extracellular domain having the amino acid sequence SEQ ID NO: 13.

[0221] In some embodiments, the CAR binds to CD38. The extracellular domain of the CD38-binding CAR is 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 extracellular domain of the CAR is derived from a commercially available anti-CD38 antibody, an anti-CD38 binding fragment, or a derivative thereof, such as daratumumab. ® Sarclisa ® (and mizetuzumab). In some embodiments, the extracellular domain of the CAR binds to the CD38 extracellular domain of the CAR-adaptor. In some embodiments, the extracellular domain of the CAR binds to the CD38 extracellular domain of the CAR-adaptor having the amino acid sequence SEQ ID NO: 14.

[0222] In some embodiments, the CAR binds to the MSLN. The extracellular domain of the CAR that binds to the MSLN is known in the art. See, for example, U.S. Patents 10,550,179, 10,640,569, 10,730,954, 11,648,268, and 11,702,472, and U.S. Patent Application Publications 2020 / 0255803, 2021 / 0079057, and 2022 / 0112263. In some embodiments, the extracellular domain of the CAR is derived from a commercially available anti-MSLN antibody, an anti-MSLN binding fragment, or a derivative thereof, such as amatuximab. In some embodiments, the extracellular domain of the CAR binds to the MSLN extracellular domain of a CAR-adaptor. In some embodiments, the extracellular domain of the CAR binds to the MSLN extracellular domain of a CAR-adaptor having the amino acid sequences SEQ ID NO: 15 and 17-18.

[0223] The intracellular domain of a CAR contains signal transduction domains capable of enabling intracellular signal transduction and immune cell function. These signal transduction domains may include primary signal transduction domains and / or co-stimulatory signal transduction domains. In some embodiments, the intracellular domains are capable of delivering signals that are natively linked to an ITAM-containing molecule or receptor complex (such as a TCR receptor complex).

[0224] In some embodiments, the signal transduction domain includes multiple (e.g., two or three) co-stimulatory signal transduction domains, selected, for example, from 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the signal transduction domain may include a CD3ζ domain as a primary signal transduction domain, and any of the following co-stimulatory signal transduction domain pairs (from 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 signal transduction domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some implementations, the co-stimulatory signal transduction domains are derived from 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.

[0225] A representative CAR with a CD3ζ stimulatory signal transduction domain is the FDA-approved CAR-expressing immune cell cytokinase (Kymriah). ® A representative CAR with co-stimulatory signaling domains of CD3ζ and 4-1BB is the FDA-approved CAR-expressing immune cell Abecma. ® Breyanzi ® ) and Carvykti ® A representative CAR with co-stimulatory signaling domains of CD28 and CD3ζ is the FDA-approved CAR-expressing immune cell line *Tecartus*. ® ) and Yescarta ® ).

[0226] In some embodiments, the CAR immune cells are T cells. In some embodiments, the CAR immune cells are NK cells. Other CAR immune cells are known in the art, such as 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.

[0227] CAR immune cells can be autologous or allogeneic. For example, immune cells or their progenitor cells can be isolated from a subject's body fluids or tissues before being administered to the same subject (autologous) or different compatible subjects (allogeneic). Most typically, CAR immune cells are administered once.

[0228] The number of CAR immune cells administered to a subject will vary widely, depending on the location, type, and severity of the cancer, the individual's age, weight, and condition. The physician will ultimately determine the appropriate number and dosage of cells to be used. Typically, CAR immune cells will be administered in a single, one-time dose.

[0229] The effective dose (e.g., quantity) of CAR immune cells for cancer treatment is known in the art. In some embodiments, the effective quantity of CAR immune cells is between approximately 1 × 10⁻⁶ per subject. 4 One to approximately 1 × 10⁹ cells 10 Between 100 cells. In some implementations, the effective number of CAR immune cells is between approximately 1 × 10⁻⁶ per subject. 5 One to approximately 1 × 10⁹ cells 10 Between cells. In some implementations, the effective number of CAR immune cells is approximately the number of cells given in FDA-approved CAR T-cell therapies, which is between approximately 1 × 10⁻⁶ cells per kg of subject body weight. 6 One to approximately 1 × 10⁹ cells 10 Between 100 cells. In some implementations, the effective number of CAR immune cells is between 1 × 10⁻⁶ per kg of subject body weight. 5 One cell to approximately 6 × 10 8 Between individual cells.

[0230] Because CAR-adaptors promote the functionality and persistence of CAR immune cells, it is envisioned that CAR therapies coordinating the administration of CAR-adaptors may require fewer CAR immune cells compared to FDA-approved CAR T-cell therapies. Therefore, the methods disclosed herein may require fewer cells, for example, approximately 1 × 10⁻⁶ cells per kg of subject body weight. 4 One to approximately 1 × 10⁹ cells 7 Each cell.

[0231] CAR immune cells can be administered to subjects to treat cancer via any medically acceptable route. CAR immune cells are typically delivered intravenously, although they can also be introduced to other convenient sites (e.g., affected organs or tissues) or modalities, as determined by the attending physician.

[0232] CAR-E administration

[0233] In a broad sense, the order in which CAR-adaptor and CAR immune cells are administered during the same treatment process can vary, provided they can interact in vivo and produce the desired effect. In some embodiments, the CAR-adaptor and CAR immune cells are administered to the subject substantially simultaneously. In some embodiments, the CAR-adaptor and CAR immune cells are brought into contact in vitro before co-administration to the subject. In some embodiments, the CAR-adaptor is administered to the subject after the administration of CAR immune cells. In some embodiments, the CAR-adaptor is administered to the subject before the administration of CAR immune cells.

[0234] Broadly speaking, these methods require administering an effective amount of a CAR-adaptor to a cancer patient who has received, is receiving, or will receive CAR-containing immune cells. This CAR contains an extracellular domain that binds to the CAR-adaptor, a transmembrane domain, and an intracellular domain containing a stimulatory domain. The order in which the CAR-adaptor and CAR immune cells are administered during the same treatment process may not be critical, provided they can interact in vivo and elicit the desired effect. In some embodiments, the CAR-adaptor and CAR immune cells are co-administered to the subject substantially simultaneously. In some embodiments, the CAR-adaptor and CAR immune cells are contacted in vitro prior to co-administration to the subject. In some embodiments, the CAR-adaptor is administered to the subject prior to the administration of the CAR immune cells.

[0235] In some embodiments, the CAR-adaptor is administered to the subject after the administration of CAR immune cells, for example, when it is determined that the CAR immune cells have lost viability or persistence in the subject. This determination can be made according to known techniques. In some embodiments, a sample is obtained from the subject, for example, after the administration of immune cells. The concentration of immune cells present in the sample can be used to calculate the difference between the concentration of immune cells administered to the subject and the concentration of immune cells measured in the sample. The CAR-adaptor can be administered once the measured immune cell concentration is less than the administered immune cell concentration. In some embodiments, the CAR-adaptor is administered once the measured immune cell concentration is less than 90%, 80%, 70%, 60%, 50%, 25%, 10%, or 5% of the administered immune cell concentration.

[0236] In some implementations, the CAR-adaptor 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 the administration of CAR-immune cells.

[0237] In some implementations, the CAR-adaptor is administered for at least one cycle, such as weekly, bi-weekly, or tri-weekly. This cycle can be repeated, for example, for two, three, five, or eight cycles. In some implementations, the CAR-adaptor is administered for several consecutive days prior to the periodic administration, for example, once daily for five days, and then every three weeks thereafter. In some implementations, the CAR-adaptor is administered as an infusion over approximately 30 to approximately 90 minutes every three weeks (a 21-day cycle). In some implementations, the CAR-adaptor is administered every 21 days for five consecutive days, repeated for eight cycles.

[0238] In some implementations, the CAR-connector is administered as an intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some implementations, the infusion time is 30 to 60 minutes. In some implementations, the first administration is infused into the patient over 90 minutes, and subsequent administrations are infused into the patient over 30 minutes.

[0239] In some implementations where the CAR-adaptor is administered following CAR immune cells, the treatment may require a first course of CAR-adaptor therapy, which may begin up to approximately 6 months after administration of CAR immune cell therapy. As used herein, the term "effective amount" refers to an amount of CAR-adaptor sufficient to provide the desired effect, such as the amount of CAR-adaptor that binds to CAR-expressing immune cells.

[0240] In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 4 years after CAR immunotherapy. In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 3 years after CAR immunotherapy. In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 2 years after CAR immunotherapy. In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 1 year after CAR immunotherapy. In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 9 months after CAR immunotherapy. In some embodiments, the first course of CAR-enhanced therapy begins at any time up to approximately 6 months after CAR immunotherapy.

[0241] In some implementations, the first course of CAR-connector therapy can begin at any time up to about 5 months after CAR immunotherapy.

[0242] In some implementations, the first course of CAR-connector therapy can begin at any time up to about 4 months after CAR immunotherapy.

[0243] In some implementations, the first course of CAR-connector therapy can begin at any time up to about 3 months after CAR immunotherapy.

[0244] In some implementations, the first course of CAR-connector therapy can begin at any time up to about 2 months after CAR immunotherapy.

[0245] In some implementations, the first course of CAR-connector therapy can begin at any time up to about one month after CAR immunotherapy.

[0246] In some implementations, the first course of CAR-connector therapy can begin at any time up to about 4 weeks after CAR immunotherapy.

[0247] In some implementations, the first course of CAR-adaptor therapy can begin at any time up to about 3 weeks after CAR immunotherapy.

[0248] In some implementations, the first course of CAR-adaptor therapy begins approximately two weeks after CAR immunotherapy.

[0249] In some implementations, the first course of CAR-adaptor therapy begins two weeks after CAR immunotherapy.

[0250] In some implementations, the first course of CAR-enhancer therapy requires the administration of a total of approximately 1 to approximately 6 doses (e.g., 2, 3, 4, 5, or 6 doses) of the CAR-enhancer. In some implementations, the first course of CAR-enhancer therapy requires the administration of approximately 1 dose / week, approximately 2 doses / week, approximately 3 doses / week, or approximately 4 doses / week.

[0251] In some implementations, the first course of CAR-encoder therapy is performed over a period of approximately 1 to 3 weeks, with approximately 1 to 3 doses of CAR-encoder administered weekly.

[0252] The dose of CAR-enhancer administered during the first course of CAR-enhancer therapy can range from approximately 1 mg to approximately 8 mg per kg of patient body weight. In some implementations, the dose (effective dose) of CAR-enhancer is approximately 1 mg / kg, 2 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, or approximately 8 mg / kg.

[0253] In some embodiments, the method of the present invention further includes administering a second follow-up course of CAR-enhanced therapy to the subject. In these embodiments, the subject may have relapsed or be at risk of relapse. The CAR-enhanced therapy administered in the second course of CAR-enhanced therapy may be the same as or different from the CAR-enhanced therapy administered in the first course of CAR-enhanced therapy. The CAR-enhanced therapy administered in the second course of CAR-enhanced therapy may be administered at the same time period following the CAR immunotherapy described above, or at the same time amount described above, but after the first course of CAR-enhanced therapy.

[0254] Combination therapy

[0255] In some embodiments, this method may include co-administering another anticancer therapy. The term "co-administering" includes administration substantially simultaneously via the same or separate dosage forms, or sequentially, such as as part of the same treatment regimen or via a sequential treatment regimen. Thus, if administered sequentially, the first of the two therapies can still be detected at the treatment site at an effective concentration when the second therapy administration begins. Sequences and time intervals can be determined such that they can act together (e.g., synergistically providing increased benefit compared to when they are administered in other ways). For example, the therapeutic agents can be administered simultaneously or sequentially at different time points in any order; however, if not administered simultaneously, they can be administered at sufficiently close times to provide the desired therapeutic effect, which may be in a synergistic manner. Therefore, these terms are not limited to administering the active agent at exactly the same time.

[0256] In some implementations, the subject may have already received additional anticancer therapy. This additional therapy may be administered (1) before CAR immunotherapy, (2) after CAR immunotherapy but before the first course of CAR-E therapy, (3) after the first course of CAR-E therapy but before the second course of CAR-E therapy, or (4) after the second course of CAR-E therapy. In some implementations, the additional anticancer therapy is chemotherapy, radiotherapy, immunotherapy, targeted therapy, apoptosis-promoting therapy or cell cycle regulation therapy, or therapy using thalidomide, lenalidomide, bortezomib, and / or melphalan.

[0257] Expansion and differentiation agents can also be provided before, during, or after the administration of CAR immune cells to increase the differentiation, expansion, and / or persistence of CAR immune cells (e.g., T cells and NK cells).

[0258] Anticancer agents that can be used in combination with IL-2 variants and / or chimeric antigen receptor (CAR) adaptors are known in the art. See, for example, U.S. Patent No. 9,101,622 (section 5.2 thereof). “Anticancer” agents are capable of negatively affecting a subject’s cancer, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or increasing the lifespan of a subject with cancer. More generally, these other compositions will be provided in combined amounts that effectively kill cancer cells or inhibit cancer cell proliferation. This process may involve simultaneously contacting cancer cells with receptor cells and an agent or multiple agents. This can be achieved by contacting cancer cells with a single composition or pharmacological formulation comprising two agents, or by simultaneously contacting cancer cells with two different compositions or formulations, one composition comprising receptor cells and the other comprising a second agent.

[0259] In some embodiments, the IL-2 variants and / or CAR-adaptors disclosed herein are used in combination with or after prior therapy, such as chemotherapy, radiotherapy, immunotherapy intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.

[0260] In some embodiments, the disclosed IL-2 variant and / or CAR-adaptor are used in combination with high-dose chemotherapy prior to the administration of genetically modified immune cells. In some embodiments, bone marrow cells or peripheral blood stem cells are administered after high-dose chemotherapy.

[0261] In some embodiments, the IL-2 variants and / or CAR-adaptors disclosed herein are used in combination with effective amounts of thalidomide, lenalidomide, bortezomib, or combinations thereof.

[0262] Additional enhancement therapies that can be used in combination with the IL-2 variants and / or CAR-adaptors disclosed herein include melphalan. ® Evomela ® Melphalan is an alkylated antitumor agent used for high-dose pretreatment prior to hematopoietic stem cell transplantation in patients with multiple myeloma, as well as for palliative care of multiple myeloma and for remission of unresectable epithelial ovarian cancer. Melphalan is also used to treat AL amyloidosis, neuroblastoma, rhabdomyosarcoma, breast cancer, retinoblastoma of the eye, some pretreatment regimens before bone marrow transplantation, and in some cases, malignant melanoma. Melphalan can be administered orally in pill form. Typically, a 2 mg dose is taken on an empty stomach. In some cases, melphalan can be administered as an injection or intravenous infusion. Dosage depends on weight, height, disease and disease state, and the subject's overall health.

[0263] Immunotherapy

[0264] Immunotherapy, including immune checkpoint inhibitors, can be used to treat diagnosed cancers. Immune checkpoint molecules include, for example, PD-1, PDL1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Clinically available examples of immune checkpoint inhibitors include divalbumin (Imfinzi). ® Atezolizumab (Tecentriq) ® ) and averumab (Bavencio) ® Clinically available examples of PD-1 inhibitors include nivolumab (Opdivo). ® ), Pembrolizumab (Keytruda) ® ) and cimipril (Libtayo ® Other inhibitors that can be used in the practice of this disclosure are known in the art. See, for example, U.S. Patent Application Publications 2012 / 0321637, 2014 / 0194442 and 2020 / 0155520.

[0265] Chemotherapy

[0266] Cancer therapy also includes various combination therapies with chemotherapy and radiation-based treatments. Combination chemotherapy includes, for example, Abraxane. ® Hexamethylmelamine, docetaxel, Herceptin® Methotrexate, Novantrone ® Zoladex ® 1. Chloramphenicol (CDDP), Carboplatin, Procarbazine, Mechlorethamine, Cyclophosphamide, Camptothecin, Ifosfamide, Melphalan, Chlorobutyrate, Busulfan, Nitrosourea, Daunorubicin, Doxorubicin, Bleomycin, Priligy, Mitomycin, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen receptor binders, Taxol ® Gescitabine, Navelbine ® Farnesyltransferase inhibitors, cisplatin, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analogues or derivative variants of the foregoing, and combinations thereof.

[0267] Radiation therapy

[0268] Cancer therapy also includes radiation-based DNA-damaging treatments. Combined radiation therapy includes the targeted delivery of what are commonly referred to as gamma rays, X-rays, and / or radioactive isotopes to tumor cells, causing extensive damage to DNA, DNA replication and repair, and chromosome assembly and maintenance. The dosage range of the radioactive isotopes varies considerably and depends on the isotope's half-life, the intensity and type of emitted radiation, and the uptake by the proliferating cells, and will be determined by the attending physician.

[0269] Radiation therapy can be divided into external or internal radiation therapy. External radiation therapy involves placing a radiation source outside the subject's body and delivering radiation to the cancerous area inside the body. Internal radiation therapy uses radioactive material sealed in needles, seeds, threads, or catheters, which are placed directly in or near the cancer.

[0270] These and other aspects of this application will be further understood by considering the following embodiments, which are intended to illustrate certain implementations of this application but are not intended to limit the scope defined by the claims.

[0271] Example

[0272] Example 1: Materials and Methods

[0273] Protein cloning and expression were performed according to standard methods. Other procedures, including flow cytometry analysis, BLI imaging, CAR T cell generation, cell culture, and animal handling, were performed according to the standard protocols briefly described below.

[0274] CAR-Adaptor Generation. All genes were codon-optimized for mammalian expression in HEK293 cells, synthesized, and inserted into a vector expression system containing a signal sequence for protein secretion into the supernatant. To facilitate product production, a stable HEK293 cell line was generated. HEK293 cells were then transfected with pPAX2, pVSVG (packaging vector), and a lentiviral plasmid containing the sequence of interest. Lentiviral cells were harvested at 48, 72, and 96 hours post-transfection, precipitated at 20,000 × g for 2 hours, and resuspended in OptiMEM medium. New batches of HEK293 cells were then subjected to three rounds of viral transduction. Cells were allowed to be recovered in DMEM complete medium and subjected to puromycin selection to retain only cells integrated with the lentiviral plasmid. Cells were then expanded in four 15 cm dishes until they reached confluence, carefully washed with PBS, and incubated in serum-free DMEM for 24 to 48 hours. The supernatant was harvested, and protein expression was confirmed by SDS-PAGE and Western blotting. The protein was purified by adsorption onto a nickel-nitroglycerin (Ni-NTA) metal affinity column. Non-specifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM to allow recovery of the protein of interest. The protein was further purified by size exclusion chromatography and stored at -80 °C in 50 mM HEPES buffer (pH 7.5) until use.

[0275] Typically, some CAR-inverting molecules are separated by passing them through an affinity chromatography resin in the presence of a neutral phosphate buffer. The affinity chromatography resin is then subjected to an acidic buffer with a pH of approximately 3 to 4 to wash the CAR-inverting molecules away from the resin. The acidic buffer can be neutralized using an alkaline buffer, and then the neutralized buffer can be subjected to tangential flow filtration using a formulated buffer to separate a concentrated and purified solution containing the CAR-inverting molecules.

[0276] CAR T cell generation. The human CD19-binding CAR construct contained an scFv derived from the anti-human CD19 antibody clone FMC69, followed by intracellular signaling domains of human CD28 and CD3ζ. The human BCMA-binding CAR construct contained an scFv derived from the anti-human BCMA antibody clone MSK54, followed by intracellular signaling domains of human 39BB and CD3ζ. The human signaling CAR construct was transduced into HeLa cells stably producing an enveloped pseudotyped γ-retrovirus containing feline endogenous virus (RD114), which has been shown to efficiently transduce human hematopoietic cells (HSCs) (Ward et al., Mol. Ther. 8(5):804-12 (2003)). High viral titer clones were isolated by limiting dilution. High-expression clones were inoculated into DMEM complete medium containing 10% FBS and grown until 80% confluence. The medium was then replaced with RPMI complete medium containing 10% FBS. Twenty-four hours later, the virus-containing culture medium was harvested, sterilely filtered using a 0.45 μm PES filter, and used to generate CAR T cells.

[0277] The generation of CAR T cells was adapted from previous research. See, for example, Li et al., Methods Mol. Biol. 1514:111-118 (2017). In short, whole blood was obtained from apheresis filter loops of platelet-healthy donors because of the high number of viable leukocytes. Whole blood was centrifuged via a Ficoll gradient to separate PBMCs. Intact PBMCs were used, without selecting CD8+. + T cells. PBMCs were prepared at 4 × 10⁻⁶ 6 Cells were resuspended at a concentration of 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) in RPMI medium, 3 mL per well in 6-well plates. After 24 hours, cells were harvested, centrifuged, and resuspended in the same volume of fresh medium containing FBS, IL-2, IL-15, and IL-7, in addition to the medium harvested from cells producing anti-human BCMA CAR γ-retrovirus, thus resulting in PBMC inoculation with γ-retrovirus. PBMCs were then inoculated at 4 × 10⁻⁶ cells / mL. 6PBMCs were seeded at 3 mL / mL into 6-well plates coated with 20 μg retronectin (coated with 1 mL of PBS containing 20 μg / mL retronectin 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 process was repeated with medium containing fresh γ-retrovirus, cytokines, and centrifugation. Flow cytometry analysis was used to assess the transduction efficiency of the CAR transgene using the dsRed reporter gene and recombinant BCMA labeled with AlexaFlour-647.

[0278] In vivo experiments. For all experiments, NOD / SCID / γ (NSG; NOD.Cg-Prkdc) was used. scid Il2rg tm1Wjl / SzJ) mice were used because they were immunocompromised and could be effectively transplanted with human cancer cell lines. A multiple myeloma mouse model was established in NSG mice using cells from the human multiple myeloma OPM2 cell line. Cells expressing GFP and firefly luciferase were injected intravenously via the tail vein at a dose of 1×10⁻⁶ cells. 6 In vivo experiments were initiated with OPM2 cells, followed by bioluminescence imaging (BLI) every two weeks. After effective transplantation at 3 weeks, CAR T cells were intravenously injected into mice via the tail vein. BLI was then performed every two weeks to assess tumor burden. Quantification was performed using Aura software, measured in photons per second.

[0279] Organ analysis. At the end of the experiment, surviving mice were euthanized, 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 1 μg / mL collagenase), and incubated at 37°C for 1 hour. The spleen was crushed, and bone marrow was aspirated using a 30-gauge insulin needle. All treated cells were passed through a 70 μm filter to produce a single-cell suspension. The cells were resuspended in 1 mL of ammonium chloride-potassium (ACK) lysis buffer to deplete the erythrocytes in the sample at room temperature for 2 minutes. The resulting single-cell suspension was washed with PBS and 0.5% BSA in fluorescence-activated single-cell sorting (FACS) buffer, stained, and analyzed by flow cytometry.

[0280] Cell lines and cultures. The OPM2 cell line, endogenously expressing BCMA, was engineered to express green fluorescent protein (GFP) and firefly luciferase. Peripheral blood mononuclear cells (PBMCs) were obtained via a Ficoll gradient using a leukocyte-depleted filter loop from healthy platelet donors. HEK293T cells were cultured in complete DMEM (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), 1% penicillin and streptomycin (Cytiva), and 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), 1% penicillin and streptomycin (Cytiva), and 10% fetal bovine serum (FBS). All cells were grown at 37°C in an incubator with 5% CO2 and 95% air humidification.

[0281] Mouse studies. All experiments were conducted in accordance with 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 paper is described in Smith et al., Mol. Ther. 26(6):1447-1456 (2018). Briefly, 8- to 12-week-old NOD-scidIL2Rγ null (NSG) and NOD-scidH2-K1 null H2-Ab1 null H2-D1 null IL2Rg null (NSG-MHC I / II double knockout (DKO)) mice were purchased from Jackson Laboratory or bred in-house. All mice were matched for sex and age. Mice were administered 1×10⁻⁶ NSG-MHC I / II double knockout (DKO) via intravenous injection in 200 mL of PBS. 6 OPM2 or Nalm6 cells expressing GFP and luciferase were used to establish a xenograft model. Mice received a specified treatment via intraperitoneal injection in 300 mL PBS. Following intraperitoneal injection of D-luciferin (150 mg / kg, from a 15 mg / mL solution) at the specified time, IVIS was used. ®Tumor burden was assessed using the Lumina series III (PerkinElmer) imaging system. At the same time point (5 minutes), up to five mice were imaged in a supine position. BLI intensity was analyzed using Aura imaging analysis software (Spectral Instruments Imaging). Peripheral blood was obtained from mice via submandibular exsanguination in EDTA-coated tubes, and CAR T cell detection and amplification were analyzed. Briefly, the blood volume was determined to calculate the absolute value. The sample was then centrifuged, and serum was harvested. The cell pellet was resuspended in 500–1000 mL of 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, i.e., PBS + 1% bovine serum albumin (BSA). The samples were then stained with anti-CD45-PacificBlue (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 (prepared internally). Samples were processed on a Sony SP6800 spectrometer. Flow rates and acquisition times were recorded for absolute values. All experiments were performed blinded and randomized. Animals were euthanized at the end of the experiment or when they reached a pre-specified endpoint according to the IACUC protocol. At the endpoint, major immune organs (spleen and bone marrow) and essential organs that may form metastatic lesions (liver, lung, and kidney) were harvested, weighed, and analyzed. Briefly, 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, lung, and kidney were cut into small pieces using surgical scissors in 3 mL of digestion buffer (1 mL RPMI + 2 mL PBS). Type I collagenase (Worthington) was added to a final concentration of 100 mg / mL and incubated at 37°C for 1 hour. The resulting samples were passed through a 40 μm filter to obtain a single-cell suspension. The samples were then stained with the same antibody used to stain the blood samples and analyzed using a Sony SP6800 spectrometer.Record the flow rate and acquisition time to calculate the absolute value.

[0282] Microscopic examination. Cells were first stained with CellTracker Blue CMAC and seeded on poly-d-lysine-coated coverslips. The samples were then incubated with a specified treatment labeled with Alexa647 at a specified time and temperature. After fixation with BD Cytofix buffer, the cells were imaged using a Leica THUNDER imager. For all images, the intensity cutoff for the AlexaFluor647 channel was set to 2000, except for the VHH-muIL2 sample shown on the right (condition 4), for which image sensitivity was enhanced 25-fold (intensity cutoff 80) to reveal the Alexa647 signal. Quantitative analysis of fluorescence intensity was performed by comparing the logarithm of the ratio of the cell membrane to the cytoplasm's integrated intensity (see y-axis in Figure 16B). 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 CARs. + T cells. For VHH-muIL2, cells with an average intensity-to-background ratio greater than 2 based on the Alexa 647 channel were selected to eliminate background artifacts. Image quantification was performed using ImageJ software.

[0283] ELISA. ELISA analysis was performed to measure the levels of human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low-dose CAR T cell therapy, as shown in Figure 19A. The protein was analyzed using ELISAMAX according to the manufacturer's specifications. ™ The Biolegend standard set analysis included cytokines such as IFN-γ, GM-CSF, and TNF-α; however, only IFN-γ was detectable in the collected samples. Serum samples were diluted 1:40. Both the standard and serum samples were incubated using the same plates, and a standard curve was plotted for each cytokine.

[0284] pSTAT5 assay. Initially, CAR T cells were incubated for 24 hours in complete RPMI medium without cytokines (resting). Cells were then incubated at 37°C under specified conditions using CellTrace. ™ Blue (Invitrogen) or CellTracker ™ Stain with Red (Invitrogen) for 30 minutes. Wash cells once with complete RPMI + 10% FBS medium. Then use CellTracker... ™Red-stained CAR T cells were blocked on ice for 20 minutes using recombinant BCMA-CH3 (100 nM), while CellTrace was used. ™ Blue-stained cells were not blocked. Cells were then washed once with complete RPMI + 10% FBS medium, followed by seeding approximately 2 × 10⁶ cells per well in a 96-well plate at 37°C in the presence of serially diluted treatment or cytokine controls. 5 Cells (co-cultured or individually cultured, two types of separately stained cells). After incubation for 5 minutes, immediately fix the cells with 1.5% formaldehyde in PBS at room temperature for 10 minutes. Then permeabilize the cells with ice-cold 100% methanol at ice 4°C for 20 minutes. Wash the fixed and permeabilized cells twice with FACS buffered PBS + 1% bovine serum albumin (BSA), and then incubate with anti-STAT5 pY694-PE / Cy7 (1:200, Biolegend) on ice for 30 minutes. Then wash the cells twice with FACS buffer and use BD FACSCanto... ™ Analysis was performed using II (Becton Dickinson). FlowJo was used. ™ (Becton Dickinson) analyzed flow cytometry data, fitted dose-response curves to a logistic sigmoid model, and calculated the half-maximum effective concentration (EC50) using Prism data analysis software (GraphPad). 50 ) and 95% confidence interval.

[0285] scRNA-seq analysis was performed. Gene counts for each sample were obtained using the CellRanger multifunction with 10x cloud computing, and merged using the CellRanger aggr function to produce a .h5 file that could be loaded into R as a Seurat object. A Seurat analysis workflow was performed for QC filtering (total counts < 20,000, molecular identifiers [nUMI] < 6,000, and ribosomal RNA < 10% of the reads). The data were then scaled and normalized using scTransform, and the original samples were traced back using the demultiplexing function HTODemux(). The phenotype of cells was then determined using the FindTransferAnchors() and FindQuery() workflows, projecting our samples onto the Seurat pbmc_multimodal dataset. The FindMarkers() function was used to find differentially expressed genes between selected groups. Heatmaps were generated using the DoHeatmap() function with downsampling of 500 cells. Gene scores were obtained by creating a list of genes of interest, which was given as a feature of the AddModuleScore() function.

[0286] Preparation of batch RNA sequencing samples. CAR T cells, containing and without intracellular domains, were incubated for 24 hours in cytokine-free complete RPMI medium. Sequencing was performed at 37°C with approximately 2 × 10⁻⁶ samples in the presence of a 10 nM treatment or control. 5 10 cells / well were seeded into 96-well plates for 2 hours. Cells were washed with FACS buffer and incubated at 37°C for 2 or 22 hours (4-hour and 24-hour time points). Cells were then treated with anti-CD8-FITC (1:50, Biolegend) and anti-CD4-PE / Dazzle594. ™ BCMA staining (1:50, Biolegend) and Alexa647 labeled cells were performed and sorted on a Sony Sorter MA900. 10,000 CD4 and 10,000 CD8 cells were sorted under each condition. mRNA libraries were generated using the SMART-Seq mRNA library preparation kit (Takara Bio), with each replicate labeled with a unique index. The libraries were pooled and sequenced using Novogene at a sequencing depth of 20 million reads per sample.

[0287] Batch RNA Seq analysis was performed. Gene counts were obtained from the samples by trimming the fastQ files and quantifying transcripts using RNAlysis software. Gene names were obtained from the Homo sapiens ensembl database using bioomaRt, and differential expression under different conditions was determined using the DESeq2 analysis pipeline. Volcano plots were generated using the Enhanced Volcano library, with cutoff values ​​of logFC > |2| and p > 10. -6 Heatmaps were generated using the pheatmap library. GSEA was performed using the fgsea package's analysis workflow, with the ordination metric being -log. 10 (p-value) The sign (multiple change) is calculated based on the hallmark path of the MSigDB collection.

[0288] Example 2: In vitro characterization of BCMA-containing CAR-adaptor .

[0289] A fusion protein composed of the extracellular domain of human BCMA was fused with two low-affinity mutant human IL-2 (muIL2) domains. Furthermore, to improve pharmacokinetics and enhance stability, the CH3 domain of human IgG1 (Feige et al., TrendsBiochem. Sci.35(4):189-198 (2010)) (approximately 14 kDa in size) 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-adaptor preferentially delivers low-affinity IL-2 to the surface of CAR T cells via antigen-CAR specific binding, thereby minimizing toxicity to normal T cells, Tregs, or systemic cells.

[0290] Recent studies have shown that IL-2 induces an alternative differentiation pathway for T cells, leading to the generation of different "better effector" CD8 cells. + T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may depend at least in part on the binding of IL-2 to IL-2Rα. Additionally, IL-2Rβγ-biased agonists can drive T cells toward terminal differentiation (Codarri et al., Nature 610(7930):161-172 (2022)). CAR-adaptors may be able to overcome the need for IL-2Rα in alternative differentiation pathways by anchoring low-affinity IL-2 to the surface of CAR T cells via antigen-CAR binding, thereby promoting the generation of memory CAR T cells. There may also be a potential synergistic effect between CAR signaling and IL-2 signaling.

[0291] To assess the binding affinity of the BCMA-containing CAR-adaptor, flow cytometry analysis was performed by staining BCMA CAR T cells with different concentrations of the BCMA CAR-adaptor. For the BCMA CAR-adaptor, approximately 0.21 nM of EC was observed. 50The binding was comparable to that of dimeric BCMA (BCMA-CH3) lacking muIL2 (Figure 2E), indicating that binding was primarily due to the extracellular domain of BCMA rather than muIL2. Figure 2E shows dose-dependent staining of BCMA CAR T cells with the CAR-adaptor using flow cytometry (n=3 for each point), with untransduced T cells serving as a control and a second Alexa647-labeled anti-FLAG antibody used for staining. Error bars represent the mean with a 95% confidence interval. Minimal binding of the BCMA CAR-adaptor to untransduced T cells and minimal binding of VHH-muIL2 were observed in the CH3-muIL2 CAR-adaptor construct, which was a control construct in which the extracellular domain of BCMA was replaced with an unrelated nanobody (VHH). This observation further suggests that the binding of the CAR-adaptor to CAR T cells is primarily driven by the BCMA antigen, and that muIL2 exhibits weak binding to both CAR T cells and untransduced T cells. The BCMA-muIL2 CAR-adaptor did not show binding to any immune cell populations in human peripheral blood mononuclear cells (PBMCs) (Figures 15A to 15B).

[0292] Next, the functional effects of the BCMA CAR-adaptor on BCMA CAR T cells were evaluated. After a 24-hour resting period without cytokines, BCMA CAR T cells were incubated with different concentrations of the BCMA-muIL2 CAR-adaptor for 24 hours, and the expression of CD69 activation markers was assessed by flow cytometry (Cibrián and Sánchez-Madrid, Eur. J. Immunol. 47(6):946-953 (2017)). The results demonstrated a dose-dependent and selective increase in CD69 expression on CAR T cells (Fig. 2F), while no effect was observed on untransduced T cells (Fig. 2G). Furthermore, treatment with the BCMA CAR-adaptor resulted in a significantly higher increase in CD69 expression compared to VHH-muIL2, BCMA-CH3, or combinations thereof, indicating that the observed effect was only significant when the low-affinity IL-2 was fused with the antigen. Unpaired t-tests showed that at concentrations of 0.1 nM or higher, activation in the BCMA-muIL2 treatment group was statistically significantly increased (P ≤ 0.0001) compared to the VHH-muIL2, BCMA-CH3, or combinations thereof control groups (error bars represent the mean with 95% confidence intervals) (Figure 2F). The zero treatment and CD3 / CD28 activation in Figure 2G were used as negative and positive controls, respectively (error bars represent the mean with 95% confidence intervals; the difference between CD3 / CD28 activation and BCMA-CH3-muIL2 treatment was p < 0.0001).

[0293] BCMA CAR-E does not inhibit the killing effect of BCMA CAR T cells. Since both CAR-adaptors and cancer antigens bind to CARs, the potential inhibitory effect of the BCMA CAR-adaptor on the cytotoxic activity of BCMA CAR T cells was investigated. To investigate this, BCMA CAR T cells and patient-derived BCMA were used in the presence of different concentrations of the BCMA CAR-adaptor. + OPM2 cancer cell killing assays were performed. Notably, the results demonstrated no killing inhibition even at the highest tested concentration (100 nM CAR-adaptor) (Figure 2H). OPM2 cells were co-incubated with BCMA-muIL2 CAR-E treatments at different concentrations (E:T ratio 1:1; 30,000 cells each) with BCMA CAR T cells (shown in red) or untransduced T cells (shown in gray) (E:T ratio 1:1; 30,000 cells each). After 48 hours, live (PI) cells were tested. - OPM2 cells were counted, with N = 3 for each experiment. The error bars in Figure 2H represent the mean versus standard deviation. Unbound by theory, this finding may be attributed to the reversibility of CAR-adaptor binding to CAR, while the killing process involving clustering effects and synaptic formation between CAR and cancer antigens is an irreversible event. Furthermore, the binding affinity of CAR to membrane-bound BCMA may exceed that to soluble antigens, contributing to this result. Notably, multiple myeloma patients exhibit high levels of soluble BCMA in their circulation due to γ-secretase-induced shedding (Laurent et al., Nat. Commun. 6:7333 1-12 (2015)). Nevertheless, BCMA CAR T cells produced a significant initial response in patients, indicating that soluble BCMA antigens do not inhibit CAR T cell activity. The experimental findings disclosed in this paper are consistent with these earlier findings.

[0294] The BCMA CAR-adaptor is selected by cis-delivering low-affinity IL-2 to the same target CAR T cells. Sexually induces STAT5 activity in CAR T cells IL-2 is known to exhibit strong activity against T cells, and STAT5 phosphorylation serves as a reliable indicator of IL-2 / IL-2R binding, as well as being 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 the BCMA CAR-adaptor on STAT5 activity, BCMA CAR T cells were exposed to different concentrations of the BCMA CAR-adaptor. After incubation at 37°C for 5 minutes, the cells were fixed and stained for pY694 STAT5. The results showed that the BCMA-muIL2 CAR-adaptor phosphorylated at approximately 0.014 nM EC5 at STAT5 levels. 50Induces phosphorylation of STAT5 in CAR T cells (Figure 2I). In contrast, the VHH-muIL2 control requires a higher concentration (EC). 50 BCMA-mediated delivery of low-affinity IL-2 to the surface of CAR T cells (3.9 nM) induced STAT5 phosphorylation, indicating that muIL-2 sensitivity was significantly enhanced by more than 200-fold through BCMA-mediated delivery of low-affinity IL-2 to the surface of CAR T cells. Wild-type IL-2 exhibited lower EC5 levels. 50 (Approximately 0.001 nM), indicating a difference in signal transduction dynamics. The two-step process involved in STAT5 activity mediated by the BCMA-muIL2 CAR-adaptor involves (i) binding of the antigen-CAR to the T cell surface and (ii) subsequent interaction of low-affinity IL-2 with nearby IL-2R, unbound by theory, which may explain the measured difference. In contrast, wild-type IL-2 only needs to bind to IL-2R, enabling it to induce STAT5 activity more rapidly. VHH-muIL2 can activate STAT5 solely through low-affinity IL-2, which could explain why it requires a higher concentration to induce STAT5 activity in T cells.

[0295] Pre-blocked BCMA CAR T cells with BCMA-CH3 showed a significant reduction in pSTAT5 levels, reaching the same level as the control VHH-muIL2, confirming that the efficacy of the CAR-adaptor is mediated by antigen-CAR binding (Figure 2I). To determine whether the CAR-adaptor binding to target cells could lead to STAT5 signaling (transactivation) in adjacent cells, unblocked and pre-blocked BCMA CAR T cells were co-cultured in the presence of different concentrations of the CAR-adaptor. Pre-blocked CAR T cells had lower pSTAT5 levels compared to co-cultured unblocked CAR T cells, indicating that the CAR-adaptor affects the targeted CAR T cells (cis-activation) but not adjacent cells. BCMA CAR T cells were treated with the specified treatment at 37°C for 5 minutes, followed by STAT5 phosphorylation assessment. For the pre-blocking experiment, BCMA CAR T cells were treated with BCMA-CH3 (100 nM) at 4°C for 20 minutes, and then exposed to the CAR-adaptor treatment at 37°C for 5 minutes (n=3 for each condition). The error bars in Figure 2I represent the mean and standard deviation. In summary, the analysis of STAT5 activity supports the view that the BCMA CAR-adaptor exerts its influence on targeted CAR T cells via cis-delivery of low-affinity IL-2, an effect mediated by antigen-CAR binding.

[0296] Example 3: CAR-adaptor immune cell effector domain independent of CAR stimulation of T cells .

[0297] To demonstrate the effector domain of the CAR-adaptor immune cell, the following experiment was performed. In this experiment, the experimental setup is shown in Figure 3A, in which peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to generate activated T cells, which were not transduced with any exogenous transgenes. Activated T cells were treated for 4 days with either: teceleukin (recombinant human IL-2 without glycosyl units), a CAR-adaptor containing two repeating sequences of a weak-affinity variant of a BCMA-binding N-terminal extracellular domain (approximately 7 kDa), a CH3 domain (approximately 14 kDa), and an IL-2 immune cell effector domain, with the overall structure BCMA-CH3-muIL2-muIL2 and referred to herein as BCMA-muIL2; or a CAR-adaptor containing a BCMA-binding extracellular domain, a CH3 domain, and a Neo-2 / 15 immune cell effector domain, with the overall structure BCMA-CH3-Neo-2 / 15 and referred to herein as BCMA-Neo-2 / 15. Following treatment, T cells were counted and stained with carboxyfluorescein succinimide (CFSE), and the mean fluorescence intensity (MFI) of CFSE was analyzed to determine T cell division.

[0298] T cells treated with the CAR-adaptor BCMA-muIL2 or BCMA-Neo-2 / 15 affected T cell counting and division (CFSE staining), similar to teceleukin, which is known to activate T cells. However, CAR-adaptor treatment resulted in lower sensitivity compared to teceleukin treatment. Systemic administration of IL-2 has been associated with serious side effects (Rosenberg, J. Immunol. 192(12):5451-5458 (2014); Dutcher et al., J. Immunother. Cancer. 2(1):26 pp. 1-23 (2014); Pachella et al., J. Adv. Pract. Oncol. 6(3):212-221 (2015)), including vascular leakage syndrome and CD4+. + CD25 + The preferential expansion of regulatory T (Treg) cells is known to lead to immunosuppression. The results disclosed in this invention demonstrate that when used at low concentrations, the CAR-adaptor does not activate normal T cells, and when the extracellular domain of the CAR-adaptor is attached to activate T cells, the stimulatory immune cell effector domain retains its normal function.

[0299] Example 4: CAR-adaptor specifically activates CAR T cells through extracellular domain

[0300] To demonstrate the effector domain of the CAR-adaptor stimulating immune cells, the following experiments were performed. As shown in Figure 4A, PBMCs were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to generate activated T cells, which were then transduced using a CAR-containing vector. The activated CAR-expressing T cells (CAR T cells) were left to rest for 24 hours and then treated with a CAR-adaptor containing an effector domain or a CAR-adaptor lacking an effector domain as an extracellular domain control.

[0301] T cell activation was tested using a CAR-adaptor containing the BCMA extracellular domain, the CH3 domain, and the 4-1BBL (BCMA-41BBL), weak-affinity IL-2 (BCMA-muIL2), or Neo-2 / 15 (BCMA-Neo-2 / 15) immune cell effector domains. To test the extracellular domain specificity of the CAR-adaptor for CAR T cells, T cell activation was tested as a control using an unrelated nanobody that binds to the intracellular protein UBC6E (VHH6E) fused to the CH3 domain and either the 4-1BBL (VHH6E-41BBL) or the weak-affinity IL-2 (VHH6E-muIL2). T cell activation was also tested as a separate extracellular domain-specific control using a nanobody containing FN1 (clone NJB2, abbreviated as NJB2-VHH) fused to the CH3 domain and the Neo-2 / 15 stimulatory domain (NJB2-VHH-Neo-2 / 15). The extracellular domain-specific control has a similar overall structure to the CAR-adaptor used in this experiment (protein domain -CH3-muIL2-muIL2 or protein domain -CH3-Neo-2 / 15). The extracellular domain-specific control and the CAR-adaptor were incubated with CAR T cells for 10 hours, and the cells were stained against CD69, an activation marker, and measured by flow cytometry.

[0302] All BCMA extracellular domain CAR-adaptors induced CD69 expression in CAR T cells (Fig. 4B). BCMA-CH3-Neo-2 / 15 had the lowest CD69 expression induction threshold in CAR T cells (0.01 nM CAR-adaptor). BCMA-CH3 protein (lacking immune cell effector domains) had the least effect on CD69 expression in CAR T cells at the highest tested concentration (10 nM BCMA-CH3 protein). Extracellular domain-specific control proteins did not induce CD69 expression in CAR T cells. These results indicate that CAR-adaptors containing cancer antigen extracellular domains specifically activate CAR T cells expressing CARs that recognize the cancer antigen extracellular domains of the CAR-adaptor.

[0303] Example 5: CAR-adaptor stimulation of CAR T cells to kill target cells

[0304] To demonstrate that the CAR-adaptor does not inhibit CAR T1 cell killing, the following experiments were performed. CAR T cells were generated as described above and reacted with the CAR-adaptor and BCMA. + Multiple myeloma cancer cells were co-incubated. CAR T cells were incubated with OPM2BCMA. + Cells were incubated at a 1:1 E:T ratio for 1 day, and target cell survival was analyzed compared with target cells that were not co-incubated with T cells (Figure 5A).

[0305] CAR-adaptors with the extracellular domain of BCMA containing either the muIL2 (BCMA-CH3-muIL2) or 4-1BBL (BCMA-CH3-41BBL) immune cell effector domains do not inhibit OPM2 cell killing (Fig. 5B). These results indicate that CAR-adaptors containing both the cancer antigen extracellular domain and the stimulating immune cell effector domain do not inhibit CAR T cell killing of target cells that also express the same cancer antigen as the CAR-adaptor.

[0306] Example 6: CAR-adaptor reduces tumor burden, prolongs survival, and extends the persistence of CAR T cells in vivo.

[0307] To demonstrate that the CAR-adaptor reduces tumor burden, prolongs the in vivo persistence of CAR T cells, and extends survival, the following experiments were performed. 1×10 6 OPM2 BCMA + Multiple myeloma cancer cells were injected intravenously (iv) with NOD-scid IL2Rγ null In (NSG) mice, for 10 days, followed by intravenous infusion of the second-optimal dose of 5 × 10⁻⁶ mg / L. 5 1 anti-BCMACAR T cell. Mice were treated twice a week for two weeks after CAR T cell infusion, and then injected once a week via intraperitoneal injection (ip) with 200 μg / mouse of a CAR-adaptor containing the extracellular domain, CH3 domain and two weakly affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) (Fig. 6A).

[0308] Bioluminescent imaging (BLI) of luciferase (indicating tumor burden of luciferase + OPM2 cells) was performed on mice at the days shown in Figures 6B to 6D. Control mice receiving OPM2 cells but without CAR T cell infusion exhibited progressively increasing tumor burden during the experiment, reaching the humanitarian endpoint at days 39 and 46. Mice receiving OPM2 cells and a suboptimal dose of CAR T cells had controlled tumor growth until day 32, at which point they also experienced progressively increasing tumor burden during the experiment, reaching the humanitarian endpoint at day 46. Mice receiving OPM2 cells, CAR T cells, and CAR-adaptor therapy had reduced tumor burden (Figures 6B to 6D). In the CAR-adaptor treatment group, all mice had complete clearance of 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 the formation of a solid tumor near the eye, reaching the humanitarian endpoint at day 42. The remaining two mice had their OPM2 tumor cells completely cleared, as no signal was detected by imaging, and they survived the experiment.

[0309] Next, the in vivo persistence of OPM2 and CAR T cells in these mice was analyzed by flow cytometry. One control mouse (OPM2 cells but no CAR T cell infusion) was sacrificed on day 46, two CAR-only mice were sacrificed on day 46, and one mouse treated with CAR T cells and a CAR-adaptor was sacrificed on day 42. GFP+ OPM2 cells (Figs. 7A–7C) and CD45 were analyzed in the blood, spleen, lymph nodes, bone marrow, and lungs of the sacrificed mice. + CAR + T cells (Figs. 8A to 8C). GFP+ OPM2 cells and CD45+ CAR cells in ocular tumor sites of mice treated with CAR T cells and CAR-adaptor were also analyzed. + T cells.

[0310] Figures 7A through 7C illustrate flow cytometry, with GFP represented on the y-axis. Similar levels of GFP were detected in the bone marrow, lung (Figure 7B), and liver (Figure 7C) of CAR-free control mice and CAR-only mice. + OPM2 cells. A CAR mouse alone had significant levels of OPM2 cells in its blood and spleen (Fig. 7A).

[0311] A mouse developing ocular tumors treated with CAR T and CAR-adaptor had no to almost no OPM2 cells in its blood or spleen (Fig. 7A), bone marrow or lung (Fig. 7B), and liver (Fig. 7C). The mouse had more OPM2 cells in its kidney (3.18% GFP). +(cells), and the majority of cells in the ocular tumor site are OPM2 cells (96.5% GFP). + cell).

[0312] Figures 8A to 8C illustrate flow cytometry, where the y-axis represents anti-CD45 and the x-axis represents the Alexa Flour assay. ™ BCMA marked 647 (AF647) + -CH3. CD45 + CAR + T cells persisted only in mice treated with CAR T cells and CAR-adaptor. Mice treated with CAR alone showed almost no CD45 in any of the tested organs. + Cells (Figs. 8A to 8C). However, mice treated with CAR T cells + CAR-adaptor exhibited CD45... + Cells that were also positive for BCMA cancer antigen labeled with AF647 (which is also a CAR binding target) are shown on the x-axis. CD45 + AF647 + Double-positive CAR T cells were detected in blood, spleen and lymph nodes (Fig. 8A), bone marrow and lungs (Fig. 8B), and liver and kidneys (Fig. 8C). CD45 + Single-positive cells were detected in large numbers only in the liver and kidneys (Fig. 8C). CD45 was almost absent. + AF647 + Double-positive CAR T cells were detected in ocular tumor sites (Figure 8C). These results indicate that the CAR-adaptor reduces tumor burden, prolongs the persistence of CAR T cells in vivo, and extends survival.

[0313] Example 7: CAR-Connector Fate

[0314] The CAR-adaptor binds to CAR T cells on the cell surface at 4°C and undergoes slow internalization at 37°C.Internalization of the CAR-adaptor was assessed using a fluorescently labeled BCMA-muIL2 CAR-adaptor. BCMA CAR T cells were exposed to 2 nM AlexaFluor647-labeled BCMA-muIL2, BCMA-CH3, or VHH-muIL2. 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 underwent rapid internalization within 30 minutes at 37°C, while the internalization of the BCMA-muIL2 CAR-adaptor was significantly slower (Figs. 16A–16C). The internalization rate of BCMA-CH3 was similarly slow, even slower than that of the BCMA-muIL2 CAR-adaptor. In Fig. 2C, CAR and dsRed transcripts are encoded within the transgene, and therefore the dsRed signal reflects the expression level of CAR. All imaging cells with average intensity above background are reported. For the dsRed channel, the average intensity in the cytoplasm is reported because dsRed is expressed inside the cell, while for the AlexaFluor 647 channel, the average intensity across the entire cell is measured.

[0315] CAR-adaptor is rapidly removed from the cycle.Treatment of pulsatile CAR T cells with CAR-adaptors (where the pulse involves a stimulation phase followed by a resting phase) is superior to prolonged exposure to CAR-adaptors, as prolonged exposure can lead to depletion or the generation of terminally differentiated CAR T cells. CAR-adaptors with short circulating half-lives are more effective in expanding CAR T cells, driving the generation of memory CAR T cells, reducing potential competition for CAR binding with tumor antigens, and enhancing safety in patients. Therefore, the CH3 domain of IgG1 was used in the CAR-adaptor platform. Pharmacokinetic studies have shown that the CAR-adaptor has a short circulating half-life (1–1.5 hours) (Figure 9A). 8 mg / kg of BCMA-muIL2 CAR-adaptor was administered to NSG mice (intraperitoneal delivery, N=3 mice). Blood samples were collected via 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 processed substance in the serum. ELISA plates were coated overnight with 5 μg / ml anti-His6 antibody and then incubated with serum at room temperature for 2 hours. Detection was then performed using anti-FLAG HRP antibody; the CAR-adaptor was engineered to have FLAG and His6 tags at the C-terminus. Based on five time points collected, the initial concentration of the processed product in serum was estimated to be 20% higher than that at the first (30 min) collection time point. BCMA CAR-E was cleared from circulation >90% and >99% within 8 hours and 24 hours, respectively. The circulating half-life of the BCMA CAR-adaptor was estimated to be approximately 1.5 hours. Error bars represent the mean versus standard deviation.

[0316] Activity and persistence of CAR T cells in a BCMA CAR-adaptor-enhanced multiple myeloma (MM) model A mouse model of MM xenograft was used, in which OPM2 cells were transplanted into immune-disabled NOD-SCID IL-2Rγ cells. null In NSG mice, OPM2 cells (human MM, 1 million cells) were injected intravenously via the tail vein. Two weeks after OPM2 cell injection, freshly prepared BCMA CAR T cells (500,000 CAR cells containing the 39BB-CD3ζCAR construct) were administered intravenously. +(Cells). A cohort of mice received BCMA-muIL2 CAR-adaptor treatment (Fig. 9B). NSG mice (n=5) were injected with OPM2 (human MM) cells according to a schedule, followed by administration of BCMA CAR (human) T cells. BCMA-muIL2 CAR-E treatment (200 μg) was administered twice weekly for two weeks, followed by weekly treatment until the endpoint. Mice were euthanized after one month or longer, and harvested organs were analyzed by flow cytometry. These results revealed significant expansion of CAR T cells in the spleen and bone marrow of the BCMA-muIL2 CAR-adaptor treatment group, demonstrating a selective expansion of over 100-fold compared to untreated animals that received CAR T cells only in the spleen (Fig. 9C, left panel) and bone marrow (Fig. 9C, right panel). These experiments were repeated multiple times with similar results (Fig. 9D and Fig. 17A to Fig. 17C; n=12 for CAR T cells only, n=22 for CAR T cells plus CAR-adaptor treatment).

[0317] BCMA CAR T cells were detected by co-staining with anti-human CD45 antibody and Alexa647-labeled BCMA antigen. Similar results were obtained in replicate experiments. An additional control cohort received VHH-muIL2 treatment at the same dose and regimen as BCMA-muIL2. Figure 9D shows pooled data from these experiments. The differences between CAR+BCMA-muIL2 CAR-E and CAR+PBS or CAR+VHH-muIL2 were significant in both spleen and bone marrow (p≤0.01 in spleen, p≤0.01 between CAR+BCMA-muIL2 and CAR+PBS in bone marrow, and p=0.025 between CAR+BCMA-muIL2 and CAR VHH-muIL2 in bone marrow). Data were analyzed by comparison of group means using one-way ANOVA and subsequent Tukey post-hoc analysis. Individual flow cytometry plots of the pooled data are shown in Figures 17A–17C. Error bars represent the mean versus standard deviation.

[0318] Compared to the control group that received only CAR T cells without treatment, the control group (n=7) treated with CAR T cells plus VHH-muIL2 did not show significant expansion or persistence of CAR T cells. These results indicate that the BCMA-muIL2 CAR-adaptor can expand CAR T cells in vivo. Further analysis revealed that BCMA-muIL2 treatment has an effect on CD8+. + CAR T cells have a more significant effect, specifically, leading to a higher proportion of them compared to total CD4+. + and CD8 +The initial CAR T cell population of approximately 30% unexpectedly increased significantly to approximately 70% (Figure 9E). Cohorts receiving CAR T cells alone or CAR T cells plus VHH-muIL2 control did not produce a sufficient number of durable CAR cells for similar analyses. Data were analyzed by comparing group means using one-way ANOVA and subsequent Tukey post-hoc analysis. Error bars represent the mean versus standard deviation.

[0319] Example 8: BCMA CAR-adaptor treatment enables low-dose CAR T cell CAR T therapy.

[0320] To further demonstrate the effectiveness of CAR-adaptor treatment and the clearance of tumor cells by CAR T cells, a similar protocol as described above was performed. However, in this study, only a lower dose of 100,000 CAR T cells was used (Fig. 10A). All mice treated with the BCMA-muIL2 CAR-adaptor achieved complete tumor clearance (5 / 5), while no single control mouse receiving CAR T cells alone (n=4) or a combination of CAR T cells and VHH-muIL2 treatment (n=4) was able to eliminate the tumor (Figs. 10B-10C).

[0321] Analysis of blood samples collected at different time points revealed substantial expansion of circulating CAR T cells following CAR-adaptor treatment, with peak expansion observed at week 4 (Fig. 10D). Flow cytometry analysis of blood samples revealed robust expansion of CAR T cells in the treatment group compared to the PBS or VHH-muIL2 cohort. In contrast, VHH-muIL2 treatment, while slightly enhancing the initial response, did not induce significant expansion of CAR T cells. Data were analyzed using two-factor ANOVA for days 7, 14, and 21. BCMA-muIL2 and VHH-muIL2 comparisons were performed at days 28 and 35 using multiple Mann-Whitney tests once all mice in the PBS cohort were euthanized. Error bars represent the mean versus standard deviation. This expansion correlated with the IFN-γ levels detected in circulation (Figs. 19A–19C). Furthermore, the treatment promoted the generation of memory CAR T cells, demonstrating a long-lasting effect (Figs. 10E and 19A–19C). P<0.05, P < 0.01. The error bars represent the mean and standard deviation.

[0322] Based on clinical observation and weight measurements, mice treated with the CAR-adaptor showed no signs of toxicity (Fig. 10H). Subsequent analysis two months after CAR T-cell injection demonstrated a high concentration of CAR T cells, including memory CAR T cells, in the CAR-adaptor-treated mice (Figs. 10F–10J, 18A–18C, and 19A–19C). In the CAR+PBS group, CAR T cells were detected in the spleen; however, these mice died approximately 20 days after CAR T-cell injection due to tumor growth. BCMA-muIL2 treatment also increased the presence of CAR T cells in the bone marrow compared to the PBS or VHH-muIL2 cohorts, but this difference was less significant than in the spleen. Data were analyzed using two-way ANOVA and Tukey's multiple comparison test. P<0.05, P<0.001, P < 0.0001. Individual flow cytometry data are shown in Figures 18A through 18C. Error bars represent the mean versus standard deviation. The data in Figure 10I show that persistent CAR T cells persist in the spleen of mice receiving the BCMA-muIL2 CAR-adaptor and exhibit CCR7. + CD45RA + CD62L + The stem cell memory phenotype is absent in the CAR+VHH-muIL2 or CAR+PBS cohorts.

[0323] tSNE analysis was based on the expression of surface markers CD8a, CD4, CD45, CD45RA, CD45RO, CD62L, CD69, PD-1, HLA-DR, CCR7, and BCMA-CAR, revealing the presence of different memory T cell populations. CAR T cells were detected in the bone marrow of the VHH-muIL2-treated group but not in the spleen. In the BCMA-muIL2 group, persistent CAR T cells were predominantly CD8 T cells, while in the VHH-muIL2 group, the majority of bone marrow CAR T cells were CD4 T cells. Further analysis is shown in Figures 19A to 19C. The Flt-SNE mapping shown in Figure 19C is derived from CAR T cells from mice treated with PBS, BCMA-muIL2, and VHH-muIL2, as shown in Figure 10A. The expression of 10 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) in spleen cells and bone marrow from 3 PBS mice, 3 BCMA-muIL2 mice, and 4 VHH-muIL2 mice was analyzed by flow cytometry. + 、⍺-BCMA-CAR + Immune cell cascades were formed to create a total of 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 atlas for each condition, where the signal intensity of various phenotypic markers defining a specific immunophenotype was represented by a blue-green-yellow-red continuous color scale. Flt-SNE was performed with the following parameters: maximum number of iterations: 1000, θ: 0.5, learning rate: 200, perplexity: 20. The number of CAR T cells in the spleen of the VHH-muIL2 cohort was insufficient for Flt-SNE. To enhance visibility, the dots representing the VHH-muIL2 bone marrow samples were magnified, as fewer cells were detectable in these mice. In BCMA-muIL2 treated mice, most CAR T cells were present in the spleen. + The cell is CD8 + Cells, while in the VHH-muIL2 sample, CD4 + Cells constitute most CARs + Cells. It is noteworthy that the CAR in the CAR+PBS cohort...+ Cells exhibited low or no expression of CD45RA, CD45RO, or CD62L, while BCMA-muIL2-treated mice showed elevated CAR levels of these memory markers. + group.

[0324] Therefore, this treatment not only promotes robust proliferation and eradication of tumor cells using low doses of CAR T cells, but also promotes the development of long-lasting memory cells, demonstrating the efficacy of BCMA-muIL2 CAR-adaptor treatment in enhancing the clearance of tumor cells by CAR T cells and generating long-lasting memory cells.

[0325] Example 9: Durable CAR T cells treated with CAR-adaptor retain function three months after infusion

[0326] Mice received 1 million OPM2 cells, followed by 500,000 BCMA CAR T cells (Fig. 11A). One group of mice received CAR-adaptor treatment twice weekly for two weeks, followed by once weekly for another two weeks (6 doses, 200 μg each on days 4, 10, 14, 17, 21, and 28; n=5). The control group received VHH-muIL2 treatment at the same dose and regimen (n=5), while another control cohort received tumor cells only (n=3). All mice receiving CAR T cells showed an initial response compared to control mice without CAR T cells (Fig. 11B). All CAR-adaptor-treated mice (n=5 of 5) and 3 of 5 mice in the VHH-muIL2 group survived for more than three months, covering the duration of the experiment. One VHH-muIL2 mouse died within approximately one month, and a second mouse died from cancer recurrence and liver metastasis (Fig. 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, mice treated with the CAR-adaptor showed significantly higher CAR T cell homing and persistence in the bone marrow and spleen compared to mice receiving VHH-muIL2-treated CAR T cells (Fig. 11C). Given the two-month treatment-free period prior to euthanasia, these results further suggest that treatment promotes the generation of memory cells within the CAR T cells.

[0327] To demonstrate the functionality of durable CAR T cells in CAR-adaptor-treated mice, an in vitro killing assay was performed using BCMA CAR T cells harvested from 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 or spleen cells were co-incubated with OPM2 target cells at various E:T ratios (1:1 and 2:1) based on CAR-expressing cells. Survival was determined at 24, 48, and 72 hours using flow cytometry analysis. Three-month-old CAR T cells demonstrated effective killing of tumor cells and long-term functionality (Fig. 11D). Only one mouse treated with VHH-muIL2 showed sufficient CAR T cells for a similar killing assay, and although it showed tumor cell killing, its efficiency was lower than that observed in CAR-adaptor-treated CAR T cells (Fig. 11D) (error bars indicate mean versus standard deviation). Therefore, CAR-adaptor treatment robustly expands and drives the durability of CAR T cells while maintaining their killing potential.

[0328] To further characterize the phenotype of these durable CAR T cells, flow cytometry analysis was performed to evaluate the expression of a range of T cell markers (CD45, BCMA CAR, CD4, CD8, CD62L, CD45RO, CD45RA, CD69, and PD-1). For ease of interpretation, t-SNE mapping maps of spleen cells and bone marrow cells were generated (Figure 11E). Flow cytometry analysis was performed on spleen cells and bone marrow cells from five BCMA-muIL2 treated mice for the expression of anti-CD45-Pacific Blue, anti-CD8-FITC, anti-CD4-PEDazzle594, BCMA (antigen)-AlexaFluor647, anti-CD69-BV421, anti-PD-1-BV605, anti-CD45RA-APC-Cy7, anti-CD45RO-PerCP-Cy5.5, anti-CD62L-PE, and CCR7-AlexaFluor700. + CD8 + 、⍺-BCMA-CAR +Cell cascades were constructed to form a total of approximately 17,600 cells (spleen) and approximately 10,800 cells (bone marrow). The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to create a single tSNE atlas, where the signal intensities of six phenotypic markers defining specific immunophenotypes were expressed using a blue-green-yellow-red continuous color scale. tSNE analysis was performed on the spleen and bone marrow using 1000 iterations, a perplexity of 30, and learning rates of 1237 and 756, respectively. The population labeled 1 appeared to exhibit a memory-like phenotype, expressing higher levels of CD45RO, CD62L, and CD45RA. The population labeled 2 appeared to exhibit an effector-like phenotype, expressing lower levels of CD45RO, CD62L, and CD45RA.

[0329] Flow cytometry analysis revealed that persistent CAR T cells are generated by CD4+ + and CD8 + Group composition. CD8 + CAR cells appear to exhibit two distinct populations: effector cells and CD45RA cells. + CD62L + Memory cells. The memory population exhibited higher levels of BCMACAR and CD45 expression (Figure 11E). Similarly, CD4... + CAR T cells exhibited two populations: effector cells and memory cells (Figure 20). The number of CAR T cells detected in VHH-muIL2-treated mice was insufficient for similar flow cytometry analysis. Therefore, CAR-adaptor treatment resulted in the generation of long-acting memory CAR T cells.

[0330] Figure 20 shows the CD4+ of five BCMA-muIL2 CAR-E treated mice, as shown in Figures 11A to 11E. + CAR + t-SNE mapping of T cells. The expression of nine immune cell markers (aCD45-Pacific Blue, aCD8-FITC, aCD4-PEDazzle594, BCMA (antigen)-AlexaFluor647, aCD69-BV421, aPD-1-BV605, aCD45RA-APC-Cy7, aCD45RO-PerCP-Cy5.5, aCD62L-PE) in spleen cells and bone marrow from five BCMA-muIL2 treated mice was analyzed by flow cytometry. + CD4 + 、⍺-BCMA-CAR +Immune cell cascades were constructed to form a total of approximately 9000 cells (spleen) and approximately 6600 cells (bone marrow). The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to create a single t-SNE atlas, where the signal intensities of six phenotypic markers defining specific immunophenotypes were expressed using a blue-green-yellow-red continuous color scale. t-SNE analyses were performed on the spleen and bone marrow using 1000 iterations, a perplexity of 30, and learning rates of 630 and 466, respectively. The population labeled "1" appeared to exhibit a memory-like phenotype, expressing higher levels of CD45RO, CD62L, and CD45RA. The population labeled "2" appeared to exhibit an effector-like phenotype, expressing lower levels of CD45RO, CD62L, and CD45RA.

[0331] CARs isolated from mice treated with BCMA-muIL2 or VHH-muIL2 control + T cells were analyzed using single-cell RNA sequencing (scRNAseq). Although the presence of CAR T cells was limited in VHH-muIL2-treated mice, a sufficient number of cells were obtained from one of the VHH-treated mice for experiments (Figure 21A). CAR cells from BCMA-muIL2 or VHH-muIL2-treated mice were analyzed. + Cells were stained with BCMA-Alexa Fluor647 and TotalSeq-C hash antibodies and then sorted, as shown in the red and green boxes in Figure 21A, respectively. 5000 CARs from the bone marrow and spleen of BCMA-muIL2 mice were used... + Cells and 2500 CARs from the bone marrow and spleen of VHH-muIL2 mice + Cells were loaded onto the 10X channel. scRNA-seq analysis revealed that the major population of durable CAR T cells in BCMA-muIL2-treated mice was composed of CD8+ cells. + T cell composition (Figs. 21B-21C) exhibits gene enrichment associated with activated T cell states (Figs. 21D-21E). The heatmap in Fig. 21D shows significantly differentially expressed genes between different relevant conditions in CD8 and CD4CAR T cells, separated by CAR-adaptor treatment and VHH conditions. The labeled genes were those that were significantly differentially expressed between BCMA-muIL2 and VHH-muIL2-treated mice in the subgroup of interest. This was evidenced by elevated expression levels 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 because the mice had cleared tumors 60 days prior. The upregulation of exhaustion markers induced by BCMA-muIL2 treatment indicated that treatment does not induce exhaustion of durable CAR T cells.

[0332] Next, the diversity of T-cell receptor (TCR) clonal types was evaluated in mice treated with BCMA-muIL2 and VHH-muIL2 (Figs. 21F to 21G). Both groups showed similar clonal diversity, indicating that BCMA-muIL2 CAR-adaptor treatment effectively promotes the generation of a diverse TCR library in durable CAR T cells, rather than promoting the advantage of a limited set of TCR clones. The pie chart in Fig. 21F illustrates the diversity of TCR clonal types, where each slice of the pie chart represents the proportion of different TCR clonal types present; colors were randomly assigned to different clonal types. Clonal diversity within the total cell count for each sample was visualized using a stacked bar chart (Fig. 21G), which combined similar clonal types with counts below 50. To assess diversity within each sample, the Simpson index was calculated, with higher values ​​indicating greater diversity. Overall, the results suggest that the BCMA CAR-adaptor not only helps CAR T cells completely clear tumor cells but also robustly induces the generation of long-lasting and functional memory CAR T cells.

[0333] Example 10: CAR-adaptor expands CAR T cells in the absence of tumor antigens

[0334] CAR T-cell expansion usually occurs after patient infusion, with peak expansion observed approximately 10–14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002–1014 (2023)).

[0335] Eradication of minimal residual disease (MRD) promotes a long-lasting and complete response. However, the limited presence of MRD-associated antigens may be insufficient 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 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 post-CAR T cell injection. The control group received VHH-muIL2 treatment (n=4 per group). On day 30, the mice were euthanized, and the presence of CAR T cells in their spleen and bone marrow was assessed. Mice treated with BCMA-muIL2 showed significantly higher numbers of CAR cells in their spleen (approximately 6.8-fold) and bone marrow (approximately 5.5-fold). + T cells, indicating that the BCMA CAR-adaptor amplifies CAR T cells in vivo, even in the absence of tumor cells (Figures 12A-12B); error bars represent the mean versus standard deviation. Overall, these findings demonstrate that the CAR-adaptor can amplify CAR T cells even in the absence of tumor antigens. Furthermore, the effectiveness of treatment is evident even at lower doses and frequencies.

[0336] Example 11: CAR-adaptor containing an IL-2 variant with an immune cell effector domain

[0337] The binding strength of IL-2 variants to CAR immune cells may significantly influence the efficacy of CAR-adaptor therapy. Therefore, CAR-adaptor IL-2 variants containing different IL-2 variants were generated and integrated into the CAR-adaptors, summarized in Table 9. Amino acid substitutions are relative to the native (wild-type) sequence of human IL-2 (NCBI accession number NP_000577; SEQ ID NO: 102). Furthermore, some CAR-adaptors contain different dimerizing domains, including CH3 and a silenced fragment crystallizable region (Fc). The silenced Fc contains at least two mutations relative to wild-type Fc that eliminate Fc-γ receptor and complement component 1q (C1q) binding while maintaining the stabilizing effect of wild-type Fc and binding to the nascent Fc receptor (FcRn). These at least two mutations include L234A and L235A, and are commonly referred to as (“LALA”). The CAR-adaptor variant E4 contains a dimerizing domain of IgG1, has silent Fc mutations of L234A and L235A (“LALA”), and also lacks an IL-2 variant. The CAR-adaptor variant E4 was developed and used as a control molecule.

[0338] CAR-receptors containing a single IL-2 variant are listed as “One-IL-2” in Table 9, while CAR-receptors containing two repeats of the IL-2 variant are listed as “IL-2” in the table below. For example, variants Q3, A8, A10, and A16 have two repeats of the IL-2 variant shown on each molecule of the CAR-receptor. Furthermore, CAR-receptors can exist as dimers due to the presence of dimerizing domains. CAR-receptors containing wild-type CD19 (SEQ ID NO: 5) are listed as “CD19 wt” in Table 9, such as variants Y9 and Y3. CAR-receptors containing CD19 variants having the amino acid sequence of SEQ ID NO: 3 are listed as “CD19” in Table 9.

[0339] Table 1: CAR-connector ICE variants

[0340] Figure 22A shows the percentage of BCMA CAR T cells positive for phosphorylated STAT5 (pSTAT5) staining, as determined by flow cytometry, after treatment with the selected CAR-adaptor ICE variants described in Table 9. BCMA CAR T cells were subjected to designated treatments at 37°C at different doses for 10 minutes, followed by assessment of pSTAT5 (n=3 for each condition). Except for U7, all newly developed IL-2 variants demonstrated the ability to induce STAT5 activity in BCMA CAR T cells after 10 minutes.

[0341] Figure 22B illustrates dose-dependent activation of resting BCMA CAR T cells 24 hours after CAR-adaptor treatment. Resting BCMA CAR T cells were incubated with different concentrations of the BCMA CAR-adaptor ICE variant, and subsequent evaluation of CD69 expression was performed by flow cytometry after 24 hours. CD69 serves as a marker of human T cell activation. The dashed line at 0 nM represents background CD69 expression in the absence of any treatment or cytokines. The dashed line “+ cytokines” indicates the CD69 expression level of non-resting CAR T cells incubated in the presence of IL-2, IL-7, and IL-15 cytokines. Dashed lines are included as additional negative and positive controls.

[0342] Figure 23A illustrates STAT5 phosphorylation in BCMA CAR T cells after treatment with CAR-adaptor ICE variants. Mean fluorescence intensity (MFI) of pSTAT5-positive BCMA CAR T cells was determined by flow cytometry after 3 hours of treatment with different doses of CAR-adaptor ICE variants at 37°C. Cells were washed to remove excess unbound CAR-adaptor before flow cytometry analysis, and pSTAT5 was assessed after 24 hours (n=3 for each condition). Several IL-2 variants demonstrated the ability to induce STAT5 activity in BCMA CAR T cells.

[0343] Figure 23B illustrates the dose-dependent activation of resting BCMA CAR T cells by BCMA CAR-adaptor ICE variants. BCMA CAR T cells were incubated with different concentrations of BCMA CAR-adaptor ICE variants for 3 hours, followed by washing. CD69 was assessed by flow cytometry 24 hours after washing. All CAR-adaptor ICE variants activated BCMA CAR T cells compared to the U5 negative control.

[0344] Figures 24A and 24B illustrate STAT5 phosphorylation in BCMA CAR cells induced by treatment with the BCMA CAR-adaptor detailed in Table 9. The CAR T cells contain the FDA-approved BCMA CAR construct Ide-cel (ABECMA). ® ) or Cilta-cel; CARVYKTI ® The percentage of BCMA CAR T cells positive for phosphorylated STAT5 (pSTAT5) staining, as determined by flow cytometry, is shown. BCMA CAR T cells were subjected to specified treatments at 37°C at different doses for 30 minutes, followed by assessment of STAT5 phosphorylation (n=3 for each condition). These results demonstrate constructs that specifically and efficiently induce pSTAT5 activity in CAR T cells but have no or limited effect on untransduced T cells.

[0345] Figures 25A through 25F illustrate STAT5 phosphorylation in BCMA CAR T cells induced by treatment with the BCMA-muIL2 CAR-adaptor detailed in Table 9. The CAR T cells contained the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel. The percentage of pSTAT5-positive BCMA CAR T cells is shown, as determined by flow cytometry. BCMA CAR T cells were subjected to designated treatments at 37°C at different doses for 15 minutes, 2 hours, or 24 hours, followed by assessment of STAT5 phosphorylation (n=3 for each condition). For the 24-hour analysis, CAR T cells were washed at 2 hours to remove unbound CAR-adaptor. These results show which constructs specifically and effectively induce pSTAT5 activity in CAR T cells and for how long (monitored over 24 hours), but have no or limited effect on untransduced T cells.

[0346] Figures 26A and 26B illustrate dose-dependent activation of resting BCMA CAR T cells treated with the BCMA CAR-adaptor detailed in Table 9. The CAR T cells contain the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel. The incubation of resting BCMA CAR T cells with different concentrations of the BCMA CAR-adaptor treatments and subsequent assessment of CD69 expression by flow cytometry after 24 hours are shown. CD69 serves as a marker of human T cell activation. These results demonstrate which constructs specifically and effectively activate CAR T cells, but have little or no effect on untransduced T cells.

[0347] Figures 27A and 28B illustrate dose-dependent activation of resting BCMA CAR T cells following treatment with additional BCMA CAR-adaptor details described in Table 9. The CAR T cells contained the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel. The incubation of resting BCMA CAR T cells treated with different concentrations of BCMA CAR-adaptor containing immune cell effector domains of the U4, X5, X6, or U5 Il-2 variants, followed by assessment of CD69 expression by flow cytometry after 24 hours, is shown. These results demonstrate which constructs specifically and effectively activate CAR T cells, but have little or no effect on untransduced T cells.

[0348] Similarly, the incubation of resting BCMA CAR T cells treated with different concentrations of BCMA CAR-adaptors containing immune cell effector domains of U4, Y2, V7, X12, X15, or U5 IL-2 variants, followed by assessment of CD69 expression or STAT5 phosphorylation by flow cytometry after 24 hours, is also shown. These results show which constructs specifically and effectively activate CAR T cells and for how long (monitored over 24 hours), but have no or limited effect on untransduced T cells. IL-2 (Teceleukin ® ) and the CAR-adaptor (BCMA-CH3) without immune cell effector domains were used as controls.

[0349] Figures 29A through 29C show that the selected CAR-adaptor induced CAR T cell proliferation in vitro, with a reduced effect on untransduced T cells compared to wild-type IL-2. Figure 29A shows the incubation of activated BCMA CAR T cells containing the Cilta-cel CAR construct treated with increasing concentrations of the BCMA CAR-adaptor, followed by flow cytometry assessment of CAR T cell proliferation after 3 days. The dashed lines in Figure 29A represent the number of untreated CAR T cells. Figure 29B shows a replication of the experiment shown in Figure 29A using a different CAR-adaptor. Figure 29C shows a similar experiment evaluating the effect of the CAR-adaptor on cell proliferation in normal untransduced T cells. The CAR-adaptor induced significantly less proliferation in untransduced T cells than wild-type IL-2. The CAR-adaptor containing triple mutant IL-2 (V7, V9, Y2, and Y9) induced significantly less proliferation in normal T cells compared to the CAR-adaptor containing double mutant IL-2 (U4).

[0350] Figure 42 shows that the CAR-adaptor induced CAR T cell proliferation in vitro with little effect on untransduced T cells. The ability of the BCMACAR-adaptor containing an immune cell effector domain with a variant of IL-2 (U4, V7, Y2, V6, X12, X15, or U8) to induce proliferation in anti-BCMA CAR T cells or untransduced T cell controls was examined. Wild-type IL-2, a non-targeted CAR-adaptor with an immune cell effector domain containing an E3 IL-2 variant (VHH-cH3-IL2), and a BCMA CAR-adaptor without an immune cell effector domain (BCMA-CH3) served as controls. Only wild-type IL-2 induced robust proliferation in untransduced T cells, and all four CAR-adaptors containing IL-2 variants induced proliferation in anti-BCMA CAR T cells (Figure 42).

[0351] Figures 30A to 30C show that the V7 CAR-adaptor exhibits a lower affinity for IL-2Rα compared to the wild-type IL-2 or U4 CAR-adaptor. Wild-type IL-2 (Teceleukin) is also shown. ® BLI analysis was performed on the association and dissociation between BCMA-muIL2 CAR-adaptor (U4; double mutant IL-2) or BCMA-muIL2 CAR-adaptor (V7; triple mutant IL-2) molecules and IL-2Rα. The dimerized IL-2Rα molecule was used for evaluation.

[0352] Figures 31A and 31B show that the newly developed CAR-adaptor containing an IL-2 variant induced CAR-T cell proliferation in vivo, and the proliferation level correlated with in vitro pSTAT5 signaling. Figure 31A schematically illustrates the experimental setup; mice received OPM2 cancer cells, followed by 500,000 CAR-T cells (Cilta-cel) one week later. Mice were bled on days 11, 18, 28, 35, and 42 for 5 weeks, and CD4 and CD8 CAR T cells were counted (Figure 31B). Compared with the control PBS-treated group, all three tested CAR-adaptors (Y2, V7, and X12) induced CAR-T cell proliferation in vivo. The V7 and Y2 CAR-adaptors induced more CAR T cell proliferation than the X12 CAR-adaptor.

[0353] Figures 32A through 32C show that the V7 CAR-adaptor enhances CAR-T cell activity when administered 3 days or even 14 days after CAR T cell injection. Figure 32A schematically illustrates the experimental setup; mice received OPM2 cancer cells, followed by 500,000 CAR-T cells (Cilta-cel) one week later. One cohort of mice received CAR T cells only (n=4). Another cohort of mice received 6 doses of the V7 CAR-adaptor starting 3 days after CAR T cell injection, administered on days 3, 6, 10, 14, 21, and 28. A third cohort of mice received 6 doses of the CAR-adaptor starting on day 14 (which is the post-CRS window in patients), and on days 14, 18, 21, 28, 35, and 42. BLI imaging was performed at the specified days to assess tumor growth in the different cohorts (Figure 32B). Starting on day 14, mice were bled weekly for 6 weeks, and CAR T cells were counted (Figure 32C). Treatment with the V7 CAR-adaptor induced CAR T cell proliferation in both cohorts compared to the control PBS-treated group.

[0354] Figures 43 to 45 show the in vitro induction of pSTAT5 signaling (Figure 43) and gene expression (Figures 44 and 45) by CAR-adaptors containing the IL-2 variant. CAR T cells (approximately 60% of the CAR...) + T cells and untransduced T cells were incubated with increasing concentrations of CAR-adaptors containing immune effector domains of IL-2 variants (including U4, V7, Y2, V6, X12, X15, and U8) and controls (wild-type IL-2 and CAR-adaptors without immune effector domains (BCMA-CH3)). pSTAT5 activity was assessed by flow cytometry 30 minutes after CAR-adaptor treatment (Figure 43). CD69 expression was assessed by flow cytometry after 2 hours of incubation with the CAR-adaptor, followed by washing and a further 22 hours of incubation (Figure 44).

[0355] After T cells were incubated with the CAR-adaptor for 2 hours, washed, and then incubated for another 22 hours and the culture medium was harvested, TNF-α and IFN-γ production was assessed by flow cytometry using multiplex ELISA (Figure 45). Each condition included three technical replicates. T cells were obtained from PBMCs from a single healthy donor.

[0356] Figures 46A and 46B show the binding of IL-2Rα and IL-2Rβγ to CAR-adaptors containing IL-2 variants via biolayer interferometry. To measure CAR-adaptor binding rates, the biosensor tip was coated with dimer IL-2Rα (Figure 46A) or IL-2Rβγ (Figure 46B) and incubated for a specified time with either a CAR-adaptor containing an immune effector domain of an IL-2 variant (including U4, V7, Y2, V6, X12, X15, and U8) or a control (monomer wild-type IL-2 (positive control), CAR-E with wild-type IL-2 (positive control), and CAR-adaptor with monomeric V7 IL-2 variant)). Release rates were assessed by transferring the biosensor tip to a washing solution. Three technical replicates were performed for each condition.

[0357] Example 12: CD19 CAR-E does not inhibit the killing effect of CD19 CAR T cells

[0358] CAR-adaptor binding to BCMA was observed to bind but not inhibit the cytotoxic efficacy of BCMA CAR T cells (Figs. 2D to 2H). To investigate the effects of CAR-Es containing other cancer antigens on antigen-specific CAR T cells, CD19 CAR T cells and patient-derived CD19 were used in the presence of different concentrations of CD19 CAR-adaptor. +A killing assay was performed on leukemia cells. Notably, the results demonstrated no killing inhibition even at the highest tested concentration (1000 nM CAR-adaptor) (Figure 2H). Nalm6 cells were co-incubated with CD19 CAR T cells (solid) or untransduced T cells (empty) (E:T ratio 1:1; 30,000 cells each) in the presence of different concentrations of CD19-muIL2 CAR-E treatment. After 48 hours, live (PI) cells were... - Nalm6 cells were counted, with N = 3 in each experiment. The experimental findings regarding CD19 disclosed in this paper are consistent with the findings in the BCMA cancer model and BCMA CAR-E described above.

[0359] Example 13: CAR-adaptor effectively enhances CAR immune cell activity at low doses during initial treatment and tumor re-challenge. Cell efficacy and durability

[0360] Next, experiments were conducted to investigate the efficacy of reduced-frequency CAR-E treatment initiated two weeks after CAR T-cell injection. Mice were intravenously injected with OPM2 cells (1 × 10⁻⁶). 6 (Intravenous administration). One week later, CAR T cells (0.5 × 10⁻⁶) were administered. 6 (Intravenous). Two weeks after CAR T cell injection, mice were divided into two cohorts, with some receiving CAR-E treatment and others not. The treatment group received four doses of CAR-E (4 mg / kg per dose) on days 14, 18, 21, and 28. Surviving mice were given 1×10⁻⁶ CAR-E on day 60. 6 OPM2 cells were re-challenged and then treated with 4 mg / kg CAR-adaptor on days 68, 70, 74, 77 and 80 (Figure 35A).

[0361] Notably, bioluminescence imaging (BLI) analysis demonstrated that all mice receiving CAR-E (5 / 5) achieved tumor clearance, while none of the control mice (0 / 4) achieved tumor clearance; all images were analyzed using the same BLI quantitative scale (photons / second) (Figs. 35B–35D). Blood analysis showed that four doses of CAR-E treatment were sufficient to induce robust expansion of CAR T cells in all treated mice compared to the control cohort (Figs. 35E and 37). Cytokine assessment also revealed elevated IFN-γ levels in the CAR-E-treated cohort by sandwich ELISA of serum samples collected on the same day as CAR-T counts, as shown in Fig. 22E (Fig. 35F).

[0362] One of the CAR-E-treated mice (M5) showed liver recurrence on day 60 (Fig. 35B). However, all five CAR-E-treated mice underwent re-excitation on day 60 using 1 million liver metastasis-derived OPM2 cells to assess the generation of memory CAR-T cells. All mice showed considerably low signal intensity compared to the treatment-naïve control mice (Fig. 35B; see day 66). While the mice exhibited liver signaling, none showed bone marrow signaling, indicating the presence of functional memory CAR T cells in the bone marrow that suppress tumor growth. To explore whether CAR-E treatment could facilitate the re-expansion of CAR T cells and control tumor growth in liver metastases, mice were retreated with CAR-E (4 mg / kg) on ​​days 68, 70, 74, 77, and 80. Impressively, all mice successfully cleared liver metastases, demonstrating that CAR-E can promote the re-expansion and transport of CAR T cells to eliminate tumor cells (Fig. 35B; see day 83). Mice showing relapse on day 60 (M5, Fig. 35B) also cleared tumors from the liver after re-stimulation, although it showed some signal on the last day of the experiment. Blood analysis confirmed that CAR-E induced robust re-expansion of CAR T cells in circulation in all mice (Fig. 35E).

[0363] On day 90 after CAR T cell injection, mice were euthanized, and organ analysis by flow cytometry revealed that all CAR-E-treated mice contained a large number of durable CAR-T cells with diverse memory phenotypes (Fig. 35G, Fig. 38A, and Fig. 38B). For analysis, human CD45 cells derived from mouse bone marrow and spleen were... + BCMA-CAR + Cells were gated and cascaded, and the merged populations were then subjected to FLOWSOM analysis to identify eight major phenotypic metaclusters. Each metacluster was qualitatively characterized using a heatmap representing the mean fluorescence intensity (MFI) of each marker within each metacluster (Figs. 35A–35D). In Fig. 35G, the proportion of each metacluster in the bone marrow and spleen of each mouse is depicted. The untreated “CAR-T only” cohort did not have enough durable CAR T cells in the bone marrow or spleen to allow for similar analyses. This experiment utilized PBMCs from a single donor.

[0364] In summary, these results demonstrate that BCMA CAR-E treatment not only promotes complete clearance of tumor cells by CAR T cells but also significantly promotes the formation of functional memory CAR T cells. Notably, even administering several doses of CAR-E two weeks after CAR T cell administration effectively expands CAR T cells and promotes the development of functional memory CAR T cells. Durable CAR T cells retain the ability to re-expand in response to CAR-E treatment.

[0365] CAR-E treatment led to the expansion and persistence of CAR T cells in the spleen and bone marrow (Figures 36B–36C and 40A–40D). These results reveal that CAR-adaptor treatment resulted in a dose-dependent expansion of CAR T cells. The error bars shown in Figures 36B–36C are mean ± standard deviation and show the bars indicating the absolute number of CAR T cells detected under each condition and the statistical analysis demonstrating that CAR-adaptor treatment led to a dose-dependent expansion of CAR T cells. The significance between the mice receiving PBS and those receiving the lowest concentration of CAR-adaptor treatment (2 mg / kg) was measured using the Mann-Whitney test. Additionally, simple linear regression was performed to demonstrate the dose-dependent effect of the treatment; the error bars on the figures represent 95% confidence intervals.

[0366] Example 14: CAR T cell expansion in the absence of tumor antigens is dose-dependent. .

[0367] Typically, CAR T-cell expansion occurs after patient infusion, with peak expansion observed approximately 10–14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002–1014 (2023)). This expansion is driven by antigen availability and tumor-killing processes, which promote CAR T-cell proliferation (Turtle et al., J. Clin. Invest. 126(6):2123–38 (2016), Gardner et al., Blood 129(25):3322–3331 (2017), Lee et al., Leukemia 35(1):255–258 (2021), Hossain et al., Blood 132(Supplement 1):490–490 (2018)). However, patients exhibiting limited CAR T-cell expansion after infusion showed poorer responses (Fraietta et al., Nat. Med. 24(5):563-571 (2018)). Furthermore, achieving a long-lasting, complete response requires the eradication of minimal residual disease. However, the limited presence of the corresponding antigens associated with minimal residual disease may not be sufficient to support CAR T-cell proliferation and efficacy.

[0368] Unbound by theoretical constraints, the mechanism of CAR-E action may not depend on tumor cells and the antigens they present, thus potentially enabling the expansion of CAR T cells in the absence of tumor cells (and therefore tumor antigens), thereby addressing the key clinical challenge of limited in vivo CAR T cell expansion after infusion. To test this hypothesis and assess the dose-dependency of CAR-E treatment, mice were injected with only 250,000 BCMA CAR T cells in the absence of tumor cells. As shown in Figure 36A, 0.25 × 10⁻⁶ BCMA CAR T cells were used. 6 NSG-DKO mice were treated with BCMA CAR T cells and assigned to different cohorts receiving different doses of BCMA-muIL2 CAR-E (2 mg / kg, 4 mg / kg, 8 mg / kg, or no CAR-E treatment; twice weekly for four weeks; n=5 per cohort). Organs were collected one month after CAR T cell injection and flow cytometry analysis was performed to assess the presence of CAR cells.

[0369] Furthermore, the results demonstrated that the effect of CAR-E on CAR T cells was dose-dependent (Figures 36B-36C). Interestingly, even at the lowest tested dose of 2 mg / kg, CAR T cells persisted extensively compared to the untreated cohort, with very few to almost no CAR T cells detected one month after CAR-E injection (Figures 36B-36C). Further analysis indicated that CAR-E induced the generation of memory CAR T cells (Figures 36D-36E). EM The cells are CD45RA - CD45RO + CCR7 - ;T EMRA The cells are CD45RA + CD45RO + CCR7 - ;T SCM The cells are CD45RA + CD45RO + CCR7 + ;T CM The cells are CD45RA - CD45RO + CCR7 + ;T Naive The cells are CD45RA + CD45RO - CCR7 + The experiments in Figures 36A to 36E utilized PBMCs from a single donor. Overall, these findings demonstrate that CAR-E treatment induces the expansion of CAR T cells, regardless of the presence of tumor cells, and enables CAR T cells to develop diverse memory phenotypes.

[0370] To further investigate the ability of CAR-E to expand CAR T cells and to confirm the major role of the low-affinity IL-2 component of BCMA-muIL-2 CAR-E in influencing CAR T cells, 4 mg / kg and 0.25 × 10⁻⁶ ions were used. 6 Similar experiments as described above were repeated for each CAR T cell. Mice received BCMA-muIL2 CAR-E treatment, were treated with control BCMA-CH3 molecules containing only the BCMA antigen and without the low-affinity mutant IL-2 component, or were untreated. As expected, the antigen-only BCMA-CH3 treatment cohort did not result in CAR T cell expansion or persistence compared to the CAR-E treatment cohort, further validating that both the extracellular domain (e.g., BCMA antigen) and immune cell effector domain (e.g., low-affinity IL-2) components of the CAR-E molecule are essential for its effect on CAR T cells (Figs. 36F–36G and 41A–41E). Mice received CAR T cells and different treatments (4 mg / kg) according to a protocol similar to that shown in Fig. 36A. BCMA-CH3 antigen, VHH-muIL2, or low-dose wild-type IL-2 treatment did not result in CAR T cell expansion or persistence compared to the CAR-adaptor treatment cohort. Error bars represent the mean versus standard deviation. The experiments in Figures 36F through 36G utilized PBMCs from two donors. The Kruskal-Wallis test was applied to each subset of CAR T cells and total T cells. A post-hoc Dunn analysis was then performed to compare each group with the treatment group. The table in Figure 23G shows the adjusted p-values. This is consistent with the in vitro and in vivo analyses disclosed herein.

[0371] To compare the efficacy of CAR-E treatment with low-dose wild-type IL-2 (which is used in combination with CAR T therapy in clinical practice), an additional cohort received low-dose wild-type IL-2 (SEQ ID NO: 102) (4.5 µg per mouse, starting on day 1 for 14 days, then twice weekly for another two weeks). The low-dose IL-2 group failed to generate a large number of durable CAR-T cells compared to the CAR-E treatment cohort (Figs. 36F to 36G). Clinical studies using low-dose IL-2 in combination with CAR-T have not produced significant benefit, and in some cases, low-dose IL-2 was discontinued due to IL-2-related toxicities (Katz et al., Clin. Cancer Res. 21(14):3149-59 (2015)). Furthermore, as expected, non-targeted, low-affinity IL-2 (VHH-muIL2) CAR-E did not lead to CAR T cell expansion or persistence (Figs. 36F to 36G), consistent with previous findings disclosed herein.

[0372] In summary, these findings demonstrate that CAR-E molecules can robustly promote the expansion of CAR T cells and the development of diverse memory phenotypes, regardless of the presence of tumor cells. Both the extracellular domain (e.g., BCMA antigen) and the immune cell effector domain (e.g., low-affinity IL-2) of the CAR-E molecule are essential for the observed effects on CAR T cells. Furthermore, the efficacy of CAR-E treatment is evident even at lower doses.

[0373] Example 15: The efficacy of CAR-E requires the signaling of both the intracellular signal transduction domains of CAR and IL-2R. Conduction

[0374] To better understand the mechanism of action of CAR-E, this study investigated whether its effect on CAR T cells is mediated solely by anchoring low-affinity IL-2 to CAR T cells via BCMA-CAR binding, or whether it involves the simultaneous binding of both IL-2R and the intracellular signaling domain of the CAR. For this purpose, BCMA CAR T cells were prepared using a BCMA CAR construct lacking the 41BB-CD3ζ intracellular signaling domain but retaining the same extracellular domain (referred to as CAR-intracellular domain deletion or CAR-ICD-Δ). In vitro analysis demonstrated that after 30 minutes of incubation with CAR-E, the BCMA-muIL2 CAR-E molecule induced pSTAT5 in CAR-ICD-Δ T cells (n=3), similar to that in intact CAR T cells (Figure 33A), indicating that the effect of low-affinity IL-2 is similar in both CAR constructs and is mediated via antigen-CAR binding. Interestingly, while CAR-E robustly activates intact CAR T cells, as evidenced by increased CD69 expression (Fig. 33B) and increased production of IFN-γ (Fig. 33C) and TNF-α (Fig. 33D), its effect on CAR-ICD-Δ CAR T cells is negligible.

[0375] Additional control conditions included non-targeted VHH-muIL2 and antigen-only BCMA-CH3 (n=3 for each condition). Error bars represent the mean versus standard deviation, and experiments were conducted using PBMCs from a single donor. Dasatinib, a lymphocyte-specific protein tyrosine kinase inhibitor (LCK), and ruxolitinib, a Janus kinase inhibitor (JAK), both alone and in combination, significantly inhibited the effects of CAR-E molecules on CAR-T cells (Figs. 33E–33G), further demonstrating that CAR-E molecules bind to both CAR and IL-2R receptors and activate their intracellular signaling pathways. CAR T cells were treated with different doses of CAR-E treatments and inhibitors alone or in combination, and evaluated 24 hours later (n=3 for each condition); error bars represent the mean versus standard deviation. Subsequently, the in vivo effects of CAR-E on intact CAR T cells and CAR-ICD-Δ T cells were compared. 0.25 × 10⁶ cells were injected in the absence of tumor cells. 6 Mice with CAR T cells were treated with CAR-E (4 mg / kg, twice weekly for four weeks; Figure 33H). One month later, the animals were euthanized, and organs were analyzed by flow cytometry. Notably, while consistent with previous results, CAR-E robustly expanded CAR T cells using intact CAR constructs, it did not lead to the expansion or persistence of CAR-ICD-Δ T cells (Figure 33I). Experiments utilized PBMCs from a single donor; statistical analysis was performed using an unpaired t-test.

[0376] To gain further insights into the mechanism of CAR-E action, their effects on the transcriptome of CAR T cells were investigated. Various treatments, including the BCMA-muIL2 CAR-E molecule, a non-targeted low-affinity IL-2 (VHH-muIL2), the BCMA-CH3 antigen, and wild-type IL-2, were added to CAR T cells. As an additional control, CAR-ICD-Δ T cells were treated with the BCMA-muIL2 CAR-E molecule. The treatments were removed after 2 hours to simulate in vivo conditions, and batch RNA sequencing of the cells was performed at 2 hours or 22 hours.

[0377] Notably, CAR-E induced substantial transcriptomic changes, quantitatively demonstrating a larger fold change compared to all other conditions, including wild-type IL-2 (Figs. 33J–33N). Differences in BCMA-muIL2 gene upregulation compared to wild-type IL-2, VHH-muIL2, and BCMA-CH3, along with GSEA under these different conditions (Fig. 34), show that while these control treatments themselves also had some effect, the stimulation induced by CAR-E molecules was significantly superior (Figs. 33L–33M). The effect of CAR-E on CAR-ICD-Δ T cells was mild, further highlighting the important role of intracellular signal transduction domains in the mechanism of action of CAR-E molecules (Figs. 33J–33K). CAR-T cells were incubated for 2 hours with 10 nM of BCMA-muIL2 CAR-E molecules or control molecules, followed by washing to remove the treatment. RNA sequencing was then performed at 2 hours and 24 hours. The gene set enrichment analysis (GSEA) shown in Figure 34 was performed using the HALLMAKR dataset from the Molecular Characteristic Database (MSigDB) and demonstrates that treatment with both the control and CAR-E molecules activated the expected pathway. A similar pathway appears to be induced between the control and CAR-E treatments, with the effect in the control being much smaller, as depicted in Figures 33J to 33M.

[0378] In summary, these findings suggest that the mechanism of action of CAR-E molecules is not merely the delivery of low-affinity IL-2 to CAR T cells. Rather, it functions by binding to and significantly bridging the intracellular signaling domains of the CAR and IL-2R receptors, thereby inducing significant T cell activation and substantial transcriptomic changes.

[0379] All patent and non-patent publications indicate the level of expertise of a person skilled in the art to which this disclosure pertains. All such publications are incorporated herein by reference to the extent that each individual publication is specifically and individually indicated as incorporated by reference.

[0380] Although the disclosure herein has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of this disclosure. Therefore, it should be understood that various modifications can be made to the exemplary embodiments, and other arrangements can be designed, without departing from the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. An IL-2 variant, wherein the IL-2 variant is selected from the amino acid residues H16, D20, R38, F42, Y45, E61, E62, L72 and V91 of SEQ ID NO: 1, and the substitutions of three to eight amino acids are different from those of wild-type IL-2 (SEQ ID NO: 102).

2. The IL-2 variant according to claim 1, wherein the first amino acid substitution is selected from H16A, H16R, H16S, D20A or D20Q, the second amino acid substitution is selected from R38D, F42A and V91H, and the third amino acid substitution is selected from Y45A and E62N.

3. The IL-2 variant according to claim 2, wherein the first amino acid substitution is H16A, H16R, or H16S.

4. The IL-2 variant according to claim 2, wherein the first amino acid substitution is D20A or D20Q.

5. The IL-2 variant according to any one of claims 2-4, wherein the third amino acid substitution is Y45A.

6. The IL-2 variant according to any one of claims 2-4, wherein the third amino acid substitution is E62N.

7. The IL-2 variant according to any one of claims 2-6, wherein the second amino acid substitution is F42A.

8. The IL-2 variant according to any one of claims 3-7, wherein the IL-2 variant further comprises a fourth amino acid substitution selected from R38D, E61A, L72G and V91H.

9. The IL-2 variant according to any one of claims 3-7, wherein the IL-2 variant further comprises a fourth amino acid substitution selected from D20A, D20Q, R38D, Y45A, E61A, L72G and V91H; and wherein the first amino acid substitution is H16A, H16R or H16S.

10. The IL-2 variant of claim 9, wherein the IL-2 variant further comprises a fifth amino acid substitution comprising R38D; and wherein the fourth amino acid substitution is selected from D20A, D20Q, Y45A, E61A, L72G, and V91H.

11. The IL-2 variant of claim 10, wherein the IL-2 variant further comprises a sixth amino acid substitution comprising L72G; and wherein the fourth amino acid substitution is selected from D20A, D20Q, Y45A, E61A, and V91H.

12. The IL-2 variant of claim 11, further comprising a seventh amino acid substitution comprising E62N; wherein the third amino acid substitution is Y45A; and wherein the fourth amino acid substitution is selected from D20A, D20Q, E61A, and V91H.

13. The IL-2 variant according to any one of claims 1-12, wherein the IL-2 variant has amino acid substitutions at H16A and F42A; or amino acid substitutions at D20Q, F42A and E62N; or amino acid substitutions at D20Q, F42A and Y45A; or amino acid substitutions at H16A, F42A and E62N; or amino acid substitutions at H16A, F42A and Y45A; or amino acid substitutions at H16R, F42A and E62N; or amino acid substitutions at H16S, F42A and Y45A; or amino acid substitutions at D20Q, V91H, The amino acid substitutions at F42A and E62N; or the amino acid substitutions at H16A, F42A, Y45A and L72G; or the amino acid substitutions at H16R, D20Q, F42A and E62N; or the amino acid substitutions at H16S, D20Q, F42A and E62N; or the amino acid substitutions at H16S, F42A, Y45A and L72G; or the amino acid substitutions at H16A, D20A, R38D, F42A and E62N; or the amino acid substitutions at H16R, D20Q, R38D, F42A and E62N are different from those of wild-type IL-2 with the amino acid sequence of SEQ ID NO:

102.

14. A chimeric antigen receptor (CAR) adaptor, the CAR adaptor comprising: a first portion of an epitope on an extracellular domain of a CAR, the first portion being connected to a second portion comprising a first immune cell effector domain, the first immune cell effector domain comprising an IL-2 variant according to any one of claims 1-13.

15. The CAR-adaptor of claim 14, wherein the first portion of the CAR-adaptor comprises an extracellular domain of an antigen present on a cancer cell.

16. The CAR-adaptor of claim 15, 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, tight junction protein 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.

17. The CAR-adaptor of claim 16, wherein the extracellular domain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

18. The CAR-adaptor of claim 17, wherein the extracellular domain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGGGSGGGSPRGSGGGSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTN (SEQ ID NO: 2).

19. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from CD19.

20. The CAR-adaptor of claim 19, wherein the extracellular domain comprises an amino acid sequence having at least about 90% sequence identity with PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDMTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGNLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 3).

21. The CAR-adaptor of claim 20, wherein the extracellular domain comprises the amino acid sequence of SEQ ID NO:

3.

22. The CAR-adaptor of claim 20, wherein the extracellular domain comprises PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISQVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWQVSDLGGLGCGLKQRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLQQSLSRDMTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGQLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 4) or PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPK LYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK ​​(SEQ ID NO: 5) amino acid sequence.

23. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from CD20.

24. The CAR-adaptor according to claim 23, wherein the extracellular domain comprises the amino acid sequence KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

25. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from SLAMF7.

26. The CAR-adaptor of claim 25, wherein the extracellular domain comprises the amino acid sequence SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSM (SEQ ID NO: 6).

27. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from PD-1.

28. The CAR-adaptor of claim 27, wherein the extracellular domain comprises the amino acid sequence FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 7).

29. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from KIT.

30. The CAR-adaptor of claim 29, wherein the extracellular domain comprises the amino acid sequence QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEK YNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFL VSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO: 8).

31. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from CD38.

32. The CAR-adaptor according to claim 31, wherein the extracellular domain comprises the amino acid sequence VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO: 9).

33. The CAR-adaptor of claim 15, wherein the extracellular domain is derived from CD22.

34. The CAR-adaptor according to claim 33, wherein the extracellular domain comprises an amino acid sequence (SEQ ID NO: 7).

35. The CAR-adaptor according to claim 33, wherein the extracellular domain comprises the amino acid sequence PHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQ (SEQ ID NO: 103).

36. The CAR-adaptor according to any one of claims 14-35, wherein the first portion is an antibody, or a derivative thereof, that binds to an epitope on the extracellular domain (ED) of the CAR.

37. The CAR-adaptor according to claim 36, wherein the CAR present on the CAR immune cells binds to AFP, AXL, B4GALNT1, BCMA, CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, CTAG1B, CEA, CLEC12A, CLDN18.2, CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, and 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, TNFRSF8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1 or ULBP2.

38. The CAR connector of claim 14, wherein the ED of the CAR further comprises a connector, and wherein the first portion is coupled to a translocate on the connector.

39. The CAR-adaptor of claim 14, wherein the CAR binds to CD19, and wherein the first portion has the amino acid sequence of SEQ ID NO:

124.

40. The CAR connector according to any one of claims 14-39, wherein the CAR connector further comprises a first connector connecting the first portion and the second portion.

41. The CAR-adaptor of claim 40, wherein the CAR-adaptor further comprises a dimerization domain disposed between the first adapter and the second portion comprising the first immune cell effector domain.

42. The CAR-connector of claim 41, further comprising a second connector connecting the dimerized domain and the second portion, wherein the first connector and the second connector may be the same or different.

43. The CAR connector of claim 42, wherein the first connector and the second connector are flexible.

44. The CAR-connector of claim 44, wherein the first connector and / or the second connector are derived from the hinge region of CD3ζ, CD4, CD8α, CD28, IgG1, IgG2 or IgG4.

45. The CAR-adaptor of claim 44, wherein the first adapter and / or the second adapter comprises the amino acid sequence GGGX, GGGGX (SEQ ID NO: 55), or GSSGSX (SEQ ID NO: 56), or GSPRG (SEQ ID NO: 58), wherein X is C or S.

46. ​​The CAR-adaptor of claim 45, wherein the first adapter has an amino acid sequence of GGGGS (SEQ ID NO: 57) or GSPRG (SEQ ID NO: 32), and the second adapter has an amino acid sequence of GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 62).

47. The CAR-adaptor according to any one of claims 41-46, wherein the dimerizing domain is derived from IgA, IgD, IgG, IgM or IgE.

48. The CAR-adaptor of claim 47, wherein the dimerizing domain comprises an IgG1 constant weight (CH)3 domain.

49. The CAR-adaptor according to claim 47, wherein the dimerizing domain further comprises an IgG CH2 domain and an IgG CH3 domain.

50. The CAR-adaptor according to any one of claims 14-49, wherein the second portion further comprises a second immune cell effector domain, the second immune cell effector domain comprising a cytokine or immune cell activation portion.

51. The CAR-adaptor of claim 50, wherein the second immune cell effector domain is different from the IL-2 variant.

52. The CAR-adaptor of claim 50, wherein the second immune cell effector domain comprises a second IL-2 variant according to any one of claims 1-13, wherein the IL-2 variant and the second immune cell effector domain are the same or different.

53. The CAR-adaptor of claim 52, wherein the IL-12 variant and the immune cell effector domain are different.

54. The CAR-adaptor according to any one of claims 14-53, wherein the CAR-adaptor is in the form of a fusion protein, and the first portion and the second portion are linked by peptide bonds.

55. The CAR-connector according to any one of claims 14-53, wherein the first portion is connected to the second portion or the first connector via an azide-alkyne link, an oxime or hydrazine link, a tetrazine-trans-cyclooctene link, an azide-nitroketone link, a thiol-olefin link, an olefin-tetrazole link, an olefin-tetrazine link, an olefin-azide link, a conjugated diene-olefin link, or an isonitrile-tetrazine link.

56. The CAR-linker according to any one of claims 40-55, wherein the CAR-linker is in the form of a homodimer comprising two of the CAR-linkers.

57. A heterodimeric CAR-adaptor comprising: a first portion, the first portion binding an epitope on an extracellular domain of the CAR connected to a first dimerizing domain; and a second portion comprising an IL-2 variant of any one of claims 1-13 connected to a second dimerizing domain; wherein the first dimerizing domain and the second dimerizing domain dimerize to form a heterodimer.

58. The CAR connector of claim 57, wherein the first dimerizing domain includes a protrusion, the second dimerizing domain includes a cavity that spatially compensates for the protrusion, and wherein the protrusion is locating within the cavity.

59. A nucleic acid encoding an IL-2 variant according to any one of claims 1-13 or a CAR-adaptor according to claim 54.

60. A nucleic acid encoding a first portion, the first portion binding to an extracellular domain of a CAR fused to a first dimerizing domain.

61. A nucleic acid encoding an IL-2 variant fused to a second dimerizing domain according to any one of claims 1-13.

62. The nucleic acid according to any one of claims 59-61, wherein the nucleic acid further comprises a sequence encoding a signal peptide.

63. A vector comprising the nucleic acid according to any one of claims 59-62.

64. A cell comprising the carrier according to claim 63.

65. The cell according to claim 64, wherein the cell is a mammalian cell.

66. The cell according to claim 65, wherein the cell is a bacterial cell.

67. A pharmaceutical composition comprising a CAR-adaptor according to any one of claims 14-58 and a pharmaceutically acceptable carrier.

68. The pharmaceutical composition of claim 67, further comprising an effective number of CAR-containing immune cells, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising a stimulatory domain, the extracellular domain of the CAR-adaptor.

69. A method for preparing an IL-2 variant or a CAR-adaptor, the method comprising: Cells according to any one of claims 64-66 are cultured in a culture medium under conditions in which nucleic acids encoding the IL-2 variant or the CAR-adaptor are expressed; And to isolate the IL-2 variant or the CAR-adaptor from the cells and / or culture medium.

70. A method for treating cancer, the method comprising: The subject is given an effective amount of a first course of CAR-adaptor therapy according to any one of claims 14-58, wherein, before, substantially simultaneously with or after the administration of the CAR-adaptor, the subject is given an effective amount of immune cells (CAR immune cells) comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain comprising a stimulatory domain of the extracellular domain of the CAR-adaptor.

71. The method of any one of claims 70, wherein the CAR immune cells are administered to the subject prior to the administration of the CAR-adaptor.

72. The method of claim 71, wherein the CAR immune cells are administered at least about 6 months, at least about 9 months, or at least about 1 year prior to the administration of the CAR-adaptor.

73. The method of claim 70, wherein the CAR-adaptor is administered substantially simultaneously with the CAR immune cells.

74. The method of claim 73, wherein co-administration comprises substantially simultaneous administration of the CAR immune cells and the CAR-adaptor.

75. The method of claim 73, further comprising contacting the CAR-adaptor with the CAR immune cells in vitro prior to administration of the CAR-adaptor and the CAR immune cells.

76. The method according to claim 73, further comprising: The concentration of CAR immune cells present in samples obtained from the subject was measured after the administration of the immune cells; And calculate the difference between the concentration of the CAR immune cells administered to the subject and the measured concentration of the CAR immune cells.

77. The method of claim 76, wherein the co-application comprises applying the CAR-adaptor when the measured CAR immune cell concentration is less than the applied CAR immune cell concentration.

78. The method according to any one of claims 70-77, wherein the CAR immune cell is a T cell or an NK cell.

79. The method of claim 78, wherein the T cell is a CD8 cell. + T cells.

80. The method according to any one of claims 70-79, wherein the cancer is a hematopoietic system cancer.

81. The method of claim 80, wherein the hematopoietic system cancer is leukemia, lymphoma, or multiple myeloma.

82. The method according to claim 81, wherein the hematopoietic system cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, or non-Hodgkin lymphoma.

83. The method according to any one of claims 70-79, wherein the cancer is characterized as a solid tumor.

84. The method according to claim 83, wherein the cancer is malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary tract cancer, serous uterine carcinoma, bile duct cancer, neuroblastoma, sarcoma, lung cancer, or melanoma.

85. The method according to any one of claims 70-84, further comprising administering a high dose of chemotherapy to the subject prior to administering the CAR immune cells.

86. The method of claim 85, further comprising administering bone marrow cells or peripheral blood stem cells to the subject.

87. The method according to any one of claims 70-86, further comprising administering to the subject an additional active agent comprising one or more of thalidomide, lenalidomide, and bortezomib.

88. The method according to any one of claims 70-87, wherein the effective number of CAR immune cells is approximately 1 × 10⁻⁶ per kg of subject body weight. 4 One to approximately 6 × 10 5 Each cell.

89. The method according to any one of claims 70-88, wherein the subject is in a state of minimal residual disease.

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