Chimeric checkpoint receptors for use in the treatment of malignant B-cell disorders

Abstract

The present invention relates to chimeric checkpoint receptor (CCR) fusion proteins, nucleic acid molecules encoding said fusion proteins, vectors comprising said nucleic acid molecules, host cells comprising said nucleic acid molecules and / or expressing said fusion proteins, methods of providing said host cells, pharmaceutical compositions comprising said fusion proteins, nucleic acid molecules or host cells, and said products for use as medicaments and in the treatment of B-cell lymphoma.

Description

[Technical Field]

[0001] The present invention relates to chimeric checkpoint receptor (CCR) fusion proteins, nucleic acid molecules encoding said fusion proteins, vectors comprising said nucleic acid molecules, host cells comprising said nucleic acid molecules and / or expressing said fusion proteins, methods of providing said host cells, pharmaceutical compositions comprising said fusion proteins, nucleic acid molecules or host cells, and said products for use as medicaments and in the treatment of B-cell lymphoma. [Background technology]

[0002] The concept and advantages of CAR T cells are well known in the art and have been the focus of pharmaceutical research and development in recent years. A prime example of the successful clinical application of CAR T cells in patients is the regulatory approval of tisagenlecleucel (Kymriah®) for the treatment of B-cell acute lymphoblastic leukemia in the United States and the European Union. However, certain side effects and variable therapeutic success in patients can complicate the use of second-generation CAR T cells such as tisagenlecleucel in patients.

[0003] The main problem associated with the use of tisagenlecleucel in patients with malignant CD19 + B lymphocytes and healthy CD19 + There is a general depletion of B lymphocytes, resulting in B cell aplasia and consequent hypogammaglobulinemia (Ali S et al.,2020 Oncologist,25(2):e321-e327).

[0004] Furthermore, significant neurotoxicity has been observed in patients treated with tisagenlecleucel, which may be due to CD19 expression on pericytes, resulting in on-target, off-tumor effects. +It has been reported that interactions with pericytes can lead to the unwanted release of IL-2 and IL-6 in these cells, which can in turn lead to neurological symptoms (Parker KR et al., 2020 Cell, 183(1):126-142.e17.).

[0005] Both IL-2 and IL-6 are considered alarm cytokines, which can further recruit and activate other immune cells, such as macrophages, and contribute to increased IL-6 release after activation. In general, increased IL-6 release can lead to cytokine release syndrome (CRS), which can be life-threatening for patients.

[0006] Increased IL-2 release has another negative effect on CAR T cell activity. IL-2 is important for the proliferation of regulatory T cells and, as a result, can reduce T cell activity (Chinen T et al., 2016 Nat Immunol, 17(11):1322-1333).

[0007] Possibly for the above reasons, patients with B-cell lymphoma (CD19) previously treated with tisagenlecleucel CAR T cells + CD80 + CD86 + The inventors found that bearer animals developed tumor recurrence in approximately 50% of cases. Re-treating these animals with tumor recurrence with tisagenlecleucel CAR T cells did not result in therapeutic success.

[0008] Therefore, based on the above-mentioned situation regarding the use of currently available CAR T cells, the inventor aimed to overcome the shortcomings of the prior art and to provide and enable advantageous improved CAR T cell therapy.

[0009] Therefore, the object of the present invention is to detect healthy B lymphocytes and CD19 + The goal is to provide safer and more efficient CAR T-cell therapy that avoids non-specific targeting of cells and cell types other than malignant B lymphocytes, such as pericytes.

[0010] It is a further object of the present invention to provide a CAR T cell therapy that allows for the maintenance of a population of healthy B lymphocytes, avoiding neurotoxic symptoms that may appear when using CAR T cell therapies described in the prior art.

[0011] It is another object of the present invention to provide a CAR T cell therapy that reduces the release of alarm cytokines such as IL-2 and IL-6 to avoid cytokine release syndrome as a significant side effect.

[0012] It is also an object of the present invention to avoid tumor recurrence in patients with B-cell lymphoma and to provide a promising treatment option for patients who have previously been treated with conventional CAR T-cell therapy and have experienced tumor recurrence. Summary of the Invention [Means for solving the problem]

[0013] The foregoing objects are solved by the aspects of the present invention specified below.

[0014] According to a first aspect of the present invention, there is provided a fusion protein comprising an extracellular domain comprising a polypeptide having specific affinity for CD80 and / or CD86, a transmembrane domain, and an intracellular domain comprising a costimulatory polypeptide.

[0015] In a preferred embodiment of the first aspect of the invention, the polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CD28 (SEQ ID NO: 2), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the CD86-binding domain of the anti-CD86 antibody commonly referred to in the art as clone hu3D1 (SEQ ID NO: 5); preferably, the polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CTLA-4 (SEQ ID NO: 1).

[0016] In another preferred embodiment of the first aspect of the invention, the transmembrane domain is suitable for insertion and anchoring of the fusion protein in the cell membrane of a mammalian cell, preferably the transmembrane domain is selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CTLA-4, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g. CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, C D134, CD137, CD154, CD200, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, VLA1, ITGA1, CD49a, ITGA4, IA4, CD49D, ITGA6, V LA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, I TGB7, TNFR2, DNAM1(CD226), SFAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD The transmembrane domain comprises the transmembrane region of one or more of human CTLA-4 (SEQ ID NO: 1) or an amino acid sequence at least 80% identical thereto, preferably the transmembrane domain comprises the transmembrane region of human CTLA-4 (SEQ ID NO: 1) or an amino acid sequence at least 80% identical thereto.

[0017] In a preferred embodiment of the first aspect of the present invention, the costimulatory polypeptide of the fusion protein comprises the intracellular domain of one or more of human 4-1BB (CD137; SEQ ID NO: 3), CD3ζ (SEQ ID NO: 25), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, Myd88-CD40, KIR2DS2, and HVEM, or an amino acid sequence at least 80% identical thereto; preferably, the costimulatory polypeptide of the fusion protein comprises the intracellular domain of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto; or preferably, the costimulatory polypeptide comprises the intracellular domain of CD3ζ (SEQ ID NO: 25), or an amino acid sequence at least 80% identical thereto.

[0018] In another preferred embodiment of the first aspect of the present invention, the polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence comprising the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an amino acid sequence at least 80% identical thereto, and the intracellular domain comprises a costimulatory peptide comprising the intracellular domain of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto.

[0019] According to a second aspect of the present invention there is provided a nucleic acid molecule encoding the fusion protein of the first aspect of the invention.

[0020] According to a third aspect of the present invention there is provided a vector comprising a nucleic acid molecule of the second aspect of the invention.

[0021] According to a fourth aspect of the present invention there is provided a host cell comprising a nucleic acid molecule of the second aspect of the invention or a vector of the third aspect of the invention.

[0022] In a preferred embodiment of the fourth aspect of the invention, the host cell is transduced with a nucleic acid molecule of the second aspect of the invention or a vector of the third aspect of the invention, or the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention is stably integrated into the genome of the host cell.

[0023] According to a fourth aspect of the invention there is also provided a host cell which stably or transiently expresses a fusion protein according to the first aspect of the invention.

[0024] In a specific preferred embodiment of the fourth aspect of the invention, the host cell stably or transiently expresses (co-expresses) a protein comprising an extracellular domain with specific affinity for CD19 and an intracellular costimulatory domain, preferably the extracellular domain with specific affinity for CD19 comprises at least an antigen-binding fragment of an anti-CD19 antibody, preferably the extracellular domain with specific affinity for CD19 comprises an anti-CD19 scFv.

[0025] In one specific preferred embodiment of the fourth aspect of the invention, the intracellular costimulatory domain of the co-expressed (CAR) protein comprises the amino acid sequence of the intracellular domain of CD3ζ (SEQ ID NO: 25), or an amino acid sequence at least 80% identical thereto. In a preferred embodiment of the fourth aspect of the invention, the host cell is a human CD8 + T cells.

[0026] According to a fifth aspect of the present invention there is provided a method of providing a host cell of the fourth aspect of the invention, the method comprising the steps of transducing a host cell with a nucleic acid molecule of the second aspect of the invention or a vector of the third aspect of the invention, culturing the transduced host cell of the previous step in a suitable medium which allows cell growth and expression of the fusion protein encoded by said nucleic acid molecule or said vector, and harvesting the host cell from the medium.

[0027] According to a fourth aspect of the invention there is also provided a host cell obtainable by the method of the fifth aspect of the invention.

[0028] According to a sixth aspect of the present invention there is provided a pharmaceutical composition comprising a fusion protein of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention and / or a host cell of the fourth aspect of the invention.

[0029] According to a seventh aspect of the present invention, there is provided a fusion protein according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention or a pharmaceutical composition according to the sixth aspect of the present invention for use as a medicament.

[0030] According to an eighth aspect of the present invention there is provided a fusion protein of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention, a host cell of the fourth aspect of the invention or a pharmaceutical composition of the sixth aspect of the invention for use in the treatment of B-cell lymphoma.

[0031] In a preferred embodiment of the eighth aspect of the invention, the B-cell lymphoma is a non-Hodgkin's lymphoma selected from the group comprising marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL), preferably the B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0032] According to a ninth aspect of the present invention there is provided a kit or kit-in-parts comprising a fusion protein of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention or a host cell of the fourth aspect of the invention. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 shows (A) a schematic diagram depicting the modular organization of the CAR / CCR expression cassette of the invention (Lκ: light chain kappa signal sequence; anti-CD19scFv: CD19-specific single-chain (heavy and light chain) variable region fragment; CD8αhinge: CD8 alpha hinge domain; CD8αtm: CD8 alpha transmembrane domain; CD3ζ(ic): CD3 zeta intracellular signaling domain; P2A: 2A peptide; CTLA-4ex: cytotoxic T-lymphocyte-associated protein extracellular domain; CTLA-4 (B) Schematic diagram depicting the expression and localization of CAR / CCR constructs on the T cell surface. (C) Expression of anti-CD19(FMC63)-CD8α(ex)-CD3ζ(tm+ic)-CAR and CTLA4(ex)-4-1BB(tm+ic)-CCR on retrovirally engineered human CD3+ T cells. CAR / CCR expression was monitored by flow cytometry using phycoerythrin-labeled anti-idiotypic antibodies and PE-Cy-7-conjugated CTLA-4-specific antibodies, with mock-transduced human CD3+ T cells as a control (gray area). [Figure 2] Figure 2 shows high expression levels of both checkpoint receptor ligands, CD80 and CD86, on the surface of relapsed B-cell lymphoma cells after CD19-CAR T-cell therapy. Tumor biopsies were collected from DLBCL patients before and during CAR T-cell treatment (Kymriah, Novartis), fixed in 10% neutral buffered formalin (NBF), processed, and embedded in paraffin (formalin-fixed, paraffin-embedded (FFPE) tissue). Fixed tissue sections were stained for CD80 and CD86 expression using HRP-conjugated CD80 and CD86 antibodies. After the detection step, the colored chromogen DAB was added, and slides were counterstained with hematoxylin. Relapsed DLBCL cells after cellular CAR T-cell immunotherapy demonstrated increased expression of immune checkpoint ligands CD80 and / or CD86 compared with CAR T-naive samples from the same patients. [Figure 3] Figure 3 shows an analysis of primary DLBCL and CLL tumor cells for expression of both CD80 and CD86. Primary DLBCL cells show increased expression of CD80 and / or CD86 in contrast to primary CLL cells and healthy B cells (controls). [Figure 4] Figure 4 shows the reduced release of IL-2 (A) and IL-6 (B) by human CD19-specific CAR / CCR T cells of the present invention when co-cultured with human CD19+ B cells in the presence of CD11b+ monocytes. Primary T cells were genetically modified to express the CD19-CAR / CTLA-4-CCR construct (first bar from the left) and co-cultured with CD19+ B cells (at a ratio of 100 CAR T cells:100 B cells:1 monocyte) for 24 hours. IL-2 released by CAR T cells and IL-6 released by monocytes were detected in the supernatant by ELISA. T cells transfected with a second-generation CD19-specific CAR (α-CD19-CD28-CD3ζ; second bar from the left), a first-generation CD19-specific CAR (α-CD19-CD3ζ; center bar), and untransduced cells (second bar from the right) were used as controls. Additionally, (C) a reduced cytotoxic effect on primary human B cells and (D) a reduced IFN-γ release by human CD19-specific CAR / CCR T cells of the present invention when co-cultured with primary human CD19+ B cells are shown. Primary T cells were genetically modified to express the CD19-CAR / CTLA-4-CCR construct (first bar from the left) and co-cultured with primary CD19+ B cells at an effector / target ratio of 1:10 (E:T) for 18 hours. T cells genetically modified to express first- and second-generation CAR constructs and unmodified T cells (mock) were used as controls. [Figure 5]Figure 5 shows the reduced release of IFN-γ by human CD19-specific CAR / CCR T cells of the present invention when cocultured with pericytes expressing human CD19 and CD248 (A). Therefore, primary human vascular wall-typical mesenchymal stem cells (VW-MSCs) were cultured in medium supplemented with TGF-β3 for 2 weeks to differentiate into pericytes expressing CD19 and CD248. Finally, differentiated CD19+CD248+ pericytes were isolated using a BD FACSAria III sorter. (B) Next, T cells were equipped with CAR (second-generation) or CAR / CCR constructs and cocultured with CD19+CD248+ pericytes (at a ratio of 1:1 CAR T cell:pericyte) for 24 hours. Finally, the supernatants from this experiment were collected and analyzed for IFN-γ by ELISA. Supernatants obtained from mock-transduced T cells and (CAR) T cells cultured without pericytes (w / o) were used as controls. [Figure 6] Figure 6 shows that CD19-CAR / CTLA-4-CCR T cells efficiently eliminate CD80+CD86+ cells from the aggressive B-cell lymphoma cell line Raji. Primary T cells were genetically modified to express the CD19-CAR / CTLA-4-CCR construct (left bar in each panel) and cocultured with CD19+CD80+CD86+ cells from the cell line Raji at a 1:4 (effector / target cell) ratio for 24–120 hours. The cytotoxic effect against tumor cells was analyzed using a HIDEX ELISA reader. T cells implanted with a second-generation CD19-specific CAR (α-CD19-CD28-CD3ζ; center bar in each panel) and a first-generation CD19-specific CAR (α-CD19-CD3ζ; right bar in each panel) were used as controls. [Figure 7]Figure 7 shows that CD19-CAR / CTLA-4-CCR T cells efficiently eliminate B-cell lymphoma cells and increase tumor-free survival. CD19+CD80+CD86+ cells from the cell line Raji were intravenously injected into RAG2- / - common γ- / - mice and treated with CD19-CAR / CTLA-4-CCR T cells on day 5. T cells genetically engineered to express a second-generation CD19-CAR (Kymriah, Novartis) and mock-transduced T cells were used as controls. [Figure 8] Figure 8 shows IFN-γ release by CD19CAR / CTLA-4CCR T cells, CD19CAR (second generation) T cells, or CD19CAR (first generation) T cells after coculture with CD19-expressing primary DLBCL cells (CD80highCD86high), the DLBCL cell line SU-DHL-10 (CD80highCD86high), the DLBCL cell line DOHH-2 (CD80highCD86low), the DLBCL cell line Oci-Ly1 (CD80lowCD86high), or the DLBCL cell line Oci-Ly19 (CD80lowCD86low). Cells were cocultured with CAR / CCR T cells, CAR (first generation) T cells, or CAR (second generation) T cells at 37°C for 48 hours. Target cells co-cultured with mock-transduced T cells (mock) or target cells without effector T cells (no T cells) served as controls (ns = not significant, *p<0.05, **p<0.005). [Figure 9]Figure 9 shows IL-2 release by CD19CAR / CTLA-4CCR T cells, CD19CAR (second generation) T cells, or CD19CAR (first generation) T cells after 48 hours of coculture with CD19-expressing primary DLBCL cells (CD80highCD86high), DLBCL cell line SU-DHL-10 (CD80highCD86high), DLBCL cell line DOHH-2 (CD80highCD86low), DLBCL cell line Oci-Ly1 (CD80lowCD86high), or DLBCL cell line Oci-Ly19 (CD80lowCD86low) at 37°C. Target cells cocultured with mock-transduced T cells (Mock) or target cells without effector T cells (No T cells) served as controls (ns = not significant, *p<0.05, **p<0.005). [Figure 10] Figure 10 shows the luminescence plots of a xenograft mouse study based on Raji-based lymphoma-bearing Rag2- / -IL-2rg- / - mice treated with 8.0 x 106 CAR / CCR T cells, CAR (second-generation) T cells, or mock T cells and assessed for tumor growth using bioluminescence. Raji expressing CD19 / CD80 / CD86 was genetically modified to express firefly luciferase and intravenously injected into immunocompromised mice. Three days after tumor implantation, mice were divided into three groups and treated with effector T cells (second-generation CAR or CAR / CCR). A group of mice treated with mock-transduced T cells served as a control. Tumor progression was monitored using an IVIS bioluminescence device. Mice were sacrificed at the tumor burden cutoff or when they exhibited paralysis. [Figure 11]Figure 11 shows IL-6 release by activated macrophages in vitro. CAR T cells were cultured in the presence of the CD19 / 80 / 86-expressing tumor cell line SU-DHL10 and isolated human CD11b+ macrophages from the same healthy donor. 1×105 CAR / CCR T cells, CAR (second-generation) T cells, or non-transduced (nt) T cells (+T) were cultured in the presence of 3.25×104 macrophages (+M) at 37°C for 24 hours. Finally, the supernatant was collected and examined for IL-6 release by ELISA. Tumor cells cultured with or without macrophages and / or (CAR / CCR, CAR, nt) T cells served as controls. [Figure 12] Figure 12 shows a schematic diagram of the design of the CAR construct and CAR / CCR construct used herein. (A) A construct of the present invention using a CD20-specific CAR (comprising an anti-CD20 scFv as the extracellular domain and CD3ζ as the intracellular signaling domain containing a costimulatory polypeptide) and a CCR comprising CTLA-4 as the extracellular domain containing a polypeptide with specific affinity for CD80 and / or CD86 and the intracellular domain of 4-1BB as the intracellular domain containing a costimulatory polypeptide. (B) A construct of the present invention using a CD19-specific CAR (comprising an anti-CD19 scFv as the extracellular domain and CD3ζ as the intracellular signaling domain containing a costimulatory polypeptide) and a CCR comprising an anti-CD86 scFv as the extracellular domain containing a polypeptide with specific affinity for CD86 and the intracellular domain of 4-1BB as the intracellular domain containing a costimulatory polypeptide. (C) A second-generation CD20-specific CAR known in the art. (D) A second-generation CD19-specific CAR known in the art. [Figure 13] FIG. 13 shows analysis of the cytotoxic effect (A; C) and release of the pro-inflammatory cytokine IFN-γ (B; D) by T cells engineered to express the constructs shown in FIG. 12 in the DLBCL (diffuse large B-cell lymphoma) cell line SUDHL-10 (A; B) and the MCL (mantle cell lymphoma) cell line JeKo-1R (C; D). [Figure 14] Figure 14 shows analysis of the cytotoxic effect (A) and release of the pro-inflammatory cytokine IFN-γ (B) by T cells engineered to express the constructs shown in Figure 12 in the JeKo-1R cell line, which has 99.9% reduced / decreased CD19 expression (verified by RT-PCR; data not shown). [Figure 15] Figure 15 shows a schematic diagram of a reverse switch design in which the intracellular signaling domain containing the costimulatory polypeptide is swapped between the CAR and CCR. (A) A construct is created by combining a CD19-specific CAR (comprising an anti-CD19 scFv as the extracellular domain and CD3ζ as the intracellular signaling domain containing the costimulatory polypeptide) with a CCR containing CTLA-4 as the extracellular domain containing a polypeptide with specific affinity for CD80 and / or CD86 and the intracellular domain of 4-1BB as the intracellular domain containing the costimulatory polypeptide. (B) A reverse switch resulting in a CAR and CCR similar to those in (A), except that the CAR contains the intracellular domain of 4-1BB as the intracellular domain containing the costimulatory polypeptide, and the CCR contains CD3ζ as the intracellular signaling domain containing the costimulatory polypeptide. (C) Flow cytometry histograms demonstrate that human T cells expressing the molecules described in (A) and (B) both induced efficient cytotoxicity against the Su-DHL-5 B cell lymphoma cell line. (D) FACS gating strategy is shown. [Figure 16]Figure 16 shows (A) a fully murine CAR / CCR construct of the invention comprising a CD19-specific CAR (comprising an anti-CD19 scFv as the extracellular domain and CD3ζ as the intracellular signaling domain comprising a costimulatory polypeptide), and a CCR comprising CTLA-4 as the extracellular domain comprising a polypeptide with specific affinity for CD80 and / or CD86, and the intracellular domain of 4-1BB as the intracellular domain comprising a costimulatory polypeptide; (B) a second-generation fully murine CD19-specific CAR design known in the art; and (C) the results regarding cell number and IFN-γ release observed when T cells expressing the constructs shown in (A) and (B) are contacted with murine CD19+CD80+CD86+ B cell lymphoma cells or primary CD19+CD80-CD86- B cells in vitro. [Figure 17] Figure 17 shows the in vivo effect of T cells expressing the constructs described in Figure 16 above in mice of the C57bl / 6 (wt) mouse strain on naive B cell populations in peripheral blood and in the spleen (right-most graph) over time as (A) ratios and (B) FACS histograms (top row: second generation CARs as in Figure 16(B) ; bottom row: CAR / CCR constructs as in Figure 16(A) ), and (C) the FACS gating strategy. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention is based on the recognition that discrimination between healthy and malignant B cells, as well as between malignant B cells and other cell types such as pericytes, is made possible by the use of costimulatory chimeric checkpoint receptors (CCRs) that recognize checkpoint ligands on malignant cells, such as CD80 and CD86.

[0035] The inventors have found that the neurotoxicity observed during therapeutic treatment of patients with Kymriah may be due to the fact that CD19 is also expressed on neural pericytes (Parker KR et al., 2020; see supra). The undesirable toxicity of CAR T cells to CD19 has previously been addressed by other researchers, in some instances using costimulatory CCRs as well (see Liao Q et al., 2020 Biomarker Research, 8:57 and Blaeschke F et al., 2021 Blood Cancer Journal, 11:108).

[0036] However, both Liao et al. (2020) and Blaeschke et al. (2021) used a CCR against PD-L1 (α-PD-L1-scFv in Liao et al.'s case, and PD-1 in Blaeschke et al.'s case) in combination with a CD19-CAR. This combination may not be suitable for eliminating neurotoxicity, because PD-L1 is also expressed on the surface of neural pericytes (Domev et al., 2014 Stem Cells Transl Med., 3(10):1169-1181), which has already been reported elsewhere to provide costimulatory signals to the aforementioned CCR fusion proteins.

[0037] In addition to reduced potential neurotoxicity, one particular advantage of using CCRs of the invention directed against CD80 and / or CD86 is that B cell lymphoma cells that have previously been treated with CD19-specific CAR T cells exhibit increased expression of CD80 and / or CD86 (see Figure 2). Thus, any moiety that targets CD80 and / or CD86 may be suitably used as part of a CCR of the invention (see Figures 12 and 13) and may serve to reduce undesired targeting of primary B cells in vitro (see Figure 16) and in immunocompetent mice of the C57bl / 6N(wt) mouse strain (see Figure 17).

[0038] Similar observations have been made by the inventors for primary B-cell lymphoma cells (i.e., primary diffuse large B-cell lymphoma (DLBCL) cells; see Figures 13(A) and 13(B)), in contrast to leukemia cells (i.e., chronic lymphocytic leukemia (CLL) cells; see Figure 3). This suggests that even primary B-cell lymphoma cells are particularly accessible to the CAR-T cells of the invention, which would benefit from costimulation by CD80 and / or CD86.

[0039] Thus, for the first time, the present invention enables a promising treatment option for patients suffering from B-cell lymphoma in general and for those whose tumors have relapsed after initial (unsuccessful) treatment, such as with Kymriah. The present strategy is based on the expression of a recombinant CTLA-4 co-receptor (CCR) in addition to a CAR, which binds to and neutralizes the checkpoint ligands CD80 and CD86 on B-cell tumor cells.

[0040] By splitting the signaling moiety between both the CD19-CAR-CD3ζ and CTLA-4-CCR-4-1BB receptors, CAR-T cells are fully activated only when CD80 and / or CD86 are expressed together with CD19 on the same target cell, thereby avoiding nonspecific off-tumor activation of T cells. Based on the experimental results provided herein, these beneficial effects are not limited to the specific constructs described above, but can also be observed with different modules as long as they meet the required binding specificity and / or signaling capabilities.

[0041] In this regard, the CD19-binding extracellular domain of the co-expressed (CAR) protein may be functionally replaced with a different CD19-, CD20-, or CD22-binding moiety, or yet another moiety that binds to a cell surface molecule or extracellular cancer biomarker found on cancer cells. Similarly, the extracellular domain of CTLA-4 may be functionally replaced with a CD80- and / or CD86-binding moiety, such as CD28, or any CD80 / CD86-binding moiety derived from an anti-CD80 or anti-CD86 antibody.

[0042] These findings, based on the experimental data disclosed herein, demonstrate that combining binding to a tumor biomarker such as CD19 with binding to CD80 or CD86 certainly leads to the benefits achieved by the present invention.

[0043] Furthermore, in addition to the advantages provided by specific embodiments of the present invention, it will be apparent to those skilled in the art that the intracellular stimulatory domain and intracellular costimulatory domain can be replaced with functional equivalents disclosed herein, as long as they are capable of providing the intracellular signal required for T cell activation. Thus, not only can (co)stimulatory moieties be exchanged between the fusion protein and the co-expressed (CAR) protein, but any (co)stimulatory domain or combination thereof known to provide the required intracellular signaling can also be used within the present invention.

[0044] However, to provide the necessary intracellular signaling within the CAR T cells required to activate T cells and kill tumor cells, at least one intracellular domain of the fusion protein and / or co-expressed (CAR) protein must contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs; see Zhang et al., 2017 Biomarker Research, 5:22). In the context of the present invention, the two most prominent and commonly used ITAM-containing domains (FcεRIγ(ic) with one ITAM and CD3ζ(ic) with three ITAMs) are preferably used, but artificially designed synthetic ITAM-containing domains that perform similar functions can also be used and are encompassed by the present invention.

[0045] In embodiments in which the ITAM-containing domain is provided as part of a fusion protein, a costimulatory domain described herein is provided as part of a co-expressed (CAR) protein. In embodiments in which the ITAM-containing domain is provided as part of a co-expressed (CAR) protein, a costimulatory domain described herein is provided as part of a fusion protein. It has also previously been reported that IL-6 can be released by antigen-presenting cells (APCs), such as macrophages or pericytes, and subsequently cause off-tumor cytokine release syndrome (CRS) in patients. The PD1-CD28 fusion CCR constructs used in Liao et al. (2020) and Blaeschke et al. (2021) have recently been reported to lead to (undesirable) increases in the release of IL-2, IFN-γ, and / or TNF.

[0046] These cytokines may act to recruit and activate other immune cells, such as macrophages, contributing to the subsequent increased release of IL-6 and the development of CRS and MAS-L (see Kennedy VE et al., Blood (2020) 136 (Supplement 1): 7-8). This is particularly evident when CD28 is used in combination with the CD3ζ intracellular domain.

[0047] Additionally, increased release of IL-2 can induce the proliferation of regulatory T cells, which can reduce the T cell activity of CAR T cells (see, for example, Chinen et al., Nat Immunol, 2016;17(11):1322-1333). This is particularly likely when using the strategies proposed by Liao et al. (2020) and Blaeschke et al. (2021).

[0048] Both of the aforementioned problems appear to be less pronounced when using the strategies of the present invention, which involve the use of alternative intracellular signaling domains such as 4-1BB (see Figures 4, 5, 8, 9 and 11 herein) or CD3ζ (see Figure 15).

[0049] The strategy of the present invention is to investigate the role of CD20 as a tumor biomarker (see Figure 14), even at the time of relapse after initial CAR T cell treatment (see Figure 6), and to investigate the role of CD19 as a tumor biomarker (see Figure 6). + , CD80 + and / or CD86 + This leads to efficient elimination of B-cell lymphoma. This efficient elimination of tumor cells was further demonstrated to lead to prolonged tumor-free survival (see Figure 7) and reduced tumor progression in xenograft mouse models (see Figure 10).

[0050] By using the strategies of the present invention, various side effects can be limited or overcome: IL-2 and IL-6 release is reduced (see Figures 4, 5, 9, and 11), off-tumor and neurotoxic effects are prevented, and a pool of healthy B lymphocytes can persist after CAR T cell therapy (in vitro, as demonstrated by the results in Figure 4(D) and Figure 16(C), and in vivo, as demonstrated by the results in Figure 17).

[0051] Thus, the present invention provides a CAR T cell therapy with increased safety and efficacy, which avoids non-specific targeting of cells other than malignant B lymphocytes, maintains a population of healthy B lymphocytes in treated patients, largely avoids neurotoxic symptoms commonly observed with prior art therapies, such as what is known as immune effector cell-associated neurotoxicity syndrome (ICAN), reduces the release of alarm cytokines and the development of cytokine release syndrome, and can specifically target and avoid tumor recurrence in patients with B cell lymphoma.

[0052] Thus, in a first aspect of the present invention there is provided a fusion protein comprising an extracellular domain comprising a polypeptide with specific affinity for CD80 and / or CD86, a transmembrane domain and an intracellular domain comprising a costimulatory polypeptide.

[0053] The extracellular domain of the fusion protein of the present invention, which comprises a polypeptide having specific affinity for CD80 and / or CD86, is preferably the extracellular domain of CTLA-4 (CD152) (UniProtKB-P16410). Alternatively, the extracellular domain of CD28 (UniProtKB-P10747) can be preferably used due to its specific affinity for CD80 and / or CD86.

[0054] Alternatively, antibodies or antibody fragments with specific affinity for CD80 and / or CD86 may be preferred. In particular, an amino acid sequence that is at least 80% identical to the amino acid sequence of the CD86-binding domain of the anti-CD86 antibody commonly referred to in the art as clone hu3D1 (SEQ ID NO: 5) may be used as a polypeptide with specific affinity for CD86.

[0055] In one embodiment, an antibody or antibody fragment with specific affinity for CD80 is used as part of the extracellular domain of the fusion protein of the invention, which is preferably derived from the CD80 binding domain of the anti-CD80 antibody commonly referred to in the art as clone hu1F1, and more preferably has the following amino acid sequence: QLVQSGAEVKKPGASVKVSCKPSGFNIKDYYMHWVRQAPGQGLEWIGWIDPENGNTLYDPKFQGKATITADTSTSTAYMELSSLRSEDTAVYYCAREGLFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQWSSYPLTFGQGTKVEIK (SEQ ID NO: 4)

[0056] In another embodiment, an antibody or antibody fragment with specific affinity for CD86 is used as part of the extracellular domain of the fusion protein of the invention, preferably derived from the CD86 binding domain of the anti-CD86 antibody commonly referred to in the art as clone hu3D1, and more preferably having the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAIQWVRQAPGQGLEWIGVINIYYDNTNYNQKFKGKATMTVDKSTSTAYMELSSLRSEDTAVYYCARAAWYMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKSSQSLLNSRTRENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSYNLYTFGQGTKVEIK (SEQ ID NO: 5)

[0057] In a preferred embodiment, a combination of an antibody or antibody fragment against CD80 and an antibody or antibody fragment against CD86 may be used as part of the extracellular domain of a fusion protein of the invention, preferably linked by a conventional linker sequence, and more preferably having the following amino acid sequence: QLVQSGAEVKKPGASVKVSCKPSGFNIKDYYMHWVRQAPGQGLEWIGWIDPENGNTLYDPKFQGKATITADTSTSTAYMELSSLRSEDTAVYYCAREGLFFAYWGQGTLVTVSSGGGGSGGGGSGG GGSDIQMTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQWSSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAIQWVRQAPGQGLEWIGVINIYYDNTNYNQKFKGKATMTVDKSTSTAYMELSSLRSEDTAVYYCARAAWYMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKSSQSLLNSRTRENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSYNLYTFGQGTKVEIK (SEQ ID NO: 6)

[0058] It would be within the ordinary skill of one in the art to select or generate antibodies or antibody fragments specific for CD80 and / or CD86.

[0059] The transmembrane domain of the fusion protein of the invention can be any type of transmembrane domain that meets the structural requirements necessary to maintain the function of the associated extracellular and intracellular domains.

[0060] Preferably, the transmembrane domain of the fusion protein of the present invention is selected from the group consisting of T cell receptor alpha chain (UniProtKB-P01848), T cell receptor beta chain (UniProtKB-P0DSE2), T cell receptor zeta chain (UniProtKB-P20963), CD28 (UniProtKB-P10747), CD3 epsilon chain (UniProtKB-P07766), CD3 zeta chain (UniProtKB-P20963), CD45 (UniProtKB-P08575), CD4 (UniProtKB-P01730), CD ... rotKB-P06127), CD8 alpha chain (UniProtKB-P01732), CD8 beta chain (UniProtKB-P10966), CD9 (UniProtKB-P21926), CD16 (UniProtKB-Q9UPY7), CD22 (UniProtKB-P20273), CD33 (UniProtKB-P20138), CD37 (UniProtKB-P11049), FCGR1A (CD64) (UniProtKB-P12314), CD80 (UniProtKB-P33681), CD86 (UniPro tKB-P42081), CD134 (UniProtKB-P43489), CD137 (UniProtKB-Q07011), CD154 (UniProtKB-P29965), CD200R1 (UniProtKB-Q8TD46), KIR2DS2 (UniPr otKB-P43631), OX40 (UniProtKB-P23510), CD2 (UniProtKB-P06729), CD27 (UniProtKB-P26842), LFA-1 (CD11a) (UniProtKB-P20701), ICOS (CD278) (UniProtKB-Q9Y6W8), CD137(4-1BB)(UniProtKB-Q07011), GITR(UniProtKB-Q9Y5U5), CD40(UniProtKB-P25942), BAFFR(UniProtKB-Q96RJ3), HVE M(UniProtKB-Q92956), SLAMF7(UniProtKB-Q9NQ25), NKp80(KLRF1)(UniProtKB-Q9NZS2), CD160(UniProtKB-O95971), CD19(UniProtKB-P15391),IL2R beta (UniProtKB-P14784), IL2R gamma (UniProtKB-P31785), IL7R alpha (UniProtKB-P16871), CD49a (UniProtKB-P56199), IA4 (UniProtKB-Q8IU71), CD49D (UniProtKB-P13612), CD49f (UniProtKB-P23229), ITGAD (CD11d) (UniProtKB-Q13349), ITGAE (CD103) (UniProtKB-P38570), CD 11a (UniProtKB-P20701), CD11b (UniProtKB-P11215), CD11c (UniProtKB-P20702), CD29 (UniProtKB-P05556), CD18 (UniProtKB-P05107), ITG B7 (UniProtKB-P26010), TNFR2 (UniProtKB-P20333), DNAM1 (CD226) (UniProtKB-Q15762), SLAMF4 (CD244) (UniProtKB-Q9BZW8), CD84 (UniPro tKB-Q9UIB8), CD96(UniProtKB-P40200), CEACAM1(UniProtKB-P13688), CRTAM(UniProtKB-O95727), Ly9(CD229)(UniProtKB-Q9HBG7), CD160 (BY55)(UniProtKB-O95971), PSGL1(UniProtKB-Q14242), CD100(SEMA4D)(UniProtKB-Q92854), SLAMF6(UniProtKB-Q96DU3), SLAM(CD150)(U The transmembrane domains are selected from the list of transmembrane domains contained in the protein sequences of the following: CD8 alpha chain (UniProtKB-P01732), CTLA-4 (CD152) (UniProtKB-P16410), preferably CD8 alpha chain (UniProtKB-P01732), CTLA-4 (CD152) (UniProtKB-P16410),The transmembrane domain is selected from the list of transmembrane domains including the transmembrane domain of CD28 (UniProtKB-P10747), and more preferably the transmembrane domain of CTLA-4 (CD152) (UniProtKB-P16410).

[0061] The intracellular domain of the fusion protein of the invention comprising a costimulatory polypeptide can comprise a signaling polypeptide or a signaling domain. Within the context of the present invention, the intracellular domain comprising a costimulatory (or stimulatory) polypeptide can also be referred to as an intracellular or cytoplasmic signaling domain, an intracellular or cytoplasmic activation domain, etc.

[0062] Intracellular signaling domains suitable for use in the fusion proteins of the invention can preferably be or comprise immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.

[0063] The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid (SEQ ID NO: 7). In certain embodiments, the intracellular signaling domain of the fusion protein contains 1, 2, 3, 4, or 5 ITAM motifs. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, and the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. For example, (YX1X2L / I)(X3) n (YX1X2L / I) (SEQ ID NO: 8), where n is an integer of 6 to 8, and each of the 6 to 8 X3 can be any amino acid. In one embodiment, the intracellular signaling domain of the fusion protein of the present invention comprises three ITAM motifs.

[0064] An intracellular signaling domain suitable for use in a fusion protein of the invention can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain of any ITAM motif-containing protein.

[0065] Thus, a suitable intracellular signaling domain or costimulatory polypeptide need not comprise the entire sequence of the entire protein from which it is derived, and it would be well within the knowledge of one of ordinary skill in the art to determine a functional intracellular signaling domain or costimulatory polypeptide contained within a given entire protein sequence.

[0066] Examples of preferred protein sequences comprising ITAM motif-containing polypeptides include, but are not limited to, DAP12 (UniProtKB-O43914), FcεRIγ (Fc epsilon receptor I gamma chain) (UniProtKB-P30273), CD3D (CD3 delta) (UniProtKB-P04234), CD3E (CD3 epsilon) (UniProtKB-P07766), CD3G (CD3 gamma) (UniProtKB-P09693), CD3Z (CD3 zeta) (UniProtKB-P20963), CD79A (antigen receptor complex-associated protein alpha chain) (UniProtKB-P11912), and CD79B (antigen receptor complex-associated protein beta chain) (UniProtKB-P40259).

[0067] In a preferred embodiment of the present invention, the co-expressed (CAR) protein comprises an intracellular domain having one or more ITAMs, preferably one of FcεRIγ(ic) and CD3ζ(ic), more preferably CD3ζ(ic), and the fusion protein comprises an intracellular domain selected from the group consisting of 4-1BB (CD137; SEQ ID NO: 3), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, Myd88-CD40, KIR2DS2, and HVEM. According to a preferred embodiment, the fusion protein comprises an intracellular domain having one or more ITAMs, preferably one of FcεRIγ(ic) and CD3ζ(ic), more preferably CD3ζ(ic), and the co-expressed (CAR) protein comprises an intracellular domain selected from the group consisting of 4-1BB (CD137; SEQ ID NO: 3), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, Myd88-CD40, KIR2DS2, and HVEM.

[0068] In a preferred embodiment of the present invention, the intracellular domain comprising the costimulatory polypeptide is selected from the group consisting of CD28 (UniProtKB-P10747), ICOS (UniProtKB-Q9Y6W8), OX-40 (UniProtKB-P43489), BTLA (UniProtKB-Q7Z6A9), CD27 (UniProtKB-P26842), CD30 (UniProtKB-P28908), CD40L (UniProtKB-P29965), GITR (UniProtKB-Q9Y5U5), Myd88 (UniProtKB-Q99836), CD40 (UniProtKB-P25942), It may be selected from the list of intracellular domains or costimulatory polypeptides contained within the protein sequences of KIR2DS2 (UniProtKB-P43631), CD137(4-1BB) (UniProtKB-Q07011), and HVEM (UniProtKB-Q92956), preferably within the protein sequence of CD137(4-1BB) (UniProtKB-Q07011), CD28 (UniProtKB-P10747), or OX-40 (UniProtKB-P43489), more preferably within the protein sequence of CD137(4-1BB) (UniProtKB-Q07011).

[0069] Within the present invention, a fusion protein may comprise a domain as defined herein and, optionally, an additional sequence that connects the domains in the fusion protein. Such linker or hinge sequences for use in CARs or CCRs are generally known in the art and can be easily determined and identified by those skilled in the art so as not to interfere with the specific function of the individual functional domains.

[0070] In a preferred embodiment of the invention, the fusion protein comprises the extracellular and transmembrane domains of CTLA-4 and the intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequence (CTLA-4 ec+tm is in gray, 4-1BB ic is in black): [ka]

[0071] Alternatively, and preferably, the fusion protein comprises the extracellular and transmembrane domains of CD28 and the intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequence (CD28 ec+tm is in grey, 4-1BB ic is in black): [ka]

[0072] Alternatively, and preferably, the fusion protein comprises a binding domain for CD80, the extracellular and transmembrane domains of CD28, and the intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequences (α-CD80 in black, CD28 ec+tm in gray, and 4-1BB ic in black, in that order): [ka]

[0073] Alternatively, and preferably, the fusion protein comprises a binding domain for CD86, the extracellular and transmembrane domains of CD28, and the intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequences (α-CD86 in black, CD28 ec+tm in gray, and 4-1BB ic in black, in that order): [ka]

[0074] Alternatively, and preferably, the fusion protein comprises a binding domain for CD80, a binding domain for CD86, the extracellular and transmembrane domains of CD28, and the intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequences (α-CD80 in gray, linker sequence in bold, α-CD86 in black, CD28 ec+tm in gray, and 4-1BB in black, in that order): [ka]

[0075] Other additional sequences may also be present in the fusion proteins of the invention, such as a leader sequence (eg, an Ig kappa light chain leader sequence such as MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 14)).

[0076] In a specific embodiment, the fusion protein of the present invention can be expressed as a protein construct combined with a given CAR expressed by the same cell, said construct comprising a proteolytic cleavage site between the two proteins, preferably a self-cleaving peptide, more preferably a 2A self-cleaving peptide, particularly preferably a 2A self-cleaving peptide of the sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 15).

[0077] According to one particular embodiment, a fusion protein is co-expressed with the respective CAR, preferably comprising the respective leader sequence and / or a self-cleaving peptide between the two protein entities, more preferably having the following general structure: leader sequence (e.g., L kappa leader sequence) - extracellular domain for a tumor-associated cell surface protein (e.g., CD19, CD20, CD22) - optionally the extracellular domain of a CD8α hinge - transmembrane domain (e.g., that of a CD8α hinge) - intracellular signaling domain (e.g., that of CD3ζ) - self-cleaving peptide (e.g., P2A self-cleaving peptide) - leader sequence (e.g., L kappa leader sequence) - extracellular domain for CD80 and / or CD86 (e.g., that of CTLA-4) - transmembrane domain (e.g., that of CTLA-4) - intracellular signaling domain (e.g., that of 4-1BB).

[0078] According to one specific embodiment, the fusion protein co-expressed with each CAR has the following amino acid sequence: (SEQ ID NO: 16)

[0079] According to the present invention, any of the functional domains referred to herein (e.g., in the case of the fusion proteins of the present invention, the extracellular domain, the transmembrane domain and / or the intracellular signaling domain) may be functionally equivalent to the domains individually listed herein.

[0080] For example, an extracellular domain that does not have the exact same sequence as the CTLA-4 extracellular domain, but which maintains specific affinity for CD80 and / or CD86, may still be suitable as the extracellular domain of a fusion protein of the invention. In this regard, it is noted that one skilled in the art can readily identify and evaluate the specific function of such domains, each of which differs in sequence.

[0081] Thus, according to one embodiment of the present invention, protein sequences consisting of or comprising a protein sequence that is at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, even more preferably at least 95% identical, even more preferably at least 98% identical, and particularly preferably at least 99% identical to a reference protein sequence disclosed herein form part of the present invention as individual domains of the protein of the invention.

[0082] The determination of percent identity between two sequences is accomplished according to the present invention by using the mathematical algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877). Such an algorithm is the basis for the BLASTN and BLASTP programs of Altschul et al. (J. Mol. Biol. (1990) 215:403-410). BLAST nucleotide searches are performed with the BLASTN program. To obtain gapped alignments for comparison, Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25:3389-3402). When utilizing the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used.

[0083] According to a particular embodiment of the present invention, the protein sequences forming part of the present invention as defined above by a given percentage identity to the individual protein sequences are protein sequences which maintain the function of the respective protein.

[0084] According to a preferred embodiment of the invention, the polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CD28 (SEQ ID NO: 2), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the CD86-binding domain of the anti-CD86 antibody commonly referred to in the art as clone hu3D1 (SEQ ID NO: 5); preferably, the polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CTLA-4 (SEQ ID NO: 1).

[0085] According to another preferred embodiment of the present invention, the transmembrane domain is suitable for insertion and anchoring of the fusion protein in the cell membrane of a mammalian cell, preferably the transmembrane domain is selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CTLA-4, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g. CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 , CD154, CD200, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, VLA1, ITGA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGA D, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1( CD226), SFAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6( The transmembrane domain comprises one or more of the transmembrane domains of human CTLA-4 (FLLWILAAVSSGLFFYSFLLT; SEQ ID NO: 17), or an amino acid sequence at least 80% identical thereto, of human CTLA-4 (FLLWILAAVSSGLFFYSFLLT; SEQ ID NO: 17), or an amino acid sequence at least 80% identical thereto.

[0086] Inclusion of a costimulatory domain, such as a 4-1BB (CD137) costimulatory domain, in the intracellular region of the fusion protein of the invention enables T cells to receive costimulatory signals that can direct and regulate specific T cell responses.

[0087] According to a preferred embodiment of the present invention, the intracellular domain can induce a costimulatory signal to T cells. Preferably, the intracellular domain of the fusion protein of the present invention comprises the intracellular domain or (co)stimulatory polypeptide of one or more of human 4-1BB (CD137; SEQ ID NO: 3), CD3ζ (SEQ ID NO: 25), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, Myd88-CD40, KIR2DS2, and HVEM, or an amino acid sequence at least 80% identical thereto. More preferably, the intracellular domain of the fusion protein of the present invention comprises the intracellular domain or costimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto.

[0088] In a preferred embodiment, the fusion protein of the present invention comprises the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an extracellular domain comprising an amino acid sequence at least 80% identical thereto, and the intracellular domain comprises the intracellular domain of human 4-1BB (CD137; SEQ ID NO: 3), or a costimulatory peptide comprising an amino acid sequence at least 80% identical thereto.

[0089] More preferably, the fusion protein comprises the amino acid sequence of CTLA-4(ec+tm) fused to 4-1BB(ic): KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18)

[0090] The fusion protein of the present invention can generally be used in combination with a chimeric antigen receptor (CAR) that targets tumor-associated cell surface proteins.In particular, the fusion protein can preferably be used in combination with a CAR directed against any of CD19, CD20 or CD22.Based on the data provided herein, it is reasonable and believable that the present invention is functional and produces advantageous results when used with a CAR directed against CD19, CD20 or CD22, or any other protein known to be expressed on the surface of tumor cells and / or to be expressed as a tumor marker in this context.

[0091] In a specific embodiment, the fusion proteins of the invention may be used in combination with a CAR directed against CD19. In another specific embodiment, the fusion proteins of the invention may be used in combination with a CAR directed against CD20. In yet another specific embodiment, the fusion proteins of the invention may be used in combination with a CAR directed against CD22.

[0092] In another aspect, the present invention further relates to nucleic acid molecules encoding the fusion proteins provided and disclosed herein. The present invention also relates to nucleic acid molecules encoding only a portion of the fusion proteins described and provided herein, for example, nucleic acid molecules encoding only the extracellular domain, only the transmembrane domain, and / or only the intracellular domain of the fusion protein.

[0093] As used herein, unless otherwise specifically defined, the terms "nucleic acid" or "nucleic acid molecule" are used interchangeably with "oligonucleotide," "nucleic acid chain," "polynucleotide," etc., and refer to a polymer containing one, two, or more nucleotides.

[0094] The term "nucleic acid molecule" refers to the sequence of bases, including purine and pyrimidine bases, contained in a polynucleotide, which thus represent the primary structure of the nucleic acid molecule.

[0095] As used herein, the term "nucleic acid molecule" includes all types of nucleic acids, such as DNA, cDNA, genomic DNA, RNA, synthetic DNA, and mixed polymers containing two or more of these molecules, and the term preferably relates to DNA and cDNA. As will be readily understood by those skilled in the art, the nucleic acid sequences provided herein represent DNA sequences, and also include the corresponding RNA sequences (wherein T is replaced by U).

[0096] The term "nucleic acid molecule" generally includes a sense strand and an antisense strand. As will be readily understood by those skilled in the art, a "nucleic acid molecule" may further include natural or artificial nucleotide analogs, as well as non-natural or derivatized nucleotide bases, for purposes such as protecting the nucleic acid molecule from endonucleases and / or exonucleases.

[0097] In yet another aspect, the present invention further relates to a vector comprising the nucleic acid molecule provided herein.

[0098] The term "vector" as used herein generally includes any kind of linear or circular nucleic acid molecule that can autonomously replicate in suitable host cells.Such vectors include, but are not limited to, plasmids, cosmids, phages, viruses (for example, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, or preferably retrovirus vectors), and other vectors or shuttles known in the art, which are suitable for carrying and introducing genes into host cells to enable stable or transient translation and constitutive or conditional expression of the fusion protein of the present invention in host cells.

[0099] Vectors usually do not integrate into the cellular genome, but may do so. The vectors of the invention comprising the nucleic acid molecules described and provided herein preferably allow stable expression of the fusion proteins of the invention in a host cell (expression vectors).

[0100] The vectors of the present invention may further comprise marker genes, promoter and / or enhancer sequences (operably linked to the nucleic acid molecules of the present invention), origins of replication suitable for each host cell, restriction sites, multicloning sites, markers, and further functional units known in the art.

[0101] Vectors may be introduced into host cells inter alia via shuttles such as viruses (which may themselves be considered vectors), or may be transformed or transduced into host cells in naked form. Vectors are preferably adapted to suit the respective host cells to be transformed or transduced.

[0102] Those skilled in the art will readily appreciate that different host cells require different types of vectors, for example, the vector (plasmid) pGEM is a vector suitable for transformation into bacterial cells, while the retroviral vector pMP71 is suitable for transducing eukaryotic cells (e.g., T cells).

[0103] In one embodiment of the invention, the vector of the invention is a viral vector, such as a retroviral vector or a lentiviral vector, such as a retroviral vector. Examples of suitable retroviral vectors are known in the art, such as pMP71-PRE (Leisegang, K Mol Med (2008), 86(5):573-583), SAMEN CMV / SRa, LZRS-id3-IHRES (Heemskerk et al., J. Exp. Med. 186 (1997), 1597-1602), FeLV (Neil et al., Nature 308 (1984), 814-820), SAX (Kantoff et al., Proc. Natl. Acad. Sci. USA 83 (1986), 6563-6567), pDOL (Desiderio, J. Exp. Med. 167 (1988), 372-388), N2 (Kasid et al., Proc. Natl. Acad. Sci. USA 87(1990), 473-477), LNL6 (Tiberghien et al., Blood 84(1994), 1333-1341), pZipNEO (Chen et al., J.Immunol. 153(1994), 3630-3638), LASN (Mullen et al., Hum.Gene Ther.7(1996),1123-1129), pGIXsNa(Taylor et al.,J.Exp.Med.184(1996),2031-2036), LCNX(Sun et al.,Hum.Gene Ther.8(1997),1041-1048), SFG(Gallardo et al.,Blood 90 (1997), LXSN (Sun et al., Hum. Gene Ther.8(1997),1041-1048), SFG(Gallardo et al.,Blood 90(1997),952-957), HMB-Hb-Hu(Vieillard et al.,Proc.Natl.Acad.Sci.USA 94(1997),11595-11600), pMV7(Cochlovius et al. al.,Cancer lmmunol.Immunother.46(1998),61-66), pSTITCH(Weitjens et al.,Gene Ther. 5(1998),1195-1203), pLZR(Yang et al.,Hum.Gene Ther.10(1999),123-132), pBAG(Wu et al.,Hum.Gene Ther.10(1999),977-982), rKat.43.267bn(Gilham et al. al., J.lmmunother.25(2002),139-151), pLGSN(Engels et al.,Hum.Gene Ther.14(2003),1155-1168), pMP71(Engels et al.,Hum.Gene Ther.14(2003),1155-1168), pGCSAM(Morgan et al. al., J.Immunol.171(2003),3287-3295), pMSGV (Zhao et al., J.Immunol.174(2005),4415-4423), or pMX (de Witte et al., J.Immunol.181(2008),5128-5136).

[0104] In a further aspect, the present invention relates to a host cell comprising the nucleic acid molecule or vector described and provided herein. In one embodiment, the host cell of the present invention is transduced or transformed with a nucleic acid molecule or vector described and provided herein.

[0105] Generally, as used herein, unless specifically defined otherwise, terms such as "transduced" or "transformed" (and "transduction" or "transformation") can be used interchangeably and generally refer to any type of introduction of nucleic acid molecules and / or vectors into a host cell, regardless of the type of host cell and regardless of the method of introduction (e.g., (chemical) transformation, (viral) transduction, electroporation, transfection, etc.).

[0106] The nucleic acid molecule and / or vector may be stably integrated into the genome of the host cell or may be extrachromosomal (i.e., transient expression). Examples of suitable methods for achieving transient expression in a host cell are known in the art and include mRNA transfection. In one embodiment, the host cell is transduced with the nucleic acid molecule and / or vector. In another embodiment, the nucleic acid molecule and / or vector is stably integrated into the genome of the host cell.

[0107] Host cells described and provided as part of the present invention, comprising the nucleic acid molecules or vectors described and provided herein, are preferably capable of stably or transiently (e.g., stably) expressing (constitutively or conditionally) a fusion protein of the invention.

[0108] Host cells generally can be transduced or transformed by any suitable nucleic acid molecule or vector by any method, hi one embodiment, host cells are transduced with a retroviral or lentiviral (e.g., retroviral) vector comprising a nucleic acid molecule encoding a fusion protein of the invention described above, or a portion thereof (e.g., the extracellular domain, transmembrane domain, and / or intracellular domain).

[0109] In one embodiment, a host cell of the invention is transduced with a retroviral vector comprising a nucleic acid molecule encoding the above-described fusion protein of the invention or a portion thereof (e.g., the extracellular domain, the transmembrane domain, and / or the intracellular domain), and stably or transiently (constitutively or conditionally) expresses the fusion protein or a portion thereof.

[0110] Preferably, the host cell then stably or transiently expresses the fusion protein within its membrane, with the extracellular domain of the fusion protein of the invention oriented towards the exterior of the host cell, the transmembrane domain (for the most part) embedded in the host cell membrane, and the intracellular domain extending into the cytoplasm of the host cell.

[0111] Within the present invention, a host cell comprising a nucleic acid molecule or vector as described and provided herein relates to a genetically modified cell that has been transduced or transformed with the nucleic acid molecule or vector, or where said nucleic acid molecule or vector has been introduced into the host cell in some other way.

[0112] As already mentioned, the host cells of the invention can be cells that transiently or stably express the fusion proteins of the invention. For example, a nucleic acid molecule encoding a fusion protein of the invention can be stably integrated into the genome of the cell by retroviral or lentiviral (e.g., retroviral) transduction.

[0113] The host cells or transduced cells of the invention can be, for example, CD8 + T cells, CD4 + The cells may be T cells, TCR, such as (but not limited to) TCR-T58 or TCR-D115 T cells, double-negative α / β T cells, NK (natural killer) cells, γδ T cells, macrophages, dendritic cells, as well as other cells suitable for storing and / or regenerating the nucleic acid molecules or vectors of the invention, including bacterial cells (e.g., E. coli) and additional eukaryotic cells. The cells may be autologous or non-autologous, but are preferably autologous. The cells may also be allogeneic or non-allogeneic, as will be readily apparent to one skilled in the art.

[0114] In one embodiment, the host cells of the invention are T cells, preferably CD8 + T cells.

[0115] The host cells of the present invention can be transduced with a nucleic acid molecule or vector encoding the fusion protein described and provided herein. Preferably, the host cells provided and described herein can be co-transduced with an additional nucleic acid molecule, such as a nucleic acid molecule encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR) (e.g., also described herein).

[0116] Such co-transduction (or other methods of introducing nucleic acid molecules into cells as described and exemplified herein) are known in the art and are also described and exemplified herein.

[0117] In one embodiment, a host cell stably or transiently expressing a fusion protein of the present invention additionally stably or transiently co-expresses a protein comprising an extracellular domain with specific affinity for CD19 and an intracellular costimulatory domain (co-expressed CAR), preferably wherein the extracellular domain with specific affinity for CD19 comprises at least an antigen-binding fragment of an anti-CD19 antibody, more preferably wherein the extracellular domain with specific affinity for CD19 comprises an anti-CD19 scFv.

[0118] In another embodiment, the host cell stably or transiently expressing the fusion protein of the present invention additionally stably or transiently co-expresses a protein comprising an extracellular domain with specific affinity for CD20 and an intracellular costimulatory domain (co-expressed CAR), preferably, the extracellular domain with specific affinity for CD20 comprises at least an antigen-binding fragment of an anti-CD20 antibody, more preferably, the extracellular domain with specific affinity for CD20 comprises an anti-CD20 scFv.

[0119] In all embodiments described herein involving stable or transient expression of proteins, stable expression of the protein is preferred in all contexts.

[0120] Preferably, the co-expressed (CAR) protein comprises, as an intracellular costimulatory domain, the amino acid sequence of CD3ζ (SEQ ID NO: 25), or an amino acid sequence that is at least 80% identical thereto.

[0121] According to one embodiment, the co-expressed (CAR) protein comprises an anti-CD19 antibody binding domain as an extracellular domain, preferably an anti-CD19 scFv, more preferably an anti-CD19 scFv commonly referred to in the art as clone FMC63, whose sequence is as follows: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 19)

[0122] Furthermore, the co-expressed (CAR) protein may alternatively or additionally comprise as an extracellular domain a binding domain of an anti-CD20 antibody, preferably a binding domain of an anti-CD20 antibody commonly referred to in the art as clone 2H7. The sequence of said binding domain is as follows: MAQVKLQESGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPTILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKRAAA (SEQ ID NO: 20)

[0123] Alternatively or additionally, the co-expressed (CAR) protein may comprise, as an extracellular domain, a binding domain of an anti-CD22 antibody, preferably a binding domain of an anti-CD22 antibody commonly referred to in the art as clone m971. The sequence of the binding domain is as follows: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 21)

[0124] In one embodiment, the co-expressed (CAR) protein may comprise the extracellular and transmembrane domains of CD8 alpha together with the intracellular domain of CD3 zeta, preferably having the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22)

[0125] In one specific embodiment, the co-expressed (CAR) protein may comprise an anti-CD19 scFv together with the extracellular and transmembrane domains of CD8 alpha and the intracellular domain of CD3 zeta, preferably having the following amino acid sequence: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT GGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY YCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 23)

[0126] In certain embodiments, the commonly known product Kymriah®, a CD19 CAR of tisagenlecleucel, may be used within the present invention.

[0127] Alternatively, the co-expressed (CAR) protein may comprise an anti-CD20 binding domain and an anti-CD19 scFv together with the extracellular and transmembrane domains of CD8 alpha and the intracellular domain of CD3 zeta, preferably having the following amino acid sequence: (SEQ ID NO: 24)

[0128] In one embodiment, the intracellular costimulatory domain of the fusion protein or co-expressed (CAR) protein comprises the amino acid sequence of the intracellular domain of CD3ζ, preferably the following amino acid sequence: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25), or an amino acid sequence that is at least 80% identical to this amino acid sequence.

[0129] In one aspect, the present invention also relates to a method for preparing the host cells of the invention as described and provided herein, comprising the steps of transducing (or transforming) the host cells described above with a nucleic acid molecule or vector as described and provided herein, culturing the transduced (or transformed) host cells in a suitable medium that allows cell growth and expression of the fusion protein encoded by said nucleic acid molecule or said vector, and harvesting the host cells from the medium.

[0130] In a preferred embodiment of the present invention, host cells are transduced or transformed ex vivo. Cells (e.g., T cells, e.g., CD8 + T cells, CD4 + Methods for obtaining, isolating, and culturing T cells, TCR, for example (but not limited to) TCR-T58 or TCR-D115 T cells, are known in the art and include, inter alia, blood sampling or bone marrow sampling.

[0131] According to the methods of the present invention, host cells may be transduced, transformed, or otherwise provided with the nucleic acid molecules or vectors described and provided herein by any method known in the art. Such methods include, inter alia, (chemical) transformation, (viral) transduction, electroporation, transfection, etc. In one embodiment, the host cells are transduced with a retroviral vector.

[0132] The present invention also relates to host cells obtainable by the preparation methods provided herein. In another aspect, the present invention further relates to pharmaceutical compositions comprising the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided by the present invention. Such pharmaceutical compositions are suitable for administration to patients (preferably human patients), particularly donors of the above-mentioned host cells.

[0133] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable carriers and additional ingredients for galenic purposes.

[0134] The fusion proteins, nucleic acid molecules, vectors, host cells and pharmaceutical compositions each have utility as a medicament, preferably for use in the treatment of diseases and disorders associated with co-expression of CD19 (or other tumor markers, such as, but not limited to, CD20 and CD22) with CD80 and / or CD86, more preferably for use in the treatment of B-cell lymphomas, even more preferably for use in the treatment of marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphoma (SMLL ... The present invention is particularly advantageous for the treatment of non-Hodgkin's lymphomas selected from the group comprising lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL), and particularly preferably for the treatment of diffuse large B-cell lymphoma (DLBCL).

[0135] Other additional diseases and disorders that can be treated using the present invention will be apparent to those skilled in the art and include various types of cancer, such as (but not limited to) lung cancer, gastric cancer, renal cell carcinoma, colon cancer, breast cancer, ovarian cancer, urothelial cancer, melanoma, pancreatic cancer, myeloma, Hodgkin's lymphoma, retinoblastoma, leukemia, cervical cancer, esophageal cancer, glioma, non-Hodgkin's lymphoma, hepatocellular carcinoma, and oral cancer, among others.

[0136] Additionally, the present invention relates to methods for treating the above-listed diseases or disorders by administering pharmaceutical compositions comprising the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided by the present invention. The present invention further relates to methods for treating the above-listed diseases or disorders by using the fusion proteins, nucleic acid molecules, vectors, and / or host cells described and provided by the present invention.

[0137] Finally, the present invention also relates to a kit or kit of parts comprising a fusion protein, a nucleic acid molecule, a vector, a host cell or a pharmaceutical composition as described and provided as part of the present invention and a container.

[0138] It is understood that all embodiments of the present invention disclosed and described herein can be combined in any combination unless a person skilled in the art would consider such combination to be technically meaningless. The present invention will be further described below by way of specific examples, which are intended to illustrate but not limit the present invention. [Example]

[0139] PBMC isolation and T cell activation Peripheral blood mononuclear cells were collected from donor buffy coats by density gradient isolation. The buffy coats were carefully pipetted onto STEMCELL Technologies Lymphoprep and centrifuged at 600 x g for 30 minutes. The resulting mononuclear cell layer was extracted and washed four times with PBS. Up to 1e9 cells were cultured in ThermoFisher RPMI 1640 medium containing 10% FCS, Pen / Strep, and HEPES buffer. Two days prior to transduction, T cells were stimulated with IL-2 (1000 U / ml), anti-human CD3 (clone OKT3) (200 ng / ml), and anti-human CD28 (100 ng / ml) (clone 15E8). Primary mouse cells were collected by dissecting the spleens of C57BL / 6N (Black 6; RRID:IMSR_JAX:000664) mice and isolating B cells with the MojoSort Mouse Pan B Cell Isolation Kit II (BioLegend catalog no. 480087) or T cells with the MojoSort Mouse CD T Cell Isolation Kit (BioLegend catalog no. 480031) according to the manufacturer's instructions. Mouse B cells were cultured in RPMI 1640 supplemented with 10% FCS and Pen / Strep, and mouse T cells were cultured in X-Vivo 15 supplemented with 5% FCS and 200 ng mL for 3 days prior to transduction. -1Anti-CD3, 100ng·mL -1 , 1000U·mL -1 IL-2, and 10 ng·mL -1 Stimulated with IL-15.

[0140] Vector transfection and transduction CAR and CAR / CCR expression was induced by retroviral transduction. Two 10 cm plates containing monolayers of HEK293t cells at 50-70% confluence were transfected with 10 μg of vector DNA and 5 μg each of GAL-V and M-MuLV helper plasmids in 500 μl of RPMI 1640 using 20 μl of Polyplus PeiPro transfection reagent. The transfection reagent was added to HEK293t cells cultured in 9 ml of RPMI 1640 containing 10% FCS, Pen / Strep, and HEPES buffer.

[0141] The culture medium containing the transducing virus was collected after 10–24 hours and added to 1.6e7 PBMC-derived T cells in poly-D-lysine-coated plates, centrifuged at 450g for 90 minutes, and cultured overnight. This process was repeated twice with the same cells to improve transduction yield. During transduction, T cells were continuously stimulated overnight with IL-2 (1000U / ml) after the first run and IL-2 (500U / ml) after the second run.

[0142] Fluorescence-based in vitro cytotoxicity assay The cytolytic activity of the tested CAR T cells and CAR / CCR T cells was assessed by measuring the fluorescence levels over time in co-cultures of T cells and eGFP-expressing tumor cell lines using a HIDEX Sense microplate reader platform equipped with a compatible digital atmosphere controller. Cytolytic activity is expressed as the inverse relative increase in fluorescence level compared to measurements in wells containing tumor alone, calculated using the following formula:

number

[0143] where Fl = fluorescence readings in co-culture wells, Tu = fluorescence readings in wells containing tumor cells alone, and Med = fluorescence readings in wells containing culture medium alone. Assay wells contained 200 μl of RPMI1640 with 10% FCS, Pen / Strep, and HEPES, varying ratios of tumor cells and CAR-T cells, normalized to total CAR-T cell and total cell numbers by adding mock-transduced T cells from the same donor.

[0144] Flow cytometry-based in vitro cytotoxicity assay The cytotoxic effect on primary human B cells was examined by flow cytometry after co-culture with CAR / CCR T cells and CAR (second generation) T cells at an effector / target ratio of 1:10 in a humidity-controlled environment for 18 hours at 37°C. Samples were stained with anti-CD3 APC (Miltenyi Biotec catalog no. 130-113-125; RRID:AB_2725953), anti-CD19 FITC (Miltenyi Biotec catalog no. 130-113-645; RRID:AB_2726198), and 7-AAD (BioLegend catalog no. 420404), washed twice, and analyzed.

[0145] In this experiment, the cytotoxic effect of mouse T cells equipped with mouse CAR / CCR constructs and mCAR (second-generation) constructs on primary mouse B cells was detected by pre-staining target cells with the CellTrace™ Violet Proliferation Kit for 24–48 hours before adding effector cells. Cells were then detected using a MACSQuant X flow cytometer (Miltenyi Biotec, Bergisch-Gladbach, Germany), and the absolute number of CellTrace™ Violet-positive cells was plotted for comparison of each construct.

[0146] Cytokine level detection in supernatants by ELISA The concentration of secreted cytokines in the assay supernatants was measured using a sandwich ELISA technique. Nunc MaxiSorp™ 96-well plates were coated with capture antibodies (detailed below), washed, blocked, and incubated with 50 μl of assay supernatant overnight at 4°C. PBS dilutions of the supernatant were applied as needed. The samples were then discarded, and the capture antibodies and enzyme-linked secondary antibodies were sequentially incubated, with four PBS-Tween (0.05% v / v) washing steps between each step.

[0147] Detection was performed using TMB substrate solution (Life Technologies), incubated in the dark for 15–30 minutes, and stopped with 0.5 M sulfuric acid. Assay OD was measured using a ThermoFisher ELISA reader (Multiscan Go), and concentrations were calculated from a standard curve, as appropriate. Human IFN-γ was recorded in the culture supernatant using a matched pair of antibodies (clones NIB42 and B133.5). Human IL-2 was recorded in the culture supernatant using a matched pair of antibodies (B33-2 and 5344.111). Human IL-6 was recorded in the culture supernatant using an IL-6 ELISA kit (BD Biosciences).

[0148] CAR T cell culture Transduced T cells were cultured in RPMI 1640 medium containing 10% FCS, HEPES buffer, Pen / Strep, and 100-300 U / ml IL-2. Culture medium was added or replaced every 3-4 days or when acidification was confirmed by a color change in phenol red.

[0149] Tumor cell line culture Tumor cell lines were cultured in RPMI 1640 medium containing 10% (Raji, DOHH-2) or 20% (SU-DHL-10, Oci-Ly1, Oci-Ly19) FCS and Pen / Strep. Cultures were split and medium was changed every 3–4 days or when acidification was detected by a color change in phenol red. Cultures were checked for mycoplasma by PCR at regular intervals and before in vivo application.

[0150] Mesenchymal stem cell culture, differentiation and characterization Mesenchymal stem cells were cultured in Mesenchymal Stem Cell Expansion Medium 2. To achieve differentiation, cells were treated with various concentrations of recombinant TGF-beta3 (10–20 ng / ml). Cells were then characterized using FACS analysis and anti-CD19 APC-conjugated antibodies (ImmunoTools #21270196) and anti-CD248 FITC-conjugated antibodies (Bioss bs-2101R-FITC). MACS isolation was achieved using the same antibodies in combination with magnetic anti-APC microbeads (Miltenyi Biotec #130-090-855) on the autoMACS Pro platform.

[0151] Magnetic-activated cell sorting: macrophages and T cells Single-cell suspensions were prepared from buffy coats from healthy donors using a CD16 microbead kit in an autoMACS Pro separator (Miltenyi Biotec). The number of contaminating cells in the isolated monocyte population was less than 2%. Human CD4+ and CD8+ T cells were isolated using a Pan T cell isolation kit (Miltenyi Biotec) for the separation of naive T cells from peripheral blood mononuclear cells (PBMCs) by non-T cell depletion.

[0152] FACS analysis of CD80 / 86 expression on B-cell lymphoma cell lines (DLBCL and CLL) and healthy B cells Antigen and receptor expression by tumor cell lines was characterized using FACS analysis using the BD Canto II platform. Cells were quantified, and 5e5–1e6 cells per FACS tube were isolated from cultures. Cells were washed twice with 4 ml of PBS before and after antibody application and incubated with 7-AAD (BD Biosciences 559925), CD5-BV510 (Biolegend 364018), CD19-FITC (Biolegend 392508), CD20-APC / Fire™ 750 (Biolegend 302358), CD80-PE (Biolegend 305208), or CD86-APC (Biolegend 374208) for 30 min at 4 °C according to the manufacturer's instructions.

[0153] Detection of CAR and CAR / CCR expression using flow cytometry Transduction efficiency was assessed by FACS analysis using a non-commercial anti-idiotypic CD19-CAR (FMC63) antibody supplied by Miltenyi Biotec and detected with anti-biotin PE (Miltenyi, catalog no. REA746), anti-CTLA4 (CD152) BV421 (BioLegend; catalog no. 369605), and anti-CD3 APC (Miltenyi, catalog no. BW264 / 56) at the manufacturer's concentration. Antibodies were incubated at 4°C for 30 min, with two PBS wash steps between primary and secondary antibodies and before analysis. CAR transduction efficiency was assessed in the lymphocyte / single cell / CD3+ gate to confirm successful transduction and normalize CAR-T cell numbers for downstream experiments.

[0154] Details of the xenograft mouse efficacy study On day -3, a total of 28 Rag2 mice aged 100-147 days were - / - γc - / -Mice (Jax mice) (13 females, 15 males) were intravenously injected with Raji-fLuc cells (5e4 cells per mouse). One female mouse was injected intraperitoneally. On day 3, tumor engraftment was assessed by injecting D-luciferin (1.5 mg / mouse) and measuring tumor luminescence in anesthetized mice using an IVIS200 instrument (PerkinElmer, Waltham, MA, USA).

[0155] Mice were divided into groups receiving 8e6 intravenous injections of CAR / CCR T cells (6 mice), second-generation CAR T cells (7 mice), or non-transduced T cells (9 mice) from the same PBMC donor. Six tumor-bearing mice were left untreated. All mice were scored daily, and luminescence measurements were repeated weekly. Mice were sacrificed at a predetermined tumor burden or score cutoff.

[0156] Details of the safety study using immunocompetent mice Mouse splenocytes were genetically modified to express either a CAR (secondary) construct or a CAR / CCR construct, as shown in Figure 16. CAR and CAR / CCR expression was analyzed by flow cytometry. CAR T cells or CAR / CCR T cells were injected intravenously into the tail of C57bl / 6N mice (1 x 10 per mouse). 7 Blood samples were collected weekly and analyzed for the presence of CD19+ cells by flow cytometry using a CD19-specific antibody. Finally, spleens were removed and similarly analyzed for CD19. + The presence of B cells was analyzed.

[0157] Immunohistological analysis Immunohistological examination of human tissues from DLBCL patients (before and during CAR T cell treatment) was performed on 4-μm-thick sections of formalin-fixed, paraffin-embedded tumor tissue in whole-section format (Institute of Pathology, University of Cologne). For human CD80 detection, slides were primarily stained with a biotin-labeled polyclonal antibody against CD80 (5.0 μg / ml) (Bioss, Woburn, MA, USA) and secondarily stained with streptavidin-horseradish peroxidase (500 mU / ml) (Roche, Basel, Switzerland).

[0158] For human CD86 detection, slides were primarily stained with a mouse monoclonal conjugated CD86-specific antibody (clone SPM600) (Novus Biologicals, Centennial, CO, USA) at 2.0 μg / ml, followed by a polyclonal HRP-conjugated mouse IgG1-specific antibody (Bioss) at 2.0 μg / ml. Both CD80- and CD86-stained sections were then further incubated with DAB chromogen substrate (Vector Laboratories, Burlingame, CA, USA) and hematoxylin (PanReac AppliChem, Cranberry, NJ, USA) according to the manufacturer's instructions for immunohistological analysis.

[0159] Slides were documented using an Olympus-UC90 4K microscope (Olympus, Tokyo, Japan) and analyzed for CD80 and CD86 expression using ImageJ version 1.53 (National Institutes of Health, USA). Finally, slides were analyzed using an Olympus FV1000 microscope (Olympus, Tokyo, Japan), and fluorescence intensity was determined using ImageJ software.

Claims

1. a) an extracellular domain comprising a polypeptide having specific affinity for CD80 and / or CD86; b) a transmembrane domain; and c) an intracellular domain comprising a costimulatory polypeptide; A fusion protein comprising:

2. The polypeptide having specific affinity for CD80 and / or CD86 has an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the extracellular domain of human CD28 (SEQ ID NO: 2), or an amino acid sequence that is at least 80% identical to the amino acid sequence of the CD86-binding domain of the anti-CD86 antibody (SEQ ID NO: 5) commonly referred to in the art as clone hu3D1, and preferably has an amino acid sequence that is at least 80% identical to the amino acid sequence of the CD80 and / or CD86-binding domain of the anti-CD86 antibody (SEQ ID NO: 5) commonly referred to in the art as clone hu3D1. and / or the transmembrane domain is suitable for insertion and anchoring of the fusion protein in the cell membrane of a mammalian cell, and preferably the transmembrane domain is selected from the group consisting of the alpha, beta or zeta chains of the T cell receptor, CTLA-4, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD 33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD200, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, VLA1, ITGA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGA7 GAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11 c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SFAMF4 ( CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (B Y55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SEAM (SLAMF1, CD150,and / or the intracellular domain comprises the transmembrane region of one or more of human 4-1BB (CD137; SEQ ID NO:3), CD3ζ (SEQ ID NO:25), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, Myd88-CD40, KIR2DS2, and HVEM, or an amino acid sequence at least 80% identical thereto. The fusion protein of claim 1, wherein the intracellular domain comprises the intracellular domain or costimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto, or the intracellular domain comprises the intracellular domain or costimulatory polypeptide of CD3ζ (SEQ ID NO: 25), or an amino acid sequence at least 80% identical thereto, preferably the intracellular domain comprises the intracellular domain or costimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto.

3. 3. The fusion protein of claim 1, wherein the extracellular domain comprises the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an amino acid sequence at least 80% identical thereto, and the intracellular domain comprises a costimulatory peptide comprising the intracellular domain of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto.

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising a nucleic acid molecule according to claim 4 or a vector according to claim 5, preferably said host cell being transduced with the nucleic acid molecule according to claim 4 or the vector according to claim 5, or preferably said nucleic acid molecule according to claim 4 or the vector according to claim 5 being stably integrated into the genome of said host cell.

7. A host cell stably or transiently expressing the fusion protein of any one of claims 1 to 3.

8. the host cell stably or transiently expresses a protein comprising an extracellular domain with specific affinity for CD19 and an intracellular costimulatory domain, preferably wherein the extracellular domain with specific affinity for CD19 comprises at least an antigen-binding fragment of an anti-CD19 antibody, more preferably wherein the extracellular domain with specific affinity for CD19 comprises an anti-CD19 scFv; Alternatively, the host cell according to any one of claims 6 or 7, wherein the host cell stably or transiently expresses a protein comprising an extracellular domain having specific affinity for CD20 and an intracellular costimulatory domain, wherein the extracellular domain having specific affinity for CD20 preferably comprises at least an antigen-binding fragment of an anti-CD20 antibody, and more preferably the extracellular domain having specific affinity for CD20 comprises an anti-CD20 scFv.

9. The host cell of claim 8, wherein the intracellular costimulatory domain of the fusion protein and / or the protein comprising an extracellular domain with specific affinity for CD19 or CD20 comprises the amino acid sequence of the intracellular domain of CD3ζ (SEQ ID NO: 25), or an amino acid sequence at least 80% identical thereto, or the amino acid sequence of the intracellular domain or costimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence at least 80% identical thereto, preferably, the intracellular costimulatory domain of the fusion protein and / or the protein comprising an extracellular domain with specific affinity for CD19 or CD20 comprises the amino acid sequence of the intracellular domain of CD3ζ (SEQ ID NO: 25), or an amino acid sequence at least 80% identical thereto.

10. The host cell according to any one of claims 6 to 9, which is a human CD8+ T cell.

11. (1) transducing a host cell with the nucleic acid molecule of claim 4 or the vector of claim 5; (2) culturing the transduced host cells of step (1) in a suitable medium that allows growth of the cells and expression of the fusion protein encoded by the nucleic acid molecule or the vector; (3) harvesting the host cells from the medium; A method for providing a host cell according to any one of claims 6 to 10, comprising:

12. A host cell obtainable by the method of claim 11.

13. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, and / or a host cell according to any one of claims 6 to 10 or 12.

14. A fusion protein according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, a host cell according to any one of claims 6 to 10 or 12, or a pharmaceutical composition according to claim 13, for use as a medicament.

15. 14. The fusion protein of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of any one of claims 6 to 10 or 12, or the pharmaceutical composition of claim 13, for use in the treatment of B-cell lymphoma, preferably for the treatment of a non-Hodgkin's lymphoma selected from the group comprising marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma, lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL), more preferably for the treatment of diffuse large B-cell lymphoma (DLBCL).

16. A kit or kit of parts comprising a fusion protein according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, a host cell according to any one of claims 6 to 10 or 12, or a pharmaceutical composition according to claim 13, and a container.

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