Chimeric checkpoint receptors for treatment of malignant b-cell diseases

Abstract

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

[0002] The concept and advantages of CAR T cells are well known in the art and have become the focus of drug research and development in recent years. The main example of successful clinical application of CAR T cells in patients is Tisagenlecleucel Regulatory approval in the US and in the EU for the treatment of B-cell acute lymphoblastic leukemia. However, certain side effects and varying degrees of treatment success in patients may complicate the use of second-generation CAR T cells such as Tisagenlecleucel in patients.

[0003] The main concern associated with the use of tisagenlecleucel in patients is the presence of malignant as well as healthy CD19 + General depletion of B lymphocytes leads to reported B cell aplasia and subsequent hypogammaglobulinemia (Ali S et al., 2020 Oncologist, 25(2): e321-e327).

[0004] In addition, substantial neurotoxicity has been observed in patients treated with tisagenlecleucel, which can be attributed to the expression of CD19 on pericytes, thus causing on-target off-tumor effects. + Interactions between pericytes lead to the induction of the undesired release of IL-2 and IL-6 in such cells, which can then 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 in turn can recruit and activate other immune cells such as macrophages, which then contribute to the increased release of IL-6 following activation. Often, increased IL-6 release can lead to cytokine release syndrome (CRS), which can cause life-threatening conditions in patients.

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

[0007] Possibly for the above reasons, the inventors realized that previously treated B-cell lymphoma (CD19 + CD80 + CD86 + ) Animals with CAR T cells developed tumor recurrence in approximately 50% of all cases. Retreatment of those animals with tumor recurrence using Tisagenlecleucel CAR T cells did not result in therapeutic success.

[0008] Based on the above-described circumstances regarding the use of currently available CAR T cells, the present inventors therefore set out to overcome the shortcomings of the prior art and to provide and achieve advantageous and improved CAR T cell therapies.

[0009] Therefore, one object of the present invention is to provide a CAR T cell therapy with increased safety and efficiency, which avoids cells and cell types other than malignant B lymphocytes, such as healthy B lymphocytes and CD19 + Nonspecific targeting of pericytes.

[0010] A further object of the present invention is to provide a CAR T-cell therapy that allows maintaining a population of healthy B lymphocytes and avoids neurotoxic symptoms that may occur when using CAR T-cell therapy as described in the prior art.

[0011] Another object of the present invention is 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 prominent side effect.

[0012] Yet another object of the present invention is to avoid tumor recurrence in B-cell lymphoma patients and to provide a promising treatment option for patients who have been previously treated with conventional CAR T-cell therapy and experience tumor recurrence. Summary of the invention

[0013] The above objects are solved by aspects of the present invention as described in detail 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 co-stimulatory polypeptide.

[0015] In a preferred embodiment of the first aspect of the invention, the polypeptide with 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 wherein the polypeptide with 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 wherein the transmembrane domain comprises the transmembrane region of one or more of the following: the α, β or ζ chain of the T cell receptor, CTLA-4, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD1 37, CD154, CD200, KIRDS2, 0X40, CD2, CD27, LFA-1(CDlla, CD18), ICOS(CD278), 4-1BB(CD137), GITR, CD40, BAFF R, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7R.α., VLA1, ITGA1, CD49a, ITGA4, IA4, C D49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB 1. CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SFAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEAC AM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SEAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp, or an amino acid sequence at least 80% identical thereto, preferably wherein 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 invention, the costimulatory polypeptide of the fusion protein comprises the intracellular domain of one or more of the following: 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 wherein 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 wherein 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 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 that is at least 80% identical thereto, and the intracellular domain includes a co-stimulatory peptide comprising the intracellular domain of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence that is at least 80% identical thereto.

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

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

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

[0022] In a preferred embodiment of the fourth aspect of the invention, the host cell is transduced with the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention, or wherein 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 present invention, a host cell is also provided, which stably or transiently expresses the fusion protein according to the first aspect of the present invention.

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

[0025] In one embodiment of the specific preferred embodiment of the fourth aspect of the present invention, the intracellular co-stimulatory 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 that is at least 80% identical thereto. In a preferred embodiment of the fourth aspect of the present invention, the host cell is a human CD8 + T cells.

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

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

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

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

[0030] According to the eighth aspect of the present invention, the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention or the pharmaceutical composition of the sixth aspect of the present invention is provided for 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 lymphoma selected from the group comprising marginal zone B cell lymphoma (MZL), mucosa-associated lymphatic tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL / CLL), mantle cell lymphoma (MCL), Burkitt's lymphoma, lymphoplasmacytic lymphoma, Macroglobulinemia ( The invention relates to leukemia, lymph node metastasis, and hematologic malignancy. The invention relates to leukemia, lymph node metastasis, and hematologic malignancy. The invention relates to leukemia, lymph node metastasis, and hematologic malignancy.

[0032] According to the ninth aspect of the present invention, a kit or a kit-in-parts is provided, which comprises the fusion protein of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention or the host cell of the fourth aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1(A) Schematic diagram depicting the modular composition of the CAR / CCR expression cassette of the present invention (Lκ, light chain κ signal sequence; anti-CD19 scFv, CD19-specific single chain (heavy and light) variable fragment; CD8α hinge, CD8α hinge domain; CD8αtm, CD8α transmembrane domain; CD3ζ(ic), CD3ζ intracellular signaling domain; P2A, 2A peptide; CTLA-4ex, cytotoxic T-Lymphocyte Associated Protein; protein) extracellular domain; CTLA-4tm, cytotoxic T lymphocyte-associated protein transmembrane domain; 4-1BB(ic), 4-1BB(CD137) intracellular signaling domain; (B) Schematic diagram depicting the expression and localization of CAR / CCR constructs on the surface of T cells; (C) Anti-CD19(FMC63)-CD8α(ex)-CD3ζ(tm+ic)-CAR and CTLA4(ex)-4-1BB(tm+ic)-CCR in retrovirally engineered human CD3 + Expression on T cells; monitoring of CAR / CCR expression by flow cytometry using phycoerythrin-labeled anti-idiotypic antibody and PE-Cy-7-conjugated CTLA-4-specific antibody; mock transduced human CD3 + T cells served as control (grey area).

[0034] Figure 2 High expression levels of both checkpoint receptor ligands CD80 and CD86 on the surface of relapsed B-cell lymphoma cells following CD19-CAR-T cell therapy were shown; tumor biopsies from patients with DLBCL were collected before and with CAR T-cell therapy (Kymriah, Novartis) and 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 coloring chromogen DAB was added and the sections were counterstained with hematoxylin; compared with CAR T initial samples ( Compared with the control group, relapsed DLBCL cells after CAR T cell immunotherapy showed increased expression of immune checkpoint ligands CD80 and / or CD86.

[0035] Figure 3Analysis of primary DLBCL and CLL tumor cells for the expression of both CD80 and CD86 is shown; primary DLBCL cells exhibit increased expression of CD80 and / or CD86 compared to primary CLL cells and healthy B cells (control).

[0036] Figure 4 Displayed on CD11b + In the presence of monocytes, in the presence of human CD19 + Reduced IL-2 (A) and IL-6 (B) release by human CD19-specific CAR / CCR T cells of the invention in the presence of B cells co-culture; primary T cells were engineered to express the CD19-CAR / CTLA-4-CCR construct (first bar from the left) and co-cultured with CD19 + B cells were co-cultured for 24 hours (at a ratio of 100 CAR T cells: 100 B cells: 1 monocyte); IL-2 released by CART cells and IL-6 released by monocytes in the supernatant were detected by ELISA; T cells engrafted with a second-generation CD19-specific CAR (α-CD19-CD28-CD3ζ, second bar from the left), T cells engrafted with a first-generation CD19-specific CAR (α-CD19-CD3ζ; center bar), and untransduced cells (second bar from the right) were used as controls; further shown in comparison with primary human CD19 + (C) Reduced cytotoxic effect and (D) reduced IFN-γ release by human CD19-specific CAR / CCR T cells of the present invention on primary human B cells in the case of B cell co-culture; primary T cells were engineered to express the CD19-CAR / CTLA-4-CCR construct (first bar from the left) and co-cultured with primary CD19 at an effector-target ratio of 1:10 (E:T). + B cells were co-cultured for 18 hours. T cells genetically modified to express first and second generation CAR constructs and non-modified T cells (Mock) were used as controls.

[0037] Figure 5Shown is the reduced IFN-γ release by human CD19-specific CAR / CCR T cells of the present invention in the case of co-culture with pericytes expressing human CD19 and CD248 (A); Therefore, primary human vascular wall typical mesenchymal stem cells (VW-MSCs) were differentiated into pericytes expressing CD19 and CD248 by culturing in medium supplemented with TGF-β3 for 2 weeks; Finally, differentiated CD19+CD248+ pericytes were isolated by BD FACSAria III sorter. (B) Then, T cells were equipped with CAR (2nd generation) or CAR / CCR constructs and co-cultured with CD19 + CD248 + Pericytes were co-cultured for 24 h (at a ratio of 1 CAR T cell:1 pericyte); finally, supernatants from this experiment were collected and analyzed for IFN-γ by ELISA; supernatants from mock-transduced T cells and (CAR) T cells cultured in the absence (w / o) of pericytes were used as controls.

[0038] Figure 6 CD19-CAR / CTLA-4-CCR T cells effectively eliminate CD80 in the aggressive B-cell lymphoma cell line Raji + CD86 + Cells; Primary T cells were engineered to express the CD19-CAR / CTLA-4-CCR construct (left bar in each figure) and co-cultured with the CD19 of the Raji cell line at a ratio of 1:4 (effector to target cell). + CD80 + CD86 + Cells were co-cultured for 24 to 120 hours; cytotoxic effects on tumor cells were analyzed using a HIDEX ELISA reader; T cells transplanted with a second-generation CD19-specific CAR (α-CD19-CD28-CD3ζ; central bars in each graph) and T cells transplanted with a first-generation CD19-specific CAR (α-CD19-CD3ζ; right bars in each graph) were used as controls.

[0039] Figure 7 CD19-CAR / CTLA-4-CCR T cells were shown to effectively eliminate B cell lymphoma cells and enhance tumor-free survival; CD19 + CD80 + CD86 + Cells were injected intravenously into RAG2 - / - Normal - / -Mice were treated with CD19-CAR / CTLA-4-CCR T cells on day 5; T cells engineered to express second-generation CD19-CAR (Kymriah, Novartis) and mock-transduced T cells were used as controls.

[0040] Figure 8 Shown in primary DLBCL cells expressing CD19 (CD80 高 CD86 高 ), DLBCL cell line SU-DHL-10 (CD80 高 CD86 高 ), DLBCL cell line DOHH-2 (CD80 高 CD86 低 ), DLBCL cell line Oci-Ly1 (CD80 低 CD86 高 ) or DLBCL cell line Oci-Ly19 (CD80 低 CD86 低 ) after co-culture by CD19CAR / CTLA-4CCR, CD19CAR (second generation) or CD19CAR (first generation) T cell IFN-γ release; Cells are co-cultured with CAR / CCR, CAR (first generation) 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 not co-cultured with effector T cells (no T cells) serve as controls (ns=not significant, *p<0.05, **p<0.005).

[0041] Fig. 9 Shown in primary DLBCL cells expressing CD19 (CD80 高 CD86 高 ), DLBCL cell line SU-DHL-10 (CD80 高 CD86 高 ), DLBCL cell line DOHH-2 (CD80 高 CD86 低 ), DLBCL cell line Oci-Ly1 (CD80 低 CD86 高 ) or DLBCL cell line Oci-Ly19 (CD80 低 CD86 低) IL-2 release by CD19CAR / CTLA-4CCR, CD19CAR (second generation) or CD19CAR (first generation) T cells after 48 hours of co-culture at 37°C. Target cells co-cultured with mock-transduced T cells (Mock) or target cells not co-cultured with effector T cells (no T cells) served as controls (ns=not significant, *p<0.05, **p<0.005).

[0042] Fig.10 A luminescent image showing a xenograft mouse study based on Raji-based lymphoma-bearing Rag2- / -IL-2rg- / - mice using 8.0x10 6 CAR / CCR, CAR (second generation) or mock T cells were treated and tumor growth was evaluated using bioluminescence; Raji expressing CD19 / CD80 / CD86 was genetically modified to express firefly luciferase and injected intravenously into immunocompromised mice; 3 days after tumor transplantation, mice were divided into 3 groups and treated with effector T cells (second generation CAR or CAR / CCR); the group of mice treated with mock transduced T cells was used as a control; tumor progression was monitored by using an IVIS bioluminescence device; mice were sacrificed at the tumor burden cutoff or when paralysis was shown.

[0043] Fig.11 Shows IL-6 release by activated macrophages in vitro; in the tumor cell line SU-DHL10 expressing CD19 / 80 / 86 and in isolated human CD11b from the same healthy donor + CAR T cells were cultured in the presence of macrophages; at 3.25x10 4 1x10 macrophages (+M) were cultured at 37°C 5 CAR / CCR, CAR (second generation) or non-transduced (nt) T cells (+T) were cultured for 24 hours; finally, the supernatant was collected and IL-6 release was explored by ELISA; tumor cells cultured in the presence / absence of macrophages and / or (CAR / CCR, CAR, nt) T cells served as controls.

[0044] Fig.12The designs of CAR and CAR / CCR constructs used in this article are schematically shown; (A) a construct according to the present invention having a CD20-specific CAR (which includes an anti-CD20 scFv as an extracellular domain and CD3ζ as an intracellular signaling domain comprising a co-stimulatory polypeptide) and a CCR (which includes CTLA-4 as an extracellular domain comprising a polypeptide having a specific affinity for CD80 and / or CD86 and an intracellular domain of 4-1BB as an intracellular domain comprising a co-stimulatory polypeptide); (B) a construct according to the present invention having a CD19-specific CAR (which includes an anti-CD19 scFv as an extracellular domain and CD3ζ as an intracellular signaling domain comprising a co-stimulatory polypeptide) and a CCR (which includes an anti-CD86 scFv as an extracellular domain comprising a polypeptide having a specific affinity for CD86 and an intracellular domain of 4-1BB as an intracellular domain comprising a co-stimulatory polypeptide); (C) a second-generation CD20-specific CAR known in the art; and (D) a second-generation CD19-specific CAR known in the art.

[0045] Fig.13 Shown in the DLBCL (diffuse large B-cell lymphoma) cell line SUDHL-10 (A; B) and in the MCL (mantle cell lymphoma) cell line JeKo-1R (C; D), modified to express Fig.12 Analysis of the T cell cytotoxic effect of the constructs shown in (A; C) and analysis of the release of the proinflammatory cytokine IFN-γ (B; D).

[0046] Fig.14 Shown in the JeKo-1R cell line with 99.9% reduction / attenuation of CD19 expression (confirmed by RT-PCR; data not shown), by modifying to express Fig.12 Analysis of T cell cytotoxic effects of the constructs shown (A) and analysis of the release of the proinflammatory cytokine IFN-γ (B).

[0047] Fig.15Schematic diagram of the design of a reverse switch in which the intracellular signaling domain containing a co-stimulatory polypeptide is exchanged between the CAR and the CCR; (A) a CD19-specific CAR (which includes an anti-CD19) in combination with a CCR (which includes CTLA-4 as an extracellular domain containing a polypeptide having specific affinity for CD80 and / or CD86 and the intracellular domain of 4-1BB as an intracellular domain containing a co-stimulatory polypeptide) (A) and (B) result in a reverse switch of the CAR and CCR that differs from (A) in that the CAR includes the intracellular domain of 4-1BB as the intracellular domain comprising a co-stimulatory polypeptide, and the CCR includes CD3ζ as the intracellular signaling domain comprising a co-stimulatory polypeptide; (C) shows in flow cytometry histograms that human T cells expressing the molecules described in (A) and (B) both result in potent cytotoxicity against the Su-DHL-5 B cell lymphoma cell line; (D) shows the FACS gating strategy.

[0048] Fig.16 Schematically showing (A) the design of a fully murine CAR / CCR construct according to the present invention, the construct comprising a CD19-specific CAR (which comprises an anti-CD19 scFv as an extracellular domain and CD3ζ as an intracellular signaling domain comprising a co-stimulatory polypeptide) and a CCR (which comprises CTLA-4 as an extracellular domain comprising a polypeptide having a specific affinity for CD80 and / or CD86 and an intracellular domain of 4-1BB as an intracellular domain comprising a co-stimulatory polypeptide); (B) the design of a second generation fully murine CD19-specific CAR known in the art; and (C) when T cells expressing the constructs shown in (A) and (B) are interacted with murine CD19 + CD80 + CD86 + B cell lymphoma cells, or with primary CD19 + CD80 - CD86 - Results of cell counts and IFN-γ release observed upon in vitro exposure of B cells.

[0049] Fig.17 It was shown that in mice of the C57bl / 6 (wt) mouse strain, the expression of the above Fig.16 Figure 2. In vivo effects of T cells containing the constructs described in on naive B cell populations in peripheral blood and spleen (rightmost panels) over time, shown as (A) ratios, and (B) FACS histograms (top: as Fig.16 (B) The second generation of CAR; bottom: Fig.16(A) CAR / CCR constructs in Figure 2), (C) FACS gating strategy. DETAILED DESCRIPTION

[0050] The present invention is based on the recognition that discrimination between healthy and malignant B cells, as well as between other cell types such as pericytes and malignant B cells, can be made by employing co-stimulatory chimeric checkpoint receptors (CCRs) that recognize checkpoint ligands such as CD80 and CD86 on malignant cells.

[0051] The inventors found that the neurotoxicity observed during therapeutic treatment of patients using Kymriah may be due to the fact that CD19 is also expressed on neuronal pericytes (see Parker KR et al., 2020; above). The undesirable toxicity of CAR T cells directly targeting CD19 has been previously addressed by other researchers, and in some cases co-stimulation of CCR is also used (see Liao Q et al., 2020 Biomarker Research, 8:57 and Blaeschke F et al., 2021 Blood Cancer Journal, 11:108).

[0052] However, Liao et al., 2020 and Blaeschke et al., 2021 have both used CCRs directed against PD-L1 in combination with CD19-CAR (α-PD-L1-scFv in the case of Liao et al., and PD-1 in the case of Blaeschke et al.). This combination does not seem to be suitable for eliminating neurotoxicity, because PD-L1 has been previously reported elsewhere to be expressed on the surface of neuronal pericytes (Domev et al., 2014 Stem Cells Transl Med., 3(10): 1169-1181), and can provide costimulatory signals for the above-mentioned CCR fusion proteins.

[0053] In addition to reducing the potential for neurotoxicity, a particular advantage of using CCRs of the invention directed against CD80 and / or CD86 is that B cell lymphoma cells that have been previously treated with CD19-specific CAR T cells show 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 according to the present invention (see Fig.12 and 13 ) and can be used in vitro (see Fig.16 ) and in immunocompetent mice of the C57bl / 6N(wt) mouse strain (see Fig.17 ) to reduce any undesired targeting of primary B cells.

[0054] Compared to leukemia cells (i.e., chronic lymphocytic leukemia (CLL) cells; see Figure 3 ), the present inventors conducted a study on primary B cell lymphoma cells (i.e., primary diffuse large B cell lymphoma (DLBCL) cells, see Fig.13 (A) and (B)) made similar observations. This suggests that even primary B cell lymphoma cells are particularly amenable to the CAR-T cells of the present invention that benefit from costimulation of CD80 and / or CD86.

[0055] Thus, the present invention makes possible for the first time a promising treatment option for patients with B-cell lymphoma in general, and for patients with tumor relapse after initial (and unsuccessful) therapy with Kymriah, etc. The strategy of the present invention is based on expressing a recombinant CTLA-4 co-receptor (CCR) in addition to the CAR, wherein the CCR binds to and neutralizes the checkpoint ligands CD80 and CD86 on B-cell tumor cells.

[0056] By splitting the signaling part into two receptors CD19-CAR-CD3ζ and CTLA-4-CCR-4-1BB, CAR-T cells are fully activated only when CD80 and / or CD86 are expressed on the same target cell together with CD19. This avoids nonspecific and off-tumor activation of T cells. Based on the experimental results provided herein, these beneficial effects are not limited to the specific constructs mentioned above, and can also be observed using different modules, as long as they meet the required binding specificity and / or signaling ability.

[0057] In this regard, the CD19 binding extracellular domain of the co-expressed (CAR) protein can be functionally replaced by a different CD19 binding portion, a CD20 binding portion, a CD22 binding portion, or another portion that binds to a cell surface molecule found on a cancer cell or an extracellular cancer biomarker. Along the same lines, the extracellular domain of CTLA-4 can also be functionally replaced by a CD80 and / or CD86 binding portion such as CD28 or any CD80 / CD86 binding portion derived from an anti-CD80 or anti-CD86 antibody.

[0058] These insights based on the experimental data disclosed herein demonstrate that the combination of binding to a tumor biomarker such as CD19 and binding to CD80 or CD86 will reliably lead to the insights into the benefits obtained with the present invention.

[0059] In addition, in addition to the effects caused by the specific examples of the present invention, it will be apparent to the technician that intracellular stimulation and costimulatory domains can be replaced by functional equivalents as disclosed herein, as long as they can provide the intracellular signals required for T cell activation. Therefore, not only can the (co) stimulatory part be exchanged between the fusion protein and the co-expressed (CAR) protein, but any (co) stimulatory domain or combination thereof known to transmit the required intracellular signaling can be used within the present invention.

[0060] However, in order to provide the required intracellular signaling in CAR T cells necessary for causing T cell activation and tumor cell killing, at least one intracellular domain of the fusion protein and / or co-expressed (CAR) protein must contain more than one immunoreceptor tyrosine-based activation motif (ITAM; see Zhang et al., 2017 Biomarker Research, 5: 22). Although the two most prominent examples and the most commonly used domains containing ITAMs (FcεRIγ(ic) with one ITAM and CD3ζ(ic) with three ITAMs) can be preferably used in the context of the present invention, domains with synthetic and artificially designed ITAMs that perform similar functions can also be appropriately used and included in the present invention.

[0061] In embodiments, the domain containing ITAM is provided as part of a fusion protein, and the costimulatory domain as described herein is provided as part of a co-expressed (CAR) protein. In embodiments, the domain containing ITAM is provided as part of a co-expressed (CAR) protein, and the costimulatory domain as described herein is provided as part of a fusion protein. In addition, it has been previously reported that IL-6 can be released by antigen presenting cells (APC) such as macrophages or pericytes, which can then cause de-tumor cytokine release syndrome (CRS) in patients. The PD1-CD28 fusion CCR construct used in Liao et al., 2020 and Blaeschke et al., 2021 has recently been reported as causing such (unexpected) increased release of IL-2, IFN-γ and / or TNF.

[0062] These cytokines can be used to recruit and activate other immune cells, such as macrophages, which then contribute to increased IL-6 release and the onset 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.

[0063] In addition, increased IL-2 release induces the proliferation of regulatory T cells, which can reduce the T cell activity of CAR T cells (see, e.g., Chinen et al., Nat Immunol, 2016; 17(11): 1322-1333). This may occur significantly when adopting the strategies proposed by Liao et al., 2020 and Blaeschke et al., 2021.

[0064] When using a construct involving an alternative intracellular signaling domain such as 4-1BB (see Figure 4 , 5 , 8, 9 and 11) or CD3ζ (see Fig.15 ) when using the strategy of the present invention, the above problems seem to be less obvious.

[0065] The strategy of the present invention leads to CD19 + 、CD80 + and / or CD86 + Effective elimination of B-cell lymphomas, even in cases of relapse after initial CAR T-cell therapy (see Figure 6 ), for example by considering CD20 as a tumor biomarker (see Fig.14 This efficient elimination of tumor cells was further demonstrated to result in increased length of tumor-free survival in a xenograft mouse model (see Figure 7 ), and reduced tumor progression (see Fig.10 ).

[0066] Various side effects can be limited or overcome by using the strategies of the present invention. Figure 4 , 5 , 9 and 11), prevent de-tumor and neurotoxic effects, and maintain a pool of healthy B lymphocytes after CAR T-cell therapy (e.g., by Figure 4 (D) and Fig.16 (C) The results were demonstrated in vitro and by Fig.17 The results were demonstrated in vivo).

[0067] Thus, the present invention provides CAR T cell therapies with increased safety and efficacy, avoids nonspecific targeting of cells other than malignant B lymphocytes, maintains a population of healthy B lymphocytes in treated patients, largely avoids neurotoxic symptoms commonly observed using prior art therapies, such as those described as immune effector cell-associated neurotoxicity syndrome (ICAN), reduces the release of alarm cytokines and the development of cytokine release syndrome, and is able to avoid and specifically target tumor recurrence in patients with B cell lymphomas.

[0068] Therefore, in a first aspect of the present invention, a fusion protein is provided, which comprises an extracellular domain comprising a polypeptide having specific affinity for CD80 and / or CD86, a transmembrane domain, and an intracellular domain comprising a co-stimulatory polypeptide.

[0069] As the extracellular domain of the fusion protein of the present invention comprising a polypeptide having a specific affinity for CD80 and / or CD86, the extracellular domain of CTLA-4 (CD152) (UniProtKB-P16410) is preferred. Alternatively, the extracellular domain of CD28 (UniProtKB-P10747) can be preferably used due to its specific affinity for CD80 and / or CD86.

[0070] Alternatively, antibodies or antibody fragments having 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) can be used as a polypeptide having specific affinity for CD86.

[0071] In one embodiment, an antibody or antibody fragment having a specific affinity for CD80 is used as part of the extracellular domain of the fusion protein of the present invention, preferably a CD80 binding domain derived from the anti-CD80 antibody commonly referred to in the art as clone hu1F1, more preferably of the following amino acid sequence: QLVQSGAEVKKPGASVKVSCKPSGFNIKDYYMHWVRQAPGQGLEWIGWIDPENGNTLYDPKFQGKATITADTSTSTAYMELSSLRSEDTAVYYCAREGLFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQWSSYPLTFGQGTKVEIK (SEQ ID NO: 4).

[0072] In another embodiment, an antibody or antibody fragment having a specific affinity for CD86 is used as part of the extracellular domain of the fusion protein of the present invention, preferably derived from the CD86 binding domain of the anti-CD86 antibody commonly referred to in the art as clone hu3D1, more preferably of the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAIQWVRQAPGQGLEWIGVINIYYDNTNYNQKFKGKATMTVDKSTSTAYMELSSLRSEDTAVYYCARAAWYMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKSSQSLLNSRTRENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSYNLYTFGQGTKVEIK (SEQ ID NO: 5).

[0073] In a preferred embodiment, a combination of an antibody or antibody fragment directed against CD80 and an antibody or antibody fragment directed against CD86 can be used as part of the extracellular domain of the fusion protein of the present invention, preferably connected by a conventional linker sequence, more preferably having the following amino acid sequence: QLVQSGAEVKKPGASVKVSCKPSGFNIKDYYMHWVRQAPGQGLEWIGWIDPENGNTLYDPKFQGKATITADTSTSTAYMELSSLRSEDTAVYYCAREGLFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPLIYGTSNLASGVPSRFSGSGSGTDYT LTISSLQPEDVATYYCQQWSSYPLTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAIQWVRQAPGQGLEWIGVINIYYDNTNYNQKFKGKATMTVDKSTSTAYMELSSLRSEDTAVYYC ARAAWYMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPDSLAVSLGERATISCKSSQSLLNSRTRENYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSYNLYTFGQGTKVEIK(SEQ ID NO:6).

[0074] It will be within the routine knowledge of the skilled artisan to select or generate antibodies or antibody fragments specifically directed against CD80 and / or CD86.

[0075] The transmembrane domain of the fusion protein of the present invention can be any type of transmembrane domain that meets the structural requirements necessary to maintain the functions of the associated extracellular and intracellular domains.

[0076] Preferably, the transmembrane domain of the fusion protein of the present invention is selected from the list of transmembrane domains contained in the protein sequences of the following: T-cell receptor α chain (UniProtKB-P01848), T-cell receptor β chain (UniProtKB-P0DSE2), T-cell receptor ζ chain (UniProtKB-P20963), CD28 (UniProtKB-P10747), CD3ε chain (UniProtKB-P07766), CD3ζ chain (UniProtKB-P20963), CD45 (UniProtKB-P08575), CD4 (UniProtKB-P01730), CD5 (UniProtKB-P08616), CD6 (UniProtKB-P08617), CD7 (UniProtKB-P08619), CD8 (UniProtKB-P08613), CD9 (UniProtKB-P08614), CD10 (UniProtKB-P08615), CD11 (UniProtKB-P08617), CD12 (UniProtKB-P08619), CD13 (UniProtKB-P08613), CD14 (UniProtKB-P08614), CD15 (UniProtKB-P08615), CD16 (UniProtKB-P08616), CD17 (UniProtKB-P08617), CD18 (UniProtKB-P08617), CD19 (UniProtKB-P08618), CD28 (UniProtKB-P10747), CD3 niProtKB-P06127), CD8α chain (UniProtKB-P01732), CD8β chain (UniProtKB-P10966), CD9 (UniProtKB-P21926), CD16 (UniProtKB-Q9UPY7), CD22 (UniProt KB-P20273), CD33 (UniProtKB-P20138), CD37 (UniProtKB-P11049), FCGR1A (CD64) (UniProtKB-P12314), CD80 (UniProtKB-P33681), CD86 (UniProt KB-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),IL2Rβ(UniProtKB-P14784), IL2Rγ(UniProtKB-P31785), IL7Rα(UniProtKB-P16871), CD49a(UniProtKB-P56199), IA4(UniProtKB-Q8IU71), CD49D (UniProtKB-P13612), CD49f (UniProtKB-P23229), ITGAD (CD11d) (UniProtKB-Q13349), ITGAE (CD103) (UniProtKB-P38570), CD11a (UniProtKB-P20 701), CD11b (UniProtKB-P11215), CD11c (UniProtKB-P20702), CD29 (UniProtKB-P05556), CD18 (UniProtKB-P05107), ITGB7 (UniProtKB-P26010), T NFR2(UniProtKB-P20333), DNAM1(CD226)(UniProtKB-Q15762), SLAMF4(CD244)(UniProtKB-Q9BZW8), CD84(UniProtKB-Q9UIB8), CD96(UniProtKB- P40200), CEACAM1(UniProtKB-P13688), CRTAM(UniProtKB-O95727), Ly9(CD229)(UniProtKB-Q9HBG7), CD160(BY55)(UniProtKB-O95971), PSGL1(U niProtKB-Q14242), CD100(SEMA4D)(UniProtKB-Q92854), SLAMF6(UniProtKB-Q96DU3), SLAM(CD150)(UniProtKB-Q13291), BLAME(SLAMF8)(UniPro tKB-Q9P0V8), SELPLG (CD162) LTBR (UniProtKB-Q14242), PAG-Cbp (UniProtKB-P06241), CTLA-4 (CD152) (UniProtKB-P16410), preferably selected from the list of transmembrane domains comprising the transmembrane domains of CD8 α chain (UniProtKB-P01732), CTLA-4 (CD152) (UniProtKB-P16410), CD28 (UniProtKB-P10747), more preferably the transmembrane domain of CTLA-4 (CD152) (UniProtKB-P16410).

[0077] The intracellular domain of the fusion protein of the present invention comprising a co-stimulatory polypeptide may comprise a signaling polypeptide or a signaling domain. In the context of the present invention, the intracellular domain comprising a co-stimulatory (or stimulatory) polypeptide may also be designated as an intracellular or cytoplasmic signaling domain, or an intracellular or cytoplasmic activation domain, etc.

[0078] The intracellular signaling domain suitable for use in the fusion protein of the present invention may preferably be an intracellular signaling polypeptide containing an immunoreceptor tyrosine-based activation motif (ITAM), or comprises an intracellular signaling polypeptide containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0079] The ITAM motif is YX1X2L / I, wherein X1 and X2 are independently any amino acid (SEQ ID NO: 7). In certain embodiments, the intracellular signaling domain of the fusion protein comprises 1, 2, 3, 4 or 5 ITAM motifs. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids, e.g., (YX1X2L / I)(X3) n (YX1X2L / I) (SEQ ID NO: 8), wherein n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid. In one embodiment, the intracellular signaling domain of the fusion protein of the present invention comprises 3 ITAM motifs.

[0080] Suitable intracellular signaling domains for the fusion proteins of the present invention may be portions containing ITAM motifs derived from polypeptides containing ITAM motifs. For example, suitable intracellular signaling domains may be domains containing ITAM motifs from any protein containing ITAM motifs.

[0081] Therefore, a suitable intracellular signaling domain or co-stimulatory polypeptide need not contain the entire sequence of the entire protein from which it is derived. It will be well within the knowledge of the skilled person to determine a functional intracellular signaling domain or co-stimulatory polypeptide contained in a given entire protein sequence.

[0082] Examples of preferred protein sequences comprising polypeptides containing ITAM motifs include, but are not limited to: DAP12 (UniProtKB-043914), FcεRIγ (Fcε receptor Iγ chain) (UniProtKB-P30273), CD3D (CD3δ) (UniProtKB-P04234); CD3E (CD3ε) (UniProtKB-P07766), CD3G (CD3γ) (UniProtKB-P09693), CD3Z (CD3ζ) (UniProtKB-P20963), CD79A (antigen receptor complex-associated protein α chain) (UniProtKB-P11912) and CD79B (antigen receptor complex-associated protein β chain) (UniProtKB-P40259).

[0083] In a preferred embodiment of the present invention, the co-expressed (CAR) protein comprises an intracellular domain having more than one ITAM, 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 more than one ITAM, 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.

[0084] In a preferred embodiment of the present invention, the intracellular domain comprising a co-stimulatory polypeptide may be selected from the list of intracellular domains or co-stimulatory polypeptides contained in the protein sequence 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), Myd8 8 (UniProtKB-Q99836), CD40 (UniProtKB-P25942), KIR2DS2 (UniProtKB-P43631), CD137 (4-1BB) (UniProtKB-Q07011), and HVEM (UniProtKB-Q92956), preferably in the protein sequence of CD137 (4-1BB) (UniProtKB-Q07011), CD28 (UniProtKB-P10747), or OX-40 (UniProtKB-P43489), more preferably CD137 (4-1BB) (UniProtKB-Q07011).

[0085] In the present invention, the fusion protein may include domains as defined herein, and optionally additional sequences connecting these domains within the fusion protein. Such joint or hinge sequences used in CAR or CCR are well 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 functions of a single functional domain.

[0086] In a preferred embodiment of the present invention, the fusion protein comprises the extracellular domain and transmembrane domain 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-4ec+tm is gray, 4-1BB ic is black):

[0087]

[0088] Optionally, the preferred fusion protein comprises the extracellular domain and transmembrane domain 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 gray, 4-1BB ic is black):

[0089]

[0090] Optionally, the preferred fusion protein comprises a binding domain directly targeting CD80, an extracellular domain and a transmembrane domain of CD28, and an intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequence (α-CD80 is black, CD28 ec+tm is gray, 4-1BB ic is black, in this order):

[0091]

[0092] Optionally, the preferred fusion protein comprises a binding domain directly targeting CD86, an extracellular domain and a transmembrane domain of CD28, and an intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequence (α-CD86 is black, CD28 ec+tm is gray, 4-1BB ic is black, in this order):

[0093]

[0094] Optionally, the preferred fusion protein comprises a binding domain directly for CD80, a binding domain directly for CD86, an extracellular domain and a transmembrane domain of CD28, and an intracellular signaling domain of 4-1BB. In a preferred embodiment, the fusion protein comprises the following amino acid sequence (α-CD80 is gray, the linker sequence is bold, α-CD86 is black, CD28ec+tm is gray, 4-1BB ic is black, in this order):

[0095]

[0096] Additional sequences such as a leader sequence (eg, an Ig kappa light chain leader sequence such as MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 14)) or others may also be present in the fusion proteins of the invention.

[0097] In a specific embodiment, the fusion protein of the present invention can be expressed as a combined protein construct with a given CAR to be expressed by the same cell, wherein the construct comprises a protein cleavage site between the two proteins, preferably a self-cleaving peptide, more preferably a 2A self-cleaving peptide, and particularly preferably the sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 15).

[0098] According to a specific embodiment, the 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 of the following general structure:

[0099] Leader sequence (e.g., Lκ leader sequence) - extracellular domain directed against tumor-associated cell surface proteins (e.g., CD19, CD20, CD22) - optionally the extracellular domain of the CD8α hinge - transmembrane domain (e.g., the transmembrane domain of the CD8α hinge) - intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ) - self-cleaving peptide (e.g., P2A self-cleaving peptide) - leader sequence (e.g., Lκ leader sequence) - extracellular domain directed against CD80 and / or CD86 (e.g., the extracellular domain of CTLA-4) - transmembrane domain (e.g., the transmembrane domain of CTLA-4) - intracellular signaling domain (e.g., the intracellular signaling domain of 4-1BB).

[0100] According to a specific embodiment, the fusion protein co-expressed with the respective CAR has the following amino acid sequence:

[0101] (SEQ ID NO: 16).

[0102] According to the present invention, any functional domain as mentioned herein (e.g., an extracellular domain, a transmembrane domain and / or an intracellular signaling domain in the case of a fusion protein of the present invention) may be a domain that is functionally equivalent to the domains listed individually herein.

[0103] For example, an extracellular domain that does not have exactly the same sequence as the CTLA-4 extracellular domain, but maintains a specific affinity for CD80 and / or CD86 may still be suitable as an extracellular domain of a fusion protein according to the invention. In this regard, it is pointed out that the specific functions of such domains with different sequences can be easily identified and evaluated by a person skilled in the art.

[0104] Therefore, according to one embodiment of the present invention, the protein sequence forming part of the present invention as a single domain of the protein of the present invention consists of, or comprises, 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, particularly preferably at least 99% identical to the reference protein sequence disclosed herein.

[0105] The determination of percent identity between two sequences is accomplished in accordance with 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 of the BLASTN and BLASTP programs of Altschul et al. (J. Mol. Biol. (1990) 215:403-410). BLAST nucleotide searches are performed using the BLASTN program. In order to obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschul et al. (Nucleic Acids Res. (1997) 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used.

[0106] According to a particular embodiment of the invention, protein sequences forming part of the invention as defined above by a given percentage identity to an individualized protein sequence are those protein sequences which maintain the function of the respective protein.

[0107] According to a preferred embodiment of the present invention, the polypeptide with 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 wherein the polypeptide with 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).

[0108] 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 wherein the transmembrane domain comprises the transmembrane region of one or more of the following: α, β or ζ chain of the T cell receptor, CTLA-4, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD15 4. CD200, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (L IGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7R.α., VLA1, ITGA1, CD49a, ITGA4, IA4, CD49D, ITGA 6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITG B2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SFAMF4(CD244, 2B4), CD84, CD96(tactile), CEACAM1, CRTAM, Ly9(CD229) , CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SEAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp, or an amino acid sequence at least 80% identical thereto, preferably wherein the transmembrane domain comprises the transmembrane region of human CTLA-4 (FLLWILAAVSSGLFFYSFLLT; SEQ ID NO: 17), or an amino acid sequence at least 80% identical thereto.

[0109] The inclusion of a costimulatory domain, such as the 4-IBB (CD 137) costimulatory domain, in the intracellular region of the fusion proteins of the invention enables T cells to receive costimulatory signals that can direct and control specific T cell responses.

[0110] According to a preferred embodiment of the present invention, the intracellular domain can send a costimulatory signal to T cells. Preferably, the intracellular domain of the fusion protein of the present invention includes the intracellular domain or (co) stimulatory polypeptide of one or more of the following: 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 at least 80% identical amino acid sequence thereto, more preferably the intracellular domain of the fusion protein of the present invention includes the intracellular domain or costimulatory polypeptide (CD137 of human 4-1BB; (SEQ ID NO: 3)), or at least 80% identical amino acid sequence thereto.

[0111] In a preferred embodiment, the fusion protein of the present invention includes an extracellular domain comprising the extracellular domain of human CTLA-4 (SEQ ID NO: 1), or an extracellular domain having an amino acid sequence at least 80% identical thereto, and the intracellular domain includes a co-stimulatory peptide comprising the intracellular domain of human 4-1BB (CD137; (SEQ ID NO: 3)), or an amino acid sequence at least 80% identical thereto.

[0112] More preferably, the fusion protein comprises the following amino acid sequence of CTLA-4(ec+tm) fused to 4-1BB(ic):

[0113] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18).

[0114] The fusion protein of the present invention can generally be used in combination with a chimeric antigen receptor (CAR) targeting a cell surface protein associated with a tumor. In particular, the fusion protein can preferably be used in combination with any CAR directed against CD19, CD20 or CD22. Based on the data provided herein, it seems reasonable and credible that when used with a CAR for CD19, CD20 or CD22, or any other protein known to be expressed on the surface of a tumor cell in this case and / or expressed as a tumor marker, the present invention will be functional and produce favorable results.

[0115] In a specific embodiment, the fusion protein of the present invention can be used in combination with a CAR directed against CD19. In another specific embodiment, the fusion protein of the present invention can be used in combination with a CAR directed against CD20. In yet another specific embodiment, the fusion protein of the present invention can be used in combination with a CAR directed against CD22.

[0116] On the other hand, the present invention further relates to nucleic acid molecules encoding fusion proteins as 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, transmembrane domain, and / or intracellular domain of the fusion protein.

[0117] As used herein, unless specifically defined otherwise, the term "nucleic acid" or "nucleic acid molecule" is used synonymously with "oligonucleotide," "nucleic acid chain," or "polynucleotide," and means a polymer comprising one, two, or more nucleotides.

[0118] The term "nucleic acid molecule" refers to a base sequence including purine bases and pyrimidine bases consisting of a polynucleotide, wherein the bases represent the primary structure of the nucleic acid molecule.

[0119] In this article, the term "nucleic acid molecule" includes all types of nucleic acids, including DNA, cDNA, genomic DNA, RNA, synthetic forms of DNA and mixed polymers containing two or more of these molecules, and preferably refers to DNA and cDNA. As those skilled in the art will readily appreciate, the nucleic acid sequences provided herein represent DNA sequences, and also include corresponding RNA sequences in which T is replaced by U.

[0120] The term "nucleic acid molecule" generally comprises a sense strand and an antisense strand. As will be readily appreciated by those skilled in the art, a "nucleic acid molecule" may further comprise non-natural or derived nucleotide bases, and natural or artificial nucleotide analogs, for example, to protect the nucleic acid molecule from the influence of endonucleases and / or exonucleases.

[0121] In yet another aspect, the present invention further relates to a vector comprising the nucleic acid molecule described and provided herein.

[0122] The term "vector" as used herein generally includes all kinds of linear or circular nucleic acid molecules that can replicate autonomously in a suitable host cell. Such vectors include, but are not limited to, plasmids, cosmids, phages, viruses (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, or preferably retroviral vectors), and other vectors or shuttles known in the art that are suitable for carrying genes and transferring genes into host cells to allow stable or transient translation and constitutive or conditional expression of the fusion protein of the present invention in host cells.

[0123] The vector usually does not integrate into the cell genome, but may also be integrated.The vector according to the invention comprising the nucleic acid molecule as described and provided herein preferably allows for stable expression of the fusion protein of the invention in a host cell (expression vector).

[0124] The vector of the present invention may further comprise a marker gene, a promoter and / or enhancer sequence (operably linked to the nucleic acid molecule of the present invention), an origin of replication suitable for the respective host cell, a restriction site, a multiple cloning site, a tag and other functional units known in the art.

[0125] Among other things, the vector can be transferred into the host cell via a shuttle such as a virus (which itself can be considered a vector), or transformed or transduced into the host cell in naked form. The vector is preferably adapted to be suitable for the respective host cell into which it is to be transformed or transduced.

[0126] The skilled person will readily appreciate that different host cells will require different types of vectors. For example, the vector (plasmid) pGEM is a suitable vector for transformation into bacterial cells, while the retroviral vector pMP71 is suitable for transduction into eukaryotic cells (e.g., T cells).

[0127] 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 and include, for example, 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., Cancer lmmunol. lmmunother. 46 (1998), 61-66), pSTlTCH (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., J. Immunother. 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., 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). .

[0128] In a further aspect, the invention relates to a host cell comprising a nucleic acid molecule or a vector as described and provided herein. In one embodiment, a host cell of the invention is transduced or transformed with a nucleic acid molecule or a vector as described and provided herein.

[0129] Generally, as used herein, unless explicitly defined otherwise, the terms "transduced" or "transformed" (as well as "transduction" or "transformation") and the like are used interchangeably and generally mean any kind of transfer of nucleic acid molecules and / or vectors into a host cell, regardless of the type of host cell and regardless of the mode of transfer (e.g., (chemical) transformation, (viral) transduction, electroporation, transfection, etc.).

[0130] Nucleic acid molecules and / or vectors can be stably integrated into the genome of the host cell, or are extrachromosomal (i.e., transient expression). Examples of suitable methods for realizing transient expression in host cells are known in the art, and include mRNA transfection. In one embodiment, host cells are transduced with nucleic acid molecules and / or vectors. In another embodiment, nucleic acid molecules and / or vectors are stably integrated into the genome of the host cell.

[0131] Host cells described and provided as part of the present invention comprising nucleic acid molecules or vectors as described and provided herein are preferably capable of stably or transiently (eg, stably) expressing (either constitutively or conditionally) a fusion protein of the present invention.

[0132] Host cells can be transduced or transformed with any suitable nucleic acid molecule or vector generally by any method. In 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 present invention as described above or a portion thereof (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain).

[0133] In one embodiment, a host cell of the invention is transduced with a retroviral vector comprising a nucleic acid molecule encoding a fusion protein of the invention as described above, or a portion thereof (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain), and stably or transiently expresses (either constitutively or conditionally) the fusion protein or one or more portions thereof.

[0134] Preferably, the host cell then stably or transiently expresses the fusion protein in its membrane, with the extracellular domain of the fusion protein of the invention facing the outside of the host cell, the transmembrane domain being (mostly) embedded in the host cell membrane, and the intracellular domain extending into the cytoplasm of the host cell.

[0135] In the present invention, a host cell comprising a nucleic acid molecule or a vector as described and provided herein relates to a genetically modified cell transduced or transformed with the nucleic acid molecule or vector, or wherein the nucleic acid molecule or vector is otherwise introduced into the host cell.

[0136] As mentioned, the host cell of the present invention can be a cell that transiently or stably expresses the fusion protein of the present invention. For example, the nucleic acid molecule encoding the fusion protein of the present invention can be stably integrated into the genome of the cell by retroviral or lentiviral (e.g., retroviral) transduction.

[0137] The host cell or transduced cell of the present invention can be, for example, a CD8 + T cells, CD4 + T cells, TCRs 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, and cells suitable for storing and / or reproducing the nucleic acid molecules or vectors of the invention, including bacterial cells (e.g., E. coli) and other eukaryotic cells. The cells can be autologous or non-autologous, but are preferably autologous. In addition, as is apparent to the skilled person, the cells can be allogeneic or non-allogeneic.

[0138] In one embodiment, the host cell of the present invention is a T cell, preferably a CD8 + T cells.

[0139] The host cell of the present invention can be transduced with a nucleic acid molecule or vector encoding a fusion protein as described and provided herein. Preferably, the host cell provided and described herein can be co-transduced with other nucleic acid molecules, for example, co-transduced with a nucleic acid molecule encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), for example, as described herein.

[0140] Such co-transduction (or other methods for introducing nucleic acid molecules into cells as described and exemplified herein) are known in the art, and are also described and exemplified herein.

[0141] In one embodiment, a host cell stably or transiently expressing a fusion protein of the present invention additionally stably or transiently co-expresses a (co-expressed CAR) protein comprising an extracellular domain with specific affinity for CD19 and an intracellular co-stimulatory 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.

[0142] 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 co-stimulatory domain (co-expressing CAR), preferably wherein the extracellular domain with specific affinity for CD20 comprises at least an antigen-binding fragment of an anti-CD20 antibody, more preferably wherein the extracellular domain with specific affinity for CD20 comprises an anti-CD20 scFv.

[0143] In all embodiments described herein as involving stably or transiently expressing a protein, stable expression of the protein is preferred in any case.

[0144] Preferably, the co-expressed (CAR) protein comprises the amino acid sequence of CD3ζ (SEQ ID NO: 25) as an intracellular co-stimulatory domain, or an amino acid sequence that is at least 80% identical thereto.

[0145] According to one embodiment, the co-expressed (CAR) protein comprises the binding domain of an anti-CD19 antibody as an extracellular domain, preferably an anti-CD19 scFv, more preferably an anti-CD19 scFv commonly referred to in the art as clone FMC63 as an extracellular domain. The sequence of the anti-CD19 scFv is as follows:

[0146] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSE VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:19).

[0147] In addition, the co-expressed (CAR) protein may optionally or additionally comprise an anti-CD20 antibody binding domain as an extracellular domain, preferably an anti-CD20 antibody binding domain commonly referred to in the art as clone 2H7. The sequence of the binding domain is as follows:

[0148] MAQVKLQESGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGQGTTVTVSS GGGGSGGGGSGGGGSDIELTQSPTILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKRAAA (SEQ ID NO:20).

[0149] In addition, the co-expressed (CAR) protein may optionally or additionally comprise a binding domain of an anti-CD22 antibody as an extracellular domain, 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:

[0150] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK(SEQ ID NO:21).

[0151] In one embodiment, the co-expressed (CAR) protein may comprise the extracellular domain and transmembrane domain of CD8α and the intracellular domain of CD3ζ, preferably having the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 22).

[0152] In a specific embodiment, the co-expressed (CAR) protein may comprise an anti-CD19 scFv as well as the extracellular domain and transmembrane domain of CD8α and the intracellular domain of CD3ζ, preferably having the following amino acid sequence:

[0153] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI TGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTA IYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSAD APAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:23).

[0154] In certain embodiments, the commonly known products CD19 CARs containing Tisagenlecleucel or Tisagenlecleucel can be used in the present invention.

[0155] Alternatively, the co-expressed (CAR) protein may comprise an anti-CD20 binding domain and an anti-CD19 scFv as well as the extracellular domain and transmembrane domain of CD8α and the intracellular domain of CD3ζ, preferably having the following amino acid sequence:

[0156] (SEQ ID NO:24).

[0157] In one aspect, the intracellular co-stimulatory domain of the fusion protein or co-expressed (CAR) protein comprises the amino acid sequence of the intracellular domain of CD3ζ, preferably having the following amino acid sequence:

[0158] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25), or an amino acid sequence that is at least 80% identical to the amino acid sequence.

[0159] In one aspect, the present invention also relates to a method for preparing a host cell of the present invention as described and provided herein, the method comprising transducing (or transforming) a host cell as described above with a nucleic acid molecule or a vector as described and provided herein, culturing the transduced (or transformed) host cell in a suitable culture medium that allows growth of the cell and expression of the fusion protein encoded by the nucleic acid molecule or the vector; and collecting the host cell from the culture medium.

[0160] In a preferred embodiment of the invention, the host cells are transduced or transformed outside the human body. For obtaining, isolating and culturing cells (e.g., T cells such as CD8 + T cells, CD4 + Methods for removing T cells, TCRs such as (but not limited to) TCR-T58 or TCR-D115 T cells) are known in the art and include, among others, blood draw or bone marrow removal.

[0161] According to the method of the present invention, host cells can be transduced or transformed or otherwise provided with nucleic acid molecules or vectors as described and provided herein by any method known in the art. Such methods include, among others, (chemical) transformation, (viral) transduction, electroporation, and transfection, etc. In one embodiment, host cells are transduced with retroviral vectors.

[0162] The present invention also relates to host cells obtainable by the preparation methods provided herein. On the other hand, the present invention further relates to pharmaceutical compositions comprising fusion proteins, nucleic acid molecules, vectors, and / or host cells as described and provided by the present invention. Such pharmaceutical compositions are suitable for administration to patients (preferably human patients), particularly to donors of host cells as described above.

[0163] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and other components for galenic purposes.

[0164] The fusion proteins, nucleic acid molecules, vectors, host cells and pharmaceutical compositions are each particularly advantageous for use as a medicament, preferably for the treatment of diseases and disorders associated with the 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 the treatment of B-cell lymphomas, even more preferably for the treatment of non-Hodgkin's lymphomas 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, Macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL) and diffuse large B-cell lymphoma (DLBCL), particularly preferably for the treatment of diffuse large B-cell lymphoma (DLBCL).

[0165] Other and additional diseases and conditions that may be treated using the present invention will be apparent to the skilled artisan, and specifically include, but are not limited to, different types of cancer such as lung cancer, gastric cancer, renal cell carcinoma, colon cancer, breast cancer, ovarian cancer, urothelial carcinoma, melanoma, pancreatic cancer, myeloma, Hodgkin's lymphoma, retinoblastoma, leukemia, cervical cancer, esophageal cancer, glioma, non-Hodgkin's lymphoma, hepatocellular carcinoma, oral cancer, and other cancers.

[0166] In addition, the present invention also relates to a method for treating a disease or condition as listed above by administering a pharmaceutical composition comprising a fusion protein, nucleic acid molecule, vector, and / or host cell as described and provided by the present invention. The present invention further relates to a method for treating a disease or condition as listed above by using a fusion protein, nucleic acid molecule, vector, and / or host cell as described and provided by the present invention.

[0167] Finally, the present invention also relates to a kit or a 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.

[0168] All embodiments of the present invention as disclosed and described herein are considered combinable in any combination, unless a skilled person deems such combination to have no technical significance. The present invention is now further explained by individual examples, which are intended to illustrate rather than limit the present invention.

[0169] Example

[0170] PBMC isolation and T cell activation

[0171] Peripheral blood mononuclear cells were harvested from the buffy coat of the donors via density gradient separation. The buffy coat was carefully pipetted onto a STEMCELL Technologies Lymphoprep and centrifuged at 600 x g for 30 minutes. The resulting mononuclear cell layer was extracted, washed 4 times in PBS, and up to 1e9 cells were placed in ThermoFisher RPMI 1640 culture medium with 10% FCS, Pen / Strep and HEPES buffer for culture. Prior to transduction, T cells were stimulated for 2 days 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 singularizing spleens of C57BL / 6N (Black 6; RRID:IMSR_JAX:000664) mice and either isolating B cells with MojoSort Mouse Pan B Cell Isolation Kit II (BioLegend Cat#480087) or isolating T cells with MojoSort Mouse CD T Cell Isolation Kit (BioLegend Cat#480031) according to the manufacturer's instructions. Murine B cells were cultured in RPMI 1640 supplemented with 10% FCS and Pen / Strep, and murine T cells were cultured in X-Vivo 15 supplemented with 5% FCS and stained with 200 ng mL prior to transduction. -1 Anti-CD3, 100 ng mL -1 、1000U mL -1 IL-2 and 10 ng mL -1 IL-15 stimulation for three days.

[0172] Vector transfection and transduction

[0173] CAR and CAR / CCR expression were induced by retroviral transduction. Two 10 cm plates of HEK293t cells with a monolayer were transfected at 50-70% confluence using 20 μl PolyplusPeiPro transfection reagent in 500 μl RPMI 1640 with 10 μg of carrier DNA and 5 μg of GAL-V and M-MuLV helper plasmids, respectively. The transfection reagent was added to HEK293t cells cultured in 9 ml of RPMI 1640 with 10% FCS, Pen / Strep and HEPES buffer.

[0174] The culture medium containing the transduced virus was harvested after 10-24 hours and added to 1,6e7 PBMC-derived T cells in a plate coated with poly-D-lysine, centrifuged at 450g for 90 minutes and cultured overnight. The process was repeated a second time with the same cells to increase the transduction yield. During transduction, T cells were kept stimulated overnight with IL-2 (1000U / ml) after the first run and kept stimulated overnight with IL-2 (500U / ml) after the second run.

[0175] Fluorescence-based in vitro cytotoxicity assay

[0176] The cytolytic activity of the studied CAR and CAR / CCR T cells was evaluated by measuring the fluorescence level over time in co-culture of T cells and tumor cell lines expressing eGFP using a HIDEX Sense microplate reader platform with compatible digital atmospheric control. The cytolytic activity was given as the inverse relative increase in fluorescence level compared to the measurement of wells containing only tumors, calculated using the following formula:

[0177]

[0178] Where Fl = fluorescence measurement in co-culture wells, Tu = fluorescence measurement in wells containing only tumor cells, and Med = fluorescence measurement in wells containing only medium. The assay wells contained 200 μl RPMI 1640 with 10% FCS, Pen / Strep and HEPES, tumor cells and CAR-T cells at different ratios, normalized to total CAR-T cell number and total cell number by adding Mock transduced T cells from the same donor.

[0179] In vitro cytotoxicity assay based on flow cytometry

[0180] Cytotoxic effects on primary human B cells were explored by flow cytometry in a humidity-controlled environment at 37°C after co-culturing with CAR / CCR and CAR (second generation) T cells at an effector-target ratio of 1:10 for 18 hours. Samples were stained with anti-CD3 APC (Miltenyi Biotec Cat#130-113-125; RRID:AB_2725953), anti-CD19 FITC (Miltenyi Biotec Cat#130-113-645; RRID:AB_2726198) and 7-AAD (BioLegend Cat#420404), washed twice and analyzed.

[0181] The cytotoxicity of mouse T cells equipped with murine CAR / CCR and mCAR (second generation) constructs against primary mouse B cells was determined by using CellTrace TM Violet proliferation kit was used to pre-stain the target cells for 24-48 hours. The cells were then detected using a MACSQuant X flow cytometer (Miltenyi Biotec, Bergisch-Gladbach, Germany) and the CellTrace TM Absolute counts of Violet-positive cells were plotted for comparison between constructs.

[0182] Detection of cytokine levels in supernatants by ELISA

[0183] The concentration of secreted cytokines in the assay supernatant was measured using a sandwich ELISA technique. TM 96-well plates were coated with capture antibody (see below), washed, blocked, and incubated with 50 μl of assay supernatant at 4°C overnight. Where necessary, PBS dilutions of supernatant were used. The samples were then discarded and incubated sequentially with capture antibody and secondary enzyme-linked antibody, with 4 PBS-Tween (0.05% v / v) washes between each step.

[0184] Detection was carried out by incubation of 15-30 minutes in the dark with TMB substrate solution (Life technologies) and stopped with 0.5M sulfuric acid. OD was measured using ThermoFisher ELISA reader (Multiscan Go), and concentration was calculated by standard curve when applicable. Human IFN-γ in culture supernatant was recorded by using matched pair antibody (matched pair antibody) (clone NIB 42 and B133.5). Human IL-2 in culture supernatant was recorded by using matched pair antibody (B33-2 and 5344.111). Human IL-6 in culture supernatant was recorded by using IL-6ELISA kit (BD Biosciences).

[0185] Culture of CAR T cells

[0186] Transduced T cells were cultured in RPMI 1640 medium with 10% FCS, HEPES buffer, Pen / Strep and 100-300 U / ml IL-2. The medium was added or replaced every 3-4 days or when the onset of acidification was observed via phenol red color change.

[0187] Cultivation of tumor cell lines

[0188] Tumor cell lines were cultured in RPMI 1640 medium with 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 the onset of acidification was observed via phenol red color change. Cultures were checked for mycoplasma at regular intervals and prior to in vivo use via PCR.

[0189] Culture, differentiation and characterization of mesenchymal stem cells

[0190] Mesenchymal stem cells were cultured in Mesenchymal Stem Cell Growth Medium 2. To achieve differentiation, cells were treated with different concentrations of recombinant TGF-β3 (10-20 ng / ml). Cells were then characterized using FACS analysis and anti-CD19 APC-linked antibodies (ImmunoTools #21270196) and anti-CD248 FITC-linked antibodies (Bioss bs-2101R-FITC). MACS separation was achieved on the autoMACS Pro platform using a combination of the same antibodies and magnetic anti-APC microbeads (Miltenyi Biotec #130-090-855). Magnetic activated cell separation Select: Macrophages and T cells

[0191] Single cell suspensions from buffy coats derived from healthy donors were prepared using a CD16 microbead kit in an autoMACS Pro separator (Miltenyi Biotec). The number of contaminating cells in the isolated mononuclear cell population was less than 2%. Human CD4+ and CD8+ T cells were isolated using a Pan T cell isolation kit (Miltenyi Biotec) for isolating untouched T cells from peripheral blood mononuclear cells (PBMCs) by depletion of non-T cells.

[0192] FACS analysis of CD80 / 86 expression on B-cell lymphoma cell lines (DLBCL and CLL) and healthy B cells

[0193] Tumor cell line expression of antigens and receptors was characterized using FACS analysis using the BD Canto II platform. Cells were quantified and isolated from culture at 5e5-1e6 cells per FACS tube, washed twice with 4 ml PBS before and after antibody application, and stained with 7-AAD (BD Biosciences 559925) CD5-BV510 (Biolegend 364018) CD19-FITC (Biolegend 392508) CD20-APC / Fire according to the manufacturer's instructions. TM 750 (Biolegend 302358) CD80-PE (Biolegend 305208) CD86-APC (Biolegend 374208) were incubated at 4°C for 30 minutes.

[0194] Detection of CAR and CAR / CCR Expression Using Flow Cytometry

[0195] Transduction efficiency was evaluated via FACS analysis using a non-commercial anti-idiotypic CD19-CAR (FMC63) antibody supplied by Miltenyi Biotec and detected with anti-biotin PE (Miltenyi, Cat#REA746), anti-CTLA4 (CD152) BV421 (BioLegend; Cat#369605), anti-CD3 APC (Miltenyi, Cat#BW264 / 56) at the supplier's concentration. Antibodies were incubated at 4°C for 30 minutes, with two PBS wash steps between the primary and secondary antibodies and before analysis. CAR-transduction efficiency was evaluated in lymphocyte / monocyte / CD3+ gates to confirm successful transduction and normalize the number of CAR-T cells in downstream experiments.

[0196] Details of the efficacy study in xenograft mice

[0197] A total of 28 Rag 2 between 100 and 147 days of age - / - γc - / - Mice (Jax mice) (13 females, 15 males) were injected intravenously with Raji-fLuc cells (5e4 cells per mouse) on day 3. One female mouse was injected intraperitoneally. Tumor implantation was evaluated on day 3 by injecting D-luciferin (1.5 mg / mouse) and measuring tumor luminescence in anesthetized mice in an IVIS200 apparatus (PerkinElmer, Waltham, Massachusetts, USA).

[0198] Mice were divided into groups receiving intravenous administration of CAR / CCR (6 mice), second-generation CAR (7 mice) or untransduced T cells (9 mice) from the same PBMC donor of 8e6 cells each. 6 tumor-bearing mice remained untreated. All mice were scored every day, and luminescence measurements were repeated weekly. Mice were killed at a predetermined tumor load or score cutoff.

[0199] Details of studies using immunocompetent mice to investigate safety.

[0200] Mouse spleen cells are genetically engineered to express CAR (second generation) or CAR / CCR constructs, such as Fig.16 CAR and CAR / CCR expression were analyzed by flow cytometry. CAR or CAR / CCR T cells were injected intravenously into the tail of C57bl / 6N mice (1x10 7 ). Blood samples were collected weekly and analyzed for the presence of CD19+ cells by flow cytometry using a CD19-specific antibody. Finally, the spleen was removed and also analyzed for the presence of CD19+ B cells.

[0201] Immunohistological analysis

[0202] Immunohistological studies of human tissues from DLBCL patients (before and in the case of CAR T cell therapy) were performed as whole sections on 4 μm thick sections of formalin-fixed paraffin-embedded tumor tissue biopsies (University of Cologne, Institute of Pathology). For human CD80 detection, slides were stained once with a biotin-labeled polyclonal antibody (5.0 μg / ml) (Bioss, Woburn, Massachusetts, USA) for CD80, and secondary staining was performed with streptavidin-horseradish peroxidase (500 mU / ml) (Roche, Basel, Suisse).

[0203] For human CD86 detection, slides were stained once with a mouse monoclonal conjugated CD86-specific antibody (2.0 μg / ml), clone SPM600 (Novus Biologicals, Centennial, CO, USA), and secondarily with a polyclonal HRP-conjugated mouse IgG1-specific antibody (2.0 μg / ml) (Bioss). Subsequently, both CD80- and CD86-stained sections were additionally incubated with DAB chromogen substrate (Vector Laboratories, Burlingame, CA, USA) and with hematoxylin (PanReac AppliChem, Cranbury, NJ, USA) for immunohistological analysis according to the manufacturer's instructions.

[0204] Slides were recorded using an Olympus-UC90 4K microscope (Olympus, Tokyo, Japan), and CD80 and CD86 expression was analyzed using ImageJ version 1.53 (National Institutes of Health, USA).Finally, slides were analyzed using an Olympus FV 1000 microscope (Olympus, Tokyo, Japan), and fluorescence intensity was determined using ImageJ software.

Claims

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

2. The fusion protein according to claim 1, wherein 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 wherein 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: NO:1) is an amino acid sequence that is at least 80% identical to that of the fusion protein, and / or wherein the transmembrane domain is suitable for insertion and anchoring of the fusion protein in the cell membrane of a mammalian cell, preferably wherein the transmembrane domain comprises one or more of the following transmembrane regions: α, β or ζ chain of a T cell receptor, CTLA-4, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64 , CD80, CD86, CD134, CD137, CD154, CD200, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1 BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7R.α., VLA1, ITGA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM , CDllb, ITGAX, CDllc, 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 (NTB-A, Ly108), SEAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp, or an amino acid sequence at least 80% identical thereto, preferably wherein the transmembrane domain comprises the transmembrane region of human CTLA-4 (SEQ ID NO: 17), or an amino acid sequence that is at least 80% identical thereto, and / or wherein the intracellular domain comprises the intracellular domain or co-stimulatory polypeptide of one or more of the following: 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 that is at least 80% identical thereto, preferably wherein the intracellular domain comprises the intracellular domain or co-stimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence that is at least 80% identical thereto, or wherein the intracellular domain comprises the intracellular domain or co-stimulatory polypeptide of CD3ζ (SEQ ID NO: 25), or an amino acid sequence that is at least 80% identical thereto, preferably wherein the intracellular domain comprises the intracellular domain or co-stimulatory polypeptide of human 4-1BB (CD137; SEQ ID NO: 3), or an amino acid sequence that is at least 80% identical thereto, NO:3), or an amino acid sequence that is at least 80% identical thereto.

3. A fusion protein according to any one of claims 1 or 2, wherein the extracellular domain comprises a polypeptide 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 co-stimulatory 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 a fusion protein according to any one of the preceding claims. A vector comprising the nucleic acid molecule according to claim 4 .

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

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

8. 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 a specific affinity for CD19 and an intracellular co-stimulatory domain, preferably wherein the extracellular domain having a specific affinity for CD19 comprises at least an antigen-binding fragment of an anti-CD19 antibody, more preferably wherein the extracellular domain having a specific affinity for CD19 comprises an anti-CD19 scFv, Or wherein the host cell stably or transiently expresses a protein comprising an extracellular domain with specific affinity for CD20 and an intracellular co-stimulatory domain, preferably wherein the extracellular domain with specific affinity for CD20 comprises at least an antigen-binding fragment of an anti-CD20 antibody, more preferably wherein the extracellular domain with specific affinity for CD20 comprises an anti-CD20 scFv.

9. A host cell according to 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 that is 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 that is at least 80% identical thereto, preferably 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 that is at least 80% identical thereto.

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

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

12. A host cell obtainable by the method according to claim 11.

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

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

15. The fusion protein according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the host cell according to any one of claims 6 to 10 or 12, or the pharmaceutical composition according to claim 13, for use in the treatment of B-cell lymphoma, preferably for the treatment of non-Hodgkin's lymphoma selected from the group consisting of 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, 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 a 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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