Anti CD276 adapter chimeric antigen receptor (CAR)

Abstract

The present invention provides a combination of compositions comprising a first composition comprising a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag and a second composition comprising an immune effector cell expressing a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of said tagged polypeptide. A second aspect of the invention provides a combination of compositions as disclosed herein for use in the treatment of a disease, preferentially cancer. Another aspect of the invention is a combination of compositions for use in a method of treating a disease, said method comprising administering to a subject in need thereof: a first composition comprising a tagged polypeptide comprising an antigen binding domain specific CD276 and a tag; and a second composition comprising an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said tag of said tagged polypeptide.

Description

Title

[0001] Anti CD276 adapter chimeric antigen receptor (CAR)Field of the invention

[0002] The invention belongs to the field of immunotherapy, more specifically cancer therapy using chimeric antigen receptors.Background of the invention

[0003] B7-H3, also known as CD276, has been frequently observed to be upregulated in numerous types of tumors, such as brain gliomas, lung cancer, and various pediatric cancers, positioning it as a potential target for immunotherapy treatments (1,2,3). CAR T cells specific for CD276 have demonstrated promising results, both pre-clinical and clinically (4,5). However, CD276 can be expressed by T-lymphocytes in addition to tumor cells or the tumor stroma (6). The expression of CD276 on non-tumor tissue e.g. T cells or CAR T cells can cause off-target toxicity. In addition to that, the expression of CD276 on activated CAR T cells can lead to fratricide of conventional CAR T cells during the manufacturing process and it competes with their ability to bind to CD276-specific tumor cells (7). In summary, this limits the persistence and fitness of the cellular product, thereby leading to a reduced anti-tumor efficacy.

[0004] Therefore, an improved or alternative approach is needed to target CD276-expressing tumor cells to overcome current limitations.Brief description of the invention

[0005] Surprisingly, it was found that the use of the adapter CAR system for targeting CD276 expressing cells overcomes the key limitations of fratricide, production issues and in vivo CAR T cell persistence (Figure 6-8 / Example 6-8). The use of CD276-specific Fab-based adapter molecules in an adapter CAR concept was compared to a conventional CAR construct having the same CD276 binding moiety but in scFv format. The use of the adapter CAR concept combined with CD276-specific Fab adapters showed enhanced tumor control in a SCLC xenograft model and better persistence (Figure 7-8 / Example 7-8). In addition, better expansion during CAR T cell manufacturing of AdCAR T cells targeting CD267 via a CD276-specific Fab adapter compared to conventional CAR T cells specific for CD276 expressing the same binding moiety as an scFv was observed (Figure 6 / Example 6)

[0006] For this reason the first aspect of the invention is a combination of compositions comprising a first composition comprising a tagged polypeptide comprising an antigen bindingdomain specific for CD276 and a tag and a second composition comprising an immune effector cell expressing a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of said tagged polypeptide.

[0007] A second aspect of the invention provides a combination of compositions as disclosed herein for use in the treatment of a disease, preferentially cancer.

[0008] Another aspect of the invention is a combination of compositions for use in a method of treating a disease, said method comprising administering to a subject in need thereof a first composition comprising a tagged polypeptide comprising an antigen binding domain specific CD276 and a tag; and a second composition comprising an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said tag of said tagged polypeptide.

[0009] In addition to that, five anti-CD276 Fab molecules eliciting superior killing activities and cytokine release of the CAR T cells upon binding, have been identified (Figure 1-2, Figure 4-5 / Example 2, 4-5).

[0010] Out of these five candidates, one candidate was identified as having the highest thermostability and protein titer, making this clone a better candidate for efficient Fab molecule production (Figure 3 / Example 3). Moreover, in vivo experiments have shown that this candidate in combination with the AdCAR system shows a high persistence in vivo and high tumor killing. In contrast to that, using this binder in direct CAR format no persistence in vivo was found. These data suggest, that a clinical translation of CAR T cells targeting CD276 expressed on tumor cells via the AdCAR system using a CD276-specific adapter is having substantial advantages over a conventional CAR T cell approach. This novel concept to target CD276 positive tumor cells but spare out CD276 positive T cells has not been described so far and allows to generate CAR T cells to treat a broad range of solid tumors expressing this antigen.Brief description of the drawings

[0011] Figure 1 : CD276-specific Fab molecules bind antigen-positive tumor cells and induced AdCAR T-cell-mediate tumor lysis. CD276 expressing GFP positive OCI-AML-2 cells were cultured in the presence of InM CD276-specific Fabs recombinantly linked to a screening tag and sAdCAR T cells. Tumor cell lysis was quantified by measuring the number of GFP-positive tumor cells via flow cytometry after 24h of co-culture. The white line indicates the mean.

[0012] Figure 2: CD276-specific Fab molecules bind antigen-positive tumor cells and induce AdCAR T-cell-mediate cytokine secretion. CD276 expressing GFP positive OCI-AML-2 cellswere cultured in the presence of InM CD276-specific Fabs recombinantly linked to a screening tag and sAdCAR T cells. Cytokine secretion was quantified by MACS Plex analysis.

[0013] Figure 3: Thermostability and protein yield of CD276- specific Fab molecules. Protein yield was quantified via absorbance at OD 280 and correlated to the melting temperature (Tm) measured via the Nanotemper Tycho system. The solid line indicates the linear regression curve with it goodness of fit R2. The dotted lines represent the 95% confidence interval bands.

[0014] Figure 4: CD276-specific Fab molecules bind antigen-positive tumor cells and induce AdCAR T-cell-mediate tumor lysis. CD276 expressing luciferase positive RH-30 cells (a), IMR-32 (b) cells and DMS-114 (c) cells were cultured in the presence or absence of 5nM CD276-specific Fabs conjugated with the LC-biotin and AdCAR T cells. Tumor cell lysis was quantified by quantifying the bioluminescence of tumor cells after 96h of co-culture. The bioluminescent signal was normalized to the target cell only control condition. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD. Statistical analysis was performed by One-way ANOVA. Correction for multiple comparisons done with Dunnett. Significance defined as: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0015] Figure 5: CD276-specific Fab molecules bind CD276-positive tumor cells and induce AdCAR T cell-mediate cytokine secretion. CD276 expressing DMS-114 cells were cultured in the presence or absence of 5nM CD276-specific Fabs conjugated with LC-biotin and AdCAR T cells Cytokine secretion was quantified by MACSPlex assay after 24h of co-culture. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD

[0016] Figure 6: Conventional CAR T cells expressing AdCD276_l constitutively as an scFv show reduced expansion during CAR T cell manufacturing when compared to non-transduced T cells or AdCAR T cells. Initially, 2xl06T cells were plated in a 24-well plate with 2 mL of TexMACS™ medium, enriched with 12.5 ng / mL of both recombinant human IL-7 and IL-15. The cells were then activated using a 1 :100 dilution of TransAct™. After 24 hours, lentiviral vectors (LVVs) were introduced to achieve a multiplicity of integration (MOI) of 10. Cell concentration was analyzed by flow cytometry using propidium iodide to discriminate between live and dead cells. Statistical analysis was performed by One-way ANOVA. Correction for multiple comparisons done with Holm-Sidak. Significance defined as: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0017] Figure 7: AdCAR T cells in the presence of AdCD276_l showed improved tumor control when compared to conventional CAR T cells expressing AdCD276_l as an scFv. NOD.Cg-PrkdcAscid I12rg tmlWjl / SzI mice were initially inoculated intravenously with 0.5xl06CD276 positive SHP-77 cells. Three days post-inoculation, mice received an injectionof IxlO7AdCAR T cells, either with or without the CD276-specific Fab adapter clone AdCD276 1. For comparison, a cohort was treated with conventional CAR T cells or nontransduced T cells. Additionally, a consistent dosage of 100 pg of the CD276-specific Fab adapter clone AdCD276_l was administered intraperitoneally to the AdCAR T cells cohort daily. Tumor growth of SHP-77 cells was analyzed by BLI imaging. At least five mice were analyzed per cohort and median values of photon flux are plotted ±SEM.

[0018] Figure 8: AdCAR T cells in the presence of AdCD276_l show improved persistance in vivo when compared to conventional CAR T cells expressing AdCD276_l as an scFv. NOD.Cg-PrkdcAscid I12rgAtmlWjl / SzJ mice were initially inoculated intravenously with 0.5xl06CD276 positive SHP-77 cells. Three days post-inoculation, mice received an injection of IxlO7AdCAR T cells, either with or without the CD276-specific Fab adapter clone AdCD276_l. For comparison, a cohort was treated with conventional CAR T cells. Additionally, a consistent dosage of 100 pg of the CD276-specific Fab adapter clone AdCD276_l was administered intraperitoneally to the AdCAR T cells cohort daily. Ex vivo analysis of viable CD45+cells in blood at day 28. Blood samples were analyzed via flow cytometry using a fluorochrome conjugate specific for human CD45. Statical analysis was performed with unpaired two-tailed t-test. Significance defined as: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.Detailed description of the invention

[0019] In a first aspect the present invention provides a combination of compositions comprising: a first composition comprising a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag; and a second composition comprising an immune effector cell expressing a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of said tagged polypeptide.First composition

[0020] As disclosed herein the antigen binding domain of said tagged polypeptide is specific for CD276. CD276 is expressed on the surface of target cells. These target cells may be cancer cells, especially cells or cellular subsets comprised in solid cancer. Commonly known examples for cancers that express CD276 are: breast cancer, prostate cancer, renal cell carcinoma, colorectal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, gastric, glioblastoma, and bladder cancer.

[0021] The antigen binding domain (specific for CD276) of said tagged polypeptide may be, may comprise or may consist of e.g. single domain antibody, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies, diabodies or other alternative antigen binding fragments thereof that bind to a target antigen CD276 expressed on the surface of the target cell.

[0022] In a preferred embodiment of the invention the antigen binding domain of said tagged polypeptide may be a Fab.

[0023] In one embodiment of the invention the antigen binding domain (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) comprising a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) and a light chain variable region of an antibody (VL) comprising a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein a the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 9, 11, 13, 2, 4 and 6 respectively, b the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 23, 25, 27, 16, 18 and 20 respectively, c. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 37, 39, 41, 30, 32 and 34 respectively, d. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 51, 53, 55, 44, 46 and 48 respectively, or e. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO 65, 67, 69, 58, 60 and 62 respectively

[0024] In a preferred embodiment of the invention, the antigen binding domain (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) comprising a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementarydetermining region 3 (HC CDR3) and a light chain variable region of an antibody (VL) comprising a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 9, 11, 13, 2, 4 and 6 respectively.

[0025] In addition to that, said antigen binding domain (specific for CD276) of said tagged polypeptide comprises the heavy chain variable region of an antibody (VH) comprising a first framework region (HFR1), a second framework region (HFR2), a third framework region (HFR3) and a fourth framework region (HFR4) and wherein the light chain variable region of an antibody (VL) comprises a first framework region (LFR1), a second framework region (LFR2), a third framework region (LFR3) and a fourth framework region (LFR4), wherein a. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 8, 10, 12 ,14, 1, 3, 5 and 7 respectively, b. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 22, 24, 26 , 28, 15, 17, 19 and 21 respectively, c. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 36, 38, 40, 42, 29, 31, 33, and 35 respectively, d. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 50, 52, 54, 56, 43, 45, 47, and 49 respectively, or e. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO 64, 66, 68, 70, 57, 59, 61, and 63 respectively

[0026] In a preferred embodiment of the invention the antigen binding domain (specific for CD276) of said tagged polypeptide comprises the heavy chain variable region of an antibody (VH) comprising a first framework region (HFR1), a second framework region (HFR2), a third framework region (HFR3) and a fourth framework region (HFR4) and wherein the light chain variable region of an antibody (VL) comprises a first framework region (LFR1), a second framework region (LFR2), a third framework region (LFR3) and a fourth framework region(LFR4), wherein The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO 8, 10, 12 ,14, 1, 3, 5 and 7 respectively.

[0027] In one embodiment of the invention the antigen binding domain (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) wherein a. VH and VL comprise the amino acid sequences of SEQ ID NO: 71 and 72, respectively, b. VH and VL comprise the amino acid sequences of SEQ ID NO: 73 and 74, respectively, c. VH and VL comprise the amino acid sequences of SEQ ID NO: 75 and 76, respectively, d. VH and VL comprise the amino acid sequences of SEQ ID NO: 77 and 78, respectively, or e. VH and VL comprise the amino acid sequences of SEQ ID NO: 79 and 80, respectively

[0028] In a preferred embodiment of the invention the antigen binding domain (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 71 and 72, respectively

[0029] In one embodiment of the invention the tagged polypeptide (preferentially a Fab) may additionally comprise a constant region of immunoglobulin light chain kappa (e.g. SEQ ID No:81) or immunoglobulin light chain lambda (e.g. SEQ ID NO: 82). In a preferred embodiment of the invention the tagged polypeptide (preferentially a Fab) may additionally comprise a constant region of an immunoglobulin light chain kappa, preferentially this immunoglobulin light chain kappa comprises SEQ ID NO: 81.

[0030] In one embodiment of the invention the tagged polypeptide (preferentially a Fab) may additionally comprise a constant region of immunoglobulin heavy chain (e.g. SEQ ID NO: 83)

[0031] In one embodiment of the invention the tagged polypeptide comprises or consists of a. SEQ ID NO: 84 and 85 b. SEQ ID NO: 86 and 87 c. SEQ ID NO: 88 and 89 d. SEQ ID NO: 90 and 91, or e. SEQ ID NO: 92 and 93

[0032] In a preferred embodiment of the invention the tagged polypeptide comprises or consist of SEQ ID NO: 84 and 85.Tag

[0033] As disclosed herein, the tagged polypeptide comprises a tag. Said tag may be a hapten tag e g. FITC, biotin, PE, streptavidin or dextran. In another embodiment of the invention the tag may be a peptide tag such as c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag. In a preferred embodiment the tag may be biotin or a derivate thereof. In another preferred embodiment the tag may be LC biotin or a derivative thereof. LC biotin is a biotin derivate which additionally comprises a C6 chain for example as disclosed in EP23192028.1 and EP23192030.7

[0034] In a preferred embodiment of the invention, said tag of said tagged polypeptide is biotin or a derivative thereof, more preferentially LC biotin or a derivative thereof. It is understood that the corresponding antigen binding domain of said CAR is specific for biotin or a derivative thereof, more preferentially LC biotin or a derivative thereof.Second composition

[0035] As disclosed herein the second composition comprises an immune effector cell. This immune effector cell may be an immune cell or an immune cell from an immune cell subset, preferentially a T cell, a tumor infiltrating lymphocyte (TIL) or NK cell. In a preferred embodiment of the invention, said immune cell is a T cell.

[0036] These immune effector cells are genetically modified to comprise a chimeric antigen receptor encoded by an exogenous nucleic acid sequence. Such modification may be done by methods known in the art. In a one embodiment of the invention, the exogenous nucleic acid encoding said CAR may be introduced into the immune effector cell by transfection, electroporation or transduction, preferentially viral transduction, more preferentially lentiviral transduction.

[0037] As disclosed herein, the CAR according to the current invention comprises an antigen binding domain specific for said tag of the tagged polypeptide.

[0038] The antigen binding domain may comprise or consist of e.g. single domain antibody, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies, each of which may be specific for the tag of the tagged polypeptide. In a preferred embodiment of the invention, said antigen binding domain may be an scFv.

[0039] Said CAR comprises an antigen binding domain specific for said tag of the tagged polypeptide, a spacer, a transmembrane domain and an intracellular signaling domain.

[0040] In one embodiment of the invention, the CAR may comprise a hinge domain as spacer, wherein the hinge domain may comprise e.g. a sequence of the hinge of CDSalpha or IgG4. In a preferred embodiment of the invention the CAR may comprise the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101).

[0041] Moreover, the CAR comprises a transmembrane domain comprising a sequence of the transmembrane domains from CDSalpha CD28, ICOS or TNFRSF19 preferentially the transmembrane domain is CD8 (SEQ ID NO: 102; SEQ ID NO: 103).

[0042] The intracellular (cytoplasmic) signaling domain comprised in the CAR may comprise at least one primary cytoplasmic signaling domain comprising an immunoreceptor tyrosinebased activation motif (IT AM) and / or at least one co-stimulatory signaling domain.

[0043] The primary cytoplasmic signaling domain of said CAR may be CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109)

[0044] Moreover the at least one co-stimulatory domain of said CAR, may be selected from the group consisting of ICOS, CD154, CD5, CD2, CD46, HVEM, CD8, CD97, TNFRSF18, CD30, SLAM, DAP10, CD64, CD16, CD89, MyD88, KIR-2DS, KIR-3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD27, 0X40, 4-1BB, and CD28. In a preferred embodiment of the invention the at least one costimulatory domain comprised in said CAR may be CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and / or 4-1BB (SEQ ID NO: 106: SEQ ID NO: 107).

[0045] In one embodiment of the invention the CAR may comprise or consist of an antigen binding domain specific for said tag of the tagged polypeptide, the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102: SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-lBB (SEQ ID NO: 106; SEQ ID NO: 107) and a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108: SEQ ID NO: 109).

[0046] In one embodiment of the invention the tag of the tagged polypeptide may be or may comprise biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Based on that, the antigen binding domain of the CAR (expressed by the immune effector cell) comprised in the second composition may be specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof) and comprises a heavy chain variable region of anantibody (VH) which may comprise the amino acid sequence SEQ ID NO: 94 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID NO: 96. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102; SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109).

[0047] Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially for LC biotin or a derivative thereof) and may comprise the amino acid sequence SEQ ID NO: 98. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102; SEQ ID NO: 103), a co-stimulatory of 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) and CD28 (SEQ ID NO: 104; SEQ ID NO: 105), a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109).

[0048] In one embodiment of the invention the CAR binding biotin or a derivative thereof (especially LC biotin or a derivative thereof) may comprise or consist of the amino acid sequence SEQ ID NO: 110.Specific embodiments

[0049] In a specific embodiment of the invention, the combination of compositions comprises a first composition and a second composition. Said first composition comprises a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag. In this embodiment the antigen binding domain, preferentially a Fab (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) comprising a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) and a light chain variable region of an antibody (VL) comprising a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein (a) the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 9, 11, 13, 2, 4 and 6 respectively, (b) theHC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 23, 25, 27, 16, 18 and 20 respectively, (c) the HC CDR1,HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 37, 39, 41, 30, 32 and 34 respectively, (d) the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 51, 53, 55, 44, 46 and 48 respectively, or (e) the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 65, 67, 69, 58, 60 and 62 respectively, and the tag of the tagged polypeptide is biotin or a derivative thereof, preferentially LC biotin or a derivate thereof. The second composition comprises an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof). The antigen binding domain of said CAR comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID NO: 94 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID NO: 96. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102; SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109). Preferentially said CAR has SEQ ID NO: 110; SEQ ID NO: 111.

[0050] In a preferred embodiment of the invention, the combination of compositions comprises a first composition and a second composition. Said first composition comprises a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag. In this embodiment of the invention the antigen binding domain, preferentially a Fab (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) comprising a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) and a light chain variable region of an antibody (VL) comprising a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 9, 11, 13, 2, 4 and 6, respectively; and the tag of the tagged polypeptide is biotin or a derivative thereof,preferentially LC biotin or a derivate thereof. The second composition comprises an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof). The antigen binding domain of said CAR comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID NO: 94 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID NO: 96. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO :102; SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109). Preferentially said CAR has SEQ ID NO: 110; SEQ ID NO: 111.

[0051] In a specific embodiment of the invention, the combination of compositions comprises a first composition and a second composition. Said first composition comprises a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag. In this embodiment of the invention the antigen binding domain, preferentially a Fab (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) wherein (a) VH and VL comprise the amino acid sequences of SEQ ID NO: 71 and 72, respectively, (b) VH and VL comprise the amino acid sequences of SEQ ID NO: 73 and 74, respectively, (c) and VL comprise the amino acid sequences of SEQ ID NO: 75 and 76, respectively, (d) VH and VL comprise the amino acid sequences of SEQ ID NO: 77 and 78, respectively, or (e) VH and VL comprise the amino acid sequences of SEQ ID NO: 79 and 80, respectively, and the tag of the tagged polypeptide is biotin or a derivative thereof, preferentially LC biotin or a derivate thereof. The second composition comprises an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof). The antigen binding domain of said CAR comprises a heavy chain variable region of an antibody (VH) which may comprisethe amino acid sequence SEQ ID NO: 94 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID NO: 96. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102; SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109). Preferentially said CAR has SEQ ID NO: 110; SEQ ID NO: 111.

[0052] In a preferred embodiment of the invention, the combination of compositions comprises a first composition and a second composition. Said first composition comprises a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tag. In this embodiment of the invention the antigen binding domain, preferentially a Fab (specific for CD276) of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) wherein VH and VL comprise the amino acid sequences of SEQ ID NO: 71 and 72, respectively and the tag of the tagged polypeptide is biotin or a derivative thereof, preferentially LC biotin or a derivate thereof. The second composition comprises an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof). The antigen binding domain of said CAR comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID NO: 94 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID NO: 96. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID NO: 100; SEQ ID NO: 101), a transmembrane domain of CD8 (SEQ ID NO: 102; SEQ ID NO: 103), a co-stimulatory of CD28 (SEQ ID NO: 104; SEQ ID NO: 105) and 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID NO: 108; SEQ ID NO: 109). Preferentially said CAR has SEQ ID NO: 110; SEQ ID NO: 111.Pharmaceutical composition

[0053] Another aspect of the invention is a combination of pharmaceutical compositions as disclosed herein.

[0054] In this aspect of the invention the combination of pharmaceutical compositions may optionally comprise a pharmaceutical acceptable carrier.

[0055] Pharmaceutical acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0056] Said combination of pharmaceutical composition may be used for the treatment of cancer in a subject suffering from cancer, wherein said cancerous cells of said cancer express CD276. Said cancer may be selected from the group breast cancer, prostate cancer, renal cell carcinoma, colorectal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, gastric, glioblastoma, and bladder cancer glioblastoma. In a preferred embodiment, said cancer is glioblastoma, ovarian cancer and pancreatic cancer .

[0057] In another embodiment the invention provides a combination of pharmaceutical compositions as disclosed herein, wherein said pharmaceutical compositions may comprise a pharmaceutically acceptable carrier and a chemotherapeutical agent for combined treatment of said cancer.Combination of compositions for use in treatment of a disease

[0058] Another aspect of the invention provides a combination of compositions for use in the treatment of a disease according to the features and embodiments disclosed herein. This aspect of the invention provides a combination of compositions for treating a disease in a subject suffering from said disease. In addition to that, this aspect of the invention provides a combination of compositions for use in the treatment of a cancer, an autoimmune disease, an allergic disease, an infectious disease or a graft rejection.

[0059] In one embodiment the invention provides a combination of compositions for use in the treatment of CD276 expressing cancer, preferentially solid cancer. In a preferred embodiment the invention provides a combination of compositions for use in the treatment of cancer, wherein said cancer may be selected from the group consisting pancreatic cancer, ovarian cancer und glioblastoma

[0060] Another embodiment of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering to a subject in need thereof: A first composition comprising a tagged polypeptide comprising an antigen binding domain specific CD276 and a tag and a second composition comprising an immune effectorcell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said tag of said tagged polypeptide.

[0061] Several treatment strategies may be beneficial for the patient.

[0062] One embodiment of the invention is a combination of compositions for use in a method of treating a disease and each composition may be administered separately to the patient. In one embodiment of the invention the first composition may be administered first to the patient. The second composition may be administered separately in specific time intervals.

[0063] In another embodiment the first and second composition may be administered at the same time.

[0064] In yet another embodiment of the invention, the second composition be administered first to the patient, followed by the administration of the first composition.

[0065] In addition to that, the initial administration of the tagged polypeptides (first composition) to the patient, may have the advantage that the tagged polypeptides may have bound the target already before administration of the CART cell expressing immune effector cell.

[0066] In one embodiment of the invention, the compositions as disclosed herein comprising immune cells expressing the CAR (anti tag CAR) as disclosed herein may be for use e.g. in the treatment of a subject suffering from cancer or an autoimmune disease or an allergic disease or an infectious disease or a graft rejection as disclosed herein. The subject may suffer from said cancer (a patient) or said autoimmune disease or may be a healthy subject. These immune cells are genetically modified (in vitro) to express said CAR as disclosed herein. These engineered cells may be activated and expanded in vitro to a therapeutically effective population of expressing cells. In cellular therapy these engineered cells may be infused to a recipient in need thereof as a pharmaceutical combination of composition as disclosed herein. The infused cells in the recipient may be e.g. able to kill (or at least stop growth of) cancerous cells expressing the antigen which is recognized by the CAR system as disclosed herein or may reduce the effect of the autoimmune disease. The recipient may be the same subject from which the cells were obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).

[0067] Populations of said CAR expressing (immune) cells may be formulated for administration to a subject using techniques known to the skilled artisan.

[0068] A formulation of a therapeutically effective population(s) of said CAR expressing cells may include one population of said CAR expressing (immune) cells, or more than one population of said CAR expressing (immune) cells. The different populations of said CARexpressing (immune) cells may vary based on the identity of the activation domain, the identity of the (sub)population of immune cells, or a combination thereof.

[0069] The formulations comprising therapeutically effective population(s) of said CAR expressing cells may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, without limitation, intratumoral, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m ), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0070] The formulations comprising therapeutically effective population(s) of said CAR expressing cells that are administered to a subject comprise a number of said CAR expressing cells such immune cells that is effective for the treatment of the specific indication or disorder.

[0071] In general, formulations may be administered that comprise between about 1 x 104and about 1 x 1010said CAR expressing cells such as immune cells. In most cases, the formulation may comprise between about 1 x 105and about 1 x 109said CAR expressing cells such as immune cells, from about 5 x 105to about 5 x 108said CAR expressing cells such as immune cells, or from about 1 x 106to about 1 x 107said CAR expressing cells such as immune cells. However, the number of said CAR expressing cells such as immune cells administered to a subject may vary between wide limits, depending upon the location, source, identity, extent and severity of the disorder, the age and condition of the individual to be treated, etc. A physician may ultimately determine appropriate dosage.

[0072] The tagged polypeptide(s) as disclosed herein may be formulated for administered to a subject using techniques known to the skilled artisan. Formulations of the tagged polypeptide(s) may include pharmaceutically acceptable excipient(s) (carriers or diluents). Excipients included in the formulations will have different purposes depending, for example, on the nature of the tag, the antigen binding domain of the tagged polypeptide, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-inj ection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0073] The tagged polypeptide(s) may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous, intraperitoneal, and intratumoral injection. Other modes include, without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intra-arterial, intramedulary,intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0074] Formulations comprising the polypeptide(s) are administered to a subject in an amount which is effective for treating the specific indication or disorder. In general, formulations comprising at least about 1 pg / kg to about 100 mg / kg body weight of the tagged polypeptide(s) may be administered to a subject in need of treatment. In most cases, the dosage may be from about 100 pg / kg to about 10 mg / kg body weight of the tagged polypeptide(s) daily, taking into account the routes of administration, symptoms, etc. The amount of tagged polypeptide(s) in formulations administered to a subject may vary between wide limits, depending upon the location, source, identity, extent and severity of the disorder, the age and condition of the individual to be treated, etc. A physician may ultimately determine appropriate dosages to be used.

[0075] The timing between the administration of the CARs expressing cell formulation and the tag polypeptide-formulation may range widely depending on factors that include the type of (immune) cells being used, the binding specificity of the CARs, the identity of the tag, the antigen binding moiety of the tagged polypeptide, the identity of said soluble antigen, the identity of the target cell, e.g. cancer cell to be treated, the location of the target cell in the subject, the means used to administer the formulations to the subject, and the health, age and weight of the subject being treated. Indeed, the tagged polypeptide formulation may be administered prior to, simultaneous with, or after the genetically engineered (immune) cell formulation.

[0076] Depending on the disorder being treated the step of administering the CARs expressing cell formulation, or the step of administering the tagged polypeptide formulation, or both, can be repeated one or more times. When two or more formulations of engineered cells such as immune cells expressing the CARs of the invention are applied to a subject, the engineered cells may be of the same cell type or of different cell types, e.g. T cells and / or NK cells. A formulation of cells such as immune cells may also comprise more than one cell type, each expressing the CARs of the invention.

[0077] All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.Definitions

[0078] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0079] The term CD276 or B7H3 (B7-H3) can be used interchangeably. It is understood that this also includes other state of the art synonyms. CD276 is predominantly expressed in cancer cells, such as cells comprised in solid cancers. CD276 expressing cells can be found in breast cancer, prostate cancer, renal cell carcinoma, colorectal cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, gastric, glioblastoma, and bladder cancer .

[0080] The term “antigen binding domain refers” to a region of a protein or a polypeptide (generally the variable region of an antibody or fragment thereof) that specifically binds to an antigen. It is composed of one constant and one variable domain of each of the heavy and the light chain.

[0081] Chimeric antigen receptors (CARs) are recombinant receptors for antigens which redirect the specificity and function of T lymphocytes and / or other immune cells in a single molecule. The concept of using CARs in immunotherapy is that CARs, which are programmed targeting tumor-associated antigens or disease associated antigens, can be replicated rapidly and homogeneously.

[0082] In general, a CAR may comprise an extracellular domain (extracellular part) comprising an antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide.

[0083] The CAR as disclosed herein may be referred to as “anti-tag” CAR or “adapterCAR” or “universal CAR” or “adCAR” as disclosed e.g. in US9233125B2. Such a CAR has a similar domain structure compared to the standard direct CAR, besides that the adapter CAR binds a target antigen indirectly by binding an adapter molecule ( tagged polypeptide) which binds to the target antigen.

[0084] A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0085] Generally, an antigen binding domain of a CAR refers to the region that specifically binds to an antigen. In this invention the CAR binds the adapter / tagged polypeptide. The CARs of the invention may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions of the CAR are localized extracellularly. The antigen binding domain may comprise an antibody, single domain antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3 -linker” or a “whitlow linker”.

[0086] “Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain of the CAR and the transmembrane domain. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge or IgG4 hinge.

[0087] The “transmembrane domain” of the CAR may be from any desired natural or synthetic source for such domain. When the source is natural the domain may be from any membranebound or transmembrane protein. The transmembrane domain may be for example from CD8alpha or CD28.

[0088] The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR).

[0089] "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions. Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

[0090] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0091] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs). Examples of ITAM containing primary cytoplasmic signaling domains often used in CARs are that those from TCRzeta (CD3zeta), FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence from CD3zeta.

[0092] The cytoplasmic domain of the CAR may be designed to comprise the CD3zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3zeta chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0093] The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0094] As mentioned above, according to the present invention, the antigen binding domain of a CAR binds a tag or hapten that is coupled to a protein or polypeptide (“haptenylated” or “tagged” polypeptide / Protein), wherein the polypeptide may bind to a disease-associated antigen such as a tumor associated antigen (TAA).

[0095] The haptens or tags may be coupled directly or indirectly to a polypeptide (the tagged polypeptide), wherein the polypeptide may bind to said disease associated antigen expressed on the (cell) surface of a target. The tag may be e.g. dextran or a hapten such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamin, but the tag may also be apeptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The tag may also be streptavidin. The tag portion of the tagged polypeptide is only constrained by being a molecule that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR. For example, when the tag is FITC (Fluorescein isothiocyanate), the tag-binding domain may constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv, respectively.

[0096] The term “tagged protein” or “tagged polypeptide” as used herein refers to a polypeptide that has bound thereto directly or indirectly at least one additional component, i.e. the tag. The tagged polypeptide as used herein is able to bind an antigen expressed on a target cell. The polypeptide may be an antibody or antigen binding fragment thereof that binds to an antigen expressed on the surface of a target cell such as a tumor associated antigen (TAA) on a cancer cell.

[0097] The terms “adapter” or “adapter molecule” or “tagged polypeptide” as used herein may be used interchangeably. The tag may be e.g. a hapten or dextran and the hapten or dextran may be bound by the antigen binding domain a CAR comprising an antigen binding domain specific for the tag. Haptens such as e.g. FITC, biotin, PE, thiamin, streptavidin or dextran are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.

[0098] But the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The peptide may be selected from the group consisting of c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag. The tag may also be streptavidin. The tag portion of the tagged polypeptide is only constrained by being a molecular that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR.

[0099] The term “engineered” as used herein can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. An “engineered cell” or “genetically modified cell” can refer to a cell with an added, deleted and / or altered gene. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. As disclosed herein, cells are also engineered to comprise an inducible nucleic acid construct.

[0100] The engineered cell expressing a CAR may be further modified by genetic engineering using methods well known in the art e.g. Meganucleases, TALEN, CrisprCas, zink finger nucleases, shRNA and / or miRNA. Said cells may be modified to reduce or lack expression of a specific gene, which is normally expressed in the cell e.g. T cell receptor (TCR), MHC, co- inhibitory molecules likePD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, HVEM.

[0101] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune effector cells are T cells and NK cells. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0102] The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody, of a fragment thereof or of a CAR refer to an antigenbinding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0103] As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, haptens and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0104] The tumor associated antigen (TAA) as used herein refers to an antigenic substance produced by tumor cells. Tumor associated antigens are useful tumor or cancer markers in identifying tumor / cancer cells with diagnostic tests and are potential candidates for use in cancer therapy. Preferentially, the TAA may be expressed on the cell surface of the tumor / cancer cell, so that it may be recognized by the antigen binding receptor as disclosed herein.

[0105] The term “target cell” as used herein refers to cell which expresses an antigen on its cell surface that should be recognized (bound) by the protein according to the current invention. Said target cell may be e.g. a cancerous cell or a cell associated with an autoimmune disease or a cell associated with an infectious disease

[0106] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.

[0107] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0108] The terms “therapeutically effective amount” or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.

[0109] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer.

[0110] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0111] The term “cancer” is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

[0112] Autoimmune diseases are a condition arising from autoimmunity or disbalance in the immune homeostasis resulting in pathologies that can affect multiple different organ systems.

[0113] The terms “nucleic acid” or “polynucleotide” can be as used interchangeably herein refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric“nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.Examples

[0114] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.Example 1 Generation of Anti-Tag CAR T CellsConstruct Design:

[0115] The AdCAR system as disclosed in EP23192028.1 and EP23192030.7 was used. This is a specific adapter CAR system which is also known as LLE CAR system. The tags and the binding moieties of the adCAR are identical to the ones disclosed in the cited patent applications. For sake of simplicity the binding moieties are referred to as biotin binding moiety and the tag moiety is referred to a biotin tag.

[0116] AdCAR T cells contained an anti-biotin scFv as a binding moiety (SEQ ID N: 98; SEQ ID NO: 99) The scFv was linked to the human CD8 (SEQ ID NO: 102; SEQ ID NO: 103) transmembrane domain via hIgG4 hinge domain (SEQ ID NO: 100; SEQ ID NO: 101). The signaling domain was composed of CD28 (SEQ ID NO: 104; SEQ ID NO: 105), 4-1BB (SEQ ID NO: 106; SEQ ID NO: 107) and 3^ (SEQ ID NO: 108; SEQ ID NO: 109) .

[0117] In the following examples the sequences of AdCD276 constructs (Fabs) AdCD276_l- 5 correspond to the sequences a-e, respectively as disclosed herein and as claimed. More specifically the sequence of AdCD276_l (a) is SEQ ID NO: 84 and 85; AdCD276_2 (b) is SEQ ID NO: 86 and 87; AdCD276_3 (c) is SEQ ID NO: 88 and 89; AdCD276_4 is SEQ ID NO: 90 and 91; AdCD276_5 is SEQ ID NO: 92 and 93). The other AdCD276 constructs (Fabs) refer to the VH / VL sequences as presented in SEQ ID NO: 114 to SEQ ID NO: 193.Generation of LV Particles and Titration:

[0118] Lentiviral vector particles were manufactured via transient transfection of HEK-293T cells. The lentiviral vector particles were pseudotyped with VSV-G. For transfection, HEK- 293T cells were seeded in T175 culture flasks in DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) 3 days before transfection. On the day oftransfection, the culture medium was removed and replaced by DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza). The cells were transfected with a three-plasmid system encoding for VSV-G, gag / pol / rev and the psi positive transfer vector. After 48 h the supernatant was collected and centrifuged for 10 min at 1000 rpm to remove cellular debris. In addition, the supernatant was filtrated through a 0.45 pm filter. The pellet was re-suspended in ice-cold PBS and stored at -80° C.

[0119] A functional titer of VSV-G pseudotyped lentiviral vector particles was determined via titration on Sup-Tl cells. 2xl05cells were seeded in 150 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) in a 96 well-round bottom plates. For transduction 50 pL of serial diluted lentiviral vector particles were added to the seeded cells. 90 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) was added after 24 h. The frequency of transduced cells was quantified after 96 h by flow cytometry using a biotin CAR detection reagent (Miltenyi Biotec). Based on the frequency of positive cells, the number of seeded cells and the volume of lentiviral particles used for transduction, the titer was calculated. The titer was expressed in transducing units per mL.Manufacturing of AdCAR T cells:

[0120] Initially, 2xl06T cells were plated in a 24-well plate with 2 mL of TexMACS™ medium, enriched with 12.5 ng / mL of both recombinant human IL-7 and IL-15. The cells were then activated using a 1 :100 dilution of TransAct™. After 24 hours, lentiviral vectors (LVVs) were introduced to achieve a multiplicity of integration (MOI) of 10, followed by a resuspension of the cells. On the third day, the old medium was completely replaced with fresh medium. Subsequently, the T cells were split at a 1 :2 ratio every other day starting from day three. This expansion process continued for a minimum of ten days before the T cells were utilized in any functional assays. Cell concentration was analyzed by flow cytometry using propidium iodide to discriminate live and dead cells.Example 2 Screening of CD276 specific polypetides with enhanced tumor cell lysis

[0121] The CD276-specific Fab molecules were used as adapter moieties for screening adapter CAR T cells (sAdCAR) specifically designed for the screening of a large number of adapter moieties. The sAdCAR T cells bind a screening tag moiety which is recombinantly attached to the Fabs. This allows the test of a large number of different CD276-specific Fab molecules. In total 47 different CD276-specific Fabs were tested to identify the most potent variants.

[0122] The functionality of CD276-specific Fab molecules was tested in vitro by co-culturing CD276 expressing tumor cell lines with CD276-specific Fab molecules and sAdCAR T cells. First, 2xl04tumor cells were seeded in a flat bottom cell culture plate. The cells were cultured for 4-6h at 37°C and 5%CO2. Next, AdCAR T cells were added at an E:T ratio of 2.5 : 1. Finally, InM of CD276-specific Fab molecules were added to the co-culture. After 24h the amount of viable tumor cells was analyzed via flow cytometry. AdCD276_l-5 induced superior tumor cell lysis compared to all other CD276-specific adapter molecules (Figure 1).

[0123] In addition to the tumor lysis of antigen positive tumor cells the cytokine secretion after co-culture was analyzed. After 24h the cell culture supernatant was harvested and analyzed using the MACSPlex cytokine Kit (Miltenyi Biotec). AdCD276_l-5 were the top five CD276- specific adapters to induce the secretion of IFN-y. The highest IFN-y was induced with AdCD276_l. The cytokine secretion validates the tumor-killing data and together this demonstrates that AdCD276_l-5 are outperforming all other tested CD276-specific adapter molecules when used in combination with AdCAR T cells (Figure 2).Example 3 Developability of CD276 specific polypeptides

[0124] CD276-specific Fab molecules were produced by using the ExpiCHO™ system according to the manufactures instructions. In short, 6xl06viable cells were re-suspended in ExpiCHO™ Expression Medium, pre-warmed to 37°C. To prepare the ExpiFectamine™ CHO / plasmid DNA complexes, the plasmid DNA was diluted with cold OptiPRO™ medium. Next, the ExpiFectamine™ CHO Reagent was diluted in the same medium, and mixed. Afterwards, the diluted ExpiFectamine™ CHO Reagent was combined with the diluted DNA, mixing gently. This mixture was stored at room temperature for 1-5 minutes. Then, the mixture was transferred to a shaker flask. Finally, the cells were incubated at 37°C in a humidified atmosphere containing 8% CO2, using an orbital shaker for agitation. One day after transfection the appropriate amount of ExpiCHO enhancer was added to the cell culture according to the manufacturer’s instructions. On day 8 the cell culture supernatant was harvested and the CD276-specific Fabs were purified using the CaptureSelect CH1-XL system according to the manufacturer’s instructions.

[0125] The developability of each Fab was evaluated based on its protein titer and thermostability.

[0126] To determine the protein yield after purification the OD280 was measured. The final yield was calculated using the protein-specific extinction coefficient. The melting point of each Fab molecule was quantified using the NanoTemper Tycho device.

[0127] The protein yield and the melting point showed a positive linear correlation. The Fab with the lowest protein yield was AdCD276 3 with approx. 3mg / mL. In contrast to this AdCD276_l showed a titer of approx. 30mg / mL. Overall, the AdCD276_l showed the highest thermostability and yield after protein production and purification indicating its superior developability for later therapeutic use compared to AdCD276 2-5 (Figure 3).Example 4 Induction of tumor cell lysis via CD276 specific polypetides

[0128] The CD276-specific Fab molecules were used as adapter moieties for biotin-specific adapter CAR T cells (AdCAR). Therefore CD276-specific Fab molecules were conjugated with the LC -biotin. The conjugation was a site-specific crosslinking of heavy and light chain.

[0129] The functionality of CD276-specific Fab molecules was tested in vitro by co-culturing CD276 expressing tumor cell lines with CD276-specific Fab molecules (AdCD276_l-5, AdCD276_l (a) SEQ ID NO: 84 and 85; AdCD276_2 (b): SEQ ID NO: 86 and 87; AdCD276_3 (c) SEQ ID NO: 88 and 89; AdCD276_4 (d) SEQ ID NO: 90 and 91; AdCD276_5 (e) SEQ ID NO: 92 and 93) and AdCAR T cells.

[0130] First, IxlO4luciferase-expressing tumor cells were seeded in a flat bottom cell culture plate. The cells were cultured for 4-6h at 37°C and 5%CO2 until they were attached. Next, AdCAR T cells were added at an E:T ratio of 4: 1.

[0131] Finally, 5nM of CD276-specific Fab molecules were added to the co-culture. As a negative control, a Fab molecule specific for CD33, an antigen that is not expressed on the surface of the tumor cells was used.

[0132] After 96h luciferin (4pg / ml) was added and the bioluminescence was measured. The lysis of CD276-expressing tumor cells was calculated by normalizing the bioluminescence to the signal of the control condition, containing only tumor cells.

[0133] CD276-specific Fab molecules induced significant AdCAR T cell-mediated lysis in a rhabdomyosarcoma cell line (Figure 4a), a neuroblastoma cell line (Figure 4b) and small cell lung cancer cell-line (Figure 4c) compared to the control Fab. The data indicate specific binding of the CD276-specific Fab molecules to antigen-positive tumor cells and subsequent induction of AdCAR-mediated tumor cell lysis. The CD276-specific Fab molecules tagged with LC- biotin induced comparable tumor lysis via AdCAR T cells. These results proof the mode of action of the CD276-specific Fab molecules.Example 5 Induction of cytokine secretion via CD276-specific polypeptides

[0134] In addition to tumor cell lysis CD276-specific Fab molecules (AdCD276 1-5, AdCD276_l (a) SEQ ID NO: 84 and 85; AdCD276_2 (b): SEQ ID NO: 86 and 87; AdCD276_3 (c) SEQ ID NO: 88 and 89; AdCD276_4 (d) SEQ ID NO: 90 and 91; CD276_5 (e) SEQ ID NO: 92 and 93) were able to induce cytokine secretion after co-culturing AdCAR T cells and CD276-postive DMS-114 cells. The secretion of IFN-y was analyzed after 24h.

[0135] First, IxlO4luciferase-expressing tumor cells were seeded in a flat bottom cell culture plate. The cells were cultured for 4-6h at 37°C and 5%CO2 until they were attached. Next, AdCAR T cells were added at an E:T ratio of 4: 1. Finally, 5nM of CD276-specific Fab molecules were added to the co-culture. As a negative control, a Fab molecule specific for CD33, an antigen that is not expressed on the surface of the tumor cells was used.

[0136] All tested CD276-specific Fab molecules showed enhanced cytokine secretion compared to the non-tumor-binding CD33 control Fab (Figure 5). These results validate the mode of action of the CD276-specific Fab molecules.Example 6 Conventional CD276-specific CAR T cells show decreased proliferation during manufacturing

[0137] Although CD276 is a promising target for CAR T cell therapy due to its widespread expression on tumor tissue the drawback of this target is its expression on activated lymphocytes such as CAR T cells e.g. during manufacturing.

[0138] To compare the targeting of CD276 either via conventional CARs directly binding to CD276 expressed on the cell surface or via the AdCAR concept the variable light chain and the variable heavy chain of clone AdCD276_l ((a) SEQ ID NO: 84 and 85; were converted into an scFv (LC-3xG4S-HC) and cloned into the same 3rd generation CAR backbone as the AdCAR (SEQ ID NO: 112; 113).

[0139] Based on the observation that all CD276-specific Fab molecules performed similarly in vitro the AdCD276_l was chosen to be tested in vivo as a representative of the other clones.

[0140] Lentiviral vectors of both CAR constructs were manufactured. Primary human T cells were transduced as described in example 1. The cell count of viable human T cells was monitored during the manufacturing process via flow cytometry. Conventional CAR T cells expressing the AdCD276_l as an scFv showed significantly fewer T cells when compared to AdCAR T cells or non-transduced T cells (Figure 6). This indicates potential fratricide induced by CAR T cells expressing the conventional CD276 CAR. On the other hand the cell growth of the AdCAR T cells in absence of a CD276-specfic adapter was not impaired. This demonstratedthe advantage of the AdCAR system when targeting CD276 as it will lead to higher cell count during the manufacturing and less pre-exhausted cells. AdCAR CAR T cells will not lyse activated CD276 expressing lymphocytes during the manufacturing of the drug product.Example 7 AdCAR T cells targeting CD276 via antigen-specific polypeptides show improved tumor control in vivo compared to conventional CAR T cells targeting the same

[0141] The tumor lysis of AdCAR T cells targeting CD276 via the CD276-specific Fab adapter clone AdCD276_l ((a) SEQ ID NO: 84 and 85)and conventional CAR T expressing the clone AdCD276_l as an scFv was compared in a SCLC xenograft model.

[0142] Therefore, female immunodeficient NOD.Cg-Prkdcscid H2rgtmlWjl / SzJ (NSG) were inoculated with 0.5xl06CD276 positive SHP-77 i.v. and 3 days later the mice were injected with IxlO7AdCAR T cells with or without CD276-specific Fab adapter clone AdCD276_l ((a) SEQ ID NO: 84 and 85), conventional CAR T cells or non-transduced T cells.

[0143] A total amount of lOOpg of CD276-specificFab adapter clone AdCD276_l was injected i.p. every day. The tumor growth was monitored via bioluminescent imaging

[0144] Both, the AdCAR T cells and the conventional CAR T cells were targeting the same CD276 epitope and had the same CAR architecture. The only difference between both approaches was the structure of the targeting moiety. In case of the AdCAR it was a Fab (AdCD276_l) in case of the conventional CAR it was a CD276-specific scFv derived from AdCD276_l covalently linked to the CAR signaling architecture. The Fab adapter and the scFv were derived from the same clone and therefore have the same paratope and bind the same epitope.

[0145] In the presence of a CD276-specific Fab adapter the adapter the AdCAR T cells showed a reduced tumor growth as indicated by a decreased BLI-signal. The conventional CAR T cell showed minor tumor control.

[0146] Surprisingly, AdCAR T cells in the presence of CD276-specific Fab adapter clone AdCD276_l showed better tumor control compared to conventional CAR T cells expressing AdCD276_l as an scFv. Although targeting the same epitope AdCAR T cells and AdCD276_l outperformed its conventional CAR counterpart (Figure 7).

[0147] These results demonstrated that targeting CD276 with conventional CAR T cells is less effective than compared the AdCAR system in combination of AdCD276_l.Example 8 AdCAR T cells targeting CD276 via antigen-specific polypeptides show improved persistence in vivo compared to conventional CAR T cells targeting the same epitope

[0148] At the end of the study described in example 7 the persistence of AdCAR T cells in the presence of AdCD276 1 ((a) SEQ ID NO: 84 and 85) and conventional CAR T cells expressing AdCD276_l as an scFv was analyzed via flow cytometry.

[0149] Animal cohorts treated with AdCAR T cells in the presence of AdCD276_l showed significantly more human CD45+cells in the blood than conventional CAR T cells expressing AdCD276_l as an scFv (Figure 8). The data indicate that CD276-specific Fab adapters can be used to target antigen-positive tumors with AdCARs but the same binding molecules are nonfunctional when used as scFv binding moieties for conventional CARs as both, tumor control and expansion are limited in vivo. These data also validate the observation seen during the manufacturing process described in example 6. That activated CD276+lymphocytes get lysed by CD276 CAR expressing T cells when an scFv is used as a target moiety. Surprisingly, the same antibody clone cloned used as a Fab adapter for AdCAR T cells does not induce killing of non- tumor cells, such as activated lymphocytes.

[0150] Description of the sequences of the sequence protocolSEQ ID NO 1 : Adi LFR1 SEQ ID NO 2: Adi CDR-L1 SEQ ID NO 3: Adi LFR2 SEQ ID NO 4: Adi CDR-L2 SEQ ID NO 5: Adi LFR3 SEQ ID NO 6: Adi CDR-L3 SEQ ID NO 7: Adi LFR4 SEQ ID NO 8: Adi HFR1 SEQ ID NO 9: Adi CDR-H1 SEQ ID NO 10: Adi HFR2 SEQ ID NO 11: Adi CDR-H2 SEQ ID NO 12: Adi HFR3 SEQ ID NO 13: Adi CDR-H3 SEQ ID NO 14: Adi HFR4 SEQ ID NO 15: Ad2 LFR1 SEQ ID NO 16 Ad2 CDR-L1 SEQ ID NO 17: Ad2 LFR2 SEQ ID NO 18: Ad2 CDR-L2 SEQ ID NO 19: Ad2 LFR3 SEQ ID NO 20: Ad2 CDR-L3 SEQ ID NO 21: Ad2 LFR4 SEQ ID NO 22: Ad2 HFR1) SEQ ID NO 23: Ad2 CDR-H1 SEQ ID NO 24: Ad2 HFR2SEQ ID NO 25: Ad2 CDR-H2 SEQ ID NO 26: Ad2 HFR3 SEQ ID NO 27: Ad2 CDR-H3 SEQ ID NO 28: Ad2 HFR4 SEQ ID NO 29: Ad3 LFR1 SEQ ID NO 30: Ad3 CDR-L1 SEQ ID NO 31: Ad3 LFR2 SEQ ID NO 32: Ad3 CDR-L2 SEQ ID NO 33: Ad3 LFR3 SEQ ID NO 34: Ad3 CDR-L3 SEQ ID NO 35: Ad3 LFR4 SEQ ID NO 36: Ad3 HFR1 SEQ ID NO 37: Ad3 CDR-H1 SEQ ID NO 38: Ad3 HFR2 SEQ ID NO 39: Ad3 CDR-H2 SEQ ID NO 40: Ad3 HFR3 SEQ ID NO 41: Ad3 CDR-H3 SEQ ID NO 42: Ad3 HFR4 SEQ ID NO 43: Ad4 LFR1 SEQ ID NO 44: Ad4 CDR-L1 SEQ ID NO 45: Ad4 LFR2 SEQ ID NO 46: Ad4 CDR-L2 SEQ ID NO 47: Ad4 LFR3 SEQ ID NO 48: Ad4 CDR-L3 SEQ ID NO 49: Ad4 LFR4 SEQ ID NO 50: Ad4 HFR1 SEQ ID NO 51: Ad4 CDR-H1 SEQ ID NO 52: Ad4 HFR2 SEQ ID NO 53: Ad4 CDR-H2 SEQ ID NO 54: Ad4 HFR3 SEQ ID NO 55: Ad4 CDR-H3 SEQ ID NO 56: Ad4 HFR4 SEQ ID NO 57: Ad5 LFR1 SEQ ID NO 58: Ad5 CDR-L1 SEQ ID NO 59: Ad5 LFR2 SEQ ID NO 60: Ad5 CDR-L2 SEQ ID NO 61: Ad5 LFR3 SEQ ID NO 62: Ad5 CDR-L3 SEQ ID NO 63: Ad5 LFR4 SEQ ID NO 64: Ad5 HFR1 SEQ ID NO 65: Ad5 CDR-H1 SEQ ID NO 66: Ad5 HFR2 SEQ ID NO 67: Ad5 CDR-H2 SEQ ID NO 68: Ad5 HFR3 SEQ ID NO 69: Ad5 CDR-H3 SEQ ID NO 70: Ad5 HFR4 SEQ ID NO 71: Adi VH SEQ ID NO 72: Adi VL SEQ ID NO 73: Ad2VH SEQ ID NO 74: Ad2VL SEQ ID NO 75: Ad3VHSEQ ID NO 76: Ad3VL SEQ ID NO 77: Ad4VH SEQ ID NO 78: Ad4VL SEQ ID NO 79: Ad5VH SEQ ID NO 80: Ad5VL SEQ ID NO 81 : kappa SEQ ID NO 82: Lambda SEQ ID NO 83: CHI SEQ ID NO 84: Adi LC SEQ ID NO 85: Adi HC SEQ ID NO 86: Ad2 LC SEQ ID NO 87: Ad2 HC SEQ ID NO 88: Ad3 LC SEQ ID NO 89: Ad3 HC SEQ ID NO 90: Ad4 LC SEQ ID NO 91: Ad4 HC SEQ ID NO 92: Ad5 LC SEQ ID NO 93: Ad5 HC SEQ ID NO 94: VH CAR SEQ ID NO 95: VH CAR SEQ ID NO 96: VL CAR SEQ ID NO 97: VL CAR SEQ ID NO 98: full binder SEQ ID NO 99: full binder SEQ ID NO 100: hIgG4 hinge SEQ ID NO 101: h!gG4 hinge SEQ ID NO 102: CD8 TM SEQ ID NO 103: CD8 TM SEQ ID NO 104: CD28 SEQ ID NO 105: CD28 SEQ ID NO 106: 4-1BB SEQ ID NO 107: 4-1BB SEQ ID NO 108: CD3 zeta SEQ ID NO 109: CD3 zeta SEQ ID NO 110: Adapter CAR SEQ ID NO 111: Adapter CAR SEQ ID NO 112: CAR directly binding to CD276 SEQ ID NO 113: CAR directly binding to CD276 SEQ ID NO 114: AdCD276_7 (VH) SEQ ID NO 115: AdCD276_7 (VL) SEQ ID NO 116: AdCD276 8 (VH) SEQ ID NO 117: AdCD276_8 (VL) SEQ ID NO 118: AdCD276_9 (VH) SEQ ID NO 119: AdCD276_9 (VL) SEQ ID NO 120: AdCD276_10 (VH) SEQ ID NO 121: AdCD276_10 (VL) SEQ ID NO 122: AdCD276_l l (VH) SEQ ID NO 123: AdCD276_l l (VL) SEQ ID NO 124: AdCD276_12 (VH) SEQ ID NO 125: AdCD276 12 (VL) SEQ ID NO 126: AdCD276_13 (VH)SEQ ID NO 127: AdCD276_13 (VL) SEQ ID NO 128: AdCD276_14 (VH) SEQ ID NO 129: AdCD276_14 (VL) SEQ ID NO 130: AdCD276_15 (VH) SEQ ID NO 131: AdCD276_15 (VL) SEQ ID NO 132: AdCD276_16 (VH) SEQ ID NO 133: AdCD276 16 (VL) SEQ ID NO 134: AdCD276_17 (VH) SEQ ID NO 135: AdCD276_17 (VL) SEQ ID NO 136: AdCD276_18 (VH) SEQ ID NO 137: AdCD276_18 (VL) SEQ ID NO 138: AdCD276_19 (VH) SEQ ID NO 139: AdCD276_19 (VL) SEQ ID NO 140: AdCD276_20 (VH) SEQ ID NO 141: AdCD276_20 (VL) SEQ ID NO 142: AdCD276_21 (VH) SEQ ID NO 143: AdCD276_21 (VL) SEQ ID NO 144: AdCD276_22 (VH) SEQ ID NO 145: AdCD276_22 (VL) SEQ ID NO 146: AdCD276_23 (VH) SEQ ID NO 147: AdCD276_23 (VL) SEQ ID NO 148: AdCD276_24 (VH) SEQ ID NO 149: AdCD276_24 (VL) SEQ ID NO 150: AdCD276 25 (VH) SEQ ID NO 151: AdCD276_25 (VL) SEQ ID NO 152: AdCD276_26 (VH) SEQ ID NO 153: AdCD276_26 (VL) SEQ ID NO 154: AdCD276_27 (VH) SEQ ID NO 155: AdCD276_27 (VL) SEQ ID NO 156: AdCD276_28 (VH) SEQ ID NO 157: AdCD276_28 (VL) SEQ ID NO 158: AdCD276_29 (VH) SEQ ID NO 159: AdCD276 29 (VL) SEQ ID NO 160: AdCD276_30 (VH) SEQ ID NO 161: AdCD276_30 (VL) SEQ ID NO 162: AdCD276_31 (VH) SEQ ID NO 163: AdCD276_31 (VL) SEQ ID NO 164: AdCD276_32 (VH) SEQ ID NO 165: AdCD276_32 (VL) SEQ ID NO 166: AdCD276_33 (VH) SEQ ID NO 167: AdCD276 33 (VL) SEQ ID NO 168: AdCD276_35 (VH) SEQ ID NO 169: AdCD276_35 (VL) SEQ ID NO 170: AdCD276_36 (VH) SEQ ID NO 171: AdCD276_36 (VL) SEQ ID NO 172: AdCD276_37 (VH) SEQ ID NO 173: AdCD276_37 (VL) SEQ ID NO 174: AdCD276_38 (VH) SEQ ID NO 175: AdCD276_38 (VL) SEQ ID NO 176: AdCD276 39 (VH) SEQ ID NO 177: AdCD276_39 (VL)SEQ ID NO: 178 AdCD276_40 (VH)SEQ ID NO: 179 AdCD276_40 (VL)SEQ ID NO: 180 AdCD276_41 (VH)SEQ ID NO: 181 AdCD276_41 (VL)SEQ ID NO: 182 AdCD276_42 (VH)SEQ ID NO: 183 AdCD276_42 (VL)SEQ ID NO: 184 AdCD276 43 (VH)SEQ ID NO: 185 AdCD276_43 (VL)SEQ ID NO: 186 AdCD276_44 (VH)SEQ ID NO: 187 AdCD276_44 (VL)SEQ ID NO: 188 AdCD276_45 (VH)SEQ ID NO: 189 AdCD276_45 (VL)SEQ ID NO: 190 AdCD276_46 (VH)SEQ ID NO: 191 AdCD276_46 (VL)SEQ ID NO: 192 AdCD276_47 (VH)SEQ ID NO: 193 AdCD276_47 (VL)

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Claims

Claims1. A combination of compositions comprisingA first composition comprising a tagged polypeptide comprising an antigen binding domain specific for CD276 and a tagA second composition comprising an of immune effector cell expressing a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of said tagged polypeptide2. A combination of compositions according to claim 1, wherein the antigen binding domain of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) comprising a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) and a light chain variable region of an antibody (VL) comprising a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3), wherein a. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 9, 11, 13, 2, 4 and 6 respectively, b. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 23, 25, 27, 16, 18 and 20 respectively, c. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 37, 39, 41, 30, 32 and 34 respectively, d. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 1, 53, 55, 44, 46 and 48 respectively, or e. the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 sequences comprise the amino sequences of SEQ ID NO: 65, 67, 69, 58, 60 and 62 respectively3. A combination of compositions according to claim 2, wherein the antigen binding domain of said tagged polypeptide comprises the heavy chain variable region of an antibody (VH) comprising a first framework region (HFR1), a second framework region (HFR2), a third framework region (HFR3) and a fourth framework region (HFR4) and wherein the light chain variable region of an antibody (VL) comprises a first framework region (LFR1), a second framework region (LFR2), a third framework region (LFR3) and a fourth framework region (LFR4), wherein a. The HFR1 , HFR2, HFR3, HFR4, LFR1 , LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 8, 10, 12 ,14, 1, 3, 5 and 7 respectively, b . The HFR1 , HFR2, HFR3, HFR4, LFR1 , LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 22, 24, 26 , 28, 15, 17, 19 and 21 respectively, c. The HFR1 , HFR2, HFR3, HFR4, LFR1 , LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 36, 38, 40, 42, 29, 31, 33, and 35 respectively, d. The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 50, 52, 54, 56, 43, 45, 47, and 49 respectively, or e. The HFR1 , HFR2, HFR3, HFR4, LFR1 , LFR2, LFR3 and LFR4 sequences comprise the amino acid sequences of SEQ ID NO: 64, 66, 68, 70, 57, 59, 61, and 63 respectively4. A combination of compositions according to any of the claims 1-3, wherein the antigen binding domain of said tagged polypeptide comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) wherein a. VH and VL comprise the amino acid sequences of SEQ ID NO: 71 and 72, respectively, b. VH and VL comprise the amino acid sequences of SEQ ID NO: 73 and 74, respectively, c. VH and VL comprise the amino acid sequences of SEQ ID NO: 75 and 76, respectively,d. VH and VL comprise the amino acid sequences of SEQ ID NO: 77 and 78, respectively, or e. VH and VL comprise the amino acid sequences of SEQ ID NO: 79 and 80, respectively5. A combination of compositions according to any of the claims 1-4, wherein the antigen binding domain of said tagged polypeptide is a Fab6. A combination of compositions according to any of the claims 1-5, wherein said tagged polypeptide additionally comprises a constant region of immunoglobulin light chain kappa (CL) or immunoglobulin light chain lambda (CH).

7. A combination of compositions according to any of the claims 1-6, wherein said tagged polypeptide comprises a. SEQ ID NO: 84 and 85, b. SEQ ID NO: 86 and 87, c. SEQ ID NO: 88 and 89, d. SEQ ID NO: 90 and 91, or e. SEQ ID NO: 92 and 938. A combination of compositions according to any of the claims 1-7, wherein said tag of said tagged polypeptide is selected from the group consisting of: FITC, biotin, PE, streptavidin or dextran9. A combination of compositions according to any of the claims 1-8 wherein said tag of said tagged polypeptide is biotin or a derivative thereof and wherein the antigen binding domain of said CAR is specific for biotin or a derivative thereof10. A combination of pharmaceutical compositions according to any of the claims 1-9I L A combination of compositions according to claims 1-10 for use in the treatment of a cancer.

12. A combination of compositions for use in a method of treating a disease, said method comprising administering to a subject in need thereofA first composition comprising a tagged polypeptide comprising an antigen binding domain specific CD276 and a tagA second composition comprising an immune effector cell expressing a chimeric antigen receptor comprising an antigen binding domain specific for said tag of said tagged polypeptide

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