COMBINATION THERAPY COMPRISING AN INFLAMMATORY IMMUNOCYTOKINE AND A CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL

Abstract

The present invention relates to a combination comprising at least fusion protein comprising a binding protein specifically recognizing a cancer-related antigen and an inflammatory cytokine, and a chimeric antigen receptor (CAR)-T cell recognizing a cancer-related antigen.

Claims

1. A combination comprising at least a) a fusion protein comprising a1) a binding protein specifically recognizing a cancer-related antigen and a2) an inflammatory cytokine, and b) a chimeric antigen receptor (CAR)-T cell recognizing a cancer-related antigen.

2. The combination according to claim 1, wherein the cancer-related antigen recognized by the binding protein is cancer stroma-related and/or the chimeric antigen receptor (CAR)-T cell recognizes a cancer cell-related antigen.

3. The combination according to claim 1, wherein the cancer-related antigen recognized by the binding protein is an angiogenesis marker.

4. The combination according to claim 1, wherein the cancer-related antigen recognized by the binding protein is a fibronectin, or a splice isoform thereof, and/or a subdomain thereof.

5. The combination according to claim 1, wherein the cancer-related antigen recognized by the binding protein is the ED.sub.B domain of fibronectin.

6. The combination according to claim 1, wherein the inflammatory cytokine is one selected from the group consisting of IL2 and IL15.

7. The combination according to claim 1, wherein the CAR-T cell recognizes disialoganglioside GD2.

8. The combination according to claim 1, wherein the binding protein comprises at least one of the group selected from antibody, modified antibody format, antibody derivative or fragment retaining target binding properties antibody-based binding protein, oligopeptide binder and/or an antibody mimetic.

9. The combination according to claim 1, wherein the binding protein contains at least one CDR sequence of the L19 antibody.

10. The combination according to claim 1, wherein the binding protein comprises the sequences according to SEQ ID No. 6 to 11.

11. The combination according to claim 1, wherein the binding protein comprises at least one V heavy chain according to SEQ ID No. 1 or at least one V light chain according to SEQ ID No. 2.

12. The combination according to claim 1, wherein the heavy and the light chain are connected by a peptide linker.

13. The combination according to claim 11, wherein the peptide linker comprises a sequence according to SEQ ID No 3, or a sequence having at least 90% identity to the sequence according to SEQ ID No 3.

14. The combination according to claim 1, wherein the IL2 or the IL15 is mammalian IL2 or IL15, preferably human IL2 or IL15, or a functional variant thereof.

15. The combination according to claim 1, wherein the IL2 comprises a sequence according to SEQ ID No 4, or a functional variant thereof.

16. The combination according to wherein the IL15 comprises a sequence according to SEQ ID No 12, or a functional variant thereof.

17. The combination according to claim 1, wherein a fusion protein linker is connecting the binding protein and the inflammatory cytokine part.

18. The combination according to claim 16, wherein the fusion protein linker has a length of between 1 and 30 amino acids.

19. The combination according to claim 16, wherein the fusion protein linker comprises a sequence according to SEQ ID No. 5.

20. The combination according to claim 1, wherein the fusion protein is PEGylated.

21. The combination according to claim 1, wherein the chimeric antigen receptor (CAR) in the T cell comprises 14.G2a-zeta, 14.G2a-BBzeta or 14.G2a-28zeta.

22. The combination according to claim 1 for use in the treatment of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a given pathologic condition.

23. The combination according to claim 22, or use thereof, wherein the pathologic condition is a neoplastic disease.

24. The combination according to claim 22, or use thereof, wherein the pathologic condition is a solid tumor, in particular a lymphoma, carcinoma, sarcoma, or a leukemia.

25. The combination according to claim 1, or use thereof, wherein the fusion protein and the chimeric antigen receptor (CAR)-T cell are to be administered as concomitant and/or adjunctive therapy.

26. The combination according to claim 1, or use thereof, wherein the fusion protein and the chimeric antigen receptor (CAR)-T cell are to be administered as sequential therapy.

Description

FIGURES

[0095] FIG. 1: Experimental design for assessing the antitumor activity of L19-IL2 and CAR-T cells cotargeting against localized Ewing sarcoma xenografts. The following therapy groups were used:

TABLE-US-00001 run antibody T cells 1 KSF-IL2 2 L19-IL2 3 non-transduced T cells 4 CAR-T (14.G2a-BBzeta) 5 L19-IL2 non-transduced T cells 6 L19-IL2 CAR-T (14.G2a-BBzeta)

[0096] FIGS. 2-5 show the results of T cell infiltration experiments by CD3 staining.

[0097] FIG. 2A: KSF-IL2; FIG. 2B: L19-IL2. No particular CAR-T cell infiltration can be detected.

[0098] FIG. 3A: Irrelevant, non-transduced T cells without L19-IL2. Intratumoral T cell infiltration of about 1% can be detected. The quantitative estimation of percentages relies on a rough estimation according to routine of an experienced pathologist.

[0099] FIG. 3B: CAR-T cells (14.G2a-BBzeta, but without L19-IL2): Intratumoral T cell infiltration is about 5%, but no intravascular and no peritumoral T cells can be found

[0100] FIG. 4: Non-transduced T cells plus L19-IL2. Intratumoral T cell infiltration is about 3% but most of the T cells were found to be peritumoral

[0101] FIG. 5: CAR-T cells (14.G2a-BBzeta) +L19-IL2. Intratumoral T cell infiltration is about 10%, intravascular: T cell infiltration is about 30%, Peritumoral T cell infiltration is about 60%.

[0102] FIG. 6: Schematical drawing of the immunocytokine Darleukin (L19-IL2). Note that a preferred version of the L19-IL15 conjugate discussed herein has a similar shape.

[0103] FIG. 7A: Different components of an exemplary 1.sup.st generation chimeric antigen receptor. In this example, the artificial TCR comprises a fusion of an antibody component, e.g., a single-chain variable fragment (scFv) derived from a given monoclonal antibody, fused to the CD3-zeta transmembrane and endodomain. Such molecules transmit a zeta signal in response to target binding of the antibody component. When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express the target detected by the antibody component.

[0104] FIG. 7B: Schematic structure of the chimeric antigen receptor 14.G2a-BBzeta (2.sup.nd generation). The construct comprises an scFv fragment of the antibody 14G2a (1A7), fused to the 4-1BB domain and the CD3-zeta (CD3) domain by means of suitable spacers or linkers. The CD3 domain transmits a proliferative signal upon binding of the scFv fragment to its target, GD2.The 4-1BB costimulatory signaling domain mimic amplifies the activation of the CAR-T cells, leading to a more robust signal to the T cell to multiply and kill the cancer cell.

[0105] FIG. 8: The T-cell receptor complex. CD3-zeta is a chain of the CD3 T-cell co-receptor, which comprises a CD3 chain, a CD3 chain, and two CD3 chains. These chains associate with TCR- and TCR- chains and the CD3-chain (zeta-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-chain, and CD3 molecules together constitute the TCR complex.

[0106] FIG. 9: Sequence alignment between IL2 and IL15. Note the structural similarity between the two cytokines.

MATERIALS AND METHODS

1. Sarcoma Xenograft Experiments

[0107] A localized Ewing sarcoma model which relies on subcutaneous xenografting of 210.sup.6 VH-64 Ewing sarcoma cells per mouse into NOD/scid gamma (NSG) mice was produced.

[0108] Upon a tumor volume of 200-300 mm.sup.3 mice received intraperitoneal treatment with L19-IL2 (30 g twice-weekly on days 1, 5, 8, 12, 14, and 20), and with intravenous injection of 3 doses of 110.sup.7 14.G2a-BBzeta-transduced T cells, or non-transduced T cells as controls (see FIG. 1). Tumor growth was monitored by caliper quantification of diameters. 2 mice were used in each cohort. Post-therapy tumor sections were used for comparative histopathological analysis with regard to (CAR)-T cell infiltration and immunocytokine localization. Furthermore, localization of L19-IL2 within the tumor tissue was evaluated using an anti-human IL2 antibody in standard immunofluorescence procedures. L19-IL2 and 14.G2a-BBzeta are described in details elsewhere herein. Control experiments were done with [0109] (1) L19-IL2 or 14.G2a-BBzeta, respectively, alone [0110] (2) KSF-IL2, which is an immunoconjugate binding to hen egg lysozyme (KSF), and serves as negative control [0111] (3) non-transduced T cells likewise serve as negative controls. Results of this experiment are shown in FIGS. 2-5.

[0112] The combination of CAR-T cells and the immunocytokine drastically increased tumor infiltrationa finding which was completely unanticipated, because none of the current theories that explain the challenges CAR-T cells face when infiltration a solid tumor (active tumor-mediated immunosuppression, functional changes in T lymphocytes after ex vivo manipulation, physical inhibition of infiltration by the desmoplastic stroma which the cells need to penetrate) would render the synergistic effect the immunocytokine has on CAR-T cell infiltration obvious.

[0113] The functional implication of a cytokine, namely to merely regulate the activity of T cells, can not explain its supportive effect in the present scenario, where tumor-mediated immunosuppression, functional changes in T lymphocytes after ex vivo manipulation and/or physical inhibition of infiltration by the desmoplastic stroma challenge the anti tumor efficacy of the T cells.

REFERENCES

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TABLE-US-00002 SequenceListing SeqNo Specification Sequence(Onelettercode) 1 VhL19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS 2 VlL19 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK 3 scFvLinker GDGSSGGSGGAS 4 humanIL2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT 5 Fusionprotein EFSSSSGSSSSGSSSSG linker 6 CDR1Vh SFSMS 7 CDR3Vh PFPYFDY 8 CDR2Vh SISGSSGTTYYADSVKG 9 CDR1Vl RASQSVSSSFLA 10 CDR2Vl YASSRAT 11 CDR3Vl QQTGRIPPT 12 humanIL15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDAS IHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS