Specific t cell receptors against epitopes of mutant MYD88L265P protein for adoptive T cell therapy

Abstract

Disclosed herein are adoptive T cell therapies or T cell receptor (TCR) gene therapies from the treatment of cancer. The therapies utilize a nucleic acid encoding at least one TCR alpha or beta chain construct of a TCR construct capable of specifically binding to a MYD88 L265P peptide of SEQ ID NO: 2 in the context of HLA-B*07:02 having a high avidity to said peptide/HLA complex. Proteins corresponding to said TCR constructs and host cells, preferably, CD8+ T cells, comprising the TCR constructs are described, as well as the medical use of such nucleic acids, proteins or host cells, in particular, in the diagnosis, prevention and/or treatment of a MyD88 L265P expressing cancer such as a non-Hodgkin B-cell lymphoma selected from the group comprising diffuse large B-cell lymphoma (DLBCL), e.g., activated B-cell-like DLBCL (ABC-DLBCL) or pCNS DLBCL, cutaneous DLBCL, leg-type DLBCL or testicular DLBCL; lymphoplasmacytic lymphoma (LPL), e.g., Waldenstrm macroglobulinemia (WM); and IgM monoclonal gammopathy (IgM MGUS).

Claims

1. A nucleic acid encoding a TCR alpha chain construct comprising a CDR1 sequence having SEQ ID NO: 91, a CDR2 sequence having SEQ ID NO: 92, and a CDR3 sequence having SEQ ID NO: 93; and a nucleic acid encoding a TCR beta chain construct comprising a CDR1 sequence having SEQ ID NO:94, a CDR2 sequence having SEQ ID NO: 95 and a CDR3 sequence having SEQ ID NO: 96; wherein the TCR alpha chain construct and the TCR beta chain construct are encoded on the same or different nucleic acids; and wherein the TCR alpha chain construct and TCR beta chain construct form a TCR construct that is capable of specifically binding to a MYD88 L265P peptide of SEQ ID NO: 2 in the context of HLA-B*07:02.

2. The nucleic acid or nucleic acids of claim 1, wherein the TCR alpha chain construct comprises a variable region having a sequence identity of at least 90% to SEQ ID NO: 97, and/or wherein the TCR beta chain construct comprises a variable region having a sequence identity of at least 90% to SEQ ID NO: 98.

3. The nucleic acid or nucleic acids of claim 1, encoding the TCR alpha chain construct and the TCR beta chain construct, wherein the TCR encoded by the construct has an avidity with KD value of 7.410.sup.9 M or lower to the peptide of SEQ ID NO: 2 in the context of HLA-B*07:02.

4. The nucleic acid or nucleic acids of claim 3, wherein the K.sub.D value is 2.410.sup.9 M or lower.

5. The nucleic acid or nucleic acids of claim 1, wherein the TCR alpha chain construct and/or the TCR beta chain construct further comprise a constant region selected from the group comprising a human constant region, a murine constant region or a chimeric constant region.

6. The nucleic acid or nucleic acids of claim 1, encoding the TCR alpha and beta chain construct of the TCR construct, wherein the nucleic acid is selected from the group comprising a viral vector, a transposon or a vector suitable for CRISPR/CAS based recombination.

7. A host cell comprising the nucleic acid or nucleic acids of claim 1.

8. The host cell of claim 7, wherein the host cell is a human CD8+ T cell.

9. A pharmaceutical composition comprising a) a nucleic acid or nucleic acids of claim 1 encoding a TCR construct capable of specifically binding to a MYD88 L265P peptide of SEQ ID NO: 2 in the context of HLA-B*07:02; or b) a protein encoded by said nucleic acid or nucleic acids; or c) a host cell comprising said nucleic acid or protein and expressing a TCR construct capable of specifically binding to a MYD88 L265P peptide comprising SEQ ID NO: 2 in the context of HLAB*07:02.

10. A method of treating non-Hodgkin B-cell lymphoma in a patient suspected of comprising cells expressing a MYD88 L265P protein comprising administering an effective amount of the pharmaceutical composition of claim 9 to said patient.

11. The method of claim 10, wherein the method is for immune therapy selected from the group consisting of adoptive T cell therapy or TCR gene therapy of the patient comprising cells expressing the MYD88 L265P protein.

12. The method of claim 10, wherein the patient has a non-Hodgkin B-cell lymphoma selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL); lymphoplasmacytic lymphoma (LPL); and IgM monoclonal gammopathy (IgM MGUS).

13. The method of claim 12, wherein the patient has activated B-cell-type DLBCL (ABC-DLBCL), or Primary CNS lymphoma, cutaneous DLBCL, leg-type DLBCL, or testicular DLBCL.

14. The method of claim 12, wherein the patient has Waldenstrom macroglobulinemia (WM).

15. A protein comprising a TCR alpha chain construct and TCR beta chain construct encoded by the nucleic acid or the nucleic acids of claim 1.

16. A host cell comprising the protein of claim 15.

17. The host cell of claim 16, wherein the host cell is a human CD8+ T cell.

Description

EXAMPLES

Example 1: Generation of Mutation Specific T Cells

(1) PBMCs were isolated from HLA-B7-positive healthy donors' blood. Monocytes were separated by plastic adherence for generation of dendritic cells (DC) and following 3 days of culture with 800 IU/ml GM-CSF and 10 ng/ml IL-4 in RPMI with 1% human serum, immature dendritic cells (imDC) were cultured overnight with addition of 10 ng/ml LPS and 50 ng/ml Interferon gamma (IFN) for maturation. Mature dendritic cells (mDC) were then loaded with mutant peptide (RPIPIKYKAM, SEQ ID NO: 2)) and used for priming autologous CD8-positive nave T cells (510.sup.5 T cells/well in 48-well culture plates, in donor-dependently varying DC-T cell ratio). After 10 days, cells from each well were stained with a custom streptamer (HLA*B07:02-RPIPIKYKAM), or stained for T cell activation markers such as CD137 (4-1BB) following a short (6 hours) peptide re-stimulation. Positively stained wells were re-stimulated with peptide-loaded autologous PBMCs for expansion, in the case it was necessary to obtain enough cells for FACS isolation.

(2) A schematic explanation of the methodology for generation of mutation-specific T cells is shown in FIG. 1A. FIG. 1B shows a representative streptamer staining from clone-10 (TCR1610) after a single re-stimulation.

(3) Bulk T-cell clones were tested for selective reactivity against mutant peptide by co-culturing with peptide loaded autologous PBMCs overnight before the FACS isolation of streptamer-positive or peptide-reactive cells. Response was measured by IFN ELISA (FIG. 1C).

Example 2: Identification of Mutation-Specific T-Cell Receptors (TCR)

(4) After final testing with an IFN ELISA, viable CD8 and streptamer-positive cells were isolated separately from each reactive T-cell clone by FACS. Total RNA isolation was performed. TCR alpha and beta genes were amplified via 5-RACE PCR and cloned. Multiple bacterial clones from each TCR-chain were sequenced for analysis of T-cell clonality. Table 1 shows CDR3, the SEQ ID Nos thereof and gene subtypes of MyD88-L265P mutation-specific TCRs and Table 2 shows a list of CDR1, and CDR2 amino acid sequences.

(5) TABLE-US-00001 TABLE1 SEQ TCR Chain IDNO CDR3 V-gene J-gene D-gene 1336 73 CAASGRYDYKLSF TRAV13-1*02 TRAJ20*01 76 CATASDLQGDRSTEAFF TRBV15*02 TRBJ1-1*01 TRBD1*01 1605 43 CAEGTGSARQLTF TRAV13-2*01 TRAJ22*01 46 CASGPFRDSVLTLVANVLTF TRBV28*01 TRBJ2-6*01 TRBD2*01 1610 33 CAPLGGGYNKLIF TRAV21*01 TRAJ4*01 36 CASRLPTTDEKLFF TRBV6-6*02 TRBJ1-4*01 TRBD1*01 2202 53 CLSLSDSNYQLIW TRAV4*01 TRAJ33*01 56 CASSVGQGSYEQYF TRBV9*01 TRBJ2-7*01 TRBD1*01 2205 23 CLVGRDGGSYIPTF TRAV4*01 TRAJ6*01 26 CASSAGQGAYEQYF TRBV9*02 TRBJ2-7*01 TRBD1*01 2207 13 CAVDVGYSTLTF TRAV1-2*01 TRAJ11*01 16 CSARDRSGTLGGELFF TRBV20-1*01 TRBJ2-2*01 TRBD2*02 2211 83 CIVRVMKTSYDKVIF TRAV26-1*01 TRAJ50*01 86 CASSEPRTSGISYNEQFF TRBV10-1*02 TRBJ2-1*01 TRBD2*02 2219 63 CGTAHLRAGSYQLTF TRAV30*01/ TRAJ28*01 TRAV30*02 66 CASSSSSGGAFNEQFF TRBV27*01 TRBJ2-1*01 TRBD2*01 2304 93 CAVRASGTYKYIF TRAV1-2*01 TRAJ40*01 96 CASQDSYEQYF TRBV12-3*01 TRBJ2-7*01 Noresult 2705 103 CAMSGTGGFKTIF TRAV12-3*01 TRAJ9*01 106 CASSQDRPNYYGYTF TRBV4-3*01 TRBJ1-2*01 TRBD1*01 2709 113 CILRDRYGGSQGNLIF TRAV26-2*01 TRAJ42*01 116 CASSYWPTTGESTDTQYF TRBV6-2*01/ TRBJ2-3*01 TRBD1*01 TRBV6-3*01 2716 123 CAFMKPYSGGGADGLTF TRAV38-1*01 TRAJ45*01 126 CASSLAGTTVYNEQFF TRBV13*01 TRBJ2-1*01 TRBD2*01 2719 133 CLVGADSNYQLIW TRAV4*01 TRAJ33*01 136 CASSPGGGAYEQYF TRBV9*01 TRBJ2-7*01 TRBD2*01

(6) TABLE-US-00002 TABLE2 SEQIDNO SEQIDNO TCR Chain (CDR1) CDR1 (CDR2) CDR2 1336 71 DSASNY 72 IRSNVGE 74 LNHNV 75 YYDKDF 1605 41 NSASDY 42 IRSNMDK 44 MDHEN 45 SYDVKM 1610 31 DSAIYN 32 IQSSQRE 34 MNHNY 35 SVGAGI 2202 51 NIATNDY 52 GYETK 54 SGDLS 55 YYNGEE 2205 21 NIATNDY 22 GYKTK 24 SGDLS 25 YYNGEE 2207 11 TSGFNG 12 NVLDGL 14 DFQATT 15 SNEGSKA 2211 81 TISGNEY 82 GLKNN 84 WNHNN 85 SYGVHD 2219 61 KALYS 62 LLKGGEQ 64 MNHEY 65 SMNVEV 2304 91 TSGFNG 92 NVLDGL 94 SGHNS 95 FNNNVP 2705 101 NSAFQY 102 TYSSGN 104 LGHNA 105 YSLEER 2709 111 TISGTDY 112 GLTSN 114 MNHEY 115 SVGEGT 2716 121 TSENNYY 122 QEAYKQQN 124 PRHDT 125 FYEKMQ 2719 131 NIATNDY 132 GYKTK 134 SGDLS 135 YYNGEE

(7) The identified variable domains were combined with murine constant domain sequences for experimental characterization of TCRs, and synthesized with codon-optimization for expression in human cells. TCR gene cassettes encoding the TRBV in combination with a murine TRBC and the TRAV in combination with a murine TRAC, separated by a p2A signal, were constructed as described in detail in Obenaus et al. 2015 and Sommermeyer et al. 2010 (cf. FIG. 2).

Example 3: Analysis of TCR Avidity

(8) Peripheral CD8+ T cells from HLA-B7 positive healthy donors were successfully transduced to express mutation specific TCRs, with no signs of fratricide, and co-cultured with K562 cells that were transduced with HLA*B07:02 and loaded with different concentrations of mutant peptide. The IFN response was determined by ELISA. FIG. 3A/C show the non-linear curve analysis of IFN response by TCR-engineered T cells against the titration of mutant peptide. A response to mutant peptide was detectable down to the concentration of 10.sup.4 g/ml with K.sub.D values within the nano molar (high-affinity) range. FIG. 3B/D show a non-linear curve analysis of IFN response to the corresponding wild type peptide titration. Mutation-specific TCRs shown more than 10000-fold higher affinity to the mutant peptide. Table 3 shows the avidities of the different TCRs analyzed.

(9) TABLE-US-00003 TABLE 3 TCR affinity (shown as K.sub.D) to SEQ ID NO: 2 in the context of HLA-B*07:02. TCR K.sub.D (g/ml) K.sub.D (M)* 2207 0.003 2.4 10.sup.9 2304 0.003 2.4 10.sup.9 2205 0.004 3.2 10.sup.9 1605 0.009 7.4 10.sup.9 1610 0.009 7.4 10.sup.9 2202 0.033 2.7 10.sup.8 2219 0.123 1 10.sup.7 1336 0.387 3.1 10.sup.7 2211 0.560 4.6 10.sup.7 2705 0.020 1.6 10.sup.8 2709 0.102 8.3 10.sup.8 2716 0.099 8.1 10.sup.8 2719 0.024 1.9 10.sup.8 *Molecular weight of peptide SEQ ID NO: 2 is 1216.54 g/mol.

(10) TCR2304 and TCR2207 show the highest avidity against mutant peptide with the KD of 0.003 g/ml, which equals to 2.4 nM, for SEQ ID NO:2.

Example 4: Mutation-Specific Activation of TCR-Engineered T Cells

(11) K562 cells with or without HLA-B7 were virally transduced to express complete length wild type or mutant (L265P) MYD88-coupled to the expression marker GFP via p2A and used as artificial target cells for evaluation of cytotoxic reactivity of TCR-engineered T cells. When co-cultured for 16 hours, six of the TCRs led to recognition of target cells expressing the mutant MyD88 without prior peptide loading, suggesting that the epitope can successfully be processed and presented by human cells.

(12) FIG. 4A shows the mutation specific activation of T cells transduced with one of the TCRs, TCR2207, by flow cytometry analysis staining the activation marker CD137. FIG. 4B/C show a comparative mutation specific IFN response by T cells transduced with different TCRs measured by ELISA, showing a mutation-specific and HLA-B7-restricted response.

Example 5: Mutation Specific Cytotoxicity of TCR-Transduced T Cells

(13) TCR-transduced T cells were co-cultured with K562 cells that express full length wild type or mutant MYD88 linked with p2A to GFP as an expression marker under the control of the same promoter with or without HLA-B7 for 16 hours. Target cells that express mutation and HLA*B07:02 were specifically killed by TCR-transduced T cells. FIG. 5A shows viability of HLA-B7-positive target cells that were co-cultured for 16 hours with T cells expressing one of the 3 highest avidity TCRs, analyzed by flow cytometry. Cells were gated on GFP-positive as reporter of wild type or mutant MYD88 expression, and viability was analyzed by intracellular staining of activated-Caspase-3 (a-Caspase-3) in combination with a fixable dead cell stain. FIG. 5B/C show viability of target cells for comparative cytotoxicity analysis of T cells transduced with different TCRs.

Example 6: Mutation-Specific Activation of TCR-Engineered T Cells Against Lymphoma Cell Lines

(14) In order to investigate their functional potential against mutation in a more natural-like expression level, activation of T cells transduced with one of the 2 highest avidity TCRs was analyzed by flow cytometry after 16-h co-culture with OCI-Ly3 (ABC-like DLBCL, homozygous MYD88-L265P) or HBL-1 (ABC-like DLBCL, heterozygous MYD88-L265P) lymphoma cell lines. Since both cell lines were negative for HLA-B7, they were virally transduced to express it (shown as: Cell line_B7). OCI-Ly3 cells transduced with HLA-B7 were strongly recognized by TCR-engineered T cells. Weaker response was observed against heterozygous mutant HBL-1 cells, which was slightly improved when target cells were pre-treated overnight with 50 ng/ml human IFN prior to co-culture. IFN is known to improve proteasomal processing of peptides and MHC presentation in some cases.

(15) FIG. 6A shows a flow cytometry analysis of T-cell response against OCI-Ly3 and HBL-1 cells. FIG. 6B shows mutation-specific and HLA-B7-restricted activation of T cells transduced with TCR2304 against OCI-Ly3 cells.

Example 7: Mutation-Specific Cytotoxicity Against Lymphoma Cell Lines

(16) T cells transduced with TCR2304 were labelled with CSFE and co-cultured with OCI-Ly3 cells with or without HLA-B7. Viability of target lymphoma cells was analyzed as explained previously in Example 5.

(17) FIG. 7A shows viability of OCI-Ly3 cells with or without HLA-B7 expression after 16-h co-culture with TCR2304-transduced T cells. FIG. 7B shows mutation-specific lymphoma cell-killing by TCR2304-transduced T cells. FIG. 7C shows antigen induced proliferation of TCR2304-transduced T cells following 72-h co-culture with HLA-B7-positive OCI-Ly3 cells, as decreasing fluorescence intensity of CSFE indicates cell division only in TCR-transduced cells.

Example 8: Characterization of Peptide-MHC Binding Behavior of TCRs Via Alanine-Scan

(18) A list of peptides was created by exchanging every amino acid in the mutant epitope (SEQ ID NO: 2) one by one with Alanine to investigate the impact of single amino acids to the peptide-MHC-TCR relation. All these peptides were separately loaded on HLA-B7 expressing K562 cells and co-cultured with TCR-transduced T cells for 16 hours. Different number and group of amino acids were observed to be essential for recognition by different TCRs (binding motif). Nevertheless, the proline in the position 2, which reflects the amino acids substitution L265P on mutant MyD88, was absolutely necessary for all TCRs, demonstrating the specificity of TCRs to the mutation (FIG. 8A). The possibility of cross-reactivity that might be caused by binding-sequence similarity to other human proteins is analyzed individually for TCRs using an online tool called Expitope (Jaravine et al. 2017) as a part of safety screening. In the case of TCR2304, this analysis has revealed 12 peptides in human proteome with binding motif similarity (up to 5 mismatch positions) and varying affinity prediction to HLA-B7 (SEQ ID NO: 141-152). All these peptides were again loaded on HLA-B7 expressing K562 cells for a co-culture with TCR-transduced T cells from 3 different donors. No TCR recognition was observed against any of the peptides (FIG. 8B).

Example 9

(19) For a better understanding of TCR recognition and T cell function against cells harboring MyD88 L265P mutation naturally, TCR2304-transduced T cells from 3 different healthy donors were co-cultured for 16-hours with DLBCL cell lines; SU-DHL-6 (wild-type MYD88), OCI-Ly3 (homozygous MYD88 L265P) or TMD8 (heterozygous MYD88 L265P) with or without HLA-B7 expression.

(20) FIG. 9A shows mutation-specific recognition of both OCI-Ly3 and TMD8 cell lines with HLAB7 expression. SU-DHL-6 control cell line with HLA-B7 is only recognized when loaded with mutant peptide before the co-culture.

(21) FIG. 9B shows efficient mutation-specific and HLA-restricted killing of OCI-Ly3 and TMD8 cell lines by TCR-transduced T cells.