Method effector cells using anti-CD127 antibodies for applications in cell therapy

Abstract

The invention relates to methods and preparations for sorting out T effector cells from human leukocytes using anti-CD127 antibodies. Accordingly the invention relates to the use of anti-CD127 antibodies enabling discriminative depletion of subpopulations of Tregulatory cells expressing CD127+ at low levels in a population of human T lymphocytes in order to enable the use of the recovered selected cell preparations comprising essentially Teffector cells as lymphocytes and preferably devoid of Tregulatory cells, for improved efficacy in particular when administered to a patient for therapy.

Claims

1. A method for in vitro sorting out T effector cells from a preparation of cells obtained from a biological fluid or tissue wherein the preparation of cells comprises human cells, wherein the method comprises: a. contacting the preparation of cells with an anti-CD127 antibody specifically binding to CD127 on said T effector cells, said anti-CD127 antibody comprising the following CDRs sequences: in a heavy chain, CDRs of a VH3 heavy chain disclosed as sequence of SEQ ID No: 2, with CDRs of VH3-CDR1, VH3-CDR2 and VH3-CDR3 comprising or consisting of sequences of SEQ ID Nos: 14, 16 and 18, respectively and, in a light chain, CDRs of a VL3 or of a VL4 light chain disclosed as sequences of SEQ ID Nos: 4 and 6, respectively, with CDRs VL3-CDR1, VL3/4-CDR2 and VL3/4-CDR3 having the sequences of SEQ ID Nos: 20, 22 and 24 respectively, or with CDRs of VL4-CDR1, VL3/4-CDR2 and VL3/4-CDR3 comprising or consisting of the sequences of SEQ ID Nos: 26, 22 and 24 respectively; b. selecting sorting out said T effector cells bound to said anti-CD127 antibody and recovering the cell preparation enriched in said T effector cells and depleted for at least Treg cells; washing the anti-CD127 antibody from said T effector cells.

2. The method according to claim 1, wherein the anti-CD127 antibody is selected from anti-CD127 antibodies comprising VH and VL sequences as follows: a. a heavy chain variable domain designated Effi3-VH3 comprising or consisting of sequence of SEQ ID No: 2 or Effi3-VH3 variant comprising or consisting of sequence SEQ ID No: 8 and, for a variant designated Effi3-VH3VL3, either a light chain variable domain designated Effi3-VL3 comprising or consisting of sequence SEQ ID No: 4 or Effi3-VL3 variant comprising or consisting of sequence SEQ ID No: 10, or alternatively b. a heavy chain variable domain designated Effi3-VH3 comprising or consisting of sequence of SEQ ID No: 2 or Effi3-VH3 variant comprising or consisting of sequence SEQ ID No: 8 and, for a variant designated Effi3-VH3VL4, either a light chain variable domain designated Effi3-VL4 comprising or consisting of sequence SEQ ID No: 6 or Effi3-VL4 variant comprising or consisting of sequence SEQ ID No: 12.

3. The method according to claim 1, wherein the step of sorting out cells is performed using magnetic cell sorting and the anti-CD127 antibody is biotinylated or otherwise marked for staining.

4. The method according to claim 1, wherein cell sorting is performed until more than 90% of Treg cells are depleted from the preparation of cells.

5. The method of claim 4 wherein more than 99% of Treg cells are depleted from the preparation of cells.

6. The method according to claim 1, wherein the anti-CD127 antibodies is provided with a composition that comprises polyclonal immunoglobulins suitable for reducing nonspecific interactions of the anti-CD127 antibody with targeted T effector cells.

7. The method according to claim 1, wherein the cell preparation is a human blood cell preparation.

8. The method according to claim 1, wherein the cell preparation is a preparation of hematopoietic stem cells for transplantation (HSCT) or T cells for adoptive therapy.

9. The method according to claim 8 wherein the cell preparation includes T cells for adoptive therapy selected from the group consisting of tumor-infiltrating cells (TILs), genetically modified cancer-specific T cells known as Chimeric-Antigen Receptor T cells (CART), or both.

10. The method according to claim 1, further comprising administering the T effector cells to a human patient to treat a solid or liquid tumor in the human patient or administering the T effector cells to a human patient to treat a chronic infection by a pathogen selected among bacteria, parasites, protozoans, yeasts and viruses.

11. The method according to claim 10, wherein the anti-CD127 antibody is selected from anti-CD127 antibodies comprising VH and VL sequences as follows: a. a heavy chain variable domain designated Effi3-VH3 comprising or consisting of sequence of SEQ ID No: 2 or Effi3-VH3 variant comprising or consisting of sequence SEQ ID No: 8 and, for a variant designated Effi3-VH3VL3, either a light chain variable domain designated Effi3-VL3 comprising or consisting of sequence SEQ ID No: 4 or Effi3-VL3 variant comprising or consisting of sequence SEQ ID No: 10, or alternatively b. a heavy chain variable domain designated Effi3-VH3 comprising or consisting of sequence of SEQ ID No: 2 or Effi3-VH3 variant comprising or consisting of sequence SEQ ID No: 8 and, for a variant designated Effi3-VH3VL4, either a light chain variable domain designated Effi3-VL4 comprising or consisting of sequence SEQ ID No: 6 or Effi3-VL4 variant comprising or consisting of sequence SEQ ID No: 12.

12. The method according to claim 10, wherein the step of sorting out cells is performed using magnetic cell sorting and the anti-CD127 antibody is biotinylated or otherwise marked for staining.

13. The method according to claim 10, wherein cell sorting is performed until more than 90% of Treg cells are depleted from the preparation of cells.

14. The method according to claim 10, wherein the anti-CD127 antibodies is provided with a composition that comprises polyclonal immunoglobulins suitable for reducing nonspecific interactions of the anti-CD127 antibody with targeted T effector cells.

15. The method according to claim 1 wherein the anti-CD127 antibody is an antibody that does not compete with interleukin-7 for binding to an interleukin-7 (IL-7) receptor.

Description

LEGEND OF THE FIGURES

(1) FIG. 1: A. T lymphocytes from the blood of human healthy volunteers were mixed with the indicated concentration of Effi-3 or eBIORDR5 antibodies for 15 min and then with 10 ng/ml recombinant IL-7 for 15 min., fixed and permeabilized and analyzed by flow cytometry with a fluorescent antibody against the phosphorylated form of STAT-5. B. T lymphocytes from the blood of human healthy volunteers were mixed with the indicated concentration of hIL7R-M21 commercial antibody (BD) for 15 min and then with 0.1 ng/ml recombinant IL-7 for 15 min., fixed and permeabilized and analyzed by flow cytometry with a fluorescent antibody against the phosphorylated form of STAT-5.

(2) FIG. 2: Mean fluorescence intensity (MFI) measured by flow cytometry of the binding of anti-CD127 antibodies on CD3+ CD25 T cells from blood from three human healthy volunteers.

(3) FIG. 3: % CD127.sup.lowFoxP3.sup.+ within the CD4+ compartment in PBMC or in positive and negative fractions after Miltenyi magnetic beads sorting of CD127+ cells using the biotinylated Effi-3 antibody with 10 and 250 g Tegeline. Three sorting from 3 individual donors are shown. * p<0.05.

EXAMPLES

Results

(4) Antibody Selection Based on Binding Capacities

(5) Several antibodies from mouse hybridoma have been generated by classical immunization techniques. The affinity of the resulting antibodies has been measured by Biacore and the binding capacity on target cells evaluated by flow cytometry on human blood cells. Clones EFFI7-1 and EFFI7-3 showed the highest binding capacity while not the highest affinity (Table 1).

(6) TABLE-US-00001 TABLE 1 mouse antibodies have been derived by immunization and classical hybridoma technology, and their affinity has been measured by plasmon resonance (Biacore). In parallel, purified antibodies at a concentration of 0.03 micrograms/10.sup.6 target cells were used to stain human blood cells from a healthy volunteer by flow cytometry. Data are % of positive cells in CD3+ cells. Target cell recognition (% CD127+CD25 Antibody KD (M) cells) Effi7-1 1.59 10.sup.8 72 Effi7-2 6.24 10.sup.10 55 Effi7-3 0.65 10.sup.8 84 Effi7-4 0.65 10.sup.8 39 Effi7-5 2.5 10.sup.9 57 Effi7-9 4.2 10.sup.9 15 Effi7-12 .sup.2 10.sup.9 46 Effi7-13 1.3 10.sup.9 54

(7) Clones EFFI7-1 and EFFI7-3 were then compared by flow cytometry with the commercial clone eBIORDR5 (Invitrogen Catalog #: 14-1278-82), using three human donors. The data demonstrated that EFFI7-1 and EFFI7-3 bound to CD3.sup.+ cells with a mean fluorescence intensity of 8229+/1124 (SEM) and 9819+/551, respectively and that eBIORDR5 bound to CD3+ cells with a mean fluorescence intensity of 4145 +/843. Therefore, EFFI7-3 demonstrated a stronger binding than EFFI7-1 and than eBIORDR5. To prepare further developments, EFFI7-3 has been humanized to become Effi3-VH3VL3 or Effi3-VH3VL4, or Effi-3, an IgG1 CDR-grafted humanized antibody. Effi3-VH3VL3 is described herein.

(8) Affinity-Binding to CD127 Protein

(9) The affinity for CD127 of the antibody Effi3-VH3L3, and antibodies of the prior art (eBioRDR5 and hIL-7R-M21) has been assessed using Biacore technology (CD127-Fc (Sino Biological; 100 g/mL; 10 L), Effi3-VH3L3 (4.35 mg/mL; 150 kDa, 30 L), eBioRDR5 (CD127 Monoclonal Antibody, eBioscience from Invitrogen/Thermo Fisher Scientific catalog # 14-1278-82; 0.025 mg/mL; 150 kDa; 50 L)). CD127-Fc was immobilized by amine coupling on a CM5 chip. The carboxylic groups were esterified by injection of an NHS/EDC mix for 7 min. The CD127-Fc proteins diluted to 20 g/ml in Na acetate buffer pH 4.6 were injected until an SPR signal of 500 RU was obtained. Free reactive sites were inactivated by injection of 1 M ethanolamine pH8.5 for 10 min.

(10) Antibodies were analyzed in Single Cycle Kinetics (SCK) on CD127 to determine kinetic and affinity parameters. The surface functionalized with CD127-Fc was regenerated between each cycle by injection of a 4 M MgCl 2 solution for 30 seconds.

(11) The results presented in table 2 show that the Effi3 antibody is much less affin to CD127 than the commercial antibody eBioRD5 from 2 log. In the method according to the invention, moderate affinity is a key parameter. In order to recover Teffector cells depleted in Treg cells, an anti-CD127 antibody with a moderate affinity has the advantageous ability to be stripped from T cells more easily, rendering the Effi3 antibody very usefull for an in vitro method for sorting out T effectors cells from a biological sample, and thereafter washing (i.e. stripping) the T cells from the antibody used for sorting out the cells, while a high affinity antibody has the disadvantage to be more difficult to strip. Hence, T cells sorted with a high affinity antibody may still comprise antibodies used during the sorting step, even after washing the sorted cells. Those cells being intented for use in cell therapy for human in need thereof, there is a need for a formulation as pure as possible, i.e. not comprising the antibodies used during the method for sorting out the Teffector cells.

(12) TABLE-US-00002 TABLE 2 Anti-CD127 antibodies affinity for CD127 protein. KD (M) Kd1 Ka1 (BIVALENT model) eBioRDR5 1.48E05 2.71E+05 6.83E11 Effi3_VH3VL3 2.15E04 1.33E+05 1.62E09
Antibody Selection Based on Antagonist/Agonist Capacities

(13) Positive cell sorting targeted at CD127 has potentially an unwanted activity: it might prevent binding of IL-7 to its receptor and therefore reduce T cell reactivity after sorting. This is unwanted in a context where sorted T cells need to be re-injected in patients to exert their immune activity.

(14) Therefore, antibodies Effi-3, eBIORDR5 and hIL7R-M21 were compared; their potential to inhibit IL-7 mediated signal in T cells has been evaluated by analyzing the phosphorylation of STATS by flow cytometry.

(15) Human PBMC were extracted from the blood of healthy donors (EFS, Nantes) by Ficoll gradient centrifugation (GE Healthcare Life Science, Paris, France). Red blood cells were then lyzed (SOP Eq3 NS20) and washed before reconstitution at appropriate concentration in culture media. Cells were cultured in a 96-plate with different concentrations of Effi3 clone VH3VL3, eBioRDR5 or hIL7R-M21 during 30 minutes at 37 C., 5% CO2 in serum-free medium. Then, PBMC were stimulated by addition of recombinant human IL-7 at 10 ng/ml (FIG. 1A) or 0.1 ng/ml (FIG. 1B) for 15 min at 37 C. CD3+ cells were stained with fluorochrome-labelled anti-CD3 antibody to gate on T cells. After permeabilization, P-STATS molecule were detected with APC-labelled anti-pSTAT5 antibody (BD). PBMC untreated with rhIL-7 were analyzed as the background signal.

(16) The data presented FIG. 1 demonstrated that eBIORDR5 and hIL7R-M21 commercial antibodies antagonize the biological activity of IL-7, whereas Effi-3 does not modify the phosphorylation of STATS induced by IL7 like the isotype control, indicating that Effi3 antibody does not interfere with IL7-pathway.

(17) Cell Sorting Capacities of Selected Antibodies

(18) EFFI7-1 and EFFI7-3 antibodies have been biotinylated for use in magnetic cell sorting, using antibiotin-magnetic beads and the MACS cell sorting system (Miltenyi) according to the manufacturer's instructions. Three independent sorting have been realized, illustrated in Table 3. The data showed an equivalence between EFFI7-1 and EFFI7-3 antibodies for the yield and the purity of the Teff sorted cells. A tendency for better results with antibody EFFI7-3 has been recorded.

(19) TABLE-US-00003 TABLE 3 sorted cells have been counted and analyzed by flow cytometry with anti-CD3, CD127 and FoxP3 antibodies Purity in % depletion of Cell Sorting CD127+ Yield CD127lowFoxP3+ experiment # Antibody cells (%) (%) Treg cells 1 EFFI7-1 89.3 56.33 92.57 EFFI7-3 91.1 51.45 93.2 2 EFFI7-1 86.1 53.45 94.88 EFFI7-3 86.7 54.84 95.17 3 EFFI7-1 89.4 51.84 88.74 EFFI7-3 89.6 52.44 96.22

(20) Effi-3 (the humanized version of Effi7-3) has been compared with the humanized version of the Effi7-2 antibody (described in WO 2015/189302), an antibody known to possess antagonist activity towards IL-7, but used here as a matter of comparison because EFFI7-2 and its humanized version have a very high affinity (see Table 1). Surprisingly, even though humanized EFFI7-2 could bind target cells with a higher efficacy than Effi-3 (FIG. 2), Effi-3 performed better in cell sorting. Indeed, use of biotinylated Effi-3 in the cell sorting process led to a purity in CD127.sup.+ cells of 73.9% with a yield of 55.36% and a depletion in FoxP3.sup.+ Treg cells of 99.9%. In comparison, use of biotinylated humanized EFFI7-2 in the cell sorting process led to a purity in CD127.sup.+ cells of 59.2% with a yield of 17.15% and a depletion in FoxP3.sup.+ Treg cells of 99.5% (Table 4).

(21) TABLE-US-00004 TABLE 4 Outcomes of cell sorting of CD127+ cells using MACS Cell Sorter (Miltenyi) apparatus using Humanized EFFI7-2 or Effi-3 antibodies. Purity of Depletion of CD127+ Yield CD127lowFoxP3+ Antibody cells (%) (%) Treg cells Humanized EFFI7-2 59.2 17.15 99.5 Effi-3 73.9 55.36 99.9

(22) To improve the sorting parameters using biotinylated Effi-3, we added Tegeline (LFB) as a blocking agent in the staining process. Tegeline is a commercial preparation of human immunoglobulins used here to reducing nonspecific interactions of the Effi-3 antibody with target cells and to improve the purity of sorted target cells. We tested two doses of Tegeline: 10 and 250 micrograms. These sorting trials demonstrated that addition of Tegeline in the sorting process improved purity of sorted cells and, more surprisingly, improved the yield (Tables 5 and 6). The % of depletion of Treg cells stayed very high (FIG. 3).

(23) TABLE-US-00005 TABLE 5 Outcomes of cell sorting of CD127+ cells using Effi-3 antibodies together with Tegeline (one experiment). Purity of Depletion of Effi-3 together CD127+ Yield CD127lowFoxP3+ with tegeline cells (%) (%) Treg cells Tegeline 10 g 87.7 50.3 97.7 Tegeline 250 g 87.7 58.23 94.3

(24) TABLE-US-00006 TABLE 6 Outcomes of cell sorting of CD127+ cells using Effi-3 antibodies together with Tegeline (mean of data from 3 experiments) Purity of Depletion of Effi-3 together CD127+ Yield CD127lowFoxP3+ with tegeline cells (%) (%) Treg cells Tegeline 10 g 87.2 47.8 90.5 Tegeline 250 g 86.7 43.1 91.4

Conclusion

(25) Sorting out active Treg cells from T cell preparations is useful in cell therapies where Teff cells need to mount an immune response against a target, most frequently a tumor. CD127 is a target expressed on Teff and less on Treg, opening the possibility to differentially bind to and sort out Teff from Treg. We have shown that Effi-3, in particular Effi3-VH3VL3, are good antibodies able to bind to and sort out Teff cells using a Macs system, even though this antibody shows a moderate binding affinity. Other available antibodies are good binders of CD127.sup.+ cells and might be used to sort out Teff cells, such as eBIORDR5 or humanized EFFI7-2. However, eBIORDR5 and humanized EFFI7-2 are antagonist antibodies preventing T cell responses induced by IL-7. Their use might therefore likely compromise immune responses that are required for the efficacy of Teff transfer. In addition, humanized EFFI7-2 in spite of its very high affinity resulted in a poor sorting yields. Thus Effi-3 antibodies are suitable for cell sorting in the conditions described here because they show efficacy in sorting out target CD127.sup.+ cells and lack antagonist activity to IL-7.

(26) The following table (Table 7) discloses the sequence described herein. Nb stands for the SEQ ID NO of each sequence; Type discloses the nature of the sequence, either DNA or amino acid sequence (PRT).

(27) The sequences of the specific domains of the antibodies are provided in reference to the Kabat nomenclature.

(28) TABLE-US-00007 Nb Nom Type Squence 1 Effi3VH3 DNA GCTGTGCAGCTGGTCGAATCTGGGGGGGGGCTGGTCCAGCCCGGCGGGTC TCTGAAAATCACTTGCGCCGCTAGTGGGTTCACCTTTACAAACGCAGCCA TGTACTGGGTCCGACAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCACGG ATCAGAACAAAGGCTAACAACTACGCAACTTACTATGCCGACTCAGTGAA GGGCAGGTTCACCATTAGCCGCGACGATAGCAAATCCACAGTCTACCTGC AGATGGACTCTGTGAAGACAGAAGATACTGCCACCTACTATTGTATTGTG GTCGTGCTGACTACTACACGGGATTACTTTGACTATTGGGGACAGGGAGT GCTGGTGACAGTGAGTTCA 2 Effi3VH3_aa PRT AVQLVESGGGLVQPGGSLKITCAASGFTFTNAAMYWVRQAPGKGLEWVAR IRTKANNYATYYADSVKGRFTISRDDSKSTVYLQMDSVKTEDTATYYCIV VVLTTTRDYFDYWGQGVLVTVSS 3 Effi3VL3 DNA GACATCGTCCTGACTCAGTCCCCCTCTTCCCTGCCAGTGACACCTGGAGA GCCAGCATCTATCAGTTGCCGAAGCTCCCAGTCACTGCTGACTGTCAAGG GAATTACCAGCCTGTACTGGTTCCTGCAGAAGCCCGGCCAGTCCCCTAAA CTGCTGATCTATCGGATGTCTAACAGAGACAGTGGGGTGCCCGATAGGTT CTCAGGCAGCGGGTCCGAAACCGACTTTACACTGAAAATTTCTCGCGTGG AGGCTGAAGATGTCGGAACCTACTATTGCGCACAGTTTCTGGAATACCCT CACACTTTCGGGGCAGGCACTAAGCTGGAGCTGAAGCGT 4 Effi3VL3_aa PRT DIVLTQSPSSLPVTPGEPASISCRSSQSLLTVKGITSLYWFLQKPGQSPK LLIYRMSNRDSGVPDRFSGSGSETDFTLKISRVEAEDVGTYYCAQFLEYP HTFGAGTKLELKR 5 Effi3VL4 DNA GACATCGTGCTGACACAGAGTCCCTCCTCCCTGCCAGTGACACCTGGAGA GCCAGCATCTATCAGTTGCCGAAGCTCCCAGGACCTGCTGACTGTCAAGG GCATTACCTCACTGTACTGGTTCCTGCAGAAGCCCGGGCAGAGCCCTAAA CTGCTGATCTATCGGATGTCTAACAGAGACAGTGGAGTGCCCGATAGGTT CTCAGGCAGCGGGTCCGGAACCGACTTTACACTGAAAATTTCTCGCGTGG AGGCTGAAGATGTCGGCACCTACTATTGCGCACAGTTTCTGGAGTATCCC CACACCTTTGGAGCAGGCACTAAGCTGGAGCTGAAGCGT 6 Effi3VL4_aa PRT DIVLTQSPSSLPVTPGEPASISCRSSQDLLTVKGITSLYWFLQKPGQS PKLLIYRMSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGTYYCAQFLE YPHTFGAGTKLELKR 7 Effi3VH3 DNA ATGCTGGTCCTGCAGTGGGTCCTGGTCACCGCTCTGTTTCAGGGGGTCCA (+signalpeptide) TTGTGCTGTGCAGCTGGTCGAATCTGGGGGGGGGCTGGTCCAGCCCGGCG GGTCTCTGAAAATCACTTGCGCCGCTAGTGGGTTCACCTTTACAAACGCA GCCATGTACTGGGTCCGACAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGC ACGGATCAGAACAAAGGCTAACAACTACGCAACTTACTATGCCGACTCAG TGAAGGGCAGGTTCACCATTAGCCGCGACGATAGCAAATCCACAGTCTAC CTGCAGATGGACTCTGTGAAGACAGAAGATACTGCCACCTACTATTGTAT TGTGGTCGTGCTGACTACTACACGGGATTACTTTGACTATTGGGGACAGG GAGTGCTGGTGACAGTGAGTTCA 8 Effi3VH3_aa PRT MLVLQWVLVTALFQGVHCAVQLVESGGGLVQPGGSLKITCAASGFTFTNA (+signalpeptide) AMYWVRQAPGKGLEWVARIRTKANNYATYYADSVKGRFTISRDDSKSTVY LQMDSVKTEDTATYYCIVVVLTTTRDYFDYWGQGVLVTVSS 9 Effi3VL3 DNA ATGAAGTTTCCTGCTCAGTTTCTGGGCCTGATTGTGCTGTGTATTCCTGG (+signalpeptide) CGCTACCGGAGACATCGTCCTGACTCAGTCCCCCTCTTCCCTGCCAGTGA CACCTGGAGAGCCAGCATCTATCAGTTGCCGAAGCTCCCAGTCACTGCTG ACTGTCAAGGGAATTACCAGCCTGTACTGGTTCCTGCAGAAGCCCGGCCA GTCCCCTAAACTGCTGATCTATCGGATGTCTAACAGAGACAGTGGGGTGC CCGATAGGTTCTCAGGCAGCGGGTCCGAAACCGACTTTACACTGAAAATT TCTCGCGTGGAGGCTGAAGATGTCGGAACCTACTATTGCGCACAGTTTCT GGAATACCCTCACACTTTCGGGGCAGGCACTAAGCTGGAGCTGAAGCGT 10 Effi3VL3_aa PRT MKFPAQFLGLIVLCIPGATGDIVLTQSPSSLPVTPGEPASISCRSSQSLL (+signalpeptide) TVKGITSLYWFLQKPGQSPKLLIYRMSNRDSGVPDRFSGSGSETDFTLKI SRVEAEDVGTYYCAQFLEYPHTFGAGTKLELKR 11 Effi3VL4 DNA ATGAAGTTCCCTGCTCAGTTCCTGGGGCTGATTGTCCTGTGCATTCCTGG (+signalpeptide) GGCAACCGGCGACATCGTGCTGACACAGAGTCCCTCCTCCCTGCCAGTGA CACCTGGAGAGCCAGCATCTATCAGTTGCCGAAGCTCCCAGGACCTGCTG ACTGTCAAGGGCATTACCTCACTGTACTGGTTCCTGCAGAAGCCCGGGCA GAGCCCTAAACTGCTGATCTATCGGATGTCTAACAGAGACAGTGGAGTGC CCGATAGGTTCTCAGGCAGCGGGTCCGGAACCGACTTTACACTGAAAATT TCTCGCGTGGAGGCTGAAGATGTCGGCACCTACTATTGCGCACAGTTTCT GGAGTATCCCCACACCTTTGGAGCAGGCACTAAGCTGGAGCTGAAGCGT 12 Effi3VL4_aa PRT MKFPAQFLGLIVLCIPGATGDIVLTQSPSSLPVTPGEPASISCRSSQDLL (+signalpeptide) TVKGITSLYWFLQKPGQSPKLLIYRMSNRDSGVPDRFSGSGSGTDFTLKI SRVEAEDVGTYYCAQFLEYPHTFGAGTKLELKR 13 Effi3VH3_CDR1 DNA TTCACCTTTACAAACGCAGCCATGTAC 14 Effi3VH3_CDR1aa PRT FTFTNAAMY 15 Effi3VH3_CDR2 DNA CGGATCAGAACAAAGGCTAACAACTACGCAACTTACTATGCCGACTCAGT GAAGGGC 16 Effi3VH3_CDR2aa PRT RIRTKANNYATYYADSVKG 17 Effi3VH3_CDR3 DNA GTCGTGCTGACTACTACACGGGATTACTTTGACTAT 18 Effi3VH3_CDR3aa PRT VVLTTTRDYFDY 19 Effi3VL3_CDR1 DNA CGAAGCTCCCAGTCACTGCTGACTGTCAAGGGAATTACCAGCCTGTAC 20 Effi3VL3CDR1_aa PRT RSSQSLLTVKGITSLY 21 Effi3VL3/4_CDR2 DNA CGGATGTCTAACAGAGACAGT 22 Effi3VL3/4_CDR2aa PRT RMSNRDS 23 Effi3VL3/4_CDR3 DNA GCACAGTTTCTGGAATACCCTCACACT 24 Effi3VL3/4_CDR3aa PRT AQFLEYPHT 25 Effi3VL4_CDR1 DNA CGAAGCTCCCAGGACCTGCTGACTGTCAAGGGCATTACCTCACTGTAC 26 Effi3VL4_CDR1_aa PRT RSSQDLLTVKGITSLY

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