Antibodies having specificity for CD38 and uses thereof

Abstract

CD38 is also expressed in a variety of malignant hematological diseases, including multiple myeloma. In the present invention, the inventors have generated a new antibody against CD38 that could be suitable for producing bispecific antibodies as well as CAR-T cells. In particular, the inventors report the development of Bi38-3, a new bispecific T cell engager that targeted CD38 on MM cells and recruited cytotoxic T cells through the CD3. Bi38-3 lacked the Fc region of natural mAb, which contributes to resistance processes, but triggered T cells to proliferate, release cytokine and lyse CD38 positive MM cells in vitro. Similarly, Bi38-3 induced autologous T cells to eliminate tumor plasma cells isolated from MM patients both at diagnosis and at relapse. The cytotoxicity triggered by Bi38-3 was restricted to cells expressing high levels of CD38 and preserved the integrity of T, B and NK lymphocytes in vitro. Importantly, Bi38-3 rapidly reduced tumor cells in an MM1.S xenograft mouse model of human MM. Taken together, the results show that the antibody of the present invention is an effective reagent to specifically eliminate CD38 positive malignant cells without significantly affecting CD38 lowly expressing cells and represents a promising novel immunotherapeutic tool for the treatment of malignant hematological diseases, and especially multiple myeloma.

Claims

1. A monoclonal antibody having binding specificity for the extracellular domain of CD38 which comprises: a heavy chain comprising i) the H-CDR1 as set forth in SEQ ID NO:5, ii) the H-CDR2 as set forth in SEQ ID NO:6 and iii) the H-CDR3 as set forth in SEQ ID NO:7, and, a light chain comprising i) the L-CDR1 as set forth in SEQ ID NO:8, ii) the L-CDR2 as set forth in SEQ ID NO:9 and iii) the L-CDR3 as set forth in SEQ ID NO:10.

2. The monoclonal antibody of claim 1 which comprises a VH domain having at least 70% of identity with the amino acid sequence as set forth in SEQ ID NO: 3.

3. The monoclonal antibody of claim 1 which comprises a VL domain having at least 70% of identity with the amino acid sequence as set forth in SEQ ID NO:4.

4. The monoclonal antibody of claim 1 which is a chimeric antibody.

5. The monoclonal antibody of claim 1 which is a humanized antibody.

6. A scFv fragment comprising the VH and the VL domain of the antibody of claim 1.

7. The scFv fragment of claim 6 which has an amino acid sequence as set forth in SEQ ID NO:11.

8. A multispecific antibody comprising a first antigen binding site from the monoclonal antibody of claim 1 and at least one second antigen binding site.

9. A The multispecific antibody of claim 8 wherein the second antigen binding site is used for recruiting T cells.

10. The multispecific antibody of claim 9 wherein the second antigen binding site has specificity for the extracellular domain of CD38.

11. The multispecific antibody of claim 8 which comprises an antigen-binding domain comprising the single chain variable fragment (scFv) of claim 6.

12. The multispecific antibody of 8 which comprises the sequence as set forth in SEQ IQ NO: 12.

13. A chimeric antigen receptor (CAR) comprising an antigen binding domain of the antibody of claim 1.

14. The chimeric antigen receptor (CAR) of claim 13 comprising an antigen-binding domain comprising the single chain variable fragment (scFv) of claim 6.

15. The chimeric antigen receptor (CAR) of claim 14 which comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3 intracellular domains.

16. The chimeric antigen receptor (CAR) of claim 14 which has an amino acid sequence as set forth in SEQ ID NO: 13 or 14.

17. A nucleic acid sequence encoding i) the monoclonal antibody of claim 1, ii) a multispecific antibody comprising a first antigen binding site from the monoclonal antibody and at least one second antigen binding site, or iii) a chimeric antigen receptor (CAR) comprising an antigen binding domain of the monoclonal antibody.

18. A nucleic acid sequence which encodes a heavy chain and/or a light chain of the monoclonal antibody of claim 1.

19. A vector comprising the nucleic acid of claim 17.

20. A host cell engineered to express i) the monoclonal antibody of claim 1, ii) a multispecific antibody comprising a first antigen binding site from the monoclonal antibody and at least one second antigen binding site, or iii) a chimeric antigen receptor (CAR) comprising an antigen binding domain of the monoclonal antibody.

21. The host cell of claim 20 which is a CAR-T cell.

22. A method of treating cancer in a patient in need comprising administering to the subject a therapeutically effective amount of i) the antibody of claim 1 and/or ii) a multispecific antibody comprising a first antigen binding site from the monoclonal antibody and at least one second antigen binding site and/or iii) a population of CAR-T cells of engineered to express the monoclonal antibody or a multispecific antibody comprising a first antigen binding site from the monoclonal antibody or a chimeric antigen receptor (CAR) comprising an antigen binding domain of the monoclonal antibody.

23. A pharmaceutical composition comprising an amount of i) the antibody of claim 1 and/or ii) a multispecific antibody comprising a first antigen binding site from the monoclonal antibody and at least one second antigen binding site and/or ii) the population of CAR-T cells of engineered to express the monoclonal antibody or a multispecific antibody comprising a first antigen binding site from the monoclonal antibody or a chimeric antigen receptor (CAR) comprising an antigen binding domain of the monoclonal antibody.

24. The monoclonal antibody of claim 4 wherein the chimeric antibody has a heavy chain as set forth in SEQ ID NO:3 and/or a light chain as set forth in SEQ ID NO: 4.

25. The method of claim 22, wherein the cancer is a CD38-positive hematological malignancy.

Description

FIGURES

(1) FIG. 1. Bi38-3 dose dependent autologous T cell-mediated lysis of MM tumor cells from patients. CD138+ plasma cells were purified from the bone marrow of the patients and co-cultured with autologous CD3+ T cells isolated from PBMC at an E:T cell ratio of 5:1 for 24 hours. Cultures were analyzed by FACS to monitor the number of CD138+ cells falling into the live gate. Shown are the mean from triplicate experiments indicating the percentages of live CD138+ cells (relative to the untreated condition) in 4 different patients at diagnosis and 3 at relapse. Histograms show the average effects of Bi38-3 alone, T cells alone and T cells with Bi38-3 (100 ng/mL) on tumor plasma cells form the same 5 patients at diagnosis (upper) and 3 at relapse (lower). Standard deviations are shown and p values were calculated with a Student's t test (*p<0.05; **p<0.01; ***p<0.001).

(2) FIG. 2. In Vivo Activity of Bi38-3 in the MM1.Sluc Xenograft mouse model. A. Treatment schedule. NSG mice were inoculated with 5.Math.10.sup.6 MM1.SLuc cells (i.v) and treatment was initiated at day 13 when similar levels of luciferase expressing MM cells were detected in all mice. Purified T cells were (5.Math.10.sup.6 cells/mouse) were infused i.v., together with Bi38-3 or PBS (blue arrows). Injections of Bi38-3 (0.1 mg/Kg) i.v. were repeated daily for 9 days (black arrows). Luciferase activity was measured with the IVIS Imaging System at 7, 11, 13, 15, 18 and 21 (or 22) days after tumor injection (red arrows). B. Serial bioluminescence imaging to assess myeloma progression/regression. Radiance was measured on the entire body of mice. Images on the left indicate luminescence at 7 days after inoculation with MM.1S myeloma cells and before the beginning of the treatment. Images on the right indicate 18 days after inoculation with MM.1S cells and 4 days after treatment with Bi38-3 (upper panel) or with vehicle (lower panel). The radiance color scale is represented on the right. C. Longitudinal radiance levels of vehicle (blue lines) and Bi38-3 (red lines) treated mice. Represents 9 mice per group inoculated with T cells from 2 independent donors. The p value was calculated at 22 days with a Student t test. (***p<0.001).

(3) FIG. 3. In Vitro Activity of anti-CD38 CAR-T cells. A. Schematic representation of the structures of the different Chimeric Antigen Receptors (CAR) and co-stimulatory receptors (CCR). First generation (1G) CAR contains the CD3 signaling domain, 3rd generation CAR (3G) contains CD28, 4-1BB and CD3z signaling domains. CCR contains CD28 and 4-1BB signaling domains, but lacks the CD3 domain. The CAR Mock is devoid of the anti-CD38 scFv region. B. Cytotoxic activity of the different CAR-T on CD38 expressing MM (MM.1S and RPMI8226) and CD38 negative fibroblastic (HEK293) cells lines in vitro. Cells expressing the luciferase have been cultured for 20 h with the indicated CAR-T cells at various effector/target ratios (E:T). The cytotoxic activity was determined by measuring the level of luciferase in the culture. Representative of 4 independent experiments.

(4) FIG. 4: Sensitivity of blood cells and bone marrow hematopoietic progenitors to Bi38-3. A. Relative Bi38-3 mediated T cell lysis of Treg versus MM1.S cells. Purified T cells from healthy donors (n=3) were co-cultured with increasing concentrations of Bi38-3 for 24 hours in the presence of MM1.S cells. B. Relative Bi38-3 mediated T cell lysis of CD34+ bone marrow hematopoietic progenitors versus MM1.S cells. Paired CD34+ hematopoietic progenitors and T cells purified from the bone marrow of healthy donors (hip surgery) (n=4) were co-cultured with increasing concentrations of Bi38-3 for 24 hours in the presence of MM1.S cells. The numbers of live CD20+ (B cells), FoxP3+ (Treg cells), CD34+ (Hematopoietic progenitors) and CD138+ (MM1.S cells) were calculated by FACS using counting beads and expressed as a ratio to untreated controls, respectively. Histograms show the ratios of B, Treg, CD34+ hematopoietic progenitor and MM.1S cells for each Bi38-3 concentrations and error bars indicate the SD. The normality of the CD34+ populations was established with a Shapiro-Wilk normality test and P-values were determined by an unpaired Student's t test (*p<0.05; **p<0.01; ***p<0.001).

EXAMPLE 1: A NOVEL CD38/CD3 BISPECIFIC T-CELL ENGAGER FOR THE TREATMENT OF MULTIPLE MYELOMA

(5) Methods:

(6) Construction and Purification of Bi38-3.

(7) Bi38-3 was generated by the fusion of two scFvs derived from mouse hybridomas producing anti-human CD38 and CD3 (BB51 and OKT3, respectively), linked by a 15-amino-acid glycine-serine (G4S13) spacer. The human CD8 leader peptide was genetically linked to the N-terminus of the fusion fragment and Myc-tag and His-tag sequences were introduced at the C-terminus. The sequence encoding for Bi38-3 was cloned into the pCDNA3 expression vector (ThermoFisher), and confirmed by sequencing. This vector was transiently transfected in HEK-293T cells and a protein of 55.6 Kd corresponding to Bi38-3 was purified from the supernatant using HisTrap HP columns (GE). The integrity of Bi38-3 was analyzed by Coomassie blue staining and western blot with an anti-Myc-tag antibody.

(8) Cell Lines

(9) MM1.S, NCI-H929 and KMS-11 MM cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 10 g/mL streptomycin, and 2 mM L-glutamine. All cell lines were monitored for mycoplasma contamination. Luciferase expressing MM1.S and KMS-11 cells (KMS11luc and MM1.Sluc) were generated by lentiviral transduction with a luciferase expressing vector (Addgene, pLenti CMV Puro LUC (w168-1) a gift from Eric Campeau & Paul Kaufman). To generate CD38 deficient MM1.S cells, two pairs of RNAs guides were designed to delete exons 2 and 3 of the CD38 gene. Annealed oligonucleotides were cloned into the pX458 vector (Addgene plasmid ID 48138, a gift from Dr Feng Zhang) and verified by sequencing. For Cas9 deletion, 210.sup.6 MM1.Sluc cells were nucleofected with 2 g of each Cas9 vector using Nucleofector-II (Lonza), incubated in culture medium for 24 h, FACS sorted for GFP positive cells and cloned in 96-well plates. Sub-clones were analyzed for CD38 expression by flow cytometry and CD38 negative clones were selected for further analysis.

(10) Blood and Bone Marrow Samples

(11) Peripheral blood samples from healthy donors were obtained from the Etablissement Francais du Sang (EFS). Fresh tumor plasma cells were collected from buffy coat of bone marrow aspirates from myeloma patients and further purified using anti-CD138 coated beads (Miltenyi). In all cases, informed consent of patients and volunteers were obtained in accordance with the Declaration of Helsinki and with approval of the Saint-Louis Hospital Internal Review Board.

(12) Flow Cytometry and Cytotoxicity

(13) To determine lysis of KMS-11luc or MM1Sluc MINI cell lines, purified peripheral T cells (effector) were incubated in culture medium (RPMI, 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 10 g/mL streptomycin, and 2 mM L-glutamine) with luciferase expressing MM1.S or KMS-11 cells (target) with an effector to target ratio of 1:5 in flat-bottomed 96 wells with various concentrations of Bi38-3. The luciferase signal produced by surviving MM cells was determined after 24 h using a CLARIOstar Plus luminometer plate reader (BMG LABTECH GmbH, Ortenberg, Germany) within 20 min after the addition of firefly luciferase substrate according to the manufacturer's instructions (Bright-Glo Luciferase assay System, Promega). T cell cytotoxicity on primary cells was assayed by flow cytometry using similar co-culture conditions. Effector T cells were co-incubated with purified target MM cells and serial dilutions of Bi38-3. After incubation, anti-human CD138-Allophycocyanin (APC) (clone 44F9Miltenyi Biotec), anti-CD20-Brilliant Violet (BV) 605 (Clone 2H7BioLegend), anti-CD4-APC/Cyanine 7 (Clone RPA-T4BioLegend) and anti-CD8-BV421 (Clone RPA-T8BioLegend) antibodies were added to the cells to distinguish target from effector cells and the numbers of live cells were determined using Brightcount beads (ThermoFisher) by flow cytometry. All FACS acquisition was performed on a Canto II (Beckon Dickinson). Analysis and calculation of proliferation indices were performed with Flowjo.

(14) T Cell Activation and Proliferation Assays

(15) For detection of activation, effector (T cells) and targets (MM1.S) cells were co-cultured at a ratio of 5:1 for 24 h, stained with anti-human CD4-APC-Cyanine 7 (Clone RPA-T4), anti-CD8-BV421 (Clone RPA-T8), anti-CD25-Phycoerythrin (Pe)/Cyanine 5 (clone BC96) and anti-CD69-BV711 (clone FN 50) antibodies (all from Biolegend) and analyzed by flow cytometry. For analysis of proliferation, T cells were labeled with CellTrace Violet dye (ThermoFisher) and subjected to stimulation with MM1.S cells or MM1.S-CD38KO with (or without) Bi38-3 (10 ng/mL) for 96 hours. Cells were stained with anti-CD4-APC/Cy7 (clone RPA-T4) and anti-CD8-BV421 (clone RPA-T8) antibodies, and analyzed by flow cytometry.

(16) Quantification of Cytokines in Cell Culture Supernatants

(17) The concentration of cytokines in supernatants from cytotoxicity assays were analyzed using the BD CBA Human Th1/Th2 Cytokine Kit II (Beckon Dickinson). Data were acquired on a Canto II and analyzed with the FCAP Array software (Beckon Dickinson).

(18) Mouse Systemic Tumor Model

(19) We used 6- to 12-week-old NOD/SCID/IL-2Rnull mice (The Jackson Laboratory), under a protocol approved by the Institutional Animal Care and Use Committee (Comite dthique Paris-Nord). Mice were inoculated with 510.sup.6 MM1.Sluc cells by tail vein injection, followed, 14 days later, by infusion of 510.sup.6 purified human T cells (purified using Pan T-cell isolation kits from Miltenyi Biotec) containing (or not) Bi38-3 at 0.08 mg/Kg. Tail vein injection of Bi38-3 (or PBS for controls) was repeated daily for 9 days. No randomization or blinding methods were used. Bioluminescence imaging was performed every 3 or 4 days. Mice were injected intraperitoneally with 240 L of D-Luciferin (15 mg/mL) (XenoLight D-Luciferin Potassium Salt, Perkin Elmer) and image acquisition was performed after 15 minutes using the IVIS Imaging System (PerkinElmer) with the Living Image software (PerkinElmer) on a 25-cm field of view at medium binning level and at various exposure times. Twenty-two days after inoculation of MM cells, all mice were sacrificed.

(20) Results:

(21) Construction, Production and Binding Properties of Bi38-3

(22) Bi38-3 consists of two scFvs derived from mouse hybridomas, producing anti-human CD38 and CD3 (BB51 and OKT3, respectively), linked by a 15-amino-acid glycine-serine (G4S13) spacer (not shown). Amino acid sequences corresponding to anti-CD38 heavy and light chains variable domains, of anti CD38 scFv as well as of Bi38-3 are depicted in Table 1.

(23) TABLE-US-00008 TABLE1 AminoacidsequencesofImmunoglobulin(IgHandIgk)fromtheBB515 Hybridoma(anti-CD38),ofthecorrespondingscFvandofBi38-3. IgkVk12.44- DIQMTQSPASLSASVGETVTITCRASENIYSFLAWYQ IgkCDR1:ENIYSF(SEQ Jk5: QKQGKSPQLLVYNTKTLTEGVPSRFSGSGSGTQFSLK IDNO:8) INNLQPEDFGSYYCQHHYGIPLTFGAGTKLELK IgkCDR2:NTK(SEQID (SEQIDNO:4) NO:9) IgkCDR3:QHHYGIPLT (SEQIDNO:10) IgHVH1.87- QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWV IgHCDR1:GYTFTSYW D1.1-J1 KQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADK (SEQIDNO:5) SSSTAYMQLSNLTSEDSAVYYCARERTTGAPRYFDVW IgHCDR2:IYPGDGDT GAGTTVTVSS(SEQIDNO:3) (SEQIDNO:6) IgHCDR3: ARERTTGAPRYFDV(SEQID NO:7) Anti-CD38 MALPVTALLLPLALLLHAARPDIQMTQSPASLSASVG LeaderCD8a: scFv ETVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKT MALPVTALLLPLALLLHAARP LTEGVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQH (SEQIDNO:19) HYGIPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQ QSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPG QGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTA YMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTT VTVSS(SEQIDNO:20) Bi38-3 MALPVTALLLPLALLLHAARPDIQMTQSPASLSASVG LeaderCD8a-scFv ETVTITCRASENIYSFLAWYQQKQGKSPQLLVYNTKT antiCD38-scFvanti LTEGVPSRFSGSGSGTQFSLKINNLQPEDFGSYYCQH CD3e- - HYGIPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQ QSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPG QGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTA YMQLSNLTSEDSAVYYCARERTTGAPRYFDVWGAGTT VTVSSGGGGSGGGGSGGGGSDIKLQQSGAELARPGAS VKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPS RGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDS AVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGG SGGGGSVDDIQLTQSPAIMSASPGEKVTMTCSASSSV SYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGS GTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKL 0

(24) The anti-CD38 scFv was positioned N-terminally, the anti-CD3c scFv C-terminally and followed by Myc-Tag and His6-Tag sequences (not shown). Western blot analysis of HEK-293 cells transiently transfected with a Bi38-3 expression vector revealed a unique protein recognized by the anti-Myc-Tag antibody with the expected size of 55.6 Kd (data not shown). Bi38-3 was purified from culture supernatants of transiently transfected HEK-293 cells with HisTrap HP columns (GE). Purity of monomeric Bi38-3 protein was demonstrated by gel electrophoresis followed by Coomassie Blue staining (data not shown). Binding of Bi38-3 to CD38 expressing MM1.S, KMS11 and NCI-H939 MM cells was analyzed by flow cytometry with an anti-Fab antibody that recognizes the scFv domains. We observed that Bi38-3 was detected at the surface of MM cell lines, with a less intense staining of KMS11 cells that express lower levels of CD38, and stronger signals on MM1.S and NCI-H929 cells that display higher levels of CD38 (data not shown). To verify the specificity of Bi38-3 to CD38, we used a CRISPR/Cas9 approach to inactivate the CD38 gene in MM1.S cells (MM1.S-KO) (data not shown). FACS analysis revealed that Bi38-3 could not be detected at the surface of CD38 negative MM1.S-KO cells (data not shown). Thus, purified Bi38-3 efficiently and specifically recognizes CD38 on MM cells.

(25) Bi38-3 Induces T-Cell Activation and Proliferation in Response to MM Cells In Vitro

(26) We next investigated the T cell responses to MM cells triggered by Bi38-3. First, we performed FACS analysis to measure the proliferation index of violet fluorescent stained T cells. Stimulation of donor effector T cells (E) with Bi38-3 in the presence MM1.S target cells (T) led to robust proliferation, with an average of 5 cell divisions (expansion index) after 4 days, a level slightly higher than induced by treatment with anti-CD3/CD28 beads (data not shown). Proliferation required CD38 expression on target cells, as T lymphocytes cultured with CD38-deficient MM1.S cells and Bi38-3 did not proliferate. Furthermore, neither culture with Bi38-3 alone nor MM1.S cells alone induced significant T cell expansion.

(27) Second, we analyzed the expression of CD69 and CD25 early activation markers on donor T cells. Following overnight co-culture with MM1.S cells, CD4 and CD8 T cells readily upregulated both markers in a Bi38-3 dose dependent manner, with up to 80% CD69 positive T cells detected at the highest concentrations (data not shown). In contrast, we observed a weaker percentage of CD25 and CD69 expressing T cells (15% and 30% respectively) upon stimulation with 100 ng/mL of Bi38-3 alone. Furthermore, co-culture with MM1.S target cells alone did not induce expression of activation markers (data not shown). In line with this, co-culture with MM1.SKO cells and Bi38-3 trigger a lower induction of CD69 and CD25 compared to co-cultures with wild type MM1.S cells (data not shown), demonstrating that upregulation of activation markers was enhanced by CD38 expression on target cells

(28) Last, we monitored the production of cytokines triggered by Bi3 8-3. Co-culture of donor T cells with MM1.S triggered production of Interferon-gamma (IFNg), Tumor necrosis factor-alpha (TNFa), Interleukine-2 (IL-2), IL-4 and IL-10 in a Bi38-3 dose dependent manner (data not shown). In contrast, stimulation with Bi38-3 alone or co-culture with MM1.S alone could not induce T cell secretion of any of these cytokines (not shown). Altogether, these results indicate that Bi38-3 directs T cell proliferation, activation and cytokine release in response to CD38 expressing MM cells in vitro.

(29) Bi38-3 Induces CD38-Dependent T-Cell Mediated Killing of MM Cells In Vitro

(30) To assess the function of Bi38-3, we performed co-culture assays to measure the cytotoxic activity of effector T cells, isolated from PBMC of healthy donors, on firefly luciferase expressing target KMS11 and MM1.S MM cell lines. The levels of luciferase, which indicate the number of live MM target cells remaining, were compared to the luciferase observed in untreated controls in order to determine the percentages of killing in the presence of various concentrations of Bi38-3. T cells readily killed KMS11 target cells in a Bi38-3 dose dependent manner, with a half maximal effective concentration (EC.sub.50) around 5 ng/mL, the equivalent of 0.09 nM for this 55.6 Kd protein (data not shown). Bi38-3 mediated T cell cytotoxic activity was also observed in co-culture with MM1.S cells. However, in this cell line, which expresses higher levels of CD38, the EC.sub.50 was tenfold lower (0.5 ng/mL), indicating a stronger efficiency of Bi38-3. In contrast, the viability of MM1.S or KMS11 MM cells was not affected by co-culture with T cells or Bi38-3 alone (data not shown). Furthermore, Bi38-3 induced poor T cell-mediated killing of MM1.S-KO cells, with around half of CD38-deficient MM1.S cells surviving the co-culture even at the highest dose of Bi38-3 (1 g/mL) data not shown). Thus, Bi38-3 directed efficient T-cell cytotoxic activity on CD38 expressing MM cells.

(31) Bi38-3 Induces Autologous T-Cell Mediated Killing of Tumor Plasma Cells In Vitro

(32) We next analyzed the potential of Bi38-3 to induce lysis of MM cells by autologous T cells. Target tumor plasma cells, isolated from patients at diagnosis, were incubated with purified autologous effector T cells in a E:T 1:5 ratio in the presence of various concentrations of Bi38-3. FACS analysis of overnight co-cultures revealed that the numbers of viable CD138 positive MM cells were reduced in a Bi38-3 dose dependent manner, with the EC.sub.50 ranging from 0.5 to 1 ng/mL, depending on the patient (FIG. 1). Importantly, in the absence of T cells, Bi38-3 exhibited no toxicity against fresh primary MM cells. Bi38-3 induced cytotoxicity of autologous T cells was further investigated on tumor plasma cells from MM patients at relapse and demonstrated similar efficacy, with EC.sub.50 ranging from 0.2 to 1 ng/mL (FIG. 1). Thus, in these in vitro experiments, Bi38-3 triggered autologous T cell-mediated killing of tumor plasma cells from patients both at diagnosis and at relapse.

(33) Specific Activity of Bi38-3 Against CD38 Highly Expressing MM Cells In Vitro

(34) While CD38 is highly expressed on plasma cells, it is also expressed on various cell types, including subsets of hematopoietic cells. To investigate the effect of Bi38-3 on blood cells, PBMC from donors were treated with various concentrations of Bi38-3 for 24 hours and the different cells populations were analyzed by FACS (data not shown). We observed that the percentages of CD14 expressing monocytes falling in the live gate were markedly reduced in a Bi38-3 dose dependent manner (data not shown). In contrast, the percentages of CD4 and CD8 T lymphocytes, that together represented around 60% of the PBMC population, slightly increased in response to Bi38-3 as the percentages of CD14 positive cells decreased. Similarly, the B (CD19+) and NK (CD56+) cell populations slightly raised or remained at similar levels (around 10% and 5% respectively), even at high concentrations of Bi38-3 (100 ng/mL) (data not shown). Next, we investigated whether expression of CD38 at the surface of blood cells was impaired by Bi38-3. FACS analysis indicated that CD38 Mean Intensity of fluorescence (MIF) on T, B and NK cells remained similar in cultures containing increasing doses of Bi38-3 (data not shown). In line with this, CD38 expression was not dramatically reduced on CD14+ myeloid cells, although because no or too few cells could be detected, analysis could not be performed at higher doses of Bi38-3 (1 and 100 ng/mL). To compare the activity of Bi38-3 on CD38 high (CD38hi) MM versus CD38 intermediate (CD38int) cells, we performed co-culture assays with MM1.S, expressing high levels of CD38 (data not shown), freshly isolated B cells, expressing intermediate amount of CD38 (data not shown) and autologous T cells. Following an overnight culture, the percentages of viable CD20 positive B cells and CD138 positive MM1.S cells were analyzed by flow cytometry. We observed that the percentages of MM1.S cells dropped at Bi38-3 concentrations of 0.1 ng/mL and this reduction was more dramatic at higher doses (data not shown). In contrast, compared to untreated conditions, the percentages of viable CD20-positive B cells remained unchanged even at high concentrations of Bi38-3 (data not shown).

(35) We developed a similar autologous tri-culture assay to investigate potential toxic effects of Bi38-3 on CD34+ bone marrow hematopoietic progenitors and on regulatory T cells (Treg), which both express low levels of CD38. While Bi38-3 readily induced MM cell killing at low concentrations (10-2 ng/mL and above), we found that it triggered no significant T cell mediated cytotoxicity on Foxp3+ Treg (FIG. 2A). Similarly, there was no significant toxicity on CD34+ hematopoietic progenitors at concentrations below 10 ng/mL and moderate toxicity (>40% survival) at the highest concentrations (FIG. 2B). Altogether, our results indicate that Bi38-3 does not impair the surface expression of CD38 and only triggers T cell mediated killing of cells expressing high levels of CD38 with no or limited toxicity against cells expressing intermediate levels of CD38, such as hematopoietic progenitors, B, T or NK cells.

(36) Altogether, our results indicate that Bi38-3 does not impair the surface expression of CD38 and triggers T cell mediated killing of CD38hi cells without significant activity against CD38int cells.

(37) Bi38-3 Controls MM Cell Expansion In Vivo

(38) The in vivo antitumor activity of Bi38-3 was assessed using a human MM xenograft mouse model. MM1.Sluc cells were injected in the tail vein of the NSG mice and luciferase levels measured by IVIS Imaging System every 4 days. Fourteen days after MM1.S injection, purified human T cells were transplanted I.V. with or without Bi38-3 (0.08 mg/kg). Treatments with Bi38-3 or vehicle were repeated daily for 7 days (FIG. 2A). Eleven days following tumor cell injection, all mice showed similar levels of Radiance (luciferase), indicating that MM cells had effectively engrafted in host animals prior to Bi38-3 treatment (FIG. 2B). While control mice showed rapid tumor progression, all Bi38-3 treated animals displayed a fivefold reduction in tumor growth within the first 4 days of Bi38-3 treatment (FIG. 2C). After 7 days, the level of luciferase expressing MM cells in Bi38-3 treated mice was only one tenth of the initial level and was fifty fold lower than untreated controls. These results indicate that Bi38-3 is efficient to control MM tumor progression in vivo.

(39) Discussion:

(40) We report here the development of Bi38-3, a new anti-CD38/CD3 Bispecific T cell-engager antibody, which triggers specific T-cell mediated lysis of CD38-positive MM cells in vitro, ex vivo and in vivo.

(41) Monoclonal antibodies (Mab) targeting CD38 have shown therapeutic efficiencies in the treatment of MM.sup.13. Daratumumab, an anti-CD38 Mab approved for MM, has shown good therapeutic efficacy, both alone.sup.14 or in combination with normal standard of care regimens.sup.2,15. These clinical data demonstrate that CD38, which is highly expressed on tumor plasma cells, is a target of choice for immune therapies in MM. However, despite marked improved survival rates, many patients treated with Daratumumab eventually relapse because of resistance mechanisms, including FcR dependent down regulation of CD38 on tumor cells as well as inhibition of complement dependent cytotoxicity, antibody-dependent cell mediated cytotoxicity and antibody dependent cellular phagocytosis.sup.16. Bi38-3 lacks the Fc region found on natural immunoglobulins, and recruits cytotoxic T cells through its anti-CD3 scFv without downregulating CD38 expression on target cells (data not shown). Thus, the Bi38-3-mediated T cell killing of MM cells will not be affected by the mechanisms of resistance to anti-CD38 mAbs such as Daratumumab, which are associated with binding of the therapeutic antibody to FcR. Similarly, up regulation of complement inhibitors CD55 and CD59 on cytotoxic cells, that are observed at relapse.sup.17 and are thought to contribute to resistances, should not occur upon Bi38-3 treatment. In addition, MM is characterized by a defective immune system, and standard of care regimens that associate IMIDs and dexamethasone potentially limit the effectiveness of cytotoxic cells. However, our data demonstrate that Bi38-3 mediates autologous T cell mediated killing of tumor plasma cell from patients at diagnosis and at relapse with similar efficiencies (FIG. 1). Together, these data suggest that Bi38-3 could efficiently eliminate MM cells in patients that are resistant to standard treatments, including those that include Daratumumab.

(42) Because CD38 is expressed at the surface of blood cells, including T, B and NK lymphocytes.sup.18, anti-CD38 mAb may potentially target them and impair their functions. Indeed, Daratumumab was shown to eliminate regulatory T cells.sup.19, a process which could be associated with increased T cell numbers and activation during the initiation phase of the treatment.sup.16. Furthermore, Daratumumab treatment results in the depletion of NK cells.sup.20 and could favor the susceptibility of patients to infections.sup.21. Our data show that Bi38-3 has no significant effect on T, B and NK cells in vitro (data not shown). We also report that, even at high doses (10 ng/mL), it readily induced T cell mediated killing of MM cells, while preserving B cells from T cell cytotoxic activity. Interestingly, these results contrast with the activity of AMG424, a recently described anti-CD38 BiTEs, which triggered T cell cytotoxicity on B, T and NK cells in vitro.sup.22. Although additional experiments are required to evaluate the toxicity of Bi38-3 in in vivo models, in particular against myeloid cells, our results suggest that Bi38-3 could efficiently induce elimination of MM cells without impacting cells expressing low levels of CD38.

(43) Recently, bispecific antibodies directed against the Fc receptor-like 5 (Fcrl5 or FcHR5) or the B-cell maturation antigen (BCMA) have been reported.sup.10,12,23. BI 836909, a BiTE targeting BCMA and CD3 was shown to eliminate MM cells in a NCI-H929 mouse xenograft model at a dose of 0.5 mg/Kg.sup.23. Similarly, EM801, an asymmetric bispecific antibody containing a mutated Fc region, efficiently eliminated NCI-H929 cells in immunocompromised mice at the same dose (0.5 mg/Kg).sup.10. Expression of BCMA is restricted to post germinal B cells, including memory B cells and both normal and malignant plasma cells.sup.24. However, although the majority of MM patients express BCMA, 6-9% of cases are negative for this marker and expression levels on tumor plasma cells is heterogeneous among patients.sup.25,26. Furthermore, in MM patients treated with T cells expressing anti-BCMA chimeric antigen receptors, BCMA is downregulated on tumor plasma cells.sup.27, a process that could contribute to tumor escape and relapse. Altogether, these data emphasize the needs to identify and evaluate additional targets in MM. Indeed the development of efficient and safer bi-specific antibody could contribute to improve the treatment of MM.

(44) Our data demonstrate that targeting CD38 with Bi38-3 is efficient in a xenograft model at 0.1 mg/Kg (FIG. 2), a dose significantly lower than the one reported for BCMA bispecific antibodies in similar mouse models.sup.10,23. Thus, Bi-38-3 may represent an attractive therapeutic option in MM cases expressing no or very low BCMA levels.

(45) Although BiTEs have proven their efficacy in several malignancies, their clinical development is hampered by short half-life in patients, requiring continuous infusion via a pump.sup.9. The CD19/CD3 BiTE Blinatumomab was recently approved for treatment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Interestingly, early phase 2 clinical trials demonstrated that the MRD negativity, which is associated with improved survival, may occur at the end of the first cycle of treatment.sup.28,29. While the optimal number of cycles of blinatumomab in the setting of MRD remains to be further studied in ALL, the clinical data demonstrate that limited BiTEs treatments over time may still improve the outcomes of MRD+ patients. In our study, we show that Bi38-3 was able to trigger a six-fold reduction in tumor burden in only 3 days in vivo, despite the use of a highly proliferating MM cell line (MM1.S) (FIG. 2C). Thus, this rapid and pronounced activity on tumor plasma cells suggests that, as with blinatumomab in ALL, Bi38-3 could eliminate MRD in MM patients after limited numbers of cycles and improve outcomes following standard treatments.

(46) In summary, the data presented in this manuscript identify Bi38-3 as a selective and efficient compound in the treatment of MM, that could be used both front line or at relapse, and support further evaluation in MM patients.

EXAMPLE 2: PRODUCTION OF CAR-T CELLS

(47) Methods:

(48) Production of Transduced CAR-T Cells

(49) HEK293 cells were calcium phosphate transfected with 10 g CAR constructs and helper plasmids (psPAX2 and pMD2.G). Twelve hours post transfection, complete medium (DMEM, 10% FVS) was refreshed, and 2 days after transfection, cell-free supernatants containing retroviral particles were collected, concentrated by centrifugation and used for transduction. T cells (purified using Pan T-cell isolation kits from Miltenyi Biotec) were stimulated with CD3/CD28 beads (ThermoFisher) in culture medium (RPMI1640, 10% FBS, penicillin; 100 U/mL, streptomycin; 100 mg/mL). After 16 hours, cells were transferred to retronectin-coated (15 mg/mL) (Takara) 6-well plates (Falcon) and transduced over night with the indicated lentiviral particles. Seventy-two hours post transduction GFP and CAR expression were measured by flow cytometry to determine transduction efficiency. Transduced CAR-T cells represented more than 80% of total cells and were used for in vitro experiments.

(50) Results:

(51) Anti-CD38 CAR-T Cells Trigger MM Cell Lysis In Vitro

(52) Since Bi38-3 induced T cells mediated lysis of MM cells (see EXAMPLE 1), we investigated whether its anti-CD38 scFv could trigger direct cytotocicity of transgenic T cells in the context of a chimeric antigen receptor (CAR). We developed a first generation anti-CD3 8 CAR construct (CAR CD38 1G) containing the BB51 derived anti-CD38 scFv, the hinge and transmembrane regions of human CD8 and the CD3 signaling domain (FIG. 3A). Because the association of the CD3 activation region with costimulatory signaling domains was shown to enhanced the activity of CARs, we also constructed a third generation anti-CD38 CAR (CAR CD38 3G) consisting of the anti-CD38 scFv, the CD28 transmembrane region and the signaling domains of CD28, CD137 (4-1BB) and CD3, in that order (FIG. 3A). As controls, we generated a CAR devoid of scFv domain (CAR Mock) as well as a CAR of co-stimulation (CCR CD38) resembling the CD38 3G but lacking the CD3 signaling domain. The protein sequences corresponding to these constructs are depicted in Table 2. The DNA sequences encoding each CAR were cloned into a lentiviral vector allowing GFP co-expression, in order to produce viral particles and to transduce donor T cells. All CAR constructs were expressed upon transduction on human T lymphocytes (CAR-T), however, CAR CD38 3G was expressed at lower levels than the other constructs (data not shown). Despite this difference, all transduced T cells could be expanded over 2 weeks in vitro with stable expression of the CAR, indicating that potential fratricide effects on CD38 expressing T-cells still allowed CAR-T culture (data not shown). To investigate the cytotoxic function of effector CAR-T cells (E), we performed co-culture experiments with various ratios of luciferase expressing targets cells (T). CD38 expressing MM1.S and RPMI8288 MM cells were readily lysed by both CAR CD38 1G and 3G compared to Mock and CCR CD38 negative controls (FIG. 3B). Even at low E/T ratios (<2.5), anti-CD38 CAR-T killed MM cells, whereas CCR CD38 and Mock transduced T cells displayed poor cytotoxicity. In contrast, CAR CD38 1G and 3G induced no or very little lysis of CD38 negative HEK293 cells (FIG. 3B). Thus, the anti-CD38 scFv derived from the BB51 hybridoma is efficient to drive T-cell cytotoxicity on MINI cells in the context of different CAR constructs.

(53) TABLE-US-00009 TABLE2 Aminoacidsequencesof1.sup.stand3.sup.rdgenerationanti-CD38CARs(CAR CD381Gand3G,respectively)andco-stimulationCAR(CCRCD38). CARCD381G MALPVTALLLPLALLLHAARPDIQMTQSPASLSA LeaderCD8a: SVGETVTITCRASENIYSFLAWYQQKQGKSPQLL MALPVTALLLPLALLLHAARP VYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPE scFvantiCD38 DFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGG GSGGGGSQVQLQQSGAELARPGASVKLSCKASGY TFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYT QKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYY (SEQIDNO:16) CARCD383G MALPVTALLLPLALLLHAARPDIQMTQSPASLSA LeaderCD8a: SVEGETVTITCRASENIYSFLAWYQQKQGKSPQLL MALPVTALLLPLALLLHAARP VYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPE scFvantiCD38 DFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGG GSGGGGSQVQLQQSGAELARPGASVKLSCKASGY 0 TFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYT QKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYY 0 (SEQIDNO:17) CCRCD38 MALPVTALLLPLALLLHAARPDIQMTQSPASLSA LeaderCD8a: SVGETVTITCRASENTYSFLAWYQQKQGKSPQLL MALPVTALLLPLALLLHAARP VYNTKTLTEGVPSRFSGSGSGTQFSLKINNLQPE scFvantiCD38 DFGSYYCQHHYGIPLTFGAGTKLELKGGGGSGGG GSGGGGSQVQLQQSGAELARPGASVKLSCKASGY TFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYT QKFKGKATLTADKSSSTAYMQLSNLTSEDSAVYY (SEQIDNO:18)

REFERENCES

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